Accurate episomal HIV 2-LTR circles quantification using optimized DNA isolation and droplet digital PCR.

Science.gov (United States)

Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; Vandekerckhove, Linos; De Spiegelaere, Ward

2014-01-01

In HIV-infected patients on combination antiretroviral therapy (cART), the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples. We compared two sample processing procedures to more accurately quantify 2-LTR circles using droplet digital PCR (ddPCR). Episomal HIV 2-LTR circles were either isolated by genomic DNA isolation or by a modified plasmid DNA isolation, to separate the small episomal circular DNA from chromosomal DNA. This was performed in a dilution series of HIV-infected cells and HIV-1 infected patient derived samples (n=59). Samples for the plasmid DNA isolation method were spiked with an internal control plasmid. Genomic DNA isolation enables robust 2-LTR circles quantification. However, in the lower ranges of detection, PCR inhibition caused by high genomic DNA load substantially limits the amount of sample input and this impacts sensitivity and accuracy. Moreover, total genomic DNA isolation resulted in a lower recovery of 2-LTR templates per isolate, further reducing its sensitivity. The modified plasmid DNA isolation with a spiked reference for normalization was more accurate in these low ranges compared to genomic DNA isolation. A linear correlation of both methods was observed in the dilution series (R2=0.974) and in the patient derived samples with 2-LTR numbers above 10 copies per million peripheral blood mononuclear cells (PBMCs), (R2=0.671). Furthermore, Bland-Altman analysis revealed an average agreement between the methods within the 27 samples in which 2-LTR circles were detectable with both methods (bias: 0.3875±1.2657 log10). 2-LTR circles quantification in HIV-infected patients proved to be more

Validation of a method for accurate and highly reproducible quantification of brain dopamine transporter SPECT studies

DEFF Research Database (Denmark)

Jensen, Peter S; Ziebell, Morten; Skouboe, Glenna

2011-01-01

In nuclear medicine brain imaging, it is important to delineate regions of interest (ROIs) so that the outcome is both accurate and reproducible. The purpose of this study was to validate a new time-saving algorithm (DATquan) for accurate and reproducible quantification of the striatal dopamine t...... transporter (DAT) with appropriate radioligands and SPECT and without the need for structural brain scanning....

Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions.

Science.gov (United States)

Nakayama, Yuki; Yamaguchi, Hiromi; Einaga, Naoki; Esumi, Mariko

2016-01-01

The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA). Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most accurate and suitable methods for quantifying DNA for quality assessment, we compared three quantification methods: NanoDrop, which measures UV absorbance; Qubit; and quantitative PCR (qPCR), which measures the abundance of a target gene. For the comparison, we used three types of DNA: 1) DNA extracted from fresh frozen liver tissues (Frozen-DNA); 2) DNA extracted from formalin-fixed, paraffin-embedded liver tissues comparable to those used for Frozen-DNA (FFPE-DNA); and 3) DNA extracted from the remaining fractions after RNA extraction with Trizol reagent (Trizol-DNA). These DNAs were serially diluted with distilled water and measured using three quantification methods. For Frozen-DNA, the Qubit values were not proportional to the dilution ratio, in contrast with the NanoDrop and qPCR values. This non-proportional decrease in Qubit values was dependent on a lower salt concentration, and over 1 mM NaCl in the DNA solution was required for the Qubit measurement. For FFPE-DNA, the Qubit values were proportional to the dilution ratio and were lower than the NanoDrop values. However, electrophoresis revealed that qPCR reflected the degree of DNA fragmentation more accurately than Qubit. Thus, qPCR is superior to Qubit for checking the quality of FFPE-DNA. For Trizol-DNA, the Qubit values were proportional to the dilution ratio and were consistently lower than the NanoDrop values, similar to FFPE-DNA. However, the qPCR values were higher than the NanoDrop values. Electrophoresis with SYBR Green I and single-stranded DNA (ssDNA) quantification demonstrated that Trizol-DNA consisted mostly of non-fragmented ssDNA. Therefore, Qubit is not always the most accurate method for

Characterizing the strand-specific distribution of non-CpG methylation in human pluripotent cells.

Science.gov (United States)

Guo, Weilong; Chung, Wen-Yu; Qian, Minping; Pellegrini, Matteo; Zhang, Michael Q

2014-03-01

DNA methylation is an important defense and regulatory mechanism. In mammals, most DNA methylation occurs at CpG sites, and asymmetric non-CpG methylation has only been detected at appreciable levels in a few cell types. We are the first to systematically study the strand-specific distribution of non-CpG methylation. With the divide-and-compare strategy, we show that CHG and CHH methylation are not intrinsically different in human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We also find that non-CpG methylation is skewed between the two strands in introns, especially at intron boundaries and in highly expressed genes. Controlling for the proximal sequences of non-CpG sites, we show that the skew of non-CpG methylation in introns is mainly guided by sequence skew. By studying subgroups of transposable elements, we also found that non-CpG methylation is distributed in a strand-specific manner in both short interspersed nuclear elements (SINE) and long interspersed nuclear elements (LINE), but not in long terminal repeats (LTR). Finally, we show that on the antisense strand of Alus, a non-CpG site just downstream of the A-box is highly methylated. Together, the divide-and-compare strategy leads us to identify regions with strand-specific distributions of non-CpG methylation in humans.

Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion sequences.

Science.gov (United States)

Malik, Afshan N; Czajka, Anna; Cunningham, Phil

2016-07-01

Mitochondria contain an extra-nuclear genome in the form of mitochondrial DNA (MtDNA), damage to which can lead to inflammation and bioenergetic deficit. Changes in MtDNA levels are increasingly used as a biomarker of mitochondrial dysfunction. We previously reported that in humans, fragments in the nuclear genome known as nuclear mitochondrial insertion sequences (NumtS) affect accurate quantification of MtDNA. In the current paper our aim was to determine whether mouse NumtS affect the quantification of MtDNA and to establish a method designed to avoid this. The existence of NumtS in the mouse genome was confirmed using blast N, unique MtDNA regions were identified using FASTA, and MtDNA primers which do not co-amplify NumtS were designed and tested. MtDNA copy numbers were determined in a range of mouse tissues as the ratio of the mitochondrial and nuclear genome using real time qPCR and absolute quantification. Approximately 95% of mouse MtDNA was duplicated in the nuclear genome as NumtS which were located in 15 out of 21 chromosomes. A unique region was identified and primers flanking this region were used. MtDNA levels differed significantly in mouse tissues being the highest in the heart, with levels in descending order (highest to lowest) in kidney, liver, blood, brain, islets and lung. The presence of NumtS in the nuclear genome of mouse could lead to erroneous data when studying MtDNA content or mutation. The unique primers described here will allow accurate quantification of MtDNA content in mouse models without co-amplification of NumtS. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Quantification and genome-wide mapping of DNA double-strand breaks.

Science.gov (United States)

Grégoire, Marie-Chantal; Massonneau, Julien; Leduc, Frédéric; Arguin, Mélina; Brazeau, Marc-André; Boissonneault, Guylain

2016-12-01

DNA double-strand breaks (DSBs) represent a major threat to the genetic integrity of the cell. Knowing both their genome-wide distribution and number is important for a better assessment of genotoxicity at a molecular level. Available methods may have underestimated the extent of DSBs as they are based on markers specific to those undergoing active repair or may not be adapted for the large diversity of naturally occurring DNA ends. We have established conditions for an efficient first step of DNA nick and gap repair (NGR) allowing specific determination of DSBs by end labeling with terminal transferase. We used DNA extracted from HeLa cells harboring an I-SceI cassette to induce a targeted nick or DSB and demonstrated by immunocapture of 3'-OH that a prior step of NGR allows specific determination of loci-specific or genome wide DSBs. This method can be applied to the global determination of DSBs using radioactive end labeling and can find several applications aimed at understanding the distribution and kinetics of DSBs formation and repair. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of 3D PET systems for accurate quantification of myocardial blood flow

OpenAIRE

Renaud, Jennifer M.; Yip, Kathy; Guimond, Jean; Trottier, Mikaël; Pibarot, Philippe; Turcotte, Éric; Maguire, Conor; Lalonde, Lucille; Gulenchyn, Karen; Farncombe, Troy; Wisenberg, Gerald; Moody, Jonathan; Lee, Benjamin; Port, Steven C.; Turkington, Timothy G

2016-01-01

Three-dimensional (3D) mode imaging is the current standard for positron emission tomography-computed tomography (PET-CT) systems. Dynamic imaging for quantification of myocardial blood flow (MBF) with short-lived tracers, such as Rb-82- chloride (Rb-82), requires accuracy to be maintained over a wide range of isotope activities and scanner count-rates. We propose new performance standard measurements to characterize the dynamic range of PET systems for accurate quantitative...

Accurate Quantification of Cardiovascular Biomarkers in Serum Using Protein Standard Absolute Quantification (PSAQ™) and Selected Reaction Monitoring*

Science.gov (United States)

Huillet, Céline; Adrait, Annie; Lebert, Dorothée; Picard, Guillaume; Trauchessec, Mathieu; Louwagie, Mathilde; Dupuis, Alain; Hittinger, Luc; Ghaleh, Bijan; Le Corvoisier, Philippe; Jaquinod, Michel; Garin, Jérôme; Bruley, Christophe; Brun, Virginie

2012-01-01

Development of new biomarkers needs to be significantly accelerated to improve diagnostic, prognostic, and toxicity monitoring as well as therapeutic follow-up. Biomarker evaluation is the main bottleneck in this development process. Selected Reaction Monitoring (SRM) combined with stable isotope dilution has emerged as a promising option to speed this step, particularly because of its multiplexing capacities. However, analytical variabilities because of upstream sample handling or incomplete trypsin digestion still need to be resolved. In 2007, we developed the PSAQ™ method (Protein Standard Absolute Quantification), which uses full-length isotope-labeled protein standards to quantify target proteins. In the present study we used clinically validated cardiovascular biomarkers (LDH-B, CKMB, myoglobin, and troponin I) to demonstrate that the combination of PSAQ and SRM (PSAQ-SRM) allows highly accurate biomarker quantification in serum samples. A multiplex PSAQ-SRM assay was used to quantify these biomarkers in clinical samples from myocardial infarction patients. Good correlation between PSAQ-SRM and ELISA assay results was found and demonstrated the consistency between these analytical approaches. Thus, PSAQ-SRM has the capacity to improve both accuracy and reproducibility in protein analysis. This will be a major contribution to efficient biomarker development strategies. PMID:22080464

Accurate joint space quantification in knee osteoarthritis: a digital x-ray tomosynthesis phantom study

Science.gov (United States)

Sewell, Tanzania S.; Piacsek, Kelly L.; Heckel, Beth A.; Sabol, John M.

2011-03-01

The current imaging standard for diagnosis and monitoring of knee osteoarthritis (OA) is projection radiography. However radiographs may be insensitive to markers of early disease such as osteophytes and joint space narrowing (JSN). Relative to standard radiography, digital X-ray tomosynthesis (DTS) may provide improved visualization of the markers of knee OA without the interference of superimposed anatomy. DTS utilizes a series of low-dose projection images over an arc of +/-20 degrees to reconstruct tomographic images parallel to the detector. We propose that DTS can increase accuracy and precision in JSN quantification. The geometric accuracy of DTS was characterized by quantifying joint space width (JSW) as a function of knee flexion and position using physical and anthropomorphic phantoms. Using a commercially available digital X-ray system, projection and DTS images were acquired for a Lucite rod phantom with known gaps at various source-object-distances, and angles of flexion. Gap width, representative of JSW, was measured using a validated algorithm. Over an object-to-detector-distance range of 5-21cm, a 3.0mm gap width was reproducibly measured in the DTS images, independent of magnification. A simulated 0.50mm (+/-0.13) JSN was quantified accurately (95% CI 0.44-0.56mm) in the DTS images. Angling the rods to represent knee flexion, the minimum gap could be precisely determined from the DTS images and was independent of flexion angle. JSN quantification using DTS was insensitive to distance from patient barrier and flexion angle. Potential exists for the optimization of DTS for accurate radiographic quantification of knee OA independent of patient positioning.

A robust computational solution for automated quantification of a specific binding ratio based on [123I]FP-CIT SPECT images

International Nuclear Information System (INIS)

Oliveira, F. P. M.; Tavares, J. M. R. S.; Borges, Faria D.; Campos, Costa D.

2014-01-01

The purpose of the current paper is to present a computational solution to accurately quantify a specific to a non-specific uptake ratio in [ 123 I]fP-CIT single photon emission computed tomography (SPECT) images and simultaneously measure the spatial dimensions of the basal ganglia, also known as basal nuclei. A statistical analysis based on a reference dataset selected by the user is also automatically performed. The quantification of the specific to non-specific uptake ratio here is based on regions of interest defined after the registration of the image under study with a template image. The computational solution was tested on a dataset of 38 [ 123 I]FP-CIT SPECT images: 28 images were from patients with Parkinson’s disease and the remainder from normal patients, and the results of the automated quantification were compared to the ones obtained by three well-known semi-automated quantification methods. The results revealed a high correlation coefficient between the developed automated method and the three semi-automated methods used for comparison (r ≥0.975). The solution also showed good robustness against different positions of the patient, as an almost perfect agreement between the specific to non-specific uptake ratio was found (ICC=1.000). The mean processing time was around 6 seconds per study using a common notebook PC. The solution developed can be useful for clinicians to evaluate [ 123 I]FP-CIT SPECT images due to its accuracy, robustness and speed. Also, the comparison between case studies and the follow-up of patients can be done more accurately and proficiently since the intra- and inter-observer variability of the semi-automated calculation does not exist in automated solutions. The dimensions of the basal ganglia and their automatic comparison with the values of the population selected as reference are also important for professionals in this area.

Sequence-specific RNA Photocleavage by Single-stranded DNA in Presence of Riboflavin

Science.gov (United States)

Zhao, Yongyun; Chen, Gangyi; Yuan, Yi; Li, Na; Dong, Juan; Huang, Xin; Cui, Xin; Tang, Zhuo

2015-10-01

Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecue, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G•U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G•U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation.

Accurate Digital Polymerase Chain Reaction Quantification of Challenging Samples Applying Inhibitor-Tolerant DNA Polymerases.

Science.gov (United States)

Sidstedt, Maja; Romsos, Erica L; Hedell, Ronny; Ansell, Ricky; Steffen, Carolyn R; Vallone, Peter M; Rådström, Peter; Hedman, Johannes

2017-02-07

Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning of the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentration is determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrixes such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. In this study, we evaluated the impact of varying the DNA polymerase in chamber-based dPCR for both pure and impure samples using the common PCR inhibitor humic acid (HA) as a model. We compared the TaqMan Universal PCR Master Mix with two alternative DNA polymerases: ExTaq HS and Immolase. By using Bayesian modeling, we show that there is no difference among the tested DNA polymerases in terms of accuracy of absolute quantification for pure template samples, i.e., without HA present. For samples containing HA, there were great differences in performance: the TaqMan Universal PCR Master Mix failed to correctly quantify DNA with more than 13 pg/nL HA, whereas Immolase (1 U) could handle up to 375 pg/nL HA. Furthermore, we found that BSA had a moderate positive effect for the TaqMan Universal PCR Master Mix, enabling accurate quantification for 25 pg/nL HA. Increasing the amount of DNA polymerase from 1 to 5 U had a strong effect for ExTaq HS, elevating HA-tolerance four times. We also show that the average Cq values of positive reactions may be used as a measure of inhibition effects, e.g., to determine whether or not a dPCR quantification result is reliable. The statistical models developed to objectively analyze the data may also be applied in quality control. We conclude that the choice of DNA polymerase in dPCR is crucial for the accuracy of quantification when analyzing challenging samples.

Development of salt-tolerance interface for an high performance liquid chromatography/inductively coupled plasma mass spectrometry system and its application to accurate quantification of DNA samples.

Science.gov (United States)

Takasaki, Yuka; Sakagawa, Shinnosuke; Inagaki, Kazumi; Fujii, Shin-Ichiro; Sabarudin, Akhmad; Umemura, Tomonari; Haraguchi, Hiroki

2012-02-03

Accurate quantification of DNA is highly important in various fields. Determination of phosphorus by ICP-MS is one of the most effective methods for accurate quantification of DNA due to the fixed stoichiometry of phosphate to this molecule. In this paper, a smart and reliable method for accurate quantification of DNA fragments and oligodeoxythymidilic acids by hyphenated HPLC/ICP-MS equipped with a highly efficient interface device is presented. The interface was constructed of a home-made capillary-attached micronebulizer and temperature-controllable cyclonic spray chamber (IsoMist). As a separation column for DNA samples, home-made methacrylate-based weak anion-exchange monolith was employed. Some parameters, which include composition of mobile phase, gradient program, inner and outer diameters of capillary, temperature of spray chamber etc., were optimized to find the best performance for separation and accurate quantification of DNA samples. The proposed system could achieve many advantages, such as total consumption for small amount sample analysis, salt-tolerance for hyphenated analysis, high accuracy and precision for quantitative analysis. Using this proposed system, the samples of 20 bp DNA ladder (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400, 500 base pairs) and oligodeoxythymidilic acids (dT(12-18)) were rapidly separated and accurately quantified. Copyright © 2011 Elsevier B.V. All rights reserved.

Enzymatic quantification of strand breaks of DNA induced by vacuum-UV radiation

International Nuclear Information System (INIS)

Ito, Takashi

1986-01-01

Hind3 digested plasmid DNA dried on an aluminum plate was irradiated by vacuum-UV at 160 and 195 nm using a synchrotron irradiation system. A change induced in the DNA, presumably a single strand break, was quantified by the aid of the strand break-derived stimulation of poly(ADP-ribose) synthetase activity. The end group of strand breaks so induced was recognized by the enzyme as effectively as that by DNase 1 treatment, suggesting a nicking as the major lesion inflicted on the DNA. The fluence (UV) dependent stimulation of poly(ADP-ribose) synthetase activity was much higher upon 160 nm irradiation than upon 195 nm irradiation. (Auth.)

AtRTD2: A Reference Transcript Dataset for accurate quantification of alternative splicing and expression changes in Arabidopsis thaliana RNA-seq data

KAUST Repository

Zhang, Runxuan

2016-05-06

Background Alternative splicing is the major post-transcriptional mechanism by which gene expression is regulated and affects a wide range of processes and responses in most eukaryotic organisms. RNA-sequencing (RNA-seq) can generate genome-wide quantification of individual transcript isoforms to identify changes in expression and alternative splicing. RNA-seq is an essential modern tool but its ability to accurately quantify transcript isoforms depends on the diversity, completeness and quality of the transcript information. Results We have developed a new Reference Transcript Dataset for Arabidopsis (AtRTD2) for RNA-seq analysis containing over 82k non-redundant transcripts, whereby 74,194 transcripts originate from 27,667 protein-coding genes. A total of 13,524 protein-coding genes have at least one alternatively spliced transcript in AtRTD2 such that about 60% of the 22,453 protein-coding, intron-containing genes in Arabidopsis undergo alternative splicing. More than 600 putative U12 introns were identified in more than 2,000 transcripts. AtRTD2 was generated from transcript assemblies of ca. 8.5 billion pairs of reads from 285 RNA-seq data sets obtained from 129 RNA-seq libraries and merged along with the previous version, AtRTD, and Araport11 transcript assemblies. AtRTD2 increases the diversity of transcripts and through application of stringent filters represents the most extensive and accurate transcript collection for Arabidopsis to date. We have demonstrated a generally good correlation of alternative splicing ratios from RNA-seq data analysed by Salmon and experimental data from high resolution RT-PCR. However, we have observed inaccurate quantification of transcript isoforms for genes with multiple transcripts which have variation in the lengths of their UTRs. This variation is not effectively corrected in RNA-seq analysis programmes and will therefore impact RNA-seq analyses generally. To address this, we have tested different genome

Identification and accurate quantification of structurally related peptide impurities in synthetic human C-peptide by liquid chromatography-high resolution mass spectrometry.

Science.gov (United States)

Li, Ming; Josephs, Ralf D; Daireaux, Adeline; Choteau, Tiphaine; Westwood, Steven; Wielgosz, Robert I; Li, Hongmei

2018-06-04

Peptides are an increasingly important group of biomarkers and pharmaceuticals. The accurate purity characterization of peptide calibrators is critical for the development of reference measurement systems for laboratory medicine and quality control of pharmaceuticals. The peptides used for these purposes are increasingly produced through peptide synthesis. Various approaches (for example mass balance, amino acid analysis, qNMR, and nitrogen determination) can be applied to accurately value assign the purity of peptide calibrators. However, all purity assessment approaches require a correction for structurally related peptide impurities in order to avoid biases. Liquid chromatography coupled to high resolution mass spectrometry (LC-hrMS) has become the key technique for the identification and accurate quantification of structurally related peptide impurities in intact peptide calibrator materials. In this study, LC-hrMS-based methods were developed and validated in-house for the identification and quantification of structurally related peptide impurities in a synthetic human C-peptide (hCP) material, which served as a study material for an international comparison looking at the competencies of laboratories to perform peptide purity mass fraction assignments. More than 65 impurities were identified, confirmed, and accurately quantified by using LC-hrMS. The total mass fraction of all structurally related peptide impurities in the hCP study material was estimated to be 83.3 mg/g with an associated expanded uncertainty of 3.0 mg/g (k = 2). The calibration hierarchy concept used for the quantification of individual impurities is described in detail. Graphical abstract ᅟ.

Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.

Science.gov (United States)

Lock, Martin; Alvira, Mauricio R; Chen, Shu-Jen; Wilson, James M

2014-04-01

Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer-probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution

Fluorescent quantification of melanin.

Science.gov (United States)

Fernandes, Bruno; Matamá, Teresa; Guimarães, Diana; Gomes, Andreia; Cavaco-Paulo, Artur

2016-11-01

Melanin quantification is reportedly performed by absorption spectroscopy, commonly at 405 nm. Here, we propose the implementation of fluorescence spectroscopy for melanin assessment. In a typical in vitro assay to assess melanin production in response to an external stimulus, absorption spectroscopy clearly overvalues melanin content. This method is also incapable of distinguishing non-melanotic/amelanotic control cells from those that are actually capable of performing melanogenesis. Therefore, fluorescence spectroscopy is the best method for melanin quantification as it proved to be highly specific and accurate, detecting even small variations in the synthesis of melanin. This method can also be applied to the quantification of melanin in more complex biological matrices like zebrafish embryos and human hair. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Development of a Novel Reference Plasmid for Accurate Quantification of Genetically Modified Kefeng6 Rice DNA in Food and Feed Samples

Directory of Open Access Journals (Sweden)

Liang Li

2013-01-01

Full Text Available Reference plasmids are an essential tool for the quantification of genetically modified (GM events. Quantitative real-time PCR (qPCR is the most commonly used method to characterize and quantify reference plasmids. However, the precision of this method is often limited by calibration curves, and qPCR data can be affected by matrix differences between the standards and samples. Here, we describe a digital PCR (dPCR approach that can be used to accurately measure the novel reference plasmid pKefeng6 and quantify the unauthorized variety of GM rice Kefeng6, eliminating the issues associated with matrix effects in calibration curves. The pKefeng6 plasmid was used as a calibrant for the quantification of Kefeng6 rice by determining the copy numbers of event- (77 bp and taxon-specific (68 bp fragments, their ratios, and their concentrations. The plasmid was diluted to five different concentrations. The third sample (S3 was optimized for the quantification range of dPCR according to previous reports. The ratio between the two fragments was 1.005, which closely approximated the value certified by sequencing, and the concentration was found to be 792 copies/μL. This method was precise, with an RSD of ~3%. These findings demonstrate the advantages of using the dPCR method to characterize reference materials.

Decision peptide-driven: a free software tool for accurate protein quantification using gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry.

Science.gov (United States)

Santos, Hugo M; Reboiro-Jato, Miguel; Glez-Peña, Daniel; Nunes-Miranda, J D; Fdez-Riverola, Florentino; Carvallo, R; Capelo, J L

2010-09-15

The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse (18)O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse (18)O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse (18)O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using (18)O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Molecular characterization of a complex site-specific radiation-induced DNA double-strand break

International Nuclear Information System (INIS)

Datta, K.; Dizdaroglu, M.; Jaruga, P.; Neumann, R.D.; Winters, T.A.

2003-01-01

Radiation lethality is a function of radiation-induced DNA double-strand breaks (DSB). Current models propose the lethality of a DSB to be a function of its structural complexity. We present here for the first time a map of damage associated with a site-specific double-strand break produced by decay of 125 I in a plasmid bound by a 125 I-labeled triplex forming oligonucleotide ( 125 I-TFO). The E. coli DNA repair enzymes, endonuclease IV (endo IV), endonuclease III (endo III), and formamidopyrimidine-DNA glycosylase (Fpg), which recognize AP sites, and pyrimidine and purine base damage respectively, were used as probes in this study. 125 I-TFO bound plasmid was incubated with and without DMSO at -80 deg C for 1 month. No significant difference in DSB yield was observed under these conditions. A 32 base pair fragment from the upstream side of the decay site was isolated by restriction digestion and enzymatically probed to identify damage sites. Endo IV treatment of the 5'-end labeled upper strand indicated clustering of AP sites within 3 bases downstream and 7 bases upstream of the targeted base. Also, repeated experiments consistently detected an AP site 4 bases upstream of the 125 Itarget base. This was further supported by complementary results with the 3'-end labeled upper strand. Endo IV analysis of the lower strand also shows clustering of AP sites near the DSB end. Endo III and Fpg probing demonstrated that base damage is also clustered near the targeted break site. DSBs produced in the absence of DMSO displayed a different pattern of enzyme sensitive damage than those produced in the presence of DMSO. Identification of specific base damage types within the restriction fragment containing the DSB end was achieved with GC/MS. Base damage consisted of 8-hydroguanine, 8-hydroxyadenine, and 5-hydroxycytosine. These lesions were observed at relative yields of 8-hydroguanine and 5-hydroxycytosine to 8-hydroxyadenine of 7.4:1 and 4.7:1, respectively, in the absence

Fragmentation in DNA double-strand breaks

International Nuclear Information System (INIS)

Wei Zhiyong; Suzhou Univ., Suzhou; Zhang Lihui; Li Ming; Fan Wo; Xu Yujie

2005-01-01

DNA double strand breaks are important lesions induced by irradiations. Random breakage model or quantification supported by this concept is suitable to analyze DNA double strand break data induced by low LET radiation, but deviation from random breakage model is more evident in high LET radiation data analysis. In this work we develop a new method, statistical fragmentation model, to analyze the fragmentation process of DNA double strand breaks. After charged particles enter the biological cell, they produce ionizations along their tracks, and transfer their energies to the cells and break the cellular DNA strands into fragments. The probable distribution of the fragments is obtained under the condition in which the entropy is maximum. Under the approximation E≅E 0 + E 1 l + E 2 l 2 , the distribution functions are obtained as exp(αl + βl 2 ). There are two components, the one proportional to exp(βl 2 ), mainly contributes to the low mass fragment yields, the other component, proportional to exp(αl), decreases slowly as the mass of the fragments increases. Numerical solution of the constraint equations provides parameters α and β. Experimental data, especially when the energy deposition is higher, support the statistical fragmentation model. (authors)

Arranging eukaryotic nuclear DNA polymerases for replication: Specific interactions with accessory proteins arrange Pols α, δ, and ϵ in the replisome for leading-strand and lagging-strand DNA replication.

Science.gov (United States)

Kunkel, Thomas A; Burgers, Peter M J

2017-08-01

Biochemical and cryo-electron microscopy studies have just been published revealing interactions among proteins of the yeast replisome that are important for highly coordinated synthesis of the two DNA strands of the nuclear genome. These studies reveal key interactions important for arranging DNA polymerases α, δ, and ϵ for leading and lagging strand replication. The CMG (Mcm2-7, Cdc45, GINS) helicase is central to this interaction network. These are but the latest examples of elegant studies performed in the recent past that lead to a much better understanding of how the eukaryotic replication fork achieves efficient DNA replication that is accurate enough to prevent diseases yet allows evolution. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Simple and accurate quantification of BTEX in ambient air by SPME and GC-MS.

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Baimatova, Nassiba; Kenessov, Bulat; Koziel, Jacek A; Carlsen, Lars; Bektassov, Marat; Demyanenko, Olga P

2016-07-01

Benzene, toluene, ethylbenzene and xylenes (BTEX) comprise one of the most ubiquitous and hazardous groups of ambient air pollutants of concern. Application of standard analytical methods for quantification of BTEX is limited by the complexity of sampling and sample preparation equipment, and budget requirements. Methods based on SPME represent simpler alternative, but still require complex calibration procedures. The objective of this research was to develop a simpler, low-budget, and accurate method for quantification of BTEX in ambient air based on SPME and GC-MS. Standard 20-mL headspace vials were used for field air sampling and calibration. To avoid challenges with obtaining and working with 'zero' air, slope factors of external standard calibration were determined using standard addition and inherently polluted lab air. For polydimethylsiloxane (PDMS) fiber, differences between the slope factors of calibration plots obtained using lab and outdoor air were below 14%. PDMS fiber provided higher precision during calibration while the use of Carboxen/PDMS fiber resulted in lower detection limits for benzene and toluene. To provide sufficient accuracy, the use of 20mL vials requires triplicate sampling and analysis. The method was successfully applied for analysis of 108 ambient air samples from Almaty, Kazakhstan. Average concentrations of benzene, toluene, ethylbenzene and o-xylene were 53, 57, 11 and 14µgm(-3), respectively. The developed method can be modified for further quantification of a wider range of volatile organic compounds in air. In addition, the new method is amenable to automation. Copyright © 2016 Elsevier B.V. All rights reserved.

The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast.

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Hartono, Stella R; Malapert, Amélie; Legros, Pénélope; Bernard, Pascal; Chédin, Frédéric; Vanoosthuyse, Vincent

2018-02-02

R-loops, which result from the formation of stable DNA:RNA hybrids, can both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loops in fission yeast, we used the S9.6 antibody-based DRIPc-seq method to sequence the RNA strand of R-loops and obtain strand-specific R-loop maps at near nucleotide resolution. Surprisingly, preliminary DRIPc-seq experiments identified mostly RNase H-resistant but exosome-sensitive RNAs that mapped to both DNA strands and resembled RNA:RNA hybrids (dsRNAs), suggesting that dsRNAs form widely in fission yeast. We confirmed in vitro that S9.6 can immuno-precipitate dsRNAs and provide evidence that dsRNAs can interfere with its binding to R-loops. dsRNA elimination by RNase III treatment prior to DRIPc-seq allowed the genome-wide and strand-specific identification of genuine R-loops that responded in vivo to RNase H levels and displayed classical features associated with R-loop formation. We also found that most transcripts whose levels were altered by in vivo manipulation of RNase H levels did not form detectable R-loops, suggesting that prolonged manipulation of R-loop levels could indirectly alter the transcriptome. We discuss the implications of our work in the design of experimental strategies to probe R-loop functions. Copyright © 2017 Elsevier Ltd. All rights reserved.

A nuclear DNA-based species determination and DNA quantification assay for common poultry species.

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Ng, J; Satkoski, J; Premasuthan, A; Kanthaswamy, S

2014-12-01

DNA testing for food authentication and quality control requires sensitive species-specific quantification of nuclear DNA from complex and unknown biological sources. We have developed a multiplex assay based on TaqMan® real-time quantitative PCR (qPCR) for species-specific detection and quantification of chicken (Gallus gallus), duck (Anas platyrhynchos), and turkey (Meleagris gallopavo) nuclear DNA. The multiplex assay is able to accurately detect very low quantities of species-specific DNA from single or multispecies sample mixtures; its minimum effective quantification range is 5 to 50 pg of starting DNA material. In addition to its use in food fraudulence cases, we have validated the assay using simulated forensic sample conditions to demonstrate its utility in forensic investigations. Despite treatment with potent inhibitors such as hematin and humic acid, and degradation of template DNA by DNase, the assay was still able to robustly detect and quantify DNA from each of the three poultry species in mixed samples. The efficient species determination and accurate DNA quantification will help reduce fraudulent food labeling and facilitate downstream DNA analysis for genetic identification and traceability.

A specific subdomain in φ29 DNA polymerase confers both processivity and strand-displacement capacity

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Rodríguez, Irene; Lázaro, José M.; Blanco, Luis; Kamtekar, Satwik; Berman, Andrea J.; Wang, Jimin; Steitz, Thomas A.; Salas, Margarita; de Vega, Miguel

2005-01-01

Recent crystallographic studies of φ29 DNA polymerase have provided structural insights into its strand displacement and processivity. A specific insertion named terminal protein region 2 (TPR2), present only in protein-primed DNA polymerases, together with the exonuclease, thumb, and palm subdomains, forms two tori capable of interacting with DNA. To analyze the functional role of this insertion, we constructed a φ29 DNA polymerase deletion mutant lacking TPR2 amino acid residues Asp-398 to Glu-420. Biochemical analysis of the mutant DNA polymerase indicates that its DNA-binding capacity is diminished, drastically decreasing its processivity. In addition, removal of the TPR2 insertion abolishes the intrinsic capacity of φ29 DNA polymerase to perform strand displacement coupled to DNA synthesis. Therefore, the biochemical results described here directly demonstrate that TPR2 plays a critical role in strand displacement and processivity. PMID:15845765

A strand-specific RNA-Seq analysis of the transcriptome of the typhoid bacillus Salmonella typhi.

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Timothy T Perkins

2009-07-01

Full Text Available High-density, strand-specific cDNA sequencing (ssRNA-seq was used to analyze the transcriptome of Salmonella enterica serovar Typhi (S. Typhi. By mapping sequence data to the entire S. Typhi genome, we analyzed the transcriptome in a strand-specific manner and further defined transcribed regions encoded within prophages, pseudogenes, previously un-annotated, and 3'- or 5'-untranslated regions (UTR. An additional 40 novel candidate non-coding RNAs were identified beyond those previously annotated. Proteomic analysis was combined with transcriptome data to confirm and refine the annotation of a number of hpothetical genes. ssRNA-seq was also combined with microarray and proteome analysis to further define the S. Typhi OmpR regulon and identify novel OmpR regulated transcripts. Thus, ssRNA-seq provides a novel and powerful approach to the characterization of the bacterial transcriptome.

Accurate quantification of tio2 nanoparticles collected on air filters using a microwave-assisted acid digestion method

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Mudunkotuwa, Imali A.; Anthony, T. Renée; Grassian, Vicki H.; Peters, Thomas M.

2016-01-01

Titanium dioxide (TiO2) particles, including nanoparticles with diameters smaller than 100 nm, are used extensively in consumer products. In a 2011 current intelligence bulletin, the National Institute of Occupational Safety and Health (NIOSH) recommended methods to assess worker exposures to fine and ultrafine TiO2 particles and associated occupational exposure limits for these particles. However, there are several challenges and problems encountered with these recommended exposure assessment methods involving the accurate quantitation of titanium dioxide collected on air filters using acid digestion followed by inductively coupled plasma optical emission spectroscopy (ICP-OES). Specifically, recommended digestion methods include the use of chemicals, such as perchloric acid, which are typically unavailable in most accredited industrial hygiene laboratories due to highly corrosive and oxidizing properties. Other alternative methods that are used typically involve the use of nitric acid or combination of nitric acid and sulfuric acid, which yield very poor recoveries for titanium dioxide. Therefore, given the current state of the science, it is clear that a new method is needed for exposure assessment. In this current study, a microwave-assisted acid digestion method has been specifically designed to improve the recovery of titanium in TiO2 nanoparticles for quantitative analysis using ICP-OES. The optimum digestion conditions were determined by changing several variables including the acids used, digestion time, and temperature. Consequently, the optimized digestion temperature of 210°C with concentrated sulfuric and nitric acid (2:1 v/v) resulted in a recovery of >90% for TiO2. The method is expected to provide for a more accurate quantification of airborne TiO2 particles in the workplace environment. PMID:26181824

Attomolar detection of proteins via cascade strand-displacement amplification and polystyrene nanoparticle enhancement in fluorescence polarization aptasensors.

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Huang, Yong; Liu, Xiaoqian; Huang, Huakui; Qin, Jian; Zhang, Liangliang; Zhao, Shulin; Chen, Zhen-Feng; Liang, Hong

2015-08-18

Extremely sensitive and accurate measurements of protein markers for early detection and monitoring of diseases pose a formidable challenge. Herein, we develop a new type of amplified fluorescence polarization (FP) aptasensor based on allostery-triggered cascade strand-displacement amplification (CSDA) and polystyrene nanoparticle (PS NP) enhancement for ultrasensitive detection of proteins. The assay system consists of a fluorescent dye-labeled aptamer hairpin probe and a PS NP-modified DNA duplex (assistant DNA/trigger DNA duplex) probe with a single-stranded part and DNA polymerase. Two probes coexist stably in the absence of target, and the dye exhibits relatively low FP background. Upon recognition and binding with a target protein, the stem of the aptamer hairpin probe is opened, after which the opened hairpin probe hybridizes with the single-stranded part in the PS NP-modified DNA duplex probe and triggers the CSDA reaction through the polymerase-catalyzed recycling of both target protein and trigger DNA. Throughout this CSDA process, numerous massive dyes are assembled onto PS NPs, which results in a substantial FP increase that provides a readout signal for the amplified sensing process. Our newly proposed amplified FP aptasensor enables the quantitative measurement of proteins with the detection limit in attomolar range, which is about 6 orders of magnitude lower than that of traditional homogeneous aptasensors. Moreover, this sensing method also exhibits high specificity for target proteins and can be performed in homogeneous solutions. In addition, the suitability of this method for the quantification of target protein in biological samples has also been shown. Considering these distinct advantages, the proposed sensing method can be expected to provide an ultrasensitive platform for the analysis of various types of target molecules.

Mechanical properties of rubberwood oriented strand lumber (OSL: The effect of strand length

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Buhnnum Kyokong

2005-09-01

Full Text Available Effect of strand length on mechanical properties (tension, compression and bending of oriented strand lumber (OSL made of rubberwood (Hevea brasiliensis Muell. Arg. was reported. Three strand lengths of 50 mm, 100 mm, and 150 mm with 1 mm thickness and 15 mm width were used. The strands were mixed with 5% pMDI glue (weight basis in a tumble mixer. The OSL specimens were formed by hot pressing process of unidirectionally aligned strands. Average specific gravity and moisture content were 0.76 and 8.34%, respectively. Tension and compression tests were carried out for directions both parallel and perpendicular to grain while bending test was performed only in parallel direction. Ultimate stresses and moduli of elasticity were examined from the stress-strain curves. It was found that for the parallel-to-grain direction, the longer strand OSL gave higher strength. The role of the strand length did not appear for the direction normal to the grain. The relationship between the mechanical properties of OSL and strand length was well described by the modified Hankinson formula.

Differential Regulation of Strand-Specific Transcripts from Arabidopsis Centromeric Satellite Repeats.

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2005-12-01

Full Text Available Centromeres interact with the spindle apparatus to enable chromosome disjunction and typically contain thousands of tandemly arranged satellite repeats interspersed with retrotransposons. While their role has been obscure, centromeric repeats are epigenetically modified and centromere specification has a strong epigenetic component. In the yeast Schizosaccharomyces pombe, long heterochromatic repeats are transcribed and contribute to centromere function via RNA interference (RNAi. In the higher plant Arabidopsis thaliana, as in mammalian cells, centromeric satellite repeats are short (180 base pairs, are found in thousands of tandem copies, and are methylated. We have found transcripts from both strands of canonical, bulk Arabidopsis repeats. At least one subfamily of 180-base pair repeats is transcribed from only one strand and regulated by RNAi and histone modification. A second subfamily of repeats is also silenced, but silencing is lost on both strands in mutants in the CpG DNA methyltransferase MET1, the histone deacetylase HDA6/SIL1, or the chromatin remodeling ATPase DDM1. This regulation is due to transcription from Athila2 retrotransposons, which integrate in both orientations relative to the repeats, and differs between strains of Arabidopsis. Silencing lost in met1 or hda6 is reestablished in backcrosses to wild-type, but silencing lost in RNAi mutants and ddm1 is not. Twenty-four-nucleotide small interfering RNAs from centromeric repeats are retained in met1 and hda6, but not in ddm1, and may have a role in this epigenetic inheritance. Histone H3 lysine-9 dimethylation is associated with both classes of repeats. We propose roles for transcribed repeats in the epigenetic inheritance and evolution of centromeres.

siRNA-like double-stranded RNAs are specifically protected against degradation in human cell extract.

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John A H Hoerter

Full Text Available RNA interference (RNAi is a set of intracellular pathways in eukaryotes that controls both exogenous and endogenous gene expression. The power of RNAi to knock down (silence any gene of interest by the introduction of synthetic small-interfering (siRNAs has afforded powerful insight into biological function through reverse genetic approaches and has borne a new field of gene therapeutics. A number of questions are outstanding concerning the potency of siRNAs, necessitating an understanding of how short double-stranded RNAs are processed by the cell. Recent work suggests unmodified siRNAs are protected in the intracellular environment, although the mechanism of protection still remains unclear. We have developed a set of doubly-fluorophore labeled RNAs (more precisely, RNA/DNA chimeras to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET. We find that these RNA probes are substrates for relevant cellular degradative processes, including the RNase H1 mediated degradation of an DNA/RNA hybrid and Dicer-mediated cleavage of a 24-nucleotide (per strand double-stranded RNA. In addition, we find that 21- and 24-nucleotide double-stranded RNAs are relatively protected in human cytosolic cell extract, but less so in blood serum, whereas an 18-nucleotide double-stranded RNA is less protected in both fluids. These results suggest that RNAi effector RNAs are specifically protected in the cellular environment and may provide an explanation for recent results showing that unmodified siRNAs in cells persist intact for extended periods of time.

Site- and strand-specific nicking of DNA by fusion proteins derived from MutH and I-SceI or TALE repeats.

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Gabsalilow, Lilia; Schierling, Benno; Friedhoff, Peter; Pingoud, Alfred; Wende, Wolfgang

2013-04-01

Targeted genome engineering requires nucleases that introduce a highly specific double-strand break in the genome that is either processed by homology-directed repair in the presence of a homologous repair template or by non-homologous end-joining (NHEJ) that usually results in insertions or deletions. The error-prone NHEJ can be efficiently suppressed by 'nickases' that produce a single-strand break rather than a double-strand break. Highly specific nickases have been produced by engineering of homing endonucleases and more recently by modifying zinc finger nucleases (ZFNs) composed of a zinc finger array and the catalytic domain of the restriction endonuclease FokI. These ZF-nickases work as heterodimers in which one subunit has a catalytically inactive FokI domain. We present two different approaches to engineer highly specific nickases; both rely on the sequence-specific nicking activity of the DNA mismatch repair endonuclease MutH which we fused to a DNA-binding module, either a catalytically inactive variant of the homing endonuclease I-SceI or the DNA-binding domain of the TALE protein AvrBs4. The fusion proteins nick strand specifically a bipartite recognition sequence consisting of the MutH and the I-SceI or TALE recognition sequences, respectively, with a more than 1000-fold preference over a stand-alone MutH site. TALE-MutH is a programmable nickase.

Subject-specific bone attenuation correction for brain PET/MR: can ZTE-MRI substitute CT scan accurately?

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Khalifé, Maya; Fernandez, Brice; Jaubert, Olivier; Soussan, Michael; Brulon, Vincent; Buvat, Irène; Comtat, Claude

2017-10-01

In brain PET/MR applications, accurate attenuation maps are required for accurate PET image quantification. An implemented attenuation correction (AC) method for brain imaging is the single-atlas approach that estimates an AC map from an averaged CT template. As an alternative, we propose to use a zero echo time (ZTE) pulse sequence to segment bone, air and soft tissue. A linear relationship between histogram normalized ZTE intensity and measured CT density in Hounsfield units (HU ) in bone has been established thanks to a CT-MR database of 16 patients. Continuous AC maps were computed based on the segmented ZTE by setting a fixed linear attenuation coefficient (LAC) to air and soft tissue and by using the linear relationship to generate continuous μ values for the bone. Additionally, for the purpose of comparison, four other AC maps were generated: a ZTE derived AC map with a fixed LAC for the bone, an AC map based on the single-atlas approach as provided by the PET/MR manufacturer, a soft-tissue only AC map and, finally, the CT derived attenuation map used as the gold standard (CTAC). All these AC maps were used with different levels of smoothing for PET image reconstruction with and without time-of-flight (TOF). The subject-specific AC map generated by combining ZTE-based segmentation and linear scaling of the normalized ZTE signal into HU was found to be a good substitute for the measured CTAC map in brain PET/MR when used with a Gaussian smoothing kernel of 4~mm corresponding to the PET scanner intrinsic resolution. As expected TOF reduces AC error regardless of the AC method. The continuous ZTE-AC performed better than the other alternative MR derived AC methods, reducing the quantification error between the MRAC corrected PET image and the reference CTAC corrected PET image.

BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction.

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Brad Thomas Townsley

2015-05-01

Full Text Available Next Generation Sequencing (NGS is driving rapid advancement in biological understanding and RNA-sequencing (RNA-seq has become an indispensable tool for biology and medicine. There is a growing need for access to these technologies although preparation of NGS libraries remains a bottleneck to wider adoption. Here we report a novel method for the production of strand specific RNA-seq libraries utilizing inherent properties of double-stranded cDNA to capture and incorporate a sequencing adapter. Breath Adapter Directional sequencing (BrAD-seq reduces sample handling and requires far fewer enzymatic steps than most available methods to produce high quality strand-specific RNA-seq libraries. The method we present is optimized for 3-prime Digital Gene Expression (DGE libraries and can easily extend to full transcript coverage shotgun (SHO type strand-specific libraries and is modularized to accommodate a diversity of RNA and DNA input materials. BrAD-seq offers a highly streamlined and inexpensive option for RNA-seq libraries.

Development of strand burner for solid propellant burning rate studies

International Nuclear Information System (INIS)

Aziz, A; Mamat, R; Ali, W K Wan

2013-01-01

It is well-known that a strand burner is an apparatus that provides burning rate measurements of a solid propellant at an elevated pressure in order to obtain the burning characteristics of a propellant. This paper describes the facilities developed by author that was used in his studies. The burning rate characteristics of solid propellant have be evaluated over five different chamber pressures ranging from 1 atm to 31 atm using a strand burner. The strand burner has a mounting stand that allows the propellant strand to be mounted vertically. The strand was ignited electrically using hot wire, and the burning time was recorded by electronic timer. Wire technique was used to measure the burning rate. Preliminary results from these techniques are presented. This study shows that the strand burner can be used on propellant strands to obtain accurate low pressure burning rate data

Quantification of low-expressed mRNA using 5' LNA-containing real-time PCR primers

International Nuclear Information System (INIS)

Malgoyre, A.; Banzet, S.; Mouret, C.; Bigard, A.X.; Peinnequin, A.

2007-01-01

Real-time RT-PCR is the most sensitive and accurate method for mRNA quantification. Using specific recombinant DNA as a template, real-time PCR allows accurate quantification within a 7-log range and increased sensitivity below 10 copies. However, when using RT-PCR to quantify mRNA in biological samples, a stochastic off-targeted amplification can occur. Classical adjustments of assay parameters have minimal effects on such amplification. This undesirable amplification appears mostly to be dependent on specific to non-specific target ratio rather than on the absolute quantity of the specific target. This drawback, which decreases assay reliability, mostly appears when quantifying low-expressed transcript in a whole organ. An original primer design using properties of LNA allows to block off-target amplification. 5'-LNA substitution strengthens 5'-hybridization. Consequently on-target hybridization is stabilized and the probability for the off-target to lead to amplification is decreased

Electron microscopy and three-dimensional reconstruction of native thin filaments reveal species-specific differences in regulatory strand densities

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Cammarato, Anthony, E-mail: acammara@burnham.org [Department of Physiology and Biophysics, Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118 (United States); Craig, Roger [Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655 (United States); Lehman, William [Department of Physiology and Biophysics, Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118 (United States)

2010-01-01

Throughout the animal kingdom striated muscle contraction is regulated by the thin filament troponin-tropomyosin complex. Homologous regulatory components are shared among vertebrate and arthropod muscles; however, unique protein extensions and/or components characterize the latter. The Troponin T (TnT) isoforms of Drosophila indirect flight and tarantula femur muscle for example contain distinct C-terminal extensions and are {approx}20% larger overall than their vertebrate counterpart. Using electron microscopy and three-dimensional helical reconstruction of native Drosophila, tarantula and frog muscle thin filaments we have identified species-specific differences in tropomyosin regulatory strand densities. The strands on the arthropod thin filaments were significantly larger in diameter than those from vertebrates, although not significantly different from each other. These findings reflect differences in the regulatory troponin-tropomyosin complex, which are likely due to the larger TnT molecules aligning and extending along much of the tropomyosin strands' length. Such an arrangement potentially alters the physical properties of the regulatory strands and may help establish contractile characteristics unique to certain arthropod muscles.

Electron microscopy and three-dimensional reconstruction of native thin filaments reveal species-specific differences in regulatory strand densities

International Nuclear Information System (INIS)

Cammarato, Anthony; Craig, Roger; Lehman, William

2010-01-01

Throughout the animal kingdom striated muscle contraction is regulated by the thin filament troponin-tropomyosin complex. Homologous regulatory components are shared among vertebrate and arthropod muscles; however, unique protein extensions and/or components characterize the latter. The Troponin T (TnT) isoforms of Drosophila indirect flight and tarantula femur muscle for example contain distinct C-terminal extensions and are ∼20% larger overall than their vertebrate counterpart. Using electron microscopy and three-dimensional helical reconstruction of native Drosophila, tarantula and frog muscle thin filaments we have identified species-specific differences in tropomyosin regulatory strand densities. The strands on the arthropod thin filaments were significantly larger in diameter than those from vertebrates, although not significantly different from each other. These findings reflect differences in the regulatory troponin-tropomyosin complex, which are likely due to the larger TnT molecules aligning and extending along much of the tropomyosin strands' length. Such an arrangement potentially alters the physical properties of the regulatory strands and may help establish contractile characteristics unique to certain arthropod muscles.

A novel synthetic quantification standard including virus and internal report targets: application for the detection and quantification of emerging begomoviruses on tomato.

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Péréfarres, Frédéric; Hoareau, Murielle; Chiroleu, Frédéric; Reynaud, Bernard; Dintinger, Jacques; Lett, Jean-Michel

2011-08-05

Begomovirus is a genus of phytopathogenic single-stranded DNA viruses, transmitted by the whitefly Bemisia tabaci. This genus includes emerging and economically significant viruses such as those associated with Tomato Yellow Leaf Curl Disease, for which diagnostic tools are needed to prevent dispersion and new introductions. Five real-time PCRs with an internal tomato reporter gene were developed for accurate detection and quantification of monopartite begomoviruses, including two strains of the Tomato yellow leaf curl virus (TYLCV; Mld and IL strains), the Tomato leaf curl Comoros virus-like viruses (ToLCKMV-like viruses) and the two molecules of the bipartite Potato yellow mosaic virus. These diagnostic tools have a unique standard quantification, comprising the targeted viral and internal report amplicons. These duplex real-time PCRs were applied to artificially inoculated plants to monitor and compare their viral development. Real-time PCRs were optimized for accurate detection and quantification over a range of 2 × 10(9) to 2 × 10(3) copies of genomic viral DNA/μL for TYLCV-Mld, TYLCV-IL and PYMV-B and 2 × 10(8) to 2 × 10(3) copies of genomic viral DNA/μL for PYMV-A and ToLCKMV-like viruses. These real-time PCRs were applied to artificially inoculated plants and viral loads were compared at 10, 20 and 30 days post-inoculation. Different patterns of viral accumulation were observed between the bipartite and the monopartite begomoviruses. Interestingly, PYMV accumulated more viral DNA at each date for both genomic components compared to all the monopartite viruses. Also, PYMV reached its highest viral load at 10 dpi contrary to the other viruses (20 dpi). The accumulation kinetics of the two strains of emergent TYLCV differed from the ToLCKMV-like viruses in the higher quantities of viral DNA produced in the early phase of the infection and in the shorter time to reach this peak viral load. To detect and quantify a wide range of begomoviruses, five duplex

A novel synthetic quantification standard including virus and internal report targets: application for the detection and quantification of emerging begomoviruses on tomato

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Lett Jean-Michel

2011-08-01

Full Text Available Abstract Background Begomovirus is a genus of phytopathogenic single-stranded DNA viruses, transmitted by the whitefly Bemisia tabaci. This genus includes emerging and economically significant viruses such as those associated with Tomato Yellow Leaf Curl Disease, for which diagnostic tools are needed to prevent dispersion and new introductions. Five real-time PCRs with an internal tomato reporter gene were developed for accurate detection and quantification of monopartite begomoviruses, including two strains of the Tomato yellow leaf curl virus (TYLCV; Mld and IL strains, the Tomato leaf curl Comoros virus-like viruses (ToLCKMV-like viruses and the two molecules of the bipartite Potato yellow mosaic virus. These diagnostic tools have a unique standard quantification, comprising the targeted viral and internal report amplicons. These duplex real-time PCRs were applied to artificially inoculated plants to monitor and compare their viral development. Results Real-time PCRs were optimized for accurate detection and quantification over a range of 2 × 109 to 2 × 103 copies of genomic viral DNA/μL for TYLCV-Mld, TYLCV-IL and PYMV-B and 2 × 108 to 2 × 103 copies of genomic viral DNA/μL for PYMV-A and ToLCKMV-like viruses. These real-time PCRs were applied to artificially inoculated plants and viral loads were compared at 10, 20 and 30 days post-inoculation. Different patterns of viral accumulation were observed between the bipartite and the monopartite begomoviruses. Interestingly, PYMV accumulated more viral DNA at each date for both genomic components compared to all the monopartite viruses. Also, PYMV reached its highest viral load at 10 dpi contrary to the other viruses (20 dpi. The accumulation kinetics of the two strains of emergent TYLCV differed from the ToLCKMV-like viruses in the higher quantities of viral DNA produced in the early phase of the infection and in the shorter time to reach this peak viral load. Conclusions To detect and

Characterization of 3-Dimensional PET Systems for Accurate Quantification of Myocardial Blood Flow.

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Renaud, Jennifer M; Yip, Kathy; Guimond, Jean; Trottier, Mikaël; Pibarot, Philippe; Turcotte, Eric; Maguire, Conor; Lalonde, Lucille; Gulenchyn, Karen; Farncombe, Troy; Wisenberg, Gerald; Moody, Jonathan; Lee, Benjamin; Port, Steven C; Turkington, Timothy G; Beanlands, Rob S; deKemp, Robert A

2017-01-01

Three-dimensional (3D) mode imaging is the current standard for PET/CT systems. Dynamic imaging for quantification of myocardial blood flow with short-lived tracers, such as 82 Rb-chloride, requires accuracy to be maintained over a wide range of isotope activities and scanner counting rates. We proposed new performance standard measurements to characterize the dynamic range of PET systems for accurate quantitative imaging. 82 Rb or 13 N-ammonia (1,100-3,000 MBq) was injected into the heart wall insert of an anthropomorphic torso phantom. A decaying isotope scan was obtained over 5 half-lives on 9 different 3D PET/CT systems and 1 3D/2-dimensional PET-only system. Dynamic images (28 × 15 s) were reconstructed using iterative algorithms with all corrections enabled. Dynamic range was defined as the maximum activity in the myocardial wall with less than 10% bias, from which corresponding dead-time, counting rates, and/or injected activity limits were established for each scanner. Scatter correction residual bias was estimated as the maximum cavity blood-to-myocardium activity ratio. Image quality was assessed via the coefficient of variation measuring nonuniformity of the left ventricular myocardium activity distribution. Maximum recommended injected activity/body weight, peak dead-time correction factor, counting rates, and residual scatter bias for accurate cardiac myocardial blood flow imaging were 3-14 MBq/kg, 1.5-4.0, 22-64 Mcps singles and 4-14 Mcps prompt coincidence counting rates, and 2%-10% on the investigated scanners. Nonuniformity of the myocardial activity distribution varied from 3% to 16%. Accurate dynamic imaging is possible on the 10 3D PET systems if the maximum injected MBq/kg values are respected to limit peak dead-time losses during the bolus first-pass transit. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Quantification of trace-level DNA by real-time whole genome amplification.

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Kang, Min-Jung; Yu, Hannah; Kim, Sook-Kyung; Park, Sang-Ryoul; Yang, Inchul

2011-01-01

Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, -2.1%, and -13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.

Strand displacement by DNA polymerase III occurs through a tau-psi-chi link to single-stranded DNA-binding protein coating the lagging strand template.

Science.gov (United States)

Yuan, Quan; McHenry, Charles S

2009-11-13

In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the single-stranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of gamma-complex to support the reaction in the absence of tau. However, if gamma-complex is present to load beta(2), a truncated tau protein containing only domains III-V will suffice. This truncated protein is sufficient to bind both the alpha subunit of DNA polymerase (Pol) III and chipsi. This is reminiscent of the minimal requirements for Pol III to replicate short single-stranded DNA-binding protein (SSB)-coated templates where tau is only required to serve as a scaffold to hold Pol III and chi in the same complex (Glover, B., and McHenry, C. (1998) J. Biol. Chem. 273, 23476-23484). We propose a model in which strand displacement by DNA polymerase III holoenzyme depends upon a Pol III-tau-psi-chi-SSB binding network, where SSB is bound to the displaced strand, stabilizing the Pol III-template interaction. The same interaction network is probably important for stabilizing the leading strand polymerase interactions with authentic replication forks. The specificity constant (k(cat)/K(m)) for the strand displacement reaction is approximately 300-fold less favorable than reactions on single-stranded templates and proceeds with a slower rate (150 nucleotides/s) and only moderate processivity (approximately 300 nucleotides). PriA, the initiator of replication restart on collapsed or misassembled replication forks, blocks the strand displacement reaction, even if added to an ongoing reaction.

Alaska Beluga Strandings

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — NMFS is mandated by the Marine Mammal Protection Act (MMPA) to collect reports of marine mammal stranding events. This particular catalogue item is specific to the...

Strand exchange of telomeric DNA catalyzed by the Werner syndrome protein (WRN) is specifically stimulated by TRF2

Science.gov (United States)

Edwards, Deanna N.; Orren, David K.; Machwe, Amrita

2014-01-01

Werner syndrome (WS), caused by loss of function of the RecQ helicase WRN, is a hereditary disease characterized by premature aging and elevated cancer incidence. WRN has DNA binding, exonuclease, ATPase, helicase and strand annealing activities, suggesting possible roles in recombination-related processes. Evidence indicates that WRN deficiency causes telomeric abnormalities that likely underlie early onset of aging phenotypes in WS. Furthermore, TRF2, a protein essential for telomere protection, interacts with WRN and influences its basic helicase and exonuclease activities. However, these studies provided little insight into WRN's specific function at telomeres. Here, we explored the possibility that WRN and TRF2 cooperate during telomeric recombination processes. Our results indicate that TRF2, through its interactions with both WRN and telomeric DNA, stimulates WRN-mediated strand exchange specifically between telomeric substrates; TRF2's basic domain is particularly important for this stimulation. Although TRF1 binds telomeric DNA with similar affinity, it has minimal effects on WRN-mediated strand exchange of telomeric DNA. Moreover, TRF2 is displaced from telomeric DNA by WRN, independent of its ATPase and helicase activities. Together, these results suggest that TRF2 and WRN act coordinately during telomeric recombination processes, consistent with certain telomeric abnormalities associated with alteration of WRN function. PMID:24880691

Distinguishing enhancing from nonenhancing renal masses with dual-source dual-energy CT: iodine quantification versus standard enhancement measurements.

Science.gov (United States)

Ascenti, Giorgio; Mileto, Achille; Krauss, Bernhard; Gaeta, Michele; Blandino, Alfredo; Scribano, Emanuele; Settineri, Nicola; Mazziotti, Silvio

2013-08-01

To compare the diagnostic accuracy of iodine quantification and standard enhancement measurements in distinguishing enhancing from nonenhancing renal masses. The Institutional Review Board approved this retrospective study conducted from data found in institutional patient databases and archives. Seventy-two renal masses were characterised as enhancing or nonenhancing using standard enhancement measurements (in HU) and iodine quantification (in mg/ml). Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of standard enhancement measurements and iodine quantification were calculated from χ (2) tests of contingency with histopathology or imaging follow-up as the reference standard. Difference in accuracy was assessed by means of McNemar analysis. Sensitivity, specificity, PPV, NPV and diagnostic accuracy for standard enhancement measurements and iodine quantification were 77.7 %, 100 %, 100 %, 81.8 %, 89 % and 100 %, 94.4 %, 94.7, 100 % and 97 %, respectively. The McNemar analysis showed that the accuracy of iodine quantification was significantly better (P < 0.001) than that of standard enhancement measurements. Compared with standard enhancement measurements, whole-tumour iodine quantification is more accurate in distinguishing enhancing from nonenhancing renal masses. • Enhancement of renal lesions is important when differentiating benign from malignant tumours. • Dual-energy CT offers measurement of iodine uptake rather than mere enhancement values. • Whole-tumour iodine quantification seems more accurate than standard CT enhancement measurements.

In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element Specific for Bromacil

Directory of Open Access Journals (Sweden)

Ryan M. Williams

2014-01-01

Full Text Available Bromacil is a widely used herbicide that is known to contaminate environmental systems. Due to the hazards it presents and inefficient detection methods, it is necessary to create a rapid and efficient sensing device. Towards this end, we have utilized a stringent in vitro selection method to identify single-stranded DNA molecular recognition elements (MRE specific for bromacil. We have identified one MRE with high affinity (Kd=9.6 nM and specificity for bromacil compared to negative targets of selection and other pesticides. The selected ssDNA MRE will be useful as the sensing element in a field-deployable bromacil detection device.

De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks

KAUST Repository

Mahfouz, Magdy M.; Li, Lixin; Shamimuzzaman, Md.; Wibowo, Anjar Tri; Fang, Xiaoyun; Zhu, Jian-Kang

2011-01-01

Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions

Accurate quantification of polycyclic aromatic hydrocarbons in dust samples using microwave-assisted solvent extraction combined with isotope-dilution mass spectrometry

International Nuclear Information System (INIS)

Itoh, Nobuyasu; Fushimi, Akihiro; Yarita, Takashi; Aoyagi, Yoshie; Numata, Masahiko

2011-01-01

Highlights: → We applied MAE-IDMS for accurate quantification of PAHs in dust samples. → Both partitioning and isotopic equilibria can be achieved using this technique. → MAE-IDMS can provide accurate concentrations even if extraction efficiencies were low. → Characteristics of samples strongly affected for low extraction efficiencies of PAHs. - Abstract: For accurate quantification of polycyclic aromatic hydrocarbons (PAHs) in dust samples, we investigated the use of microwave-assisted solvent extraction (MAE) combined with isotope-dilution mass spectrometry (IDMS) using deuterium-labelled PAHs (D-PAHs). Although MAE with a methanol/toluene mixture (1:3 by volume) at 160 deg. C for 40 min was best for extracting PAHs from tunnel dust among examined, the recovery yields of D-PAHs decreased with increasing molecular weight (<40% for MW ≥ 264; that of deuterium-labelled indeno[123-cd]pyrene (D-IcdP) was only 7.1%). Although the residues were extracted a second time, the observed concentrations did not change dramatically (<5%), and the recovery yields of heavier D-PAHs (i.e., MW ≥ 264) were approximately half of those of the first extract, including D-IcdP (3.4%). These results suggest that both partitioning and isotopic equilibria of PAHs and D-PAHs between sample and solvent were achieved for extractable heavier PAHs under the condition. Thus, the observed concentrations of PAHs obtained by MAE-IDMS were reasonable, even though recovery yields of D-PAHs were <50%. From the results of carbon analyses and extractable contents, lower recovery yields of D-PAHs from the tunnel dust were due to a large content of char with low extractable contents.

Determination and analysis of site-specific 125I decay-induced DNA double-strand break end-group structures.

Science.gov (United States)

Datta, Kamal; Weinfeld, Michael; Neumann, Ronald D; Winters, Thomas A

2007-02-01

End groups contribute to the structural complexity of radiation-induced DNA double-strand breaks (DSBs). As such, end-group structures may affect a cell's ability to repair DSBs. The 3'-end groups of strand breaks caused by gamma radiation, or oxidative processes, under oxygenated aqueous conditions have been shown to be distributed primarily between 3'-phosphoglycolate and 3'-phosphate, with 5'-phosphate ends in both cases. In this study, end groups of the high-LET-like DSBs caused by 125I decay were investigated. Site-specific DNA double-strand breaks were produced in plasmid pTC27 in the presence or absence of 2 M DMSO by 125I-labeled triplex-forming oligonucleotide targeting. End-group structure was assessed enzymatically as a function of the DSB end to serve as a substrate for ligation and various forms of end labeling. Using this approach, we have demonstrated 3'-hydroxyl (3'-OH) and 3'-phosphate (3'-P) end groups and 5'-ends (> or = 42%) terminated by phosphate. A 32P postlabeling assay failed to detect 3'-phosphoglycolate in a restriction fragment terminated by the 125I-induced DNA double-strand break, and this is likely due to restricted oxygen diffusion during irradiation as a frozen aqueous solution. Even so, end-group structure and relative distribution varied as a function of the free radical scavenging capacity of the irradiation buffer.

DNA Double-Strand Break Rejoining in Complex Normal Tissues

International Nuclear Information System (INIS)

Ruebe, Claudia E.; Dong, Xiaorong; Kuehne, Martin; Fricke, Andreas; Kaestner, Lars; Lipp, Peter; Ruebe, Christian

2008-01-01

Purpose: The clinical radiation responses of different organs vary widely and likely depend on the intrinsic radiosensitivities of their different cell populations. Double-strand breaks (DSBs) are the most deleterious form of DNA damage induced by ionizing radiation, and the cells' capacity to rejoin radiation-induced DSBs is known to affect their intrinsic radiosensitivity. To date, only little is known about the induction and processing of radiation-induced DSBs in complex normal tissues. Using an in vivo model with repair-proficient mice, the highly sensitive γH2AX immunofluorescence was established to investigate whether differences in DSB rejoining could account for the substantial differences in clinical radiosensitivity observed among normal tissues. Methods and Materials: After whole body irradiation of C57BL/6 mice (0.1, 0.5, 1.0, and 2.0 Gy), the formation and rejoining of DSBs was analyzed by enumerating γH2AX foci in various organs representative of both early-responding (small intestine) and late-responding (lung, brain, heart, kidney) tissues. Results: The linear dose correlation observed in all analyzed tissues indicated that γH2AX immunofluorescence allows for the accurate quantification of DSBs in complex organs. Strikingly, the various normal tissues exhibited identical kinetics for γH2AX foci loss, despite their clearly different clinical radiation responses. Conclusion: The identical kinetics of DSB rejoining measured in different organs suggest that tissue-specific differences in radiation responses are independent of DSB rejoining. This finding emphasizes the fundamental role of DSB repair in maintaining genomic integrity, thereby contributing to cellular viability and functionality and, thus, tissue homeostasis

Equilibrious Strand Exchange Promoted by DNA Conformational Switching

Science.gov (United States)

Wu, Zhiguo; Xie, Xiao; Li, Puzhen; Zhao, Jiayi; Huang, Lili; Zhou, Xiang

2013-01-01

Most of DNA strand exchange reactions in vitro are based on toehold strategy which is generally nonequilibrium, and intracellular strand exchange mediated by proteins shows little sequence specificity. Herein, a new strand exchange promoted by equilibrious DNA conformational switching is verified. Duplexes containing c-myc sequence which is potentially converted into G-quadruplex are designed in this strategy. The dynamic equilibrium between duplex and G4-DNA is response to the specific exchange of homologous single-stranded DNA (ssDNA). The SER is enzyme free and sequence specific. No ATP is needed and the displaced ssDNAs are identical to the homologous ssDNAs. The SER products and exchange kenetics are analyzed by PAGE and the RecA mediated SER is performed as the contrast. This SER is a new feature of G4-DNAs and a novel strategy to utilize the dynamic equilibrium of DNA conformations.

Pore REconstruction and Segmentation (PORES) method for improved porosity quantification of nanoporous materials

Energy Technology Data Exchange (ETDEWEB)

Van Eyndhoven, G., E-mail: geert.vaneyndhoven@uantwerpen.be [iMinds-Vision Lab, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk (Belgium); Kurttepeli, M. [EMAT, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp (Belgium); Van Oers, C.J.; Cool, P. [Laboratory of Adsorption and Catalysis, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk (Belgium); Bals, S. [EMAT, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp (Belgium); Batenburg, K.J. [iMinds-Vision Lab, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk (Belgium); Centrum Wiskunde and Informatica, Science Park 123, NL-1090 GB Amsterdam (Netherlands); Mathematical Institute, Universiteit Leiden, Niels Bohrweg 1, NL-2333 CA Leiden (Netherlands); Sijbers, J. [iMinds-Vision Lab, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk (Belgium)

2015-01-15

Electron tomography is currently a versatile tool to investigate the connection between the structure and properties of nanomaterials. However, a quantitative interpretation of electron tomography results is still far from straightforward. Especially accurate quantification of pore-space is hampered by artifacts introduced in all steps of the processing chain, i.e., acquisition, reconstruction, segmentation and quantification. Furthermore, most common approaches require subjective manual user input. In this paper, the PORES algorithm “POre REconstruction and Segmentation” is introduced; it is a tailor-made, integral approach, for the reconstruction, segmentation, and quantification of porous nanomaterials. The PORES processing chain starts by calculating a reconstruction with a nanoporous-specific reconstruction algorithm: the Simultaneous Update of Pore Pixels by iterative REconstruction and Simple Segmentation algorithm (SUPPRESS). It classifies the interior region to the pores during reconstruction, while reconstructing the remaining region by reducing the error with respect to the acquired electron microscopy data. The SUPPRESS reconstruction can be directly plugged into the remaining processing chain of the PORES algorithm, resulting in accurate individual pore quantification and full sample pore statistics. The proposed approach was extensively validated on both simulated and experimental data, indicating its ability to generate accurate statistics of nanoporous materials. - Highlights: • An electron tomography reconstruction/segmentation method for nanoporous materials. • The method exploits the porous nature of the scanned material. • Validated extensively on both simulation and real data experiments. • Results in increased image resolution and improved porosity quantification.

Characterizing the strand-specific distribution of non-CpG methylation in human pluripotent cells

OpenAIRE

Guo, Weilong; Chung, Wen-Yu; Qian, Minping; Pellegrini, Matteo; Zhang, Michael Q.

2013-01-01

DNA methylation is an important defense and regulatory mechanism. In mammals, most DNA methylation occurs at CpG sites, and asymmetric non-CpG methylation has only been detected at appreciable levels in a few cell types. We are the first to systematically study the strand-specific distribution of non-CpG methylation. With the divide-and-compare strategy, we show that CHG and CHH methylation are not intrinsically different in human embryonic stem cells (ESCs) and induced pluripotent stem cells...

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

Science.gov (United States)

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. Copyright © 2015 Elsevier B.V. All rights reserved.

Structural features of single-stranded integron cassette attC sites and their role in strand selection.

Directory of Open Access Journals (Sweden)

Marie Bouvier

2009-09-01

Full Text Available We recently showed that cassette integration and deletion in integron platforms were occurring through unconventional site-specific recombination reactions involving only the bottom strand of attC sites. The lack of sequence conservation among attC sites led us to hypothesize that sequence-independent structural recognition determinants must exist within attC sites. The structural data obtained from a synaptic complex of the Vibrio cholerae integrase with the bottom strand of an attC site has shown the importance of extra helical bases (EHB inside the stem-loop structure formed from the bottom strand. Here, we systematically determined the contribution of three structural elements common to all known single-stranded attC site recombination substrates (the EHBs, the unpaired central spacer (UCS, and the variable terminal structure (VTS to strand choice and recombination. Their roles have been evaluated in vivo in the attIxattC reaction context using the suicide conjugation assay we previously developed, but also in an attCxattC reaction using a deletion assay. Conjugation was used to deliver the attC sites in single-stranded form. Our results show that strand choice is primarily directed by the first EHB, but the presence of the two other EHBs also serves to increase this strand selection. We found that the structure of the central spacer is essential to achieve high level recombination of the bottom strand, suggesting a dual role for this structure in active site exclusion and for hindering the reverse reaction after the first strand exchange. Moreover, we have shown that the VTS has apparently no role in strand selectivity.

Single-stranded γPNAs for in vivo site-specific genome editing via Watson-Crick recognition.

Science.gov (United States)

Bahal, Raman; Quijano, Elias; McNeer, Nicole A; Liu, Yanfeng; Bhunia, Dinesh C; Lopez-Giraldez, Francesco; Fields, Rachel J; Saltzman, William M; Ly, Danith H; Glazer, Peter M

2014-01-01

Triplex-forming peptide nucleic acids (PNAs) facilitate gene editing by stimulating recombination of donor DNAs within genomic DNA via site-specific formation of altered helical structures that further stimulate DNA repair. However, PNAs designed for triplex formation are sequence restricted to homopurine sites. Herein we describe a novel strategy where next generation single-stranded gamma PNAs (γPNAs) containing miniPEG substitutions at the gamma position can target genomic DNA in mouse bone marrow at mixed-sequence sites to induce targeted gene editing. In addition to enhanced binding, γPNAs confer increased solubility and improved formulation into poly(lactic-co-glycolic acid) (PLGA) nanoparticles for efficient intracellular delivery. Single-stranded γPNAs induce targeted gene editing at frequencies of 0.8% in mouse bone marrow cells treated ex vivo and 0.1% in vivo via IV injection, without detectable toxicity. These results suggest that γPNAs may provide a new tool for induced gene editing based on Watson-Crick recognition without sequence restriction.

Second-strand cDNA synthesis: classical method

International Nuclear Information System (INIS)

Gubler, U.

1987-01-01

The classical scheme for the synthesis of double-stranded cDNA as it was reported in 1976 is described. Reverse transcription of mRNA with oligo(dT) as the primer generates first strands with a small loop at the 3' end of the cDNA (the end that corresponds to the 5' end of the mRNA). Subsequent removal of the mRNA by alkaline hydrolysis leaves single-stranded cDNA molecules again with a small 3' loop. This loop can be used by either reverse transcriptase or Klenow fragment of DNA polymerase I as a primer for second-strand synthesis. The resulting products are double-stranded cDNA molecules that are covalently closed at the end corresponding to the 5' end of the original mRNA. Subsequent cleavage of the short piece of single-stranded cDNA within the loop with the single-strand-specific S 1 nuclease generate open double-stranded molecules that can be used for molecular cloning in plasmids or in phage. Useful variations of this scheme have been described

De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks

KAUST Repository

Mahfouz, Magdy M.

2011-01-24

Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.

Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses

Directory of Open Access Journals (Sweden)

Barik Sailen

2001-12-01

Full Text Available Abstract Background Post-transcriptional gene silencing (PTGS by short interfering RNA has opened up new directions in the phenotypic mutation of cellular genes. However, its efficacy on non-nuclear genes and its effect on the interferon pathway remain unexplored. Since directed mutation of RNA genomes is not possible through conventional mutagenesis, we have tested sequence-specific 21-nucleotide long double-stranded RNAs (dsRNAs for their ability to silence cytoplasmic RNA genomes. Results Short dsRNAs were generated against specific mRNAs of respiratory syncytial virus, a nonsegmented negative-stranded RNA virus with a cytoplasmic life cycle. At nanomolar concentrations, the dsRNAs specifically abrogated expression of the corresponding viral proteins, and produced the expected mutant phenotype ex vivo. The dsRNAs did not induce an interferon response, and did not inhibit cellular gene expression. The ablation of the viral proteins correlated with the loss of the specific mRNAs. In contrast, viral genomic and antigenomic RNA, which are encapsidated, were not directly affected. Conclusions Synthetic inhibitory dsRNAs are effective in specific silencing of RNA genomes that are exclusively cytoplasmic and transcribed by RNA-dependent RNA polymerases. RNA-directed RNA gene silencing does not require cloning, expression, and mutagenesis of viral cDNA, and thus, will allow the generation of phenotypic null mutants of specific RNA viral genes under normal infection conditions and at any point in the infection cycle. This will, for the first time, permit functional genomic studies, attenuated infections, reverse genetic analysis, and studies of host-virus signaling pathways using a wild type RNA virus, unencumbered by any superinfecting virus.

Accurate and reliable quantification of total microalgal fuel potential as fatty acid methyl esters by in situ transesterification

Energy Technology Data Exchange (ETDEWEB)

Laurens, Lieve M.L.; Quinn, Matthew; Wychen, Stefanie van; Templeton, David W.; Wolfrum, Edward J. [National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO (United States)

2012-04-15

In the context of algal biofuels, lipids, or better aliphatic chains of the fatty acids, are perhaps the most important constituents of algal biomass. Accurate quantification of lipids and their respective fuel yield is crucial for comparison of algal strains and growth conditions and for process monitoring. As an alternative to traditional solvent-based lipid extraction procedures, we have developed a robust whole-biomass in situ transesterification procedure for quantification of algal lipids (as fatty acid methyl esters, FAMEs) that (a) can be carried out on a small scale (using 4-7 mg of biomass), (b) is applicable to a range of different species, (c) consists of a single-step reaction, (d) is robust over a range of different temperature and time combinations, and (e) tolerant to at least 50% water in the biomass. Unlike gravimetric lipid quantification, which can over- or underestimate the lipid content, whole biomass transesterification reflects the true potential fuel yield of algal biomass. We report here on the comparison of the yield of FAMEs by using different catalysts and catalyst combinations, with the acid catalyst HCl providing a consistently high level of conversion of fatty acids with a precision of 1.9% relative standard deviation. We investigate the influence of reaction time, temperature, and biomass water content on the measured FAME content and profile for 4 different samples of algae (replete and deplete Chlorella vulgaris, replete Phaeodactylum tricornutum, and replete Nannochloropsis sp.). We conclude by demonstrating a full mass balance closure of all fatty acids around a traditional lipid extraction process. (orig.)

Detection of lead(II) ions with a DNAzyme and isothermal strand displacement signal amplification.

Science.gov (United States)

Li, Wenying; Yang, Yue; Chen, Jian; Zhang, Qingfeng; Wang, Yan; Wang, Fangyuan; Yu, Cong

2014-03-15

A DNAzyme based method for the sensitive and selective quantification of lead(II) ions has been developed. A DNAzyme that requires Pb(2+) for activation was selected. An RNA containing DNA substrate was cleaved by the DNAzyme in the presence of Pb(2+). The 2',3'-cyclic phosphate of the cleaved 5'-part of the substrate was efficiently removed by Exonuclease III. The remaining part of the single stranded DNA (9 or 13 base long) was subsequently used as the primer for the strand displacement amplification reaction (SDAR). The method is highly sensitive, 200 pM lead(II) could be easily detected. A number of interference ions were tested, and the sensor showed good selectivity. Underground water samples were also tested, which demonstrated the feasibility of the current approach for real sample applications. It is feasible that our method could be used for DNAzyme or aptazyme based new sensing method developments for the quantification of other target analytes with high sensitivity and selectivity. © 2013 Elsevier B.V. All rights reserved.

A multivariate shape quantification approach for sickle red blood cell in patient-specific microscopy image data

Science.gov (United States)

Xu, Mengjia; Yang, Jinzhu; Zhao, Hong

2017-07-01

The morphological change of red blood cells(RBCs) plays an important role in revealing the biomechanical and biorheological characteristics of RBCs. Aiming to extract the shape indices for the sickle RBCs, an automated ex-vivo RBC shape quantification method is proposed. First, single RBC regions (ROIs) are extracted from raw microscopy image via an automatic hierarchical ROI extraction method. Second, an improved random walk method is used to detect the RBC outline. Finally, three types of RBC shape factors are calculated based on the elliptical fitting RBC contour. Experiments indicate that the proposed method can accurately segment the RBCs from the microscopy images with low contrast and prevent the disturbance of artifacts. Moreover, it can provide an efficient shape quantification means for diverse RBC shapes in a batch manner.

Direct qPCR quantification using the Quantifiler(®) Trio DNA quantification kit.

Science.gov (United States)

Liu, Jason Yingjie

2014-11-01

The effectiveness of a direct quantification assay is essential to the adoption of the combined direct quantification/direct STR workflow. In this paper, the feasibility of using the Quantifiler(®) Trio DNA quantification kit for the direct quantification of forensic casework samples was investigated. Both low-level touch DNA samples and blood samples were collected on PE swabs and quantified directly. The increased sensitivity of the Quantifiler(®) Trio kit enabled the detection of less than 10pg of DNA in unprocessed touch samples and also minimizes the stochastic effect experienced by different targets in the same sample. The DNA quantity information obtained from a direct quantification assay using the Quantifiler(®) Trio kit can also be used to accurately estimate the optimal input DNA quantity for a direct STR amplification reaction. The correlation between the direct quantification results (Quantifiler(®) Trio kit) and the direct STR results (GlobalFiler™ PCR amplification kit(*)) for low-level touch DNA samples indicates that direct quantification using the Quantifiler(®) Trio DNA quantification kit is more reliable than the Quantifiler(®) Duo DNA quantification kit for predicting the STR results of unprocessed touch DNA samples containing less than 10pg of DNA. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Excess single-stranded DNA inhibits meiotic double-strand break repair.

Directory of Open Access Journals (Sweden)

Rebecca Johnson

2007-11-01

Full Text Available During meiosis, self-inflicted DNA double-strand breaks (DSBs are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE, in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects

The application of strand invasion phenomenon, directed by peptide nucleic acid (PNA) and single-stranded DNA binding protein (SSB) for the recognition of specific sequences of human endogenous retroviral HERV-W family.

Science.gov (United States)

Machnik, Grzegorz; Bułdak, Łukasz; Ruczyński, Jarosław; Gąsior, Tomasz; Huzarska, Małgorzata; Belowski, Dariusz; Alenowicz, Magdalena; Mucha, Piotr; Rekowski, Piotr; Okopień, Bogusław

2017-05-01

The HERV-W family of human endogenous retroviruses represents a group of numerous sequences that show close similarity in genetic composition. It has been documented that some members of HERV-W-derived expression products are supposed to play significant role in humans' pathology, such as multiple sclerosis or schizophrenia. Other members of the family are necessary to orchestrate physiological processes (eg, ERVWE1 coding syncytin-1 that is engaged in syncytiotrophoblast formation). Therefore, an assay that would allow the recognition of particular form of HERV-W members is highly desirable. A peptide nucleic acid (PNA)-mediated technique for the discrimination between multiple sclerosis-associated retrovirus and ERVWE1 sequence has been developed. The assay uses a PNA probe that, being fully complementary to the ERVWE1 but not to multiple sclerosis-associated retrovirus (MSRV) template, shows high selective potential. Single-stranded DNA binding protein facilitates the PNA-mediated, sequence-specific formation of strand invasion complex and, consequently, local DNA unwinding. The target DNA may be then excluded from further analysis in any downstream process such as single-stranded DNA-specific exonuclease action. Finally, the reaction conditions have been optimized, and several PNA probes that are targeted toward distinct loci along whole HERV-W env sequences have been evaluated. We believe that PNA/single-stranded DNA binding protein-based application has the potential to selectively discriminate particular HERV-W molecules as they are at least suspected to play pathogenic role in a broad range of medical conditions, from psycho-neurologic disorders (multiple sclerosis and schizophrenia) and cancers (breast cancer) to that of an auto-immunologic background (psoriasis and lupus erythematosus). Copyright © 2016 John Wiley & Sons, Ltd.

Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand*

Science.gov (United States)

Teasley, Daniel C.; Parajuli, Shankar; Nguyen, Mai; Moore, Hayley R.; Alspach, Elise; Lock, Ying Jie; Honaker, Yuchi; Saharia, Abhishek; Piwnica-Worms, Helen; Stewart, Sheila A.

2015-01-01

The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer. PMID:25922071

World-Wide Benchmarking of ITER Nb$_{3}$Sn Strand Test Facilities

CERN Document Server

Jewell, MC; Takahashi, Yoshikazu; Shikov, Alexander; Devred, Arnaud; Vostner, Alexander; Liu, Fang; Wu, Yu; Jewell, Matthew C; Boutboul, Thierry; Bessette, Denis; Park, Soo-Hyeon; Isono, Takaaki; Vorobieva, Alexandra; Martovetsky, Nicolai; Seo, Kazutaka

2010-01-01

The world-wide procurement of Nb$_{3}$Sn and NbTi for the ITER superconducting magnet systems will involve eight to ten strand suppliers from six Domestic Agencies (DAs) on three continents. To ensure accurate and consistent measurement of the physical and superconducting properties of the composite strand, a strand test facility benchmarking effort was initiated in August 2008. The objectives of this effort are to assess and improve the superconducting strand test and sample preparation technologies at each DA and supplier, in preparation for the more than ten thousand samples that will be tested during ITER procurement. The present benchmarking includes tests for critical current (I-c), n-index, hysteresis loss (Q(hys)), residual resistivity ratio (RRR), strand diameter, Cu fraction, twist pitch, twist direction, and metal plating thickness (Cr or Ni). Nineteen participants from six parties (China, EU, Japan, South Korea, Russia, and the United States) have participated in the benchmarking. This round, cond...

Accurate quantification of endogenous androgenic steroids in cattle's meat by gas chromatography mass spectrometry using a surrogate analyte approach

International Nuclear Information System (INIS)

Ahmadkhaniha, Reza; Shafiee, Abbas; Rastkari, Noushin; Kobarfard, Farzad

2009-01-01

Determination of endogenous steroids in complex matrices such as cattle's meat is a challenging task. Since endogenous steroids always exist in animal tissues, no analyte-free matrices for constructing the standard calibration line will be available, which is crucial for accurate quantification specially at trace level. Although some methods have been proposed to solve the problem, none has offered a complete solution. To this aim, a new quantification strategy was developed in this study, which is named 'surrogate analyte approach' and is based on using isotope-labeled standards instead of natural form of endogenous steroids for preparing the calibration line. In comparison with the other methods, which are currently in use for the quantitation of endogenous steroids, this approach provides improved simplicity and speed for analysis on a routine basis. The accuracy of this method is better than other methods at low concentration and comparable to the standard addition at medium and high concentrations. The method was also found to be valid according to the ICH criteria for bioanalytical methods. The developed method could be a promising approach in the field of compounds residue analysis

Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand.

Science.gov (United States)

Teasley, Daniel C; Parajuli, Shankar; Nguyen, Mai; Moore, Hayley R; Alspach, Elise; Lock, Ying Jie; Honaker, Yuchi; Saharia, Abhishek; Piwnica-Worms, Helen; Stewart, Sheila A

2015-06-12

The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

New views on strand asymmetry in insect mitochondrial genomes.

Directory of Open Access Journals (Sweden)

Shu-Jun Wei

Full Text Available Strand asymmetry in nucleotide composition is a remarkable feature of animal mitochondrial genomes. Understanding the mutation processes that shape strand asymmetry is essential for comprehensive knowledge of genome evolution, demographical population history and accurate phylogenetic inference. Previous studies found that the relative contributions of different substitution types to strand asymmetry are associated with replication alone or both replication and transcription. However, the relative contributions of replication and transcription to strand asymmetry remain unclear. Here we conducted a broad survey of strand asymmetry across 120 insect mitochondrial genomes, with special reference to the correlation between the signs of skew values and replication orientation/gene direction. The results show that the sign of GC skew on entire mitochondrial genomes is reversed in all species of three distantly related families of insects, Philopteridae (Phthiraptera, Aleyrodidae (Hemiptera and Braconidae (Hymenoptera; the replication-related elements in the A+T-rich regions of these species are inverted, confirming that reversal of strand asymmetry (GC skew was caused by inversion of replication origin; and finally, the sign of GC skew value is associated with replication orientation but not with gene direction, while that of AT skew value varies with gene direction, replication and codon positions used in analyses. These findings show that deaminations during replication and other mutations contribute more than selection on amino acid sequences to strand compositions of G and C, and that the replication process has a stronger affect on A and T content than does transcription. Our results may contribute to genome-wide studies of replication and transcription mechanisms.

Hepatitis B virus DNA quantification with the three-in-one (3io) method allows accurate single-step differentiation of total HBV DNA and cccDNA in biopsy-size liver samples.

Science.gov (United States)

Taranta, Andrzej; Tien Sy, Bui; Zacher, Behrend Johan; Rogalska-Taranta, Magdalena; Manns, Michael Peter; Bock, Claus Thomas; Wursthorn, Karsten

2014-08-01

Hepatitis B virus (HBV) replicates via reverse transcription converting its partially double stranded genome into the covalently closed circular DNA (cccDNA). The long-lasting cccDNA serves as a replication intermediate in the nuclei of hepatocytes. It is an excellent, though evasive, parameter for monitoring the course of liver disease and treatment efficiency. To develop and test a new approach for HBV DNA quantification in serum and small-size liver samples. The p3io plasmid contains an HBV fragment and human β-actin gene (hACTB) as a standard. Respective TaqMan probes were labeled with different fluorescent dyes. A triplex real-time PCR for simultaneous quantification of total HBV DNA, cccDNA and hACTB could be established. Three-in-one method allows simultaneous analysis of 3 targets with a lower limit of quantification of 48 copies per 20 μl PCR reaction and a wide range of linearity (R(2)>0.99, pDNA samples from HBV infected patients. Total HBV DNA and cccDNA could be quantified in 32 and 22 of 33 FFPE preserved liver specimens, respectively. Total HBV DNA concentrations quantified by the 3io method remained comparable with Cobas TaqMan HBV Test v2.0. The three-in-one protocol allows the single step quantification of viral DNA in samples from different sources. Therefore lower sample input, faster data acquisition, a lowered error and significantly lower costs are the advantages of the method. Copyright © 2014 Elsevier B.V. All rights reserved.

Development, optimization, and single laboratory validation of an event-specific real-time PCR method for the detection and quantification of Golden Rice 2 using a novel taxon-specific assay.

Science.gov (United States)

Jacchia, Sara; Nardini, Elena; Savini, Christian; Petrillo, Mauro; Angers-Loustau, Alexandre; Shim, Jung-Hyun; Trijatmiko, Kurniawan; Kreysa, Joachim; Mazzara, Marco

2015-02-18

In this study, we developed, optimized, and in-house validated a real-time PCR method for the event-specific detection and quantification of Golden Rice 2, a genetically modified rice with provitamin A in the grain. We optimized and evaluated the performance of the taxon (targeting rice Phospholipase D α2 gene)- and event (targeting the 3' insert-to-plant DNA junction)-specific assays that compose the method as independent modules, using haploid genome equivalents as unit of measurement. We verified the specificity of the two real-time PCR assays and determined their dynamic range, limit of quantification, limit of detection, and robustness. We also confirmed that the taxon-specific DNA sequence is present in single copy in the rice genome and verified its stability of amplification across 132 rice varieties. A relative quantification experiment evidenced the correct performance of the two assays when used in combination.

GMO quantification: valuable experience and insights for the future.

Science.gov (United States)

Milavec, Mojca; Dobnik, David; Yang, Litao; Zhang, Dabing; Gruden, Kristina; Zel, Jana

2014-10-01

Cultivation and marketing of genetically modified organisms (GMOs) have been unevenly adopted worldwide. To facilitate international trade and to provide information to consumers, labelling requirements have been set up in many countries. Quantitative real-time polymerase chain reaction (qPCR) is currently the method of choice for detection, identification and quantification of GMOs. This has been critically assessed and the requirements for the method performance have been set. Nevertheless, there are challenges that should still be highlighted, such as measuring the quantity and quality of DNA, and determining the qPCR efficiency, possible sequence mismatches, characteristics of taxon-specific genes and appropriate units of measurement, as these remain potential sources of measurement uncertainty. To overcome these problems and to cope with the continuous increase in the number and variety of GMOs, new approaches are needed. Statistical strategies of quantification have already been proposed and expanded with the development of digital PCR. The first attempts have been made to use new generation sequencing also for quantitative purposes, although accurate quantification of the contents of GMOs using this technology is still a challenge for the future, and especially for mixed samples. New approaches are needed also for the quantification of stacks, and for potential quantification of organisms produced by new plant breeding techniques.

1H NMR quantification in very dilute toxin solutions: application to anatoxin-a analysis.

Science.gov (United States)

Dagnino, Denise; Schripsema, Jan

2005-08-01

A complete procedure is described for the extraction, detection and quantification of anatoxin-a in biological samples. Anatoxin-a is extracted from biomass by a routine acid base extraction. The extract is analysed by GC-MS, without the need of derivatization, with a detection limit of 0.5 ng. A method was developed for the accurate quantification of anatoxin-a in the standard solution to be used for the calibration of the GC analysis. 1H NMR allowed the accurate quantification of microgram quantities of anatoxin-a. The accurate quantification of compounds in standard solutions is rarely discussed, but for compounds like anatoxin-a (toxins with prices in the range of a million dollar a gram), of which generally only milligram quantities or less are available, this factor in the quantitative analysis is certainly not trivial. The method that was developed can easily be adapted for the accurate quantification of other toxins in very dilute solutions.

The European Nb3Sn advanced strand development programme

International Nuclear Information System (INIS)

Vostner, A.; Salpietro, E.

2005-01-01

Strands relevant for fusion with high critical current densities and moderate hysteresis losses were developed and already produced on industrial scale. Based on these achievements EFDA-CSU Garching has launched a Nb 3 Sn strand development and procurement action inside Europe in order to assess the current status of the Nb 3 Sn strand production capability. All six addressed companies have replied positively to the strand R and D programme which includes the three major Nb 3 Sn production techniques namely the bronze, internal-tin and powder-in-tube (PIT) route. According to the strand requirements for the ITER TF conductor a critical current density of 800 A/mm 2 (at 12 T, 4.2 K and 10 μV/m) and overall strand hysteresis losses below 500 kJ/m 3 have been specified as the minimum guaranteed strand performance. The second major objective of this programme is to motivate the strand manufacturers to develop and design high performance Nb 3 Sn strands optimised for the ITER conductor. For this purpose, a target critical current density of 1100 A/mm 2 has been added to the specification. This paper describes the strategy behind the strand development programme, the actual status of the strand production as well as first preliminary results obtained from the strand suppliers

Corrosion characteristics of post-tensioning strands in ungrouted ducts : summary.

Science.gov (United States)

2011-01-01

To prevent corrosion of post-tensioning strands, FDOT construction specifications currently require post-tensioning ducts to be grouted within seven calendar days of strand installation. This period challenges construction schedules on large projects...

Strand-specific RNA-seq reveals widespread occurrence of novel cis-natural antisense transcripts in rice

Directory of Open Access Journals (Sweden)

Lu Tingting

2012-12-01

Full Text Available Abstract Background Cis-natural antisense transcripts (cis-NATs are RNAs transcribed from the antisense strand of a gene locus, and are complementary to the RNA transcribed from the sense strand. Common techniques including microarray approach and analysis of transcriptome databases are the major ways to globally identify cis-NATs in various eukaryotic organisms. Genome-wide in silico analysis has identified a large number of cis-NATs that may generate endogenous short interfering RNAs (nat-siRNAs, which participate in important biogenesis mechanisms for transcriptional and post-transcriptional regulation in rice. However, the transcriptomes are yet to be deeply sequenced to comprehensively investigate cis-NATs. Results We applied high-throughput strand-specific complementary DNA sequencing technology (ssRNA-seq to deeply sequence mRNA for assessing sense and antisense transcripts that were derived under salt, drought and cold stresses, and normal conditions, in the model plant rice (Oryza sativa. Combined with RAP-DB genome annotation (the Rice Annotation Project Database build-5 data set, 76,013 transcripts corresponding to 45,844 unique gene loci were assembled, in which 4873 gene loci were newly identified. Of 3819 putative rice cis-NATs, 2292 were detected as expressed and giving rise to small RNAs from their overlapping regions through integrated analysis of ssRNA-seq data and small RNA data. Among them, 503 cis-NATs seemed to be associated with specific conditions. The deep sequence data from isolated epidermal cells of rice seedlings further showed that 54.0% of cis-NATs were expressed simultaneously in a population of homogenous cells. Nearly 9.7% of rice transcripts were involved in one-to-one or many-to-many cis-NATs formation. Furthermore, only 17.4-34.7% of 223 many-to-many cis-NAT groups were all expressed and generated nat-siRNAs, indicating that only some cis-NAT groups may be involved in complex regulatory networks. Conclusions

Financial restructuring and stranded costs: quantification and effect on market rules

Energy Technology Data Exchange (ETDEWEB)

Baird, F.; Marcus, W.

1999-03-01

Fred Baird, Director of Putnam, Hayes and Bartlett Inc., predicted that the province`s market rules would not inhibit new investment, an important point, since the prime test of real competition is the possibility of new entries into the market. Ontario`s approach to competition will ensure that marginal prices are competitive, and will limit Genco`s ability to charge above-market rents or practice volume accommodation strategies that would lead to unnecessary investment. Genco will not have control over the residual market, since the company will not be the supplier of last resort. These are crucial factors of success since the point of dealing with market power is to move towards an economically efficient outcome. Bill Marcus, Principal Economist of JBS Energy Inc. of Sacramento, California discussed strategies for calculating stranded debt and the treatment of Ontario Hydro`s nuclear recovery costs. He identified two critical differences in Ontario`s administrative method for calculating stranded costs: (1) past costs (such as decommissioning) are lumped in with future costs, and (2) the overall figures are based on aggregate calculations, covering the whole corporation. In most other jurisdictions `going forward costs` like fuel costs, operating and maintenance, capital additions, and retubing, are excluded. The difference is important because operating subsidies (which is what is involved in this method of calculation) harm both competitors as well as ratepayers. With respect to nuclear assets he prophesied that Ontario Hydro`s nuclear optimization program, if successful, will add only limited value to the nuclear assets, but will cannibalize the value of the rest of Ontario Hydro. He recommended that the Ministry of Finance pursue a strategy that maximizes the value of the whole company, not just the nuclear assets.

Base damage within single-strand DNA underlies in vivo hypermutability induced by a ubiquitous environmental agent.

Directory of Open Access Journals (Sweden)

Kin Chan

Full Text Available Chromosomal DNA must be in single-strand form for important transactions such as replication, transcription, and recombination to occur. The single-strand DNA (ssDNA is more prone to damage than double-strand DNA (dsDNA, due to greater exposure of chemically reactive moieties in the nitrogenous bases. Thus, there can be agents that damage regions of ssDNA in vivo while being inert toward dsDNA. To assess the potential hazard posed by such agents, we devised an ssDNA-specific mutagenesis reporter system in budding yeast. The reporter strains bear the cdc13-1 temperature-sensitive mutation, such that shifting to 37°C results in telomere uncapping and ensuing 5' to 3' enzymatic resection. This exposes the reporter region, containing three closely-spaced reporter genes, as a long 3' ssDNA overhang. We validated the ability of the system to detect mutagenic damage within ssDNA by expressing a modified human single-strand specific cytosine deaminase, APOBEC3G. APOBEC3G induced a high density of substitutions at cytosines in the ssDNA overhang strand, resulting in frequent, simultaneous inactivation of two reporter genes. We then examined the mutagenicity of sulfites, a class of reactive sulfur oxides to which humans are exposed frequently via respiration and food intake. Sulfites, at a concentration similar to that found in some foods, induced a high density of mutations, almost always as substitutions at cytosines in the ssDNA overhang strand, resulting in simultaneous inactivation of at least two reporter genes. Furthermore, sulfites formed a long-lived adducted 2'-deoxyuracil intermediate in DNA that was resistant to excision by uracil-DNA N-glycosylase. This intermediate was bypassed by error-prone translesion DNA synthesis, frequently involving Pol ζ, during repair synthesis. Our results suggest that sulfite-induced lesions in DNA can be particularly deleterious, since cells might not possess the means to repair or bypass such lesions

Quantification of massively parallel sequencing libraries - a comparative study of eight methods

DEFF Research Database (Denmark)

Hussing, Christian; Kampmann, Marie-Louise; Mogensen, Helle Smidt

2018-01-01

Quantification of massively parallel sequencing libraries is important for acquisition of monoclonal beads or clusters prior to clonal amplification and to avoid large variations in library coverage when multiple samples are included in one sequencing analysis. No gold standard for quantification...... estimates followed by Qubit and electrophoresis-based instruments (Bioanalyzer, TapeStation, GX Touch, and Fragment Analyzer), while SYBR Green and TaqMan based qPCR assays gave the lowest estimates. qPCR gave more accurate predictions of sequencing coverage than Qubit and TapeStation did. Costs, time......-consumption, workflow simplicity, and ability to quantify multiple samples are discussed. Technical specifications, advantages, and disadvantages of the various methods are pointed out....

On the biophysics and kinetics of toehold-mediated DNA strand displacement.

Science.gov (United States)

Srinivas, Niranjan; Ouldridge, Thomas E; Sulc, Petr; Schaeffer, Joseph M; Yurke, Bernard; Louis, Ard A; Doye, Jonathan P K; Winfree, Erik

2013-12-01

Dynamic DNA nanotechnology often uses toehold-mediated strand displacement for controlling reaction kinetics. Although the dependence of strand displacement kinetics on toehold length has been experimentally characterized and phenomenologically modeled, detailed biophysical understanding has remained elusive. Here, we study strand displacement at multiple levels of detail, using an intuitive model of a random walk on a 1D energy landscape, a secondary structure kinetics model with single base-pair steps and a coarse-grained molecular model that incorporates 3D geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Two factors explain the dependence of strand displacement kinetics on toehold length: (i) the physical process by which a single step of branch migration occurs is significantly slower than the fraying of a single base pair and (ii) initiating branch migration incurs a thermodynamic penalty, not captured by state-of-the-art nearest neighbor models of DNA, due to the additional overhang it engenders at the junction. Our findings are consistent with previously measured or inferred rates for hybridization, fraying and branch migration, and they provide a biophysical explanation of strand displacement kinetics. Our work paves the way for accurate modeling of strand displacement cascades, which would facilitate the simulation and construction of more complex molecular systems.

Electrophoresis examination of strand breaks in plasmid DNA induced by low-energy nitrogen ion irradiation

International Nuclear Information System (INIS)

Zhao Yong; Tan Zheng; Du Yanhua; Qiu Guanying

2003-01-01

To study the effect on plasmid DNA of heavy ion in the energy range of keV where nuclear stopping interaction becomes more important or even predominant, thin film of plasmid pGEM-3Zf(-) DNA was prepared on aluminum surface and irradiated in vacuum ( -3 Pa) by low-energy nitrogen ions with energy of 30 keV (LET=285 keV/μm) at various fluence ranging from 2 x 10 10 to 8.2 x 10 13 ions/cm 2 . DNA strand breaks were analyzed by neutral electrophoresis followed by quantification with image analysis software. Low-energy nitrogen ion irradiation induced single-, double- and multiple double-strand breaks (DSB) and multiple DSB as the dominating form of DNA damages. Moreover, the linear fluence-response relationship at a low fluence range suggests that DSBs are induced predominantly by single ion track. However, strand break production is limited to a short range in the irradiated samples

Hybrid quadrupole-orbitrap mass spectrometry analysis with accurate-mass database and parallel reaction monitoring for high-throughput screening and quantification of multi-xenobiotics in honey.

Science.gov (United States)

Li, Yi; Zhang, Jinzhen; Jin, Yue; Wang, Lin; Zhao, Wen; Zhang, Wenwen; Zhai, Lifei; Zhang, Yaping; Zhang, Yongxin; Zhou, Jinhui

2016-01-15

This study reports a rapid, automated screening and quantification method for the determination of multi-xenobiotic residues in honey using ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap) with a user-built accurate-mass database plus parallel reaction monitoring (PRM). The database contains multi-xenobiotic information including formulas, adduct types, theoretical exact mass and retention time, characteristic fragment ions, ion ratios, and mass accuracies. A simple sample preparation method was developed to reduce xenobiotic loss in the honey samples. The screening method was validated based on retention time deviation, mass accuracy via full scan-data-dependent MS/MS (full scan-ddMS2), multi-isotope ratio, characteristic ion ratio, sensitivity, and positive/negative switching performance between the spiked sample and corresponding standard solution. The quantification method based on the PRM mode is a promising new quantitative tool which we validated in terms of selectivity, linearity, recovery (accuracy), repeatability (precision), decision limit (CCα), detection capability (CCβ), matrix effects, and carry-over. The optimized methods proposed in this study enable the automated screening and quantification of 157 compounds in less than 15 min in honey. The results of this study, as they represent a convenient protocol for large-scale screening and quantification, also provide a research approach for analysis of various contaminants in other matrices. Copyright © 2015 Elsevier B.V. All rights reserved.

Accurate quantification of 5 German cockroach (GCr) allergens in complex extracts using multiple reaction monitoring mass spectrometry (MRM MS).

Science.gov (United States)

Mindaye, S T; Spiric, J; David, N A; Rabin, R L; Slater, J E

2017-12-01

German cockroach (GCr) allergen extracts are complex and heterogeneous products, and methods to better assess their potency and composition are needed for adequate studies of their safety and efficacy. The objective of this study was to develop an assay based on liquid chromatography and multiple reaction monitoring mass spectrometry (LC-MRM MS) for rapid, accurate, and reproducible quantification of 5 allergens (Bla g 1, Bla g 2, Bla g 3, Bla g 4, and Bla g 5) in crude GCr allergen extracts. We first established a comprehensive peptide library of allergens from various commercial extracts as well as recombinant allergens. Peptide mapping was performed using high-resolution MS, and the peptide library was then used to identify prototypic and quantotypic peptides to proceed with MRM method development. Assay development included a systematic optimization of digestion conditions (buffer, digestion time, and trypsin concentration), chromatographic separation, and MS parameters. Robustness and suitability were assessed following ICH (Q2 [R1]) guidelines. The method is precise (RSD  0.99, 0.01-1384 fmol/μL), and sensitive (LLOD and LLOQ MS, we quantified allergens from various commercial GCr extracts and showed considerable variability that may impact clinical efficacy. Our data demonstrate that the LC-MRM MS method is valuable for absolute quantification of allergens in GCr extracts and likely has broader applicability to other complex allergen extracts. Definitive quantification provides a new standard for labelling of allergen extracts, which will inform patient care, enable personalized therapy, and enhance the efficacy of immunotherapy for environmental and food allergies. © 2017 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Disease quantification in dermatology

DEFF Research Database (Denmark)

Greve, Tanja Maria; Kamp, Søren; Jemec, Gregor B E

2013-01-01

Accurate documentation of disease severity is a prerequisite for clinical research and the practice of evidence-based medicine. The quantification of skin diseases such as psoriasis currently relies heavily on clinical scores. Although these clinical scoring methods are well established and very ...

Collagen Quantification in Tissue Specimens.

Science.gov (United States)

Coentro, João Quintas; Capella-Monsonís, Héctor; Graceffa, Valeria; Wu, Zhuning; Mullen, Anne Maria; Raghunath, Michael; Zeugolis, Dimitrios I

2017-01-01

Collagen is the major extracellular protein in mammals. Accurate quantification of collagen is essential in the biomaterials (e.g., reproducible collagen scaffold fabrication), drug discovery (e.g., assessment of collagen in pathophysiologies, such as fibrosis), and tissue engineering (e.g., quantification of cell-synthesized collagen) fields. Although measuring hydroxyproline content is the most widely used method to quantify collagen in biological specimens, the process is very laborious. To this end, the Sircol™ Collagen Assay is widely used due to its inherent simplicity and convenience. However, this method leads to overestimation of collagen content due to the interaction of Sirius red with basic amino acids of non-collagenous proteins. Herein, we describe the addition of an ultrafiltration purification step in the process to accurately determine collagen content in tissues.

Sequence specific electronic conduction through polyion-stabilized double-stranded DNA in nanoscale break junctions

International Nuclear Information System (INIS)

Mahapatro, Ajit K; Jeong, Kyung J; Lee, Gil U; Janes, David B

2007-01-01

This paper presents a study of sequence specific electronic conduction through short (15-base-pair) double-stranded (ds) DNA molecules, measured by immobilizing 3 ' -thiol-derivatized DNAs in nanometre scale gaps between gold electrodes. The polycation spermidine was used to stabilize the ds-DNA structure, allowing electrical measurements to be performed in a dry state. For specific sequences, the conductivity was observed to scale with the surface density of immobilized DNA, which can be controlled by the buffer concentration. A series of 15-base DNA oligonucleotide pairs, in which the centre sequence of five base pairs was changed from G:C to A:T pairs, has been studied. The conductivity per molecule is observed to decrease exponentially with the number of adjacent A:T pairs replacing G:C pairs, consistent with a barrier at the A:T sites. Conductance-based devices for short DNA sequences could provide sensing approaches with direct electrical readout, as well as label-free detection

Sequential strand displacement beacon for detection of DNA coverage on functionalized gold nanoparticles.

Science.gov (United States)

Paliwoda, Rebecca E; Li, Feng; Reid, Michael S; Lin, Yanwen; Le, X Chris

2014-06-17

Functionalizing nanomaterials for diverse analytical, biomedical, and therapeutic applications requires determination of surface coverage (or density) of DNA on nanomaterials. We describe a sequential strand displacement beacon assay that is able to quantify specific DNA sequences conjugated or coconjugated onto gold nanoparticles (AuNPs). Unlike the conventional fluorescence assay that requires the target DNA to be fluorescently labeled, the sequential strand displacement beacon method is able to quantify multiple unlabeled DNA oligonucleotides using a single (universal) strand displacement beacon. This unique feature is achieved by introducing two short unlabeled DNA probes for each specific DNA sequence and by performing sequential DNA strand displacement reactions. Varying the relative amounts of the specific DNA sequences and spacing DNA sequences during their coconjugation onto AuNPs results in different densities of the specific DNA on AuNP, ranging from 90 to 230 DNA molecules per AuNP. Results obtained from our sequential strand displacement beacon assay are consistent with those obtained from the conventional fluorescence assays. However, labeling of DNA with some fluorescent dyes, e.g., tetramethylrhodamine, alters DNA density on AuNP. The strand displacement strategy overcomes this problem by obviating direct labeling of the target DNA. This method has broad potential to facilitate more efficient design and characterization of novel multifunctional materials for diverse applications.

Accurate quantification of endogenous androgenic steroids in cattle's meat by gas chromatography mass spectrometry using a surrogate analyte approach

Energy Technology Data Exchange (ETDEWEB)

Ahmadkhaniha, Reza; Shafiee, Abbas [Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14174 (Iran, Islamic Republic of); Rastkari, Noushin [Center for Environmental Research, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Kobarfard, Farzad [Department of Medicinal Chemistry, School of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tavaneer Ave., Valieasr St., Tehran (Iran, Islamic Republic of)], E-mail: farzadkf@yahoo.com

2009-01-05

Determination of endogenous steroids in complex matrices such as cattle's meat is a challenging task. Since endogenous steroids always exist in animal tissues, no analyte-free matrices for constructing the standard calibration line will be available, which is crucial for accurate quantification specially at trace level. Although some methods have been proposed to solve the problem, none has offered a complete solution. To this aim, a new quantification strategy was developed in this study, which is named 'surrogate analyte approach' and is based on using isotope-labeled standards instead of natural form of endogenous steroids for preparing the calibration line. In comparison with the other methods, which are currently in use for the quantitation of endogenous steroids, this approach provides improved simplicity and speed for analysis on a routine basis. The accuracy of this method is better than other methods at low concentration and comparable to the standard addition at medium and high concentrations. The method was also found to be valid according to the ICH criteria for bioanalytical methods. The developed method could be a promising approach in the field of compounds residue analysis.

Higher 5-hydroxymethylcytosine identifies immortal DNA strand chromosomes in asymmetrically self-renewing distributed stem cells.

Science.gov (United States)

Huh, Yang Hoon; Cohen, Justin; Sherley, James L

2013-10-15

Immortal strands are the targeted chromosomal DNA strands of nonrandom sister chromatid segregation, a mitotic chromosome segregation pattern unique to asymmetrically self-renewing distributed stem cells (DSCs). By nonrandom segregation, immortal DNA strands become the oldest DNA strands in asymmetrically self-renewing DSCs. Nonrandom segregation of immortal DNA strands may limit DSC mutagenesis, preserve DSC fate, and contribute to DSC aging. The mechanisms responsible for specification and maintenance of immortal DNA strands are unknown. To discover clues to these mechanisms, we investigated the 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) content on chromosomes in mouse hair follicle DSCs during nonrandom segregation. Although 5-methylcytosine content did not differ significantly, the relative content of 5hmC was significantly higher in chromosomes containing immortal DNA strands than in opposed mitotic chromosomes containing younger mortal DNA strands. The difference in relative 5hmC content was caused by the loss of 5hmC from mortal chromosomes. These findings implicate higher 5hmC as a specific molecular determinant of immortal DNA strand chromosomes. Because 5hmC is an intermediate during DNA demethylation, we propose a ten-eleven translocase enzyme mechanism for both the specification and maintenance of nonrandomly segregated immortal DNA strands. The proposed mechanism reveals a means by which DSCs "know" the generational age of immortal DNA strands. The mechanism is supported by molecular expression data and accounts for the selection of newly replicated DNA strands when nonrandom segregation is initiated. These mechanistic insights also provide a possible basis for another characteristic property of immortal DNA strands, their guanine ribonucleotide dependency.

A methodology to identify stranded generation facilities and estimate stranded costs for Louisiana's electric utility industry

Science.gov (United States)

Cope, Robert Frank, III

1998-12-01

The electric utility industry in the United States is currently experiencing a new and different type of growing pain. It is the pain of having to restructure itself into a competitive business. Many industry experts are trying to explain how the nation as a whole, as well as individual states, will implement restructuring and handle its numerous "transition problems." One significant transition problem for federal and state regulators rests with determining a utility's stranded costs. Stranded generation facilities are assets which would be uneconomic in a competitive environment or costs for assets whose regulated book value is greater than market value. At issue is the methodology which will be used to estimate stranded costs. The two primary methods are known as "Top-Down" and "Bottom-Up." The "Top-Down" approach simply determines the present value of the losses in revenue as the market price for electricity changes over a period of time into the future. The problem with this approach is that it does not take into account technical issues associated with the generation and wheeling of electricity. The "Bottom-Up" approach computes the present value of specific strandable generation facilities and compares the resulting valuations with their historical costs. It is regarded as a detailed and difficult, but more precise, approach to identifying stranded assets and their associated costs. This dissertation develops a "Bottom-Up" quantitative, optimization-based approach to electric power wheeling within the state of Louisiana. It optimally evaluates all production capabilities and coordinates the movement of bulk power through transmission interconnections of competing companies in and around the state. Sensitivity analysis to this approach is performed by varying seasonal consumer demand, electric power imports, and transmission inter-connection cost parameters. Generation facility economic dispatch and transmission interconnection bulk power transfers, specific

QUESP and QUEST revisited - fast and accurate quantitative CEST experiments.

Science.gov (United States)

Zaiss, Moritz; Angelovski, Goran; Demetriou, Eleni; McMahon, Michael T; Golay, Xavier; Scheffler, Klaus

2018-03-01

Chemical exchange saturation transfer (CEST) NMR or MRI experiments allow detection of low concentrated molecules with enhanced sensitivity via their proton exchange with the abundant water pool. Be it endogenous metabolites or exogenous contrast agents, an exact quantification of the actual exchange rate is required to design optimal pulse sequences and/or specific sensitive agents. Refined analytical expressions allow deeper insight and improvement of accuracy for common quantification techniques. The accuracy of standard quantification methodologies, such as quantification of exchange rate using varying saturation power or varying saturation time, is improved especially for the case of nonequilibrium initial conditions and weak labeling conditions, meaning the saturation amplitude is smaller than the exchange rate (γB 1  exchange rate using varying saturation power/time' (QUESP/QUEST) equations allow for more accurate exchange rate determination, and provide clear insights on the general principles to execute the experiments and to perform numerical evaluation. The proposed methodology was evaluated on the large-shift regime of paramagnetic chemical-exchange-saturation-transfer agents using simulated data and data of the paramagnetic Eu(III) complex of DOTA-tetraglycineamide. The refined formulas yield improved exchange rate estimation. General convergence intervals of the methods that would apply for smaller shift agents are also discussed. Magn Reson Med 79:1708-1721, 2018. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

Techniques for quantification of liver fat in risk stratification of diabetics

International Nuclear Information System (INIS)

Kuehn, J.P.; Spoerl, M.C.; Mahlke, C.; Hegenscheid, K.

2015-01-01

Fatty liver disease plays an important role in the development of type 2 diabetes. Accurate techniques for detection and quantification of liver fat are essential for clinical diagnostics. Chemical shift-encoded magnetic resonance imaging (MRI) is a simple approach to quantify liver fat content. Liver fat quantification using chemical shift-encoded MRI is influenced by several bias factors, such as T2* decay, T1 recovery and the multispectral complexity of fat. The confounder corrected proton density fat fraction is a simple approach to quantify liver fat with comparable results independent of the software and hardware used. The proton density fat fraction is an accurate biomarker for assessment of liver fat. An accurate and reproducible quantification of liver fat using chemical shift-encoded MRI requires a calculation of the proton density fat fraction. (orig.) [de

Specificity and affinity quantification of protein-protein interactions.

Science.gov (United States)

Yan, Zhiqiang; Guo, Liyong; Hu, Liang; Wang, Jin

2013-05-01

Most biological processes are mediated by the protein-protein interactions. Determination of the protein-protein structures and insight into their interactions are vital to understand the mechanisms of protein functions. Currently, compared with the isolated protein structures, only a small fraction of protein-protein structures are experimentally solved. Therefore, the computational docking methods play an increasing role in predicting the structures and interactions of protein-protein complexes. The scoring function of protein-protein interactions is the key responsible for the accuracy of the computational docking. Previous scoring functions were mostly developed by optimizing the binding affinity which determines the stability of the protein-protein complex, but they are often lack of the consideration of specificity which determines the discrimination of native protein-protein complex against competitive ones. We developed a scoring function (named as SPA-PP, specificity and affinity of the protein-protein interactions) by incorporating both the specificity and affinity into the optimization strategy. The testing results and comparisons with other scoring functions show that SPA-PP performs remarkably on both predictions of binding pose and binding affinity. Thus, SPA-PP is a promising quantification of protein-protein interactions, which can be implemented into the protein docking tools and applied for the predictions of protein-protein structure and affinity. The algorithm is implemented in C language, and the code can be downloaded from http://dl.dropbox.com/u/1865642/Optimization.cpp.

A multicenter study benchmarks software tools for label-free proteome quantification.

Science.gov (United States)

Navarro, Pedro; Kuharev, Jörg; Gillet, Ludovic C; Bernhardt, Oliver M; MacLean, Brendan; Röst, Hannes L; Tate, Stephen A; Tsou, Chih-Chiang; Reiter, Lukas; Distler, Ute; Rosenberger, George; Perez-Riverol, Yasset; Nesvizhskii, Alexey I; Aebersold, Ruedi; Tenzer, Stefan

2016-11-01

Consistent and accurate quantification of proteins by mass spectrometry (MS)-based proteomics depends on the performance of instruments, acquisition methods and data analysis software. In collaboration with the software developers, we evaluated OpenSWATH, SWATH 2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from sequential window acquisition of all theoretical fragment-ion spectra (SWATH)-MS, which uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test data sets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation-window setups. For consistent evaluation, we developed LFQbench, an R package, to calculate metrics of precision and accuracy in label-free quantitative MS and report the identification performance, robustness and specificity of each software tool. Our reference data sets enabled developers to improve their software tools. After optimization, all tools provided highly convergent identification and reliable quantification performance, underscoring their robustness for label-free quantitative proteomics.

Protective role of 3-nitrotyrosine against gamma radiation-induced DNA strand breaks: A comparison study with tyrosine

International Nuclear Information System (INIS)

Shi Weiqun; Ni Meinan; Kong Fuquan; Sui Li; Hu Jia; Xu Diandou; Li Yanmei

2008-01-01

3-Nitrotyrosine(3-NY) has been reported as a potential source of reactive oxygen species (ROSs). In this work, plasmid pBR322 DNA was irradiated by gamma rays in aqueous solution in presence and absence of 3-NY, DNA strand breaks were analyzed by neutral electrophoresis followed by quantification with image analysis software. It was found that the presence of 3-NY could effectively reduce radiation-induced DNA strand breaks. A side-by-side comparison was performed between 3-NY and tyrosine, the results showed that the protective role 3-NY was comparable with tyrosine, which might imply that protein tyrosine nitration might not significantly decrease its ability as a free radical scavenger

Tissue strands as "bioink" for scale-up organ printing.

Science.gov (United States)

Yu, Yin; Ozbolat, Ibrahim T

2014-01-01

Organ printing, takes tissue spheroids as building blocks together with additive manufacturing technique to engineer tissue or organ replacement parts. Although a wide array of cell aggregation techniques has been investigated, and gained noticeable success, the application of tissue spheroids for scale-up tissue fabrication is still worth investigation. In this paper, we introduce a new micro-fabrication technique to create tissue strands at the scale of 500-700μm as a "bioink" for future robotic tissue printing. Printable alginate micro-conduits are used as semi-permeable capsules for tissue strand fabrication. Mouse insulinoma beta TC3 cell tissue strands were formed upon 4 days post fabrication with reasonable mechanical strength, high cell viability close to 90%, and tissue specific markers expression. Fusion was readily observed between strands when placing them together as early as 24h. Also, tissue strands were deposited with human umbilical vein smooth muscle cells (HUVSMCs) vascular conduits together to fabricated miniature pancreatic tissue analog. Our study provided a novel technique using tissue strands as "bioink" for scale-up bioprinting of tissues or organs.

On the direct quantification of celecoxib in commercial solid drugs using the TT-PIXE and TT-PIGE techniques

International Nuclear Information System (INIS)

Nsouli, B.; Zahraman, K.; Bejjani, A.; Assi, S.; El-Yazbi, F.; Roumie, M.

2006-01-01

The quantification of the active ingredient (AI) in drugs is a crucial and important step in the drug quality control process. This is usually performed by using wet chemical techniques like LC-MS, UV spectrophotometry and other appropriate organic analytical methods. In the case of an active ingredient contains specific heteroatoms (F, S, Cl, etc.,), elemental IBA technique can be explored for molecular quantification. IBA techniques permit the analysis of the sample under solid form, without any laborious sample preparation. This is an advantage when the number of sample is relatively large. In this work, we demonstrate the ability of the thick target PIXE (TT-PIXE) and the TT-PIGE techniques for rapid and accurate quantification of celecoxib in commercial drugs. The experimental aspects related to the quantification validity are presented and discussed

Localization of specific sequences and DNA single-strand breaks in individual UV-A-irradiated human lymphocytes by COMET FISH

Science.gov (United States)

Bock, Claudia; Rapp, Alexander; Dittmar, Heike; Monajembashi, Shamci; Greulich, Karl-Otto

1999-01-01

The COMET assay, a single cell electrophoresis technique which allows to separate electrophoretically fractionated DNA according to size has been combined with fluorescence in situ hybridization (FISH) which allows to localize specific genes or gene regions. This combination (COMET FISH) allows the detection of DNA single strand breaks in specific regions of the genome of human lymphocytes at the single cell level. Various types of DNA probes, e.g. centromere-, (alpha) - satellite-, telomere-, whole chromosome-, single copy- and region specific DNA probes have been used to investigate whether the UV-A induced DNA single strand breaks are distributed randomly all over the human genome or induced at specific sites ('hot spots'). In the investigated human peripheral blood lymphocytes all but one centromere reveal low sensitivity for UV-A irradiation (500 kJ/m2), while telomeres are randomly distributed over COMET heads and tails. The human chromosome 1 is fractionated by irradiation, but remains in the COMET head, indicating an only moderate degree of fractionation. Among three tested single copy probes, c- myc, p53 and p58, the p53 gene located on chromosome 17p13.1 and the p58 gene (1p36) appear to be located in UV-A stable regions of the human genome in 95% of 65 investigated lymphocytes. In contrast, the c-myc proto-oncogene (8q24) is found in the COMET tail in 90% of the 27 investigated lymphocytes and thus appears to be more sensitive to UV-A irradiation.

Detection of negative and positive RNA strand of poliovirus Sabin 1 and echovirus E19 by a stem-loop reverse transcription PCR.

Science.gov (United States)

Fikatas, A; Dimitriou, T G; Kyriakopoulou, Z; Moschonas, G D; Amoutzias, G D; Mossialos, D; Gartzonika, C; Levidiotou-Stefanou, S; Markoulatos, P

2017-09-01

In this report a strand specific RT-PCR was established for the detection of the replicative negative RNA strand of poliovirus sabin 1 (Sabin1) and Echovirus 19 (E19) strains. The key for the successful conduction of the assay was the use of a specific reverse transcription primer targeting the 5'-UTR of enteroviruses that consisted of a stem-loop structure at the 5'-end and an enteroviral-specific sequence at the 3'-end. The stem loop RT-PCR was found to be an accurate and sensitive method, detecting even 10 -2 CCID 50 of poliovirus sabin 1 (Sabin1) and E19 strains 6 h postinfection (p.i.), while CPE appeared 3 days later. This assay was also validated in SiHa and Caski cell lines that are not used for the detection of enteroviruses. The negative RNA strand was detected 6 h and 12 h p.i. in SiHa and Caski cells, when these cell lines were inoculated with 10 5 and 1 CCID 50 respectively, whereas CPE was observed 5 days p.i for SiHa cells and 8 days p.i for Caski cells and that only at 10 5 CCID 50 . The results show that this approach may be used for replacing the time-consuming cell cultures in order to detect the active replication of enteroviruses. Enteroviruses are positive stranded RNA viruses that may cause severe diseases. The conventional method for detection of active viral replication involves virus isolation in sensitive cell cultures followed by titration and seroneutralization. In this report, we describe the use of a stem-loop secondary structured oligonucleotide in RT-PCR assay for the detection of the replicative negative strand of the positive-stranded RNA of poliovirus sabin 1 and E19 strains. This approach proved to be a useful tool that may be used for replacing the time-consuming cell culture assays in order to detect the active replication of enteroviruses. © 2017 The Society for Applied Microbiology.

Fingerprinting and quantification of GMOs in the agro-food sector.

Science.gov (United States)

Taverniers, I; Van Bockstaele, E; De Loose, M

2003-01-01

Most strategies for analyzing GMOs in plants and derived food and feed products, are based on the polymerase chain reaction (PCR) technique. In conventional PCR methods, a 'known' sequence between two specific primers is amplified. To the contrary, with the 'anchor PCR' technique, unknown sequences adjacent to a known sequence, can be amplified. Because T-DNA/plant border sequences are being amplified, anchor PCR is the perfect tool for unique identification of transgenes, including non-authorized GMOs. In this work, anchor PCR was applied to characterize the 'transgene locus' and to clarify the complete molecular structure of at least six different commercial transgenic plants. Based on sequences of T-DNA/plant border junctions, obtained by anchor PCR, event specific primers were developed. The junction fragments, together with endogeneous reference gene targets, were cloned in plasmids. The latter were then used as event specific calibrators in real-time PCR, a new technique for the accurate relative quantification of GMOs. We demonstrate here the importance of anchor PCR for identification and the usefulness of plasmid DNA calibrators in quantification strategies for GMOs, throughout the agro-food sector.

Accurate measurement of mitochondrial DNA deletion level and copy number differences in human skeletal muscle.

Directory of Open Access Journals (Sweden)

John P Grady

Full Text Available Accurate and reliable quantification of the abundance of mitochondrial DNA (mtDNA molecules, both wild-type and those harbouring pathogenic mutations, is important not only for understanding the progression of mtDNA disease but also for evaluating novel therapeutic approaches. A clear understanding of the sensitivity of mtDNA measurement assays under different experimental conditions is therefore critical, however it is routinely lacking for most published mtDNA quantification assays. Here, we comprehensively assess the variability of two quantitative Taqman real-time PCR assays, a widely-applied MT-ND1/MT-ND4 multiplex mtDNA deletion assay and a recently developed MT-ND1/B2M singleplex mtDNA copy number assay, across a range of DNA concentrations and mtDNA deletion/copy number levels. Uniquely, we provide a specific guide detailing necessary numbers of sample and real-time PCR plate replicates for accurately and consistently determining a given difference in mtDNA deletion levels and copy number in homogenate skeletal muscle DNA.

Strand-Specific RNA-Seq Analyses of Fruiting Body Development in Coprinopsis cinerea.

Directory of Open Access Journals (Sweden)

Hajime Muraguchi

Full Text Available The basidiomycete fungus Coprinopsis cinerea is an important model system for multicellular development. Fruiting bodies of C. cinerea are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC. To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.

Swift Quantification of Fenofibrate and Tiemonium methylsulfate Active Ingredients in Solid Drugs Using Particle Induced X-Ray Emission

International Nuclear Information System (INIS)

Bejjani, A.; Nsouli, B.; Zahraman, K.; Assi, S.; Younes, Gh.; Yazbi, F.

2011-01-01

The quantification of active ingredients (AI) in drugs is a crucial and important step in the drug quality control process. This is usually performed by using wet chemical techniques like LC-MS, UV spectrophotometry and other appropriate organic analytical methods. However, if the active ingredient contains specific heteroatoms (F, S, Cl), elemental IBA like PIXE and PIGE techniques, using small tandem accelerator of 1-2 MV, can be explored for molecular quantification. IBA techniques permit the analysis of the sample under solid form, without any laborious sample preparations. In this work, we demonstrate the ability of the Thick Target PIXE technique for rapid and accurate quantification of both low and high concentrations of active ingredients in different commercial drugs. Fenofibrate, a chlorinated active ingredient, is present in high amounts in two different commercial drugs, its quantification was done using the relative approach to an external standard. On the other hand, Tiemonium methylsulfate which exists in relatively low amount in commercial drugs, its quantification was done using GUPIX simulation code (absolute quantification). The experimental aspects related to the quantification validity (use of external standards, absolute quantification, matrix effect,...) are presented and discussed. (author)

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

Science.gov (United States)

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

The percentage of bacterial genes on leading versus lagging strands is influenced by multiple balancing forces

Science.gov (United States)

Mao, Xizeng; Zhang, Han; Yin, Yanbin; Xu, Ying

2012-01-01

The majority of bacterial genes are located on the leading strand, and the percentage of such genes has a large variation across different bacteria. Although some explanations have been proposed, these are at most partial explanations as they cover only small percentages of the genes and do not even consider the ones biased toward the lagging strand. We have carried out a computational study on 725 bacterial genomes, aiming to elucidate other factors that may have influenced the strand location of genes in a bacterium. Our analyses suggest that (i) genes of some functional categories such as ribosome have higher preferences to be on the leading strands; (ii) genes of some functional categories such as transcription factor have higher preferences on the lagging strands; (iii) there is a balancing force that tends to keep genes from all moving to the leading and more efficient strand and (iv) the percentage of leading-strand genes in an bacterium can be accurately explained based on the numbers of genes in the functional categories outlined in (i) and (ii), genome size and gene density, indicating that these numbers implicitly contain the information about the percentage of genes on the leading versus lagging strand in a genome. PMID:22735706

Stranded costs and exit fees

International Nuclear Information System (INIS)

2002-01-01

The New Brunswick Market Design Committee has been directed to examine the issue of stranded costs since it is a major component of restructuring within the electricity sector. When regulated monopolies are faced with competition, they could find that some of their embedded costs cannot be recovered. These costs are referred to as stranded costs. Common sources include large capital investments in uneconomic plants or expensive power purchase contracts or fuel supply contracts. In general, stranded costs do not include gains or losses associated with normal business risks experienced by regulated utilities. This report presents recommendations for mitigation of stranded costs, valuation methodologies and cost-recovery mechanisms. It also presents a summary of experience with stranded costs in other jurisdictions such as California, Rhode Island, Pennsylvania and Ontario. Stranded costs are often recovered through an obligatory charge on all customers, particularly in jurisdictions where retail competition exists. In the New Brunswick market, however, the only customers who can create stranded costs are those eligible to choose their own suppliers. It is argued that since most customers will not have a choice of electricity suppliers, they cannot generate stranded costs and therefore, should not have to pay costs stranded by others. A method to quantify stranded costs is presented, along with a review of transmission-related stranded costs in New Brunswick. Expansion of self-generation in New Brunswick could strand transmission assets. Currently, self-generators only contribute a small amount to fixed charges of the transmission system. However, under new recommended tariffs, the amount could increase. It is likely that the net amount of stranded transmission costs will not be large. 2 refs., 1 fig

Stable isotope dilution HILIC-MS/MS method for accurate quantification of glutamic acid, glutamine, pyroglutamic acid, GABA and theanine in mouse brain tissues.

Science.gov (United States)

Inoue, Koichi; Miyazaki, Yasuto; Unno, Keiko; Min, Jun Zhe; Todoroki, Kenichiro; Toyo'oka, Toshimasa

2016-01-01

In this study, we developed the stable isotope dilution hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS) technique for the accurate, reasonable and simultaneous quantification of glutamic acid (Glu), glutamine (Gln), pyroglutamic acid (pGlu), γ-aminobutyric acid (GABA) and theanine in mouse brain tissues. The quantification of these analytes was accomplished using stable isotope internal standards and the HILIC separating mode to fully correct the intramolecular cyclization during the electrospray ionization. It was shown that linear calibrations were available with high coefficients of correlation (r(2)  > 0.999, range from 10 pmol/mL to 50 mol/mL). For application of the theanine intake, the determination of Glu, Gln, pGlu, GABA and theanine in the hippocampus and central cortex tissues was performed based on our developed method. In the region of the hippocampus, the concentration levels of Glu and pGlu were significantly reduced during reality-based theanine intake. Conversely, the concentration level of GABA increased. This result showed that transited theanine has an effect on the metabolic balance of Glu analogs in the hippocampus. Copyright © 2015 John Wiley & Sons, Ltd.

Strand Displacement by DNA Polymerase III Occurs through a τ-ψ-χ Link to Single-stranded DNA-binding Protein Coating the Lagging Strand Template*

OpenAIRE

Yuan, Quan; McHenry, Charles S.

2009-01-01

In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the single-stranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of γ-complex to support the reaction in the absence of τ. However, if γ-complex is p...

A Novel Computational Method to Reduce Leaky Reaction in DNA Strand Displacement

Directory of Open Access Journals (Sweden)

Xin Li

2015-01-01

Full Text Available DNA strand displacement technique is widely used in DNA programming, DNA biosensors, and gene analysis. In DNA strand displacement, leaky reactions can cause DNA signals decay and detecting DNA signals fails. The mostly used method to avoid leakage is cleaning up after upstream leaky reactions, and it remains a challenge to develop reliable DNA strand displacement technique with low leakage. In this work, we address the challenge by experimentally evaluating the basic factors, including reaction time, ratio of reactants, and ion concentration to the leakage in DNA strand displacement. Specifically, fluorescent probes and a hairpin structure reporting DNA strand are designed to detect the output of DNA strand displacement, and thus can evaluate the leakage of DNA strand displacement reactions with different reaction time, ratios of reactants, and ion concentrations. From the obtained data, mathematical models for evaluating leakage are achieved by curve derivation. As a result, it is obtained that long time incubation, high concentration of fuel strand, and inappropriate amount of ion concentration can weaken leaky reactions. This contributes to a method to set proper reaction conditions to reduce leakage in DNA strand displacement.

Mathematical modelling of the automated FADU assay for the quantification of DNA strand breaks and their repair in human peripheral mononuclear blood cells

International Nuclear Information System (INIS)

Junk, Michael; Salzwedel, Judy; Sindlinger, Thilo; Bürkle, Alexander; Moreno-Villanueva, Maria

2014-01-01

Cells continuously undergo DNA damage from exogenous agents like irradiation or genotoxic chemicals or from endogenous radicals produced by normal cellular metabolic activities. DNA strand breaks are one of the most common genotoxic lesions and they can also arise as intermediates of DNA repair activity. Unrepaired DNA damage can lead to genomic instability, which can massively compromise the health status of organisms. Therefore it is important to measure and quantify DNA damage and its repair. We have previously published an automated method for measuring DNA strand breaks based on fluorimetric detection of alkaline DNA unwinding [1], and here we present a mathematical model of the FADU assay, which enables to an analytic expression for the relation between measured fluorescence and the number of strand breaks. Assessment of the formation and also the repair of DNA strand breaks is a crucial functional parameter to investigate genotoxicity in living cells. A reliable and convenient method to quantify DNA strand breakage is therefore of significant importance for a wide variety of scientific fields, e.g. toxicology, pharmacology, epidemiology and medical sciences

Collaborative framework for PIV uncertainty quantification: comparative assessment of methods

International Nuclear Information System (INIS)

Sciacchitano, Andrea; Scarano, Fulvio; Neal, Douglas R; Smith, Barton L; Warner, Scott O; Vlachos, Pavlos P; Wieneke, Bernhard

2015-01-01

A posteriori uncertainty quantification of particle image velocimetry (PIV) data is essential to obtain accurate estimates of the uncertainty associated with a given experiment. This is particularly relevant when measurements are used to validate computational models or in design and decision processes. In spite of the importance of the subject, the first PIV uncertainty quantification (PIV-UQ) methods have been developed only in the last three years. The present work is a comparative assessment of four approaches recently proposed in the literature: the uncertainty surface method (Timmins et al 2012), the particle disparity approach (Sciacchitano et al 2013), the peak ratio criterion (Charonko and Vlachos 2013) and the correlation statistics method (Wieneke 2015). The analysis is based upon experiments conducted for this specific purpose, where several measurement techniques are employed simultaneously. The performances of the above approaches are surveyed across different measurement conditions and flow regimes. (paper)

MEIOB targets single-strand DNA and is necessary for meiotic recombination.

Directory of Open Access Journals (Sweden)

Benoit Souquet

Full Text Available Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB. This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1 (-/- spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob (-/- meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination.

Strand displacement activated peroxidase activity of hemin for fluorescent DNA sensing.

Science.gov (United States)

Wang, Quanbo; Xu, Nan; Gui, Zhen; Lei, Jianping; Ju, Huangxian; Yan, Feng

2015-10-07

To efficiently regulate the catalytic activity of the peroxidase mimic hemin, this work designs a double-stranded DNA probe containing an intermolecular dimer of hemin, whose peroxidase activity can be activated by a DNA strand displacement reaction. The double-stranded probe is prepared by annealing two strands of hemin labelled DNA oligonucleotides. Using the fluorescent oxidation product of tyramine by H2O2 as a tracing molecule, the low peroxidase activity of the hemin dimer ensures a low fluorescence background. The strand displacement reaction of the target DNA dissociates the hemin dimer and thus significantly increases the catalytic activity of hemin to produce a large amount of dityramine for fluorescence signal readout. Based on the strand displacement regulated peroxidase activity, a simple and sensitive homogeneous fluorescent DNA sensing method is proposed. The detection can conveniently be carried out in a 96-well plate within 20 min with a detection limit of 0.18 nM. This method shows high specificity, which can effectively distinguish single-base mismatched DNA from perfectly matched target DNA. The DNA strand displacement regulated catalytic activity of hemin has promising application in the determination of various DNA analytes.

Highly specific detection of genetic modification events using an enzyme-linked probe hybridization chip.

Science.gov (United States)

Zhang, M Z; Zhang, X F; Chen, X M; Chen, X; Wu, S; Xu, L L

2015-08-10

The enzyme-linked probe hybridization chip utilizes a method based on ligase-hybridizing probe chip technology, with the principle of using thio-primers for protection against enzyme digestion, and using lambda DNA exonuclease to cut multiple PCR products obtained from the sample being tested into single-strand chains for hybridization. The 5'-end amino-labeled probe was fixed onto the aldehyde chip, and hybridized with the single-stranded PCR product, followed by addition of a fluorescent-modified probe that was then enzymatically linked with the adjacent, substrate-bound probe in order to achieve highly specific, parallel, and high-throughput detection. Specificity and sensitivity testing demonstrated that enzyme-linked probe hybridization technology could be applied to the specific detection of eight genetic modification events at the same time, with a sensitivity reaching 0.1% and the achievement of accurate, efficient, and stable results.

A multi-center study benchmarks software tools for label-free proteome quantification

Science.gov (United States)

Gillet, Ludovic C; Bernhardt, Oliver M.; MacLean, Brendan; Röst, Hannes L.; Tate, Stephen A.; Tsou, Chih-Chiang; Reiter, Lukas; Distler, Ute; Rosenberger, George; Perez-Riverol, Yasset; Nesvizhskii, Alexey I.; Aebersold, Ruedi; Tenzer, Stefan

2016-01-01

The consistent and accurate quantification of proteins by mass spectrometry (MS)-based proteomics depends on the performance of instruments, acquisition methods and data analysis software. In collaboration with the software developers, we evaluated OpenSWATH, SWATH2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra), a method that uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test datasets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation windows setups. For consistent evaluation we developed LFQbench, an R-package to calculate metrics of precision and accuracy in label-free quantitative MS, and report the identification performance, robustness and specificity of each software tool. Our reference datasets enabled developers to improve their software tools. After optimization, all tools provided highly convergent identification and reliable quantification performance, underscoring their robustness for label-free quantitative proteomics. PMID:27701404

A specific endogenous reference for genetically modified common bean (Phaseolus vulgaris L.) DNA quantification by real-time PCR targeting lectin gene.

Science.gov (United States)

Venturelli, Gustavo L; Brod, Fábio C A; Rossi, Gabriela B; Zimmermann, Naíra F; Oliveira, Jaison P; Faria, Josias C; Arisi, Ana C M

2014-11-01

The Embrapa 5.1 genetically modified (GM) common bean was approved for commercialization in Brazil. Methods for the quantification of this new genetically modified organism (GMO) are necessary. The development of a suitable endogenous reference is essential for GMO quantification by real-time PCR. Based on this, a new taxon-specific endogenous reference quantification assay was developed for Phaseolus vulgaris L. Three genes encoding common bean proteins (phaseolin, arcelin, and lectin) were selected as candidates for endogenous reference. Primers targeting these candidate genes were designed and the detection was evaluated using the SYBR Green chemistry. The assay targeting lectin gene showed higher specificity than the remaining assays, and a hydrolysis probe was then designed. This assay showed high specificity for 50 common bean samples from two gene pools, Andean and Mesoamerican. For GM common bean varieties, the results were similar to those obtained for non-GM isogenic varieties with PCR efficiency values ranging from 92 to 101 %. Moreover, this assay presented a limit of detection of ten haploid genome copies. The primers and probe developed in this work are suitable to detect and quantify either GM or non-GM common bean.

freeQuant: A Mass Spectrometry Label-Free Quantification Software Tool for Complex Proteome Analysis.

Science.gov (United States)

Deng, Ning; Li, Zhenye; Pan, Chao; Duan, Huilong

2015-01-01

Study of complex proteome brings forward higher request for the quantification method using mass spectrometry technology. In this paper, we present a mass spectrometry label-free quantification tool for complex proteomes, called freeQuant, which integrated quantification with functional analysis effectively. freeQuant consists of two well-integrated modules: label-free quantification and functional analysis with biomedical knowledge. freeQuant supports label-free quantitative analysis which makes full use of tandem mass spectrometry (MS/MS) spectral count, protein sequence length, shared peptides, and ion intensity. It adopts spectral count for quantitative analysis and builds a new method for shared peptides to accurately evaluate abundance of isoforms. For proteins with low abundance, MS/MS total ion count coupled with spectral count is included to ensure accurate protein quantification. Furthermore, freeQuant supports the large-scale functional annotations for complex proteomes. Mitochondrial proteomes from the mouse heart, the mouse liver, and the human heart were used to evaluate the usability and performance of freeQuant. The evaluation showed that the quantitative algorithms implemented in freeQuant can improve accuracy of quantification with better dynamic range.

Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

International Nuclear Information System (INIS)

Tak, Yu Kyung; Kim, Won Young; Kim, Min Jung; Han, Eunyoung; Han, Myun Soo; Kim, Jong Jin; Kim, Wook; Lee, Jong Eun; Song, Joon Myong

2012-01-01

Highlights: ► Genomic DNA quantification were performed using a quantum dot-labeled Alu sequence. ► This probe provided PCR-free determination of human genomic DNA. ► Qdot-labeled Alu probe-hybridized genomic DNAs had a 2.5-femtogram detection limit. ► Qdot-labeled Alu sequence was used to assess DNA samples for human identification. - Abstract: Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.

Accurate and precise DNA quantification in the presence of different amplification efficiencies using an improved Cy0 method.

Science.gov (United States)

Guescini, Michele; Sisti, Davide; Rocchi, Marco B L; Panebianco, Renato; Tibollo, Pasquale; Stocchi, Vilberto

2013-01-01

Quantitative real-time PCR represents a highly sensitive and powerful technology for the quantification of DNA. Although real-time PCR is well accepted as the gold standard in nucleic acid quantification, there is a largely unexplored area of experimental conditions that limit the application of the Ct method. As an alternative, our research team has recently proposed the Cy0 method, which can compensate for small amplification variations among the samples being compared. However, when there is a marked decrease in amplification efficiency, the Cy0 is impaired, hence determining reaction efficiency is essential to achieve a reliable quantification. The proposed improvement in Cy0 is based on the use of the kinetic parameters calculated in the curve inflection point to compensate for efficiency variations. Three experimental models were used: inhibition of primer extension, non-optimal primer annealing and a very small biological sample. In all these models, the improved Cy0 method increased quantification accuracy up to about 500% without affecting precision. Furthermore, the stability of this procedure was enhanced integrating it with the SOD method. In short, the improved Cy0 method represents a simple yet powerful approach for reliable DNA quantification even in the presence of marked efficiency variations.

On the direct characterization and quantification of active ingredients in commercial solid drugs using PIXE, PIGE and ToF-SIMS techniques

Energy Technology Data Exchange (ETDEWEB)

Nsouli, B; Zahraman, K; Bejjani, A; Roumie, M; Noun, M [Ion Beam Analysis Laboratory, Lebanese Atomic Energy Commission - CNRS, P.O.Box: 11-8281 Beirut (Lebanon); Younes, G [Faculty of Sciences, Department of Chemistry - Beirut Arab University, Beirut (Lebanon); Assi, S; El-Yazbi, F; Mahmoud, R [Faculty of Pharmacy, Department of Pharmaceutical Analytical Chemistry - Beirut Arab University, Beirut (Lebanon); Thomas, J P [Institut de Physique Nucleaire de Lyon - Universite Claude Bernard Lyon 1 (France)

2010-01-15

The quantification of the active ingredient (AI) in drugs is a crucial and important step in the drug quality control process. This is usually performed by using wet chemical techniques like HPLC, LC-MS/MS, UV spectrophotometry and other appropriate organic analytical methods. In the case of an active ingredient contains specific heteroatoms (F, S, Cl, Br, ...), elemental IBA technique can be explored for molecular quantification. IBA techniques permit the analysis of the sample under solid form, without any laborious sample preparation. This is an advantage when the number of sample is relatively large. In this work, we demonstrate the ability of the Thick target PIXE (TT-PIXE) and the TT-PIGE techniques for rapid and accurate quantification of celecoxib and atorvastatin in commercial solid drugs. The experimental aspects related to the quantification validity are presented and discussed. (author)

On the direct characterization and quantification of active ingredients in commercial solid drugs using PIXE, PIGE and ToF-SIMS techniques

International Nuclear Information System (INIS)

Nsouli, B.; Zahraman, K.; Bejjani, A.; Roumie, M.; Noun, M.; Younes, G.; Assi, S.; El-Yazbi, F.; Mahmoud, R.; Thomas, J.P.

2010-01-01

The quantification of the active ingredient (AI) in drugs is a crucial and important step in the drug quality control process. This is usually performed by using wet chemical techniques like HPLC, LC-MS/MS, UV spectrophotometry and other appropriate organic analytical methods. In the case of an active ingredient contains specific heteroatoms (F, S, Cl, Br, ...), elemental IBA technique can be explored for molecular quantification. IBA techniques permit the analysis of the sample under solid form, without any laborious sample preparation. This is an advantage when the number of sample is relatively large. In this work, we demonstrate the ability of the Thick target PIXE (TT-PIXE) and the TT-PIGE techniques for rapid and accurate quantification of celecoxib and atorvastatin in commercial solid drugs. The experimental aspects related to the quantification validity are presented and discussed. (author)

Bioelectrochemical sensing of promethazine with bamboo-type multiwalled carbon nanotubes dispersed in calf-thymus double stranded DNA.

Science.gov (United States)

Primo, Emiliano N; Oviedo, M Belén; Sánchez, Cristián G; Rubianes, María D; Rivas, Gustavo A

2014-10-01

We report the quantification of promethazine (PMZ) using glassy carbon electrodes (GCE) modified with bamboo-like multi-walled carbon nanotubes (bCNT) dispersed in double stranded calf-thymus DNA (dsDNA) (GCE/bCNT-dsDNA). Cyclic voltammetry measurements demonstrated that PMZ presents a thin film-confined redox behavior at GCE/bCNT-dsDNA, opposite to the irreversibly-adsorbed behavior obtained at GCE modified with bCNT dispersed in ethanol (GCE/bCNT). Differential pulse voltammetry-adsorptive stripping with medium exchange experiments performed with GCE/bCNT-dsDNA and GCE modified with bCNTs dispersed in single-stranded calf-thymus DNA (ssDNA) confirmed that the interaction between PMZ and bCNT-dsDNA is mainly hydrophobic. These differences are due to the intercalation of PMZ within the dsDNA that supports the bCNTs, as evidenced from the bathochromic displacement of UV-Vis absorption spectra of PMZ and quantum dynamics calculations at DFTB level. The efficient accumulation of PMZ at GCE/bCNT-dsDNA made possible its sensitive quantification at nanomolar levels (sensitivity: (3.50±0.05)×10(8) μA·cm(-2)·M(-1) and detection limit: 23 nM). The biosensor was successfully used for the determination of PMZ in a pharmaceutical product with excellent correlation. Copyright © 2014 Elsevier B.V. All rights reserved.

Strategy study of quantification harmonization of SUV in PET/CT images

International Nuclear Information System (INIS)

Fischer, Andreia Caroline Fischer da Silveira

2014-01-01

In clinical practice, PET/CT images are often analyzed qualitatively by visual comparison of tumor lesions and normal tissues uptake; and semi-quantitatively by means of a parameter called SUV (Standardized Uptake Value). To ensure that longitudinal studies acquired on different scanners are interchangeable, and information of quantification is comparable, it is necessary to establish a strategy to harmonize the quantification of SUV. The aim of this study is to evaluate the strategy to harmonize the quantification of PET/CT images, performed with different scanner models and manufacturers. For this purpose, a survey of the technical characteristics of equipment and acquisition protocols of clinical images of different services of PET/CT in the state of Rio Grande do Sul was conducted. For each scanner, the accuracy of SUV quantification, and the Recovery Coefficient (RC) curves were determined, using the reconstruction parameters clinically relevant and available. From these data, harmonized performance specifications among the evaluated scanners were identified, as well as the algorithm that produces, for each one, the most accurate quantification. Finally, the most appropriate reconstruction parameters to harmonize the SUV quantification in each scanner, either regionally or internationally were identified. It was found that the RC values of the analyzed scanners proved to be overestimated by up to 38%, particularly for objects larger than 17mm. These results demonstrate the need for further optimization, through the reconstruction parameters modification, and even the change of the reconstruction algorithm used in each scanner. It was observed that there is a decoupling between the best image for PET/CT qualitative analysis and the best image for quantification studies. Thus, the choice of reconstruction method should be tied to the purpose of the PET/CT study in question, since the same reconstruction algorithm is not adequate, in one scanner, for qualitative

KARAKTERISTIK ORIENTED STRAND BOARD DARI KAYU AKASIA DAN AFRIKA BERDASARKAN PENYUSUNAN ARAH STRAND

Directory of Open Access Journals (Sweden)

Nurhaida

2008-04-01

Full Text Available The research objectives arc to evaluate physical and mechanical properties of OSB based on strands orientation; and to evaluate physical and mechanical properties of OSB made from akasia wood (Acacia mangium Wild and afrika wood (Maesopsis eminii Engl. Akasia and afrika wood are used for OSB strand material with phenol formaldehyde (PF as adhesives and addition of paraffin. OSB made in this research is consist of three plies whereas are differed into eight (8 strand orientations. In the making process, hot press was carried out at 160OC and pressure 25kg.cm-2 for 15 minutes. Determination of OSB physical and mechanical properties is referred to JIS A 5908-2003. Result showed that strand orientations has no affect to OSB physical properties except for linicr swelling 24h, but it significantly influence all mechanical properties of OSB. Wood species have an effect on mechanical properties of OSB in the dry test, wet MOE lengthwise test and OSB physical properties, particularly to OSB density and water absorbing capability at 2h and 24h. All of OSB physical properties arc meet JIS A 5908-2003 standard, but not all of the mechanical properties such as dry MOE lengthwise, dry MOE and MOR widthwise. The best physical and mechanical properties is presented by OSB made from akasia wood in strand orientation F, G, Band C whereas all parameters meet JIS A 5908-2003 standard. In comparation with strand orientation B that is frequent used in industry, strand orientation F and G arc proficient to raise the modulus elasticity value (MOE and strength (MOR as much as 167.81-231.65% and 89.73-109.87%, respectively; especially in widthwise board application. Furthermore, strand orientation F and G arc more flexible as structural components

Critical points of DNA quantification by real-time PCR--effects of DNA extraction method and sample matrix on quantification of genetically modified organisms.

Science.gov (United States)

Cankar, Katarina; Stebih, Dejan; Dreo, Tanja; Zel, Jana; Gruden, Kristina

2006-08-14

evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated. The crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR.

End-specific strategies of attachment of long double stranded DNA onto gold-coated nanofiber arrays

International Nuclear Information System (INIS)

Peckys, Diana B; De Jonge, Niels; Simpson, Michael L; McKnight, Timothy E

2008-01-01

We report the effective and site-specific binding of long double stranded (ds)DNA to high aspect ratio carbon nanofiber arrays. The carbon nanofibers were first coated with a thin gold layer to provide anchorage for two controllable binding methods. One method was based on the direct binding of thiol end-labeled dsDNA. The second and enhanced method used amine end-labeled dsDNA bound with crosslinkers to a carboxyl-terminated self-assembled monolayer. The bound dsDNA was first visualized with a fluorescent, dsDNA-intercalating dye. The specific binding onto the carbon nanofiber was verified by a high resolution detection method using scanning electron microscopy in combination with the binding of neutravidin-coated fluorescent microspheres to the immobilized and biotinylated dsDNA. Functional activity of thiol end-labeled dsDNA on gold-coated nanofiber arrays was verified with a transcriptional assay, whereby Chinese hamster lung cells (V79) were impaled upon the DNA-modified nanofibers and scored for transgene expression of the tethered template. Thiol end-labeled dsDNA demonstrated significantly higher expression levels than nanofibers prepared with control dsDNA that lacked a gold-binding end-label. Employing these site-specific and robust techniques of immobilization of dsDNA onto nanodevices can be of advantage for the study of DNA/protein interactions and for gene delivery applications.

Five-Strand versus Four-Strand Hamstring Tendon Graft Technique for Anterior Cruciate Ligament Reconstruction: A Biomechanical Comparison.

Science.gov (United States)

Vaillant, Eric R; Parks, Brent G; Camire, Lyn M; Hinton, Richard Y

2017-11-01

The aim of this article is to compare diameter and stiffness, displacement, and strain in a five-strand versus four-strand hamstring graft for anterior cruciate ligament reconstruction. Eight matched pairs of lower extremities underwent four-strand or five-strand hamstring graft reconstruction. Diameter was significantly higher in the five-strand versus the four-strand construct ( p  = 0.002). No significant difference was found between the groups in construct displacement or stiffness. Significantly higher strain was observed in the inner limb versus the outer limb in the four-strand construct ( p  = 0.001) and in the inner limb versus the fifth limb in the 5-strand construct ( p  = 0.004). A fifth limb added to a four-strand hamstring graft significantly increased graft diameter but did not significantly change stiffness or displacement, suggesting that attachment of additional graft material via suture did not provide for full incorporation of the added limb into the graft at time zero. The inner limb in both constructs absorbed significantly greater load than did other limbs. The use of suture to attach additional material to a four-strand hamstring graft may not contribute to improved biomechanical qualities of the graft at time zero. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Modelling Toehold-Mediated RNA Strand Displacement

OpenAIRE

Šulc, Petr; Ouldridge, Thomas E.; Romano, Flavio; Doye, Jonathan P.K.; Louis, Ard A.

2015-01-01

We study the thermodynamics and kinetics of an RNA toehold-mediated strand displacement reaction with a recently developed coarse-grained model of RNA. Strand displacement, during which a single strand displaces a different strand previously bound to a complementary substrate strand, is an essential mechanism in active nucleic acid nanotechnology and has also been hypothesized to occur in vivo. We study the rate of displacement reactions as a function of the length of the toehold and temperat...

Knotting probabilities after a local strand passage in unknotted self-avoiding polygons

International Nuclear Information System (INIS)

Szafron, M L; Soteros, C E

2011-01-01

We investigate, both theoretically and numerically, the knotting probabilities after a local strand passage is performed in an unknotted self-avoiding polygon (SAP) on the simple cubic lattice. In the polygons studied, it is assumed that two polygon segments have already been brought close together for the purpose of performing a strand passage. This restricts the polygons considered to those that contain a specific pattern called Θ at a fixed location; an unknotted polygon containing Θ is called a Θ-SAP. It is proved that the number of n-edge Θ-SAPs grows exponentially (with n) at the same rate as the total number of n-edge unknotted SAPs (those with no prespecified strand passage structure). Furthermore, it is proved that the same holds for subsets of n-edge Θ-SAPs that yield a specific after-strand-passage knot-type. Thus, the probability of a given after-strand-passage knot-type does not grow (or decay) exponentially with n. Instead, it is conjectured that these after-strand-passage knot probabilities approach, as n goes to infinity, knot-type dependent amplitude ratios lying strictly between 0 and 1. This conjecture is supported by numerical evidence from Monte Carlo data generated using a composite (aka multiple) Markov chain Monte Carlo BFACF algorithm developed to study Θ-SAPs. A new maximum likelihood method is used to estimate the critical exponents relevant to this conjecture. We also obtain strong numerical evidence that the after-strand-passage knotting probability depends on the local structure around the strand-passage site. If the local structure and the crossing sign at the strand-passage site are considered, then we observe that the more 'compact' the local structure, the less likely the after-strand-passage polygon is to be knotted. This trend for compactness versus knotting probability is consistent with results obtained for other strand-passage models; however, we are the first to note the influence of the crossing-sign information. We

Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

Science.gov (United States)

Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

2015-01-01

Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

Strand Invasion Based Amplification (SIBA®): a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

Science.gov (United States)

Hoser, Mark J; Mansukoski, Hannu K; Morrical, Scott W; Eboigbodin, Kevin E

2014-01-01

Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2'-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

Strand Invasion Based Amplification (SIBA®: a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

Directory of Open Access Journals (Sweden)

Mark J Hoser

Full Text Available Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA. SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2'-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

A Micropillar Compression Methodology for Ductile Damage Quantification

NARCIS (Netherlands)

Tasan, C.C.; Hoefnagels, J.P.M.; Geers, M.G.D.

2012-01-01

Microstructural damage evolution is reported to influence significantly the failures of new high-strength alloys. Its accurate quantification is, therefore, critical for (1) microstructure optimization and (2) continuum damage models to predict failures of these materials. As existing methodologies

A micropillar compression methodology for ductile damage quantification

NARCIS (Netherlands)

Tasan, C.C.; Hoefnagels, J.P.M.; Geers, M.G.D.

2012-01-01

Microstructural damage evolution is reported to influence significantly the failures of new high-strength alloys. Its accurate quantification is, therefore, critical for (1) microstructure optimization and (2) continuum damage models to predict failures of these materials. As existing methodologies

Enzyme-free colorimetric detection systems based on the DNA strand displacement competition reaction

Science.gov (United States)

Zhang, Z.; Birkedal, V.; Gothelf, K. V.

2016-05-01

The strand displacement competition assay is based on the dynamic equilibrium of the competitive hybridization of two oligonucleotides (A and B) to a third oligonucleotide (S). In the presence of an analyte that binds to a specific affinity-moiety conjugated to strand B, the equilibrium shifts, which can be detected by a shift in the fluorescence resonance energy transfer signal between dyes attached to the DNA strands. In the present study we have integrated an ATP aptamer in the strand B and demonstrated the optical detection of ATP. Furthermore we explore a new readout method using a split G-quadruplex DNAzyme for colorimetric readout of the detection of streptavidin by the naked eye. Finally, we integrate the whole G-quadruplex DNAzyme system in a single DNA strand and show that it is applicable to colorimetric detection.

Enzyme-free colorimetric detection systems based on the DNA strand displacement competition reaction

DEFF Research Database (Denmark)

Zhang, Zhao; Birkedal, Victoria; Gothelf, Kurt Vesterager

2016-01-01

The strand displacement competition assay is based on the dynamic equilibrium of the competitive hybridization of two oligonucleotides (A and B) to a third oligonucleotide (S). In the presence of an analyte that binds to a specific affinity-moiety conjugated to strand B, the equilibrium shifts, w...... G-quadruplex DNAzyme for colorimetric readout of the detection of streptavidin by the naked eye. Finally, we integrate the whole G-quadruplex DNAzyme system in a single DNA strand and show that it is applicable to colorimetric detection......., which can be detected by a shift in the fluorescence resonance energy transfer signal between dyes attached to the DNA strands. In the present study we have integrated an ATP aptamer in the strand B and demonstrated the optical detection of ATP. Furthermore we explore a new readout method using a split...

Transverse load optimization in Nb3Sn CICC design; influence of cabling, void fraction and strand stiffness

International Nuclear Information System (INIS)

Nijhuis, A; Ilyin, Y

2006-01-01

We have developed a model that describes the transverse load degradation in Nb 3 Sn CICCs, based on strand and cable properties, and that is capable of predicting how such degradation can be prevented. The Nb 3 Sn cable in conduit conductors (CICCs) for the International Thermonuclear Experimental Reactor (ITER) show a significant degradation in their performance with increasing electromagnetic load. Not only do the differences in the thermal contraction of the composite materials affect the critical current and temperature margin, but mostly electromagnetic forces cause significant transverse strand contact and bending strain in the Nb 3 Sn layers. Here, we present the model for transverse electro-magnetic load optimization (TEMLOP) and report the first results of computations for the ITER type of conductors, based on the measured properties of the internal tin strand used for the toroidal field model coil (TFMC). As input, the model uses data describing the behaviour of single strands under periodic bending and contact loads, measured with the TARSIS set-up, enabling a discrimination in performance reduction per specific load and strand type. The most important conclusion of the model computations is that the problem of the severe degradation of large CICCs can be drastically and straightforwardly improved by increasing the pitch length of subsequent cabling stages. It is the first time that an increase of the pitches has been proposed and no experimental data are available yet to confirm this beneficial outcome of the TEMLOP model. Larger pitch lengths will result in a more homogeneous distribution of the stresses and strains in the cable by significantly moderating the local peak stresses associated with the intermediate-length twist pitches. The twist pitch scheme of the present conductor layout turns out to be unfortunately close to a worst-case scenario. The model also makes clear that strand bending is the dominant mechanism causing degradation. The

Correlation Coefficients Between Different Methods of Expressing Bacterial Quantification Using Real Time PCR

Directory of Open Access Journals (Sweden)

Bahman Navidshad

2012-02-01

Full Text Available The applications of conventional culture-dependent assays to quantify bacteria populations are limited by their dependence on the inconsistent success of the different culture-steps involved. In addition, some bacteria can be pathogenic or a source of endotoxins and pose a health risk to the researchers. Bacterial quantification based on the real-time PCR method can overcome the above-mentioned problems. However, the quantification of bacteria using this approach is commonly expressed as absolute quantities even though the composition of samples (like those of digesta can vary widely; thus, the final results may be affected if the samples are not properly homogenized, especially when multiple samples are to be pooled together before DNA extraction. The objective of this study was to determine the correlation coefficients between four different methods of expressing the output data of real-time PCR-based bacterial quantification. The four methods were: (i the common absolute method expressed as the cell number of specific bacteria per gram of digesta; (ii the Livak and Schmittgen, ΔΔCt method; (iii the Pfaffl equation; and (iv a simple relative method based on the ratio of cell number of specific bacteria to the total bacterial cells. Because of the effect on total bacteria population in the results obtained using ΔCt-based methods (ΔΔCt and Pfaffl, these methods lack the acceptable consistency to be used as valid and reliable methods in real-time PCR-based bacterial quantification studies. On the other hand, because of the variable compositions of digesta samples, a simple ratio of cell number of specific bacteria to the corresponding total bacterial cells of the same sample can be a more accurate method to quantify the population.

Real-Time PCR Quantification of Chloroplast DNA Supports DNA Barcoding of Plant Species.

Science.gov (United States)

Kikkawa, Hitomi S; Tsuge, Kouichiro; Sugita, Ritsuko

2016-03-01

Species identification from extracted DNA is sometimes needed for botanical samples. DNA quantification is required for an accurate and effective examination. If a quantitative assay provides unreliable estimates, a higher quantity of DNA than the estimated amount may be used in additional analyses to avoid failure to analyze samples from which extracting DNA is difficult. Compared with conventional methods, real-time quantitative PCR (qPCR) requires a low amount of DNA and enables quantification of dilute DNA solutions accurately. The aim of this study was to develop a qPCR assay for quantification of chloroplast DNA from taxonomically diverse plant species. An absolute quantification method was developed using primers targeting the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene using SYBR Green I-based qPCR. The calibration curve was generated using the PCR amplicon as the template. DNA extracts from representatives of 13 plant families common in Japan. This demonstrates that qPCR analysis is an effective method for quantification of DNA from plant samples. The results of qPCR assist in the decision-making will determine the success or failure of DNA analysis, indicating the possibility of optimization of the procedure for downstream reactions.

Numerical and Experimental Identification of Seven-Wire Strand Tensions Using Scale Energy Entropy Spectra of Ultrasonic Guided Waves

Directory of Open Access Journals (Sweden)

Ji Qian

2018-01-01

Full Text Available Accurate identification of tension in multiwire strands is a key issue to ensure structural safety and durability of prestressed concrete structures, cable-stayed bridges, and hoist elevators. This paper proposes a method to identify strand tensions based on scale energy entropy spectra of ultrasonic guided waves (UGWs. A numerical method was first developed to simulate UGW propagation in a seven-wire strand, employing the wavelet transform to extract UGW time-frequency energy distributions for different loadings. Mode separation and frequency band loss of L(0,1 were then found for increasing tension, and UGW scale energy entropy spectra were extracted to establish a tension identification index. A good linear relationship was found between the proposed identification index and tensile force, and effects of propagation distance and propagation path were analyzed. Finally, UGWs propagation was examined experimentally for a long seven-wire strand to investigate attenuation and long distance propagation. Numerical and experimental results verified that the proposed method not only can effectively identify strand tensions but can also adapt to long distance tests for practical engineering.

Quantification of miRNAs by a simple and specific qPCR method

DEFF Research Database (Denmark)

Cirera Salicio, Susanna; Busk, Peter K.

2014-01-01

MicroRNAs (miRNAs) are powerful regulators of gene expression at posttranscriptional level and play important roles in many biological processes and in disease. The rapid pace of the emerging field of miRNAs has opened new avenues for development of techniques to quantitatively determine mi...... in miRNA quantification. Furthermore, the method is easy to perform with common laboratory reagents, which allows miRNA quantification at low cost....

Droplet digital PCR improves absolute quantification of viable lactic acid bacteria in faecal samples.

Science.gov (United States)

Gobert, Guillaume; Cotillard, Aurélie; Fourmestraux, Candice; Pruvost, Laurence; Miguet, Jean; Boyer, Mickaël

2018-03-14

Analysing correlations between the observed health effects of ingested probiotics and their survival in digestive tract allows adapting their preparations for food. Tracking ingested probiotic in faecal samples requires accurate and specific tools to quantify live vs dead cells at strain level. Traditional culture-based methods are simpler to use but they do not allow quantifying viable but non-cultivable (VBNC) cells and they are poorly discriminant below the species level. We have set up a viable PCR (vPCR) assay combining propidium monoazide (PMA) treatment and either real time quantitative PCR (qPCR) or droplet digital PCR (ddPCR) to quantify a Lactobacillus rhamnosus and two Lactobacillus paracasei subsp. paracasei strains in piglet faeces. Adjustments of the PMA treatment conditions and reduction of the faecal sample size were necessary to obtain accurate discrimination between dead and live cells. The study also revealed differences of PMA efficiency among the two L. paracasei strains. Both PCR methods were able to specifically quantify each strain and provided comparable total bacterial counts. However, quantification of lower numbers of viable cells was best achieved with ddPCR, which was characterized by a reduced lower limit of quantification (improvement of up to 1.76 log 10 compared to qPCR). All three strains were able to survive in the piglets' gut with viability losses between 0.78 and 1.59 log 10 /g faeces. This study shows the applicability of PMA-ddPCR to specific quantification of small numbers of viable bacterial cells in the presence of an important background of unwanted microorganisms, and without the need to set up standard curves. It also illustrates the need to adapt PMA protocols according to the final matrix and target strain, even for closely related strains. The PMA-ddPCR approach provides a new tool to quantify bacterial survival in faecal samples from a preclinical and clinical trial. Copyright © 2018 The Authors. Published by

On the direct characterization and quantification of active ingredients in commercial solid drugs using PIXE, PIGE and TOF-SIMS techniques

Energy Technology Data Exchange (ETDEWEB)

Nsouli, B. [IBA laboratory, Lebanese Atomic Energy Commission (CNRS), Beirut (Lebanon)], E-mail: bnsouli@cnrs.edu.lb; Zahraman, K; Roumie, M [IBA laboratory, Lebanese Atomic Energy Commission (CNRS), Beirut (Lebanon); Yazbi, F [Faculty of Pharmacy, Department of Pharmaceutical Analytical Chemistry, Beirut Arab University, Beirut (Lebanon); Thomas, J P [Institut de Physique Nucleaire de Lyon, Universite Claude Bernard Lyon, Villeurbanne (France)

2009-07-01

The quantification of the active ingredient (AI) in drugs is a crucial and important step in the drug quality control process. This is usually performed by using wet chemical techniques like LC-MS, UV spectrophotometry and other appropriate organic analytical methods. In the case of an active ingredient contains specific heteroatoms (F, S, Cl, . . .), elemental IBA like PIXE and PIGE techniques, using small tandem accelerator of 1 - 2 MV, can be explored for molecular quantification. IBA techniques permit the analysis of the sample under solid form, without any laborious sample preparations. This is an advantage when the number of sample is relatively large. In this work, we demonstrate the ability of the Thick Target PIXE and PIGE technique for rapid and accurate quantification of low concentration of different fluorinated, sulfured and chlorinated active ingredients in several commercial anti-hyperlipidemic and anti-inflammatory commercial drugs. In this work we will demonstrate the ability of PIXE and PIGE techniques for rapid and accurate quantification of Celecoxib and Atorvastatin active ingredients contained in several solid commercial drugs. The experimental aspects related to the quantification validity are presented and discussed. In addition, the Time of Flight Secondary Ion Emission using multicharged Ar ions with {approx} 10 MeV energy, delivered by a 4 MV Vander Graaf single stage accelerator, was used for structural and chemical analysis for some cases of binary commercial drugs containing two different active ingredients. The aspect of sample preparation and the role of excipient will be highlighted and discussed. (author)

CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles.

Science.gov (United States)

Lemos, Brenda R; Kaplan, Adam C; Bae, Ji Eun; Ferrazzoli, Alexander E; Kuo, James; Anand, Ranjith P; Waterman, David P; Haber, James E

2018-02-27

Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

Genome-wide mapping of DNA strand breaks.

Directory of Open Access Journals (Sweden)

Frédéric Leduc

Full Text Available Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP, uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.

The effects of metal ion PCR inhibitors on results obtained with the Quantifiler(®) Human DNA Quantification Kit.

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Combs, Laura Gaydosh; Warren, Joseph E; Huynh, Vivian; Castaneda, Joanna; Golden, Teresa D; Roby, Rhonda K

2015-11-01

Forensic DNA samples may include the presence of PCR inhibitors, even after extraction and purification. Studies have demonstrated that metal ions, co-purified at specific concentrations, inhibit DNA amplifications. Metal ions are endogenous to sample types, such as bone, and can be introduced from environmental sources. In order to examine the effect of metal ions as PCR inhibitors during quantitative real-time PCR, 2800 M DNA was treated with 0.0025-18.750 mM concentrations of aluminum, calcium, copper, iron, nickel, and lead. DNA samples, both untreated and metal-treated, were quantified using the Quantifiler(®) Human DNA Quantification Kit. Quantification cycle (Cq) values for the Quantifiler(®) Human DNA and internal PCR control (IPC) assays were measured and the estimated concentrations of human DNA were obtained. Comparisons were conducted between metal-treated and control DNA samples to determine the accuracy of the quantification estimates and to test the efficacy of the IPC inhibition detection. This kit is most resistant to the presence of calcium as compared to all metals tested; the maximum concentration tested does not affect the amplification of the IPC or quantification of the sample. This kit is most sensitive to the presence of aluminum; concentrations greater than 0.0750 mM negatively affected the quantification, although the IPC assay accurately assessed the presence of PCR inhibition. The Quantifiler(®) Human DNA Quantification Kit accurately quantifies human DNA in the presence of 0.5000 mM copper, iron, nickel, and lead; however, the IPC does not indicate the presence of PCR inhibition at this concentration of these metals. Unexpectedly, estimates of DNA quantity in samples treated with 18.750 mM copper yielded values in excess of the actual concentration of DNA in the samples; fluorescence spectroscopy experiments indicated this increase was not a direct interaction between the copper metal and 6-FAM dye used to label the probe that

Using DNA origami nanostructures to determine absolute cross sections for UV photon-induced DNA strand breakage.

Science.gov (United States)

Vogel, Stefanie; Rackwitz, Jenny; Schürman, Robin; Prinz, Julia; Milosavljević, Aleksandar R; Réfrégiers, Matthieu; Giuliani, Alexandre; Bald, Ilko

2015-11-19

We have characterized ultraviolet (UV) photon-induced DNA strand break processes by determination of absolute cross sections for photoabsorption and for sequence-specific DNA single strand breakage induced by photons in an energy range from 6.50 to 8.94 eV. These represent the lowest-energy photons able to induce DNA strand breaks. Oligonucleotide targets are immobilized on a UV transparent substrate in controlled quantities through attachment to DNA origami templates. Photon-induced dissociation of single DNA strands is visualized and quantified using atomic force microscopy. The obtained quantum yields for strand breakage vary between 0.06 and 0.5, indicating highly efficient DNA strand breakage by UV photons, which is clearly dependent on the photon energy. Above the ionization threshold strand breakage becomes clearly the dominant form of DNA radiation damage, which is then also dependent on the nucleotide sequence.

Histamine quantification in human plasma using high resolution accurate mass LC-MS technology.

Science.gov (United States)

Laurichesse, Mathieu; Gicquel, Thomas; Moreau, Caroline; Tribut, Olivier; Tarte, Karin; Morel, Isabelle; Bendavid, Claude; Amé-Thomas, Patricia

2016-01-01

Histamine (HA) is a small amine playing an important role in anaphylactic reactions. In order to identify and quantify HA in plasma matrix, different methods have been developed but present several disadvantages. Here, we developed an alternative method using liquid chromatography coupled with an ultra-high resolution and accurate mass instrument, Q Exactive™ (Thermo Fisher) (LCHRMS). The method includes a protein precipitation of plasma samples spiked with HA-d4 as internal standard (IS). LC separation was performed on a C18 Accucore column (100∗2.1mm, 2.6μm) using a mobile phase containing nonafluoropentanoic acid (3nM) and acetonitrile with 0.1% (v/v) formic acid on gradient mode. Separation of analytes was obtained within 10min. Analysis was performed from full scan mode and targeted MS2 mode using a 5ppm mass window. Ion transitions monitored for targeted MS2 mode were 112.0869>95.0607m/z for HA and 116.1120>99.0855m/z for HA-d4. Calibration curves were obtained by adding standard calibration dilution at 1 to 180nM in TrisBSA. Elution of HA and IS occurred at 4.1min. The method was validated over a range of concentrations from 1nM to 100nM. The intra- and inter-run precisions were <15% for quality controls. Human plasma samples from 30 patients were analyzed by LCHRMS, and the results were highly correlated with those obtained using the gold standard radioimmunoassay (RIA) method. Overall, we demonstrate here that LCHRMS is a sensitive method for histamine quantification in biological human plasmas, suitable for routine use in medical laboratories. In addition, LCHRMS is less time-consuming than RIA, avoids the use of radioactivity, and could then be considered as an alternative quantitative method. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Radiobiology of DNA strand breakage

International Nuclear Information System (INIS)

Johansen, I.

1975-01-01

The yield of single-strand breaks in lambda DNA within lysogenic host bacteria was measured after exposure to 4-MeV electrons (50 msec) and rapid transfer (45 msec) to alkaline detergent. In nitrogen anoxia the yield was 1.2 x 10 -12 DNA single-strand breaks per rad per dalton, and under full oxygenation the yield increased to 5 x 10 -12 breaks per rad per dalton. A search for the presence of fast repair mechanisms failed to demonstrate the presence of any mechanism for repair of strand breaks operating within a fraction of a second. Strand breaks produced in the presence of oxygen were repaired in 30--40 sec, while breaks produced under anoxia were rejoined even slower. A functional product from the polAl gene was needed for the rejoining of the broken molecules. Intermediate levels of DNA strand breakage seen at low concentrations of oxygen are dependent on the concentration of cellular sulfhydryl compounds, suggesting that in strand breakage oxygen and hydrogen donors compete for reactions with radiation-induced transients in the DNA. Intercomparisons of data on radiation-induced lethality of cells and single-strand breaks in episomal DNA allow the distinction between two classes of radiation-induced radicals, R 1 and R 2 , with different chemical properties; R 1 reacts readily with oxygen and N-oxyls under formation of potentially lethal products. The reactivity of oxygen in this reaction is 30--40 times higher than that of TMPN. R 2 reacts 16 times more readily than R 1 with oxygen under formation of single-strand breaks in the DNA. R 2 does not react with N-oxyls

Theoretical analysis on ac loss properties of two-strand parallel conductors composed of superconducting multifilamentary strands

CERN Document Server

Iwakuma, M; Funaki, K

2002-01-01

The ac loss properties of two-strand parallel conductors composed of superconducting multifilamentary strands were theoretically investigated. The constituent strands generally need to be insulated and transposed for the sake of uniform current distribution and low ac loss. In case the transposition points deviate from the optimum ones, shielding current is induced according to the interlinkage magnetic flux of the twisted loop enclosed by the insulated strands and the contact resistances at the terminals. It produces an additional ac loss. Supposing a simple situation where a two-strand parallel conductor with one-point transposition is exposed to a uniform ac magnetic field, the basic equations for the magnetic field were proposed and the theoretical expressions of the additional ac losses derived. As a result, the following features were shown. The additional ac loss in the non-saturation case, where the induced shielding current is less than the critical current of a strand, is proportional to the square ...

Reference Materials for Calibration of Analytical Biases in Quantification of DNA Methylation.

Science.gov (United States)

Yu, Hannah; Hahn, Yoonsoo; Yang, Inchul

2015-01-01

Most contemporary methods for the quantification of DNA methylation employ bisulfite conversion and PCR amplification. However, many reports have indicated that bisulfite-mediated PCR methodologies can result in inaccurate measurements of DNA methylation owing to amplification biases. To calibrate analytical biases in quantification of gene methylation, especially those that arise during PCR, we utilized reference materials that represent exact bisulfite-converted sequences with 0% and 100% methylation status of specific genes. After determining relative quantities using qPCR, pairs of plasmids were gravimetrically mixed to generate working standards with predefined DNA methylation levels at 10% intervals in terms of mole fractions. The working standards were used as controls to optimize the experimental conditions and also as calibration standards in melting-based and sequencing-based analyses of DNA methylation. Use of the reference materials enabled precise characterization and proper calibration of various biases during PCR and subsequent methylation measurement processes, resulting in accurate measurements.

hnRNP-U is a specific DNA-dependent protein kinase substrate phosphorylated in response to DNA double-strand breaks

International Nuclear Information System (INIS)

Berglund, Fredrik M.; Clarke, Paul R.

2009-01-01

Cellular responses to DNA damage are orchestrated by the large phosphoinositol-3-kinase related kinases ATM, ATR and DNA-PK. We have developed a cell-free system to dissect the biochemical mechanisms of these kinases. Using this system, we identify heterogeneous nuclear ribonucleoprotein U (hnRNP-U), also termed scaffold attachment factor A (SAF-A), as a specific substrate for DNA-PK. We show that hnRNP-U is phosphorylated at Ser59 by DNA-PK in vitro and in cells in response to DNA double-strand breaks. Phosphorylation of hnRNP-U suggests novel functions for DNA-PK in the response to DNA damage.

Accurate and rapid modeling of iron-bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis.

Science.gov (United States)

Harsch, A; Marzilli, L A; Bunt, R C; Stubbe, J; Vouros, P

2000-05-01

Bleomycin B(2)(BLM) in the presence of iron [Fe(II)] and O(2)catalyzes single-stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe-BLM was incorporated into each analyte. One of these core sequences, GTAC, is a known hot-spot for ds cleavage, while the other sequence, GGCC, is a hot-spot for ss cleavage. Incubation of each oligo-nucleotide under anaerobic conditions with Fe(II)-BLM allowed detection of the non-covalent ternary Fe-BLM/oligonucleotide complex in the gas phase. Cleavage studies were then performed utilizing O(2)-activated Fe(II)-BLM. No work-up or separation steps were required and direct MS and MS/MS analyses of the crude reaction mixtures confirmed sequence-specific Fe-BLM-induced cleavage. Comparison of the cleavage patterns for both oligonucleotides revealed sequence-dependent preferences for ss and ds cleavages in accordance with previously established gel electrophoresis analysis of hairpin oligonucleotides. This novel methodology allowed direct, rapid and accurate determination of cleavage profiles of model duplex oligonucleotides after exposure to activated Fe-BLM.

Asymmetric strand segregation: epigenetic costs of genetic fidelity?

Directory of Open Access Journals (Sweden)

Diane P Genereux

2009-06-01

Full Text Available Asymmetric strand segregation has been proposed as a mechanism to minimize effective mutation rates in epithelial tissues. Under asymmetric strand segregation, the double-stranded molecule that contains the oldest DNA strand is preferentially targeted to the somatic stem cell after each round of DNA replication. This oldest DNA strand is expected to have fewer errors than younger strands because some of the errors that arise on daughter strands during their synthesis fail to be repaired. Empirical findings suggest the possibility of asymmetric strand segregation in a subset of mammalian cell lineages, indicating that it may indeed function to increase genetic fidelity. However, the implications of asymmetric strand segregation for the fidelity of epigenetic information remain unexplored. Here, I explore the impact of strand-segregation dynamics on epigenetic fidelity using a mathematical-modelling approach that draws on the known molecular mechanisms of DNA methylation and existing rate estimates from empirical methylation data. I find that, for a wide range of starting methylation densities, asymmetric -- but not symmetric -- strand segregation leads to systematic increases in methylation levels if parent strands are subject to de novo methylation events. I found that epigenetic fidelity can be compromised when enhanced genetic fidelity is achieved through asymmetric strand segregation. Strand segregation dynamics could thus explain the increased DNA methylation densities that are observed in structured cellular populations during aging and in disease.

Automatic generation of a subject-specific model for accurate markerless motion capture and biomechanical applications.

Science.gov (United States)

Corazza, Stefano; Gambaretto, Emiliano; Mündermann, Lars; Andriacchi, Thomas P

2010-04-01

A novel approach for the automatic generation of a subject-specific model consisting of morphological and joint location information is described. The aim is to address the need for efficient and accurate model generation for markerless motion capture (MMC) and biomechanical studies. The algorithm applied and expanded on previous work on human shapes space by embedding location information for ten joint centers in a subject-specific free-form surface. The optimal locations of joint centers in the 3-D mesh were learned through linear regression over a set of nine subjects whose joint centers were known. The model was shown to be sufficiently accurate for both kinematic (joint centers) and morphological (shape of the body) information to allow accurate tracking with MMC systems. The automatic model generation algorithm was applied to 3-D meshes of different quality and resolution such as laser scans and visual hulls. The complete method was tested using nine subjects of different gender, body mass index (BMI), age, and ethnicity. Experimental training error and cross-validation errors were 19 and 25 mm, respectively, on average over the joints of the ten subjects analyzed in the study.

Quantification of the sequestration of indium 111 labelled platelets

International Nuclear Information System (INIS)

Najean, Y.; Picard, N.; Dufour, V.; Rain, J.D.

1988-01-01

A simple method is proposed for an accurate quantification of the splenic and/or hepatic sequestration of the 111 In-labelled platelets. It could be allow a better prediction of the efficiency of splenectomy in idiopathic thrombocytopenic purpura [fr

Properties of targeted preamplification in DNA and cDNA quantification.

Science.gov (United States)

Andersson, Daniel; Akrap, Nina; Svec, David; Godfrey, Tony E; Kubista, Mikael; Landberg, Göran; Ståhlberg, Anders

2015-01-01

Quantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR). To evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR. The preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification. On the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing.

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange*

Science.gov (United States)

Borgogno, María V.; Monti, Mariela R.; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E.; Pezza, Roberto J.

2016-01-01

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. PMID:26709229

Differential extraction of endogenous and exogenous 25-OH-vitamin D from serum makes the accurate quantification in liquid chromatography-tandem mass spectrometry assays challenging.

Science.gov (United States)

Lankes, Ulrich; Elder, Peter A; Lewis, John G; George, Peter

2015-01-01

Extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is the method of choice when it comes to the accurate quantification of 25-OH-vitamin D in blood samples. It is generally assumed that the addition of exogenous internal standard allows for the determination of the endogenous analyte concentration. In this study we investigated the extraction properties of endogenous and exogenous 25-OH-vitamin D. Eight samples were used for the evaluation of the extraction procedure and 59 patients' samples for a method comparison. The methanol-to-sample ratio (v/v) and the sample-to-hexane ratio (v/v) were varied and the LC-MS/MS signals of endogenous 25-OH-vitamin D3, spiked 25-OH-vitamin D2 and internal standard of the extracts recorded. The optimized 'in-house' LC-MS/MS assay was compared to two automated chemiluminescence immunoassays from DiaSorin and Abbott. Mathematical analysis of the data revealed a differential extraction of endogenous 25-OH-vitamin D3, spiked 25-OH-vitamin D2 and non-equilibrated internal standard. Exogenous 25-OH-vitamin D can be measured accurately if a definite methanol-to-sample ratio is used. Endogenous 25-OH-vitamin D is affected by critical quantification issues due to a differential slope in the extraction profile. The actual 25-OH-vitamin D concentration can be one-third above the measured extractable concentration. Results confirm that the 'in-house' LC-MS/MS assay provides reproducible 25-OH-vitamin D results. Discordant concentrations of 25-OH-vitamin D from LC-MS/MS assays can be caused by selection of suboptimal extraction conditions. Furthermore, a different sample pretreatment or solvent extraction system may result in a different dissociation and extraction yield of endogenous 25-OH-vitamin D and therefore contribute to variations of LC-MS/MS results. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Development of a three-dimensional high-order strand-grids approach

Science.gov (United States)

Tong, Oisin

Development of a novel high-order flux correction method on strand grids is presented. The method uses a combination of flux correction in the unstructured plane and summation-by-parts operators in the strand direction to achieve high-fidelity solutions. Low-order truncation errors are cancelled with accurate flux and solution gradients in the flux correction method, thereby achieving a formal order of accuracy of 3, although higher orders are often obtained, especially for highly viscous flows. In this work, the scheme is extended to high-Reynolds number computations in both two and three dimensions. Turbulence closure is achieved with a robust version of the Spalart-Allmaras turbulence model that accommodates negative values of the turbulence working variable, and the Menter SST turbulence model, which blends the k-epsilon and k-o turbulence models for better accuracy. A major advantage of this high-order formulation is the ability to implement traditional finite volume-like limiters to cleanly capture shocked and discontinuous flows. In this work, this approach is explored via a symmetric limited positive (SLIP) limiter. Extensive verification and validation is conducted in two and three dimensions to determine the accuracy and fidelity of the scheme for a number of different cases. Verification studies show that the scheme achieves better than third order accuracy for low and high-Reynolds number flows. Cost studies show that in three-dimensions, the third-order flux correction scheme requires only 30% more walltime than a traditional second-order scheme on strand grids to achieve the same level of convergence. In order to overcome meshing issues at sharp corners and other small-scale features, a unique approach to traditional geometry, coined "asymptotic geometry," is explored. Asymptotic geometry is achieved by filtering out small-scale features in a level set domain through min/max flow. This approach is combined with a curvature based strand shortening

Molecular quantification of environmental DNA using microfluidics and digital PCR.

Science.gov (United States)

Hoshino, Tatsuhiko; Inagaki, Fumio

2012-09-01

Real-time PCR has been widely used to evaluate gene abundance in natural microbial habitats. However, PCR-inhibitory substances often reduce the efficiency of PCR, leading to the underestimation of target gene copy numbers. Digital PCR using microfluidics is a new approach that allows absolute quantification of DNA molecules. In this study, digital PCR was applied to environmental samples, and the effect of PCR inhibitors on DNA quantification was tested. In the control experiment using λ DNA and humic acids, underestimation of λ DNA at 1/4400 of the theoretical value was observed with 6.58 ng μL(-1) humic acids. In contrast, digital PCR provided accurate quantification data with a concentration of humic acids up to 9.34 ng μL(-1). The inhibitory effect of paddy field soil extract on quantification of the archaeal 16S rRNA gene was also tested. By diluting the DNA extract, quantified copy numbers from real-time PCR and digital PCR became similar, indicating that dilution was a useful way to remedy PCR inhibition. The dilution strategy was, however, not applicable to all natural environmental samples. For example, when marine subsurface sediment samples were tested the copy number of archaeal 16S rRNA genes was 1.04×10(3) copies/g-sediment by digital PCR, whereas real-time PCR only resulted in 4.64×10(2) copies/g-sediment, which was most likely due to an inhibitory effect. The data from this study demonstrated that inhibitory substances had little effect on DNA quantification using microfluidics and digital PCR, and showed the great advantages of digital PCR in accurate quantifications of DNA extracted from various microbial habitats. Copyright © 2012 Elsevier GmbH. All rights reserved.

Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

Directory of Open Access Journals (Sweden)

Žel Jana

2006-08-01

chosen as the primary criterion by which to evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated. Conclusion The crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR.

Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

Science.gov (United States)

Cankar, Katarina; Štebih, Dejan; Dreo, Tanja; Žel, Jana; Gruden, Kristina

2006-01-01

criterion by which to evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated. Conclusion The crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR. PMID:16907967

Modelling toehold-mediated RNA strand displacement.

Science.gov (United States)

Šulc, Petr; Ouldridge, Thomas E; Romano, Flavio; Doye, Jonathan P K; Louis, Ard A

2015-03-10

We study the thermodynamics and kinetics of an RNA toehold-mediated strand displacement reaction with a recently developed coarse-grained model of RNA. Strand displacement, during which a single strand displaces a different strand previously bound to a complementary substrate strand, is an essential mechanism in active nucleic acid nanotechnology and has also been hypothesized to occur in vivo. We study the rate of displacement reactions as a function of the length of the toehold and temperature and make two experimentally testable predictions: that the displacement is faster if the toehold is placed at the 5' end of the substrate; and that the displacement slows down with increasing temperature for longer toeholds. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Nucleolin forms a specific complex with a fragment of the viral (minus) strand of minute virus of mice DNA.

Science.gov (United States)

Barrijal, S; Perros, M; Gu, Z; Avalosse, B L; Belenguer, P; Amalric, F; Rommelaere, J

1992-01-01

Nucleolin, a major nucleolar protein, forms a specific complex with the genome (a single-stranded DNA molecule of minus polarity) of parvovirus MVMp in vitro. By means of South-western blotting experiments, we mapped the binding site to a 222-nucleotide motif within the non-structural transcription unit, referred to as NUBE (nucleolin-binding element). The specificity of the interaction was confirmed by competitive gel retardation assays. DNaseI and nuclease S1 probing showed that NUBE folds into a secondary structure, in agreement with a computer-assisted conformational prediction. The whole NUBE may be necessary for the interaction with nucleolin, as suggested by the failure of NUBE subfragments to bind the protein and by the nuclease footprinting experiments. The present work extends the previously reported ability of nucleolin to form a specific complex with ribosomal RNA, to a defined DNA substrate. Considering the tropism of MVMp DNA replication for host cell nucleoli, these data raise the possibility that nucleolin may contribute to the regulation of the parvoviral life-cycle. Images PMID:1408821

A link between double-strand break-related repair and V(D)J recombination: the scid mutation

International Nuclear Information System (INIS)

Hendrickson, E.A.; Qin, X.Q.; Bump, E.A.; Schatz, D.G.; Oettinger, M.; Weaver, D.T.

1991-01-01

We show here that mammalian site-specific recombination and DNA-repair pathways share a common factor. The effects of DNA-damaging agents on cell lines derived from mice homozygous for the scid (severe combined immune deficiency) mutation were studied. Surprisingly, all scid cell lines exhibited a profound hypersensitivity to DNA-damaging agents that caused double-strand breaks (x-irradiation and bleomycin) but not to other chemicals that caused single-strand breaks or cross-links. Neutral filter elution assays demonstrated that the x-irradiation hypersensitivity could be correlated with a deficiency in repairing double-strand breaks. These data suggest that the scid gene product is involved in two pathways: DNA repair of random double-strand breaks and the site-specific and lymphoid-restricted variable-(diversity)-joining [V(D)J] DNA rearrangement process. We propose that the scid gene product performs a similar function in both pathways and may be a ubiquitous protein

Recruitment of RecA homologs Dmc1p and Rad51p to the double-strand break repair site initiated by meiosis-specific endonuclease VDE (PI-SceI).

Science.gov (United States)

Fukuda, Tomoyuki; Ohya, Yoshikazu

2006-02-01

During meiosis, VDE (PI-SceI), a homing endonuclease in Saccharomyces cerevisiae, introduces a double-strand break (DSB) at its recognition sequence and induces homologous recombinational repair, called homing. Meiosis-specific RecA homolog Dmc1p, as well as mitotic RecA homolog Rad51p, acts in the process of meiotic recombination, being required for strand invasion and exchange. In this study, recruitment of Dmc1p and Rad51p to the VDE-induced DSB repair site is investigated by chromatin immunoprecipitation assay. It is revealed that Dmc1p and Rad51p are loaded to the repair site in an independent manner. Association of Rad51p requires other DSB repair proteins of Rad52p, Rad55p, and Rad57p, while loading of Dmc1p is facilitated by the different protein, Sae3p. Absence of Tid1p, which can bind both RecA homologs, appears specifically to cause an abnormal distribution of Dmc1p. Lack of Hop2, Mnd1p, and Sae1p does not impair recruitment of both RecA homologs. These findings reveal the discrete functions of each strand invasion protein in VDE-initiated homing, confirm the similarity between VDE-initiated homing and Spo11p-initiated meiotic recombination, and demonstrate the availability of VDE-initiated homing for the study of meiotic recombination.

The multiple personalities of Watson and Crick strands.

Science.gov (United States)

Cartwright, Reed A; Graur, Dan

2011-02-08

In genetics it is customary to refer to double-stranded DNA as containing a "Watson strand" and a "Crick strand." However, there seems to be no consensus in the literature on the exact meaning of these two terms, and the many usages contradict one another as well as the original definition. Here, we review the history of the terminology and suggest retaining a single sense that is currently the most useful and consistent. The Saccharomyces Genome Database defines the Watson strand as the strand which has its 5'-end at the short-arm telomere and the Crick strand as its complement. The Watson strand is always used as the reference strand in their database. Using this as the basis of our standard, we recommend that Watson and Crick strand terminology only be used in the context of genomics. When possible, the centromere or other genomic feature should be used as a reference point, dividing the chromosome into two arms of unequal lengths. Under our proposal, the Watson strand is standardized as the strand whose 5'-end is on the short arm of the chromosome, and the Crick strand as the one whose 5'-end is on the long arm. Furthermore, the Watson strand should be retained as the reference (plus) strand in a genomic database. This usage not only makes the determination of Watson and Crick unambiguous, but also allows unambiguous selection of reference stands for genomics. This article was reviewed by John M. Logsdon, Igor B. Rogozin (nominated by Andrey Rzhetsky), and William Martin.

The multiple personalities of Watson and Crick strands

Directory of Open Access Journals (Sweden)

Graur Dan

2011-02-01

Full Text Available Abstract Background In genetics it is customary to refer to double-stranded DNA as containing a "Watson strand" and a "Crick strand." However, there seems to be no consensus in the literature on the exact meaning of these two terms, and the many usages contradict one another as well as the original definition. Here, we review the history of the terminology and suggest retaining a single sense that is currently the most useful and consistent. Proposal The Saccharomyces Genome Database defines the Watson strand as the strand which has its 5'-end at the short-arm telomere and the Crick strand as its complement. The Watson strand is always used as the reference strand in their database. Using this as the basis of our standard, we recommend that Watson and Crick strand terminology only be used in the context of genomics. When possible, the centromere or other genomic feature should be used as a reference point, dividing the chromosome into two arms of unequal lengths. Under our proposal, the Watson strand is standardized as the strand whose 5'-end is on the short arm of the chromosome, and the Crick strand as the one whose 5'-end is on the long arm. Furthermore, the Watson strand should be retained as the reference (plus strand in a genomic database. This usage not only makes the determination of Watson and Crick unambiguous, but also allows unambiguous selection of reference stands for genomics. Reviewers This article was reviewed by John M. Logsdon, Igor B. Rogozin (nominated by Andrey Rzhetsky, and William Martin.

Robust high-resolution quantification of time signals encoded by in vivo magnetic resonance spectroscopy

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Belkić, Dževad; Belkić, Karen

2018-01-01

This paper on molecular imaging emphasizes improving specificity of magnetic resonance spectroscopy (MRS) for early cancer diagnostics by high-resolution data analysis. Sensitivity of magnetic resonance imaging (MRI) is excellent, but specificity is insufficient. Specificity is improved with MRS by going beyond morphology to assess the biochemical content of tissue. This is contingent upon accurate data quantification of diagnostically relevant biomolecules. Quantification is spectral analysis which reconstructs chemical shifts, amplitudes and relaxation times of metabolites. Chemical shifts inform on electronic shielding of resonating nuclei bound to different molecular compounds. Oscillation amplitudes in time signals retrieve the abundance of MR sensitive nuclei whose number is proportional to metabolite concentrations. Transverse relaxation times, the reciprocal of decay probabilities of resonances, arise from spin-spin coupling and reflect local field inhomogeneities. In MRS single voxels are used. For volumetric coverage, multi-voxels are employed within a hybrid of MRS and MRI called magnetic resonance spectroscopic imaging (MRSI). Common to MRS and MRSI is encoding of time signals and subsequent spectral analysis. Encoded data do not provide direct clinical information. Spectral analysis of time signals can yield the quantitative information, of which metabolite concentrations are the most clinically important. This information is equivocal with standard data analysis through the non-parametric, low-resolution fast Fourier transform and post-processing via fitting. By applying the fast Padé transform (FPT) with high-resolution, noise suppression and exact quantification via quantum mechanical signal processing, advances are made, presented herein, focusing on four areas of critical public health importance: brain, prostate, breast and ovarian cancers.

Quantification of Lignin and Its Structural Features in Plant Biomass Using 13C Lignin as Internal Standard for Pyrolysis-GC-SIM-MS.

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van Erven, Gijs; de Visser, Ries; Merkx, Donny W H; Strolenberg, Willem; de Gijsel, Peter; Gruppen, Harry; Kabel, Mirjam A

2017-10-17

Understanding the mechanisms underlying plant biomass recalcitrance at the molecular level can only be achieved by accurate analyses of both the content and structural features of the molecules involved. Current quantification of lignin is, however, majorly based on unspecific gravimetric analysis after sulfuric acid hydrolysis. Hence, our research aimed at specific lignin quantification with concurrent characterization of its structural features. Hereto, for the first time, a polymeric 13 C lignin was used as internal standard (IS) for lignin quantification via analytical pyrolysis coupled to gas chromatography with mass-spectrometric detection in selected ion monitoring mode (py-GC-SIM-MS). In addition, relative response factors (RRFs) for the various pyrolysis products obtained were determined and applied. First, 12 C and 13 C lignin were isolated from nonlabeled and uniformly 13 C labeled wheat straw, respectively, and characterized by heteronuclear single quantum coherence (HSQC), nuclear magnetic resonance (NMR), and py-GC/MS. The two lignin isolates were found to have identical structures. Second, 13 C-IS based lignin quantification by py-GC-SIM-MS was validated in reconstituted biomass model systems with known contents of the 12 C lignin analogue and was shown to be extremely accurate (>99.9%, R 2 > 0.999) and precise (RSD corn stover, and sugar cane bagasse), and lignin contents were in good agreement with the total gravimetrically determined lignin contents. Our robust method proves to be a promising alternative for the high-throughput quantification of lignin in milled biomass samples directly and simultaneously provides a direct insight into the structural features of lignin.

Simultaneous quantification of amoxicillin and potassium clavulanate in different commercial drugs using PIXE technique

International Nuclear Information System (INIS)

Bejjani, A.; Roumié, M.; Akkad, S.; El-Yazbi, F.; Nsouli, B.

2016-01-01

We have demonstrated, in previous studies that Particle Induced X-ray Emission (PIXE) is one of the most rapid and accurate choices for quantification of an active ingredient, in a solid drug, from the reactions induced on its specific heteroatom using pellets made from original tablets. In this work, PIXE is used, for the first time, for simultaneous quantification of two active ingredients, amoxicillin trihydrate and potassium clavulanate, in six different commercial antibiotic type of drugs. Since the quality control process of a drug covers a large number of samples, the scope of this study was also to found the most rapid and low cost sample preparation needed to analyze these drugs with a good precision. The chosen drugs were analyzed in their tablets’ “as received” form, in pellets made from the powder of the tablets and also in pellets made from the powder of the tablets after being heated up to 70 °C to avoid any molecular destruction until constant weight and removal of humidity. The quantification validity related to the aspects of each sample preparation (homogeneity of the drug components and humidity) are presented and discussed.

Simultaneous quantification of amoxicillin and potassium clavulanate in different commercial drugs using PIXE technique

Energy Technology Data Exchange (ETDEWEB)

Bejjani, A., E-mail: abejjani@cnrs.edu.lb [IBA Laboratory, Lebanese Atomic Energy Commission-CNRS, P.O. Box: 11-8281, Beirut (Lebanon); Roumié, M. [IBA Laboratory, Lebanese Atomic Energy Commission-CNRS, P.O. Box: 11-8281, Beirut (Lebanon); Akkad, S. [Facutly of Pharmacy, Department of Pharmaceutical Analytical Chemistry, Beirut Arab University, Beirut (Lebanon); El-Yazbi, F. [Faculty of Pharmacy, Alexandria University, P.O. Box: 21521, Elmesalla, Alexandria (Egypt); Nsouli, B. [IBA Laboratory, Lebanese Atomic Energy Commission-CNRS, P.O. Box: 11-8281, Beirut (Lebanon)

2016-03-15

We have demonstrated, in previous studies that Particle Induced X-ray Emission (PIXE) is one of the most rapid and accurate choices for quantification of an active ingredient, in a solid drug, from the reactions induced on its specific heteroatom using pellets made from original tablets. In this work, PIXE is used, for the first time, for simultaneous quantification of two active ingredients, amoxicillin trihydrate and potassium clavulanate, in six different commercial antibiotic type of drugs. Since the quality control process of a drug covers a large number of samples, the scope of this study was also to found the most rapid and low cost sample preparation needed to analyze these drugs with a good precision. The chosen drugs were analyzed in their tablets’ “as received” form, in pellets made from the powder of the tablets and also in pellets made from the powder of the tablets after being heated up to 70 °C to avoid any molecular destruction until constant weight and removal of humidity. The quantification validity related to the aspects of each sample preparation (homogeneity of the drug components and humidity) are presented and discussed.

Sea Turtle Stranding Network Reports

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — The Sea Turtle Stranding and Salvage Network (STSSN) was formally established in 1980 to collect information on and document the stranding of marine turtles along...

Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks.

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Satoshi Nakajima

Full Text Available During the DNA damage response (DDR, ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5, a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair.

Application of Neesler reagent in the ammonium quantification used in the fermentations of biotechnology products

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Dinorah Torres-Idavoy

2015-08-01

Full Text Available The ammonium salts are used in fermentations to supplement the deficient amounts of nitrogen and stabilize the pH of the culture medium. The excess ammonium ion exerts a detrimental effect on the fermentation process inhibiting microbial growth. An analytical method based on Neesler reagent was developed for monitoring and controlling the concentration of ammonium during the fermentation process. The test was standardized, by means of the selection of measuring equipment, and the reaction time as well as comparing standards of ammonium salts. The method was characterized with the evaluation of the next parameters: Specificity, Linearity and Range, Quantification Limit, Accuracy and Precision. The method proved to be specific. Two linear curves were defined in the ranges of concentrations of ammonium chloride salt (2-20 μg/ml and ammonium sulfate salt (5-30 μg/ml. The limits of quantification were the lowest points of each one. The method proved to be accurate and precise. This assay was applied to samples of the yeast culture and bacteria of the genus Saccharomyces and E. coli respectively. A novel method in micro plate for quantification and analytical control of ammonia was developed. This method is used to control this fundamental chemical component in the fermentations, to optimize the culture medium. Thus, an appropriate expression of recombinant proteins and proper vaccine candidates for clinical use are achieved

Development of a VHH-Based Erythropoietin Quantification Assay

DEFF Research Database (Denmark)

Kol, Stefan; Beuchert Kallehauge, Thomas; Adema, Simon

2015-01-01

Erythropoietin (EPO) quantification during cell line selection and bioreactor cultivation has traditionally been performed with ELISA or HPLC. As these techniques suffer from several drawbacks, we developed a novel EPO quantification assay. A camelid single-domain antibody fragment directed against...... human EPO was evaluated as a capturing antibody in a label-free biolayer interferometry-based quantification assay. Human recombinant EPO can be specifically detected in Chinese hamster ovary cell supernatants in a sensitive and pH-dependent manner. This method enables rapid and robust quantification...

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange.

Science.gov (United States)

Borgogno, María V; Monti, Mariela R; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E; Pezza, Roberto J

2016-03-04

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Review of the quantification techniques for polycyclic aromatic hydrocarbons (PAHs) in food products.

Science.gov (United States)

Bansal, Vasudha; Kumar, Pawan; Kwon, Eilhann E; Kim, Ki-Hyun

2017-10-13

There is a growing need for accurate detection of trace-level PAHs in food products due to the numerous detrimental effects caused by their contamination (e.g., toxicity, carcinogenicity, and teratogenicity). This review aims to discuss the up-to-date knowledge on the measurement techniques available for PAHs contained in food or its related products. This article aims to provide a comprehensive outline on the measurement techniques of PAHs in food to help reduce their deleterious impacts on human health based on the accurate quantification. The main part of this review is dedicated to the opportunities and practical options for the treatment of various food samples and for accurate quantification of PAHs contained in those samples. Basic information regarding all available analytical measurement techniques for PAHs in food samples is also evaluated with respect to their performance in terms of quality assurance.

A method to accurately estimate the muscular torques of human wearing exoskeletons by torque sensors.

Science.gov (United States)

Hwang, Beomsoo; Jeon, Doyoung

2015-04-09

In exoskeletal robots, the quantification of the user's muscular effort is important to recognize the user's motion intentions and evaluate motor abilities. In this paper, we attempt to estimate users' muscular efforts accurately using joint torque sensor which contains the measurements of dynamic effect of human body such as the inertial, Coriolis, and gravitational torques as well as torque by active muscular effort. It is important to extract the dynamic effects of the user's limb accurately from the measured torque. The user's limb dynamics are formulated and a convenient method of identifying user-specific parameters is suggested for estimating the user's muscular torque in robotic exoskeletons. Experiments were carried out on a wheelchair-integrated lower limb exoskeleton, EXOwheel, which was equipped with torque sensors in the hip and knee joints. The proposed methods were evaluated by 10 healthy participants during body weight-supported gait training. The experimental results show that the torque sensors are to estimate the muscular torque accurately in cases of relaxed and activated muscle conditions.

A Method to Accurately Estimate the Muscular Torques of Human Wearing Exoskeletons by Torque Sensors

Directory of Open Access Journals (Sweden)

Beomsoo Hwang

2015-04-01

Full Text Available In exoskeletal robots, the quantification of the user’s muscular effort is important to recognize the user’s motion intentions and evaluate motor abilities. In this paper, we attempt to estimate users’ muscular efforts accurately using joint torque sensor which contains the measurements of dynamic effect of human body such as the inertial, Coriolis, and gravitational torques as well as torque by active muscular effort. It is important to extract the dynamic effects of the user’s limb accurately from the measured torque. The user’s limb dynamics are formulated and a convenient method of identifying user-specific parameters is suggested for estimating the user’s muscular torque in robotic exoskeletons. Experiments were carried out on a wheelchair-integrated lower limb exoskeleton, EXOwheel, which was equipped with torque sensors in the hip and knee joints. The proposed methods were evaluated by 10 healthy participants during body weight-supported gait training. The experimental results show that the torque sensors are to estimate the muscular torque accurately in cases of relaxed and activated muscle conditions.

Gene expression signatures of radiation response are specific, durable and accurate in mice and humans.

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Sarah K Meadows

2008-04-01

Full Text Available Previous work has demonstrated the potential for peripheral blood (PB gene expression profiling for the detection of disease or environmental exposures.We have sought to determine the impact of several variables on the PB gene expression profile of an environmental exposure, ionizing radiation, and to determine the specificity of the PB signature of radiation versus other genotoxic stresses. Neither genotype differences nor the time of PB sampling caused any lessening of the accuracy of PB signatures to predict radiation exposure, but sex difference did influence the accuracy of the prediction of radiation exposure at the lowest level (50 cGy. A PB signature of sepsis was also generated and both the PB signature of radiation and the PB signature of sepsis were found to be 100% specific at distinguishing irradiated from septic animals. We also identified human PB signatures of radiation exposure and chemotherapy treatment which distinguished irradiated patients and chemotherapy-treated individuals within a heterogeneous population with accuracies of 90% and 81%, respectively.We conclude that PB gene expression profiles can be identified in mice and humans that are accurate in predicting medical conditions, are specific to each condition and remain highly accurate over time.

Real-time polymerase chain reaction assay for endogenous reference gene for specific detection and quantification of common wheat-derived DNA (Triticum aestivum L.).

Science.gov (United States)

Vautrin, Sonia; Zhang, David

2007-01-01

A species-specific endogenous reference gene system was developed for polymerase chain reaction (PCR)-based analysis in common wheat (Triticum aestivum L.) by targeting the ALMT1 gene, an aluminium-activated malate transporter. The primers and probe were elaborated for real-time PCR-based qualitative and quantitative assay. The size of amplified product is 95 base pairs. The specificity was assessed on 17 monocot and dicot plant species. The established real-time PCR assay amplified only T. aestivum-derived DNA; no amplification occurred on other phylogenetically related species, including durum wheat (T. durum). The robustness of the system was tested on the DNA of 15 common wheat cultivars using 20 000 genomic copies per PCR the mean cycle threshold (Ct) values of 24.02 +/- 0.251 were obtained. The absolute limits of detection and quantification of the real-time PCR assay were estimated to 2 and 20 haploid genome copies of common wheat, respectively. The linearity was experimentally validated on 2-fold serial dilutions of DNA from 650 to 20 000 haploid genome copies. All these results show that the real-time PCR assay developed on the ALMT1 gene is suitable to be used as an endogenous reference gene for PCR-based specific detection and quantification of T. aestivum-derived DNA in various applications, in particular for the detection and quantification of genetically modified materials in common wheat.

Comparison of quantification algorithms for circulating cell-free DNA methylation biomarkers in blood plasma from cancer patients.

Science.gov (United States)

de Vos, Luka; Gevensleben, Heidrun; Schröck, Andreas; Franzen, Alina; Kristiansen, Glen; Bootz, Friedrich; Dietrich, Dimo

2017-01-01

SHOX2 and SEPT9 methylation in circulating cell-free DNA (ccfDNA) in blood are established powerful and clinically valuable biomarkers for diagnosis, staging, prognosis, and monitoring of cancer patients. The aim of the present study was to evaluate different quantification algorithms (relative quantification, absolute quantification, quasi-digital PCR) with regard to their clinical performance. Methylation analyses were performed in a training cohort (141 patients with head and neck squamous cell carcinoma [HNSCC], 170 control cases) and a testing cohort (137 HNSCC cases, 102 controls). DNA was extracted from plasma samples, bisulfite-converted, and analyzed via quantitative real-time PCR. SHOX2 and SEPT9 methylations were assessed separately and as panel [mean SEPT9 / SHOX2 ] using the ΔCT method for absolute quantification and the ΔΔCT-method for relative quantification. Quasi-digital PCR was defined as the number of amplification-positive PCR replicates. The diagnostic (sensitivity, specificity, area under the curve (AUC) of the receiver operating characteristic (ROC)) and prognostic accuracy (hazard ratio (HR) from Cox regression) were evaluated. Sporadic methylation in control samples necessitated the introduction of cutoffs resulting in 61-63% sensitivity/90-92% specificity ( SEPT9 /training), 53-57% sensitivity/87-90% specificity ( SHOX2 /training), and 64-65% sensitivity/90-91% specificity (mean SEPT9 / SHOX2 /training). Results were confirmed in a testing cohort with 54-56% sensitivity/88-90% specificity ( SEPT9 /testing), 43-48% sensitivity/93-95% specificity ( SHOX2 /testing), and 49-58% sensitivity/88-94% specificity (mean SEPT9 / SHOX2 /testing). All algorithms showed comparable cutoff-independent diagnostic accuracy with largely overlapping 95% confidence intervals ( SEPT9 : AUC training  = 0.79-0.80; AUC testing  = 0.74-0.75; SHOX2 : AUC training  = 0.78-0.81, AUC testing  = 0.77-0.79; mean SEPT9 / SHOX2 : AUC training  = 0

Multiplex cDNA quantification method that facilitates the standardization of gene expression data

Science.gov (United States)

Gotoh, Osamu; Murakami, Yasufumi; Suyama, Akira

2011-01-01

Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h. PMID:21415008

Survey of stranded gas and delivered costs to Europe of selected gas resources

Science.gov (United States)

Attanasi, E.D.; Freeman, P.A.

2011-01-01

Two important trends affecting the expected growth of global gas markets are (1) the shift by many industrialized countries from coal-fired electricity generation to the use of natural gas to generate electricity and (2) the industrialization of the heavily populated Asian countries of India and China. This paper surveys discovered gas in stranded conventional gas accumulations and presents estimates of the cost of developing and producing stranded gas in selected countries. Stranded gas is natural gas in discovered or identified fields that is not currently commercially producible for either physical or economic reasons. Published reserves of gas at the global level do not distinguish between volumes of gas in producing fields and volumes in nonproducing fields. Data on stranded gas reported here-that is the volumes, geographical distribution, and size distributions of stranded gas fields at the country and regional level-are based on the examination of individual-field data and represent a significant improvement in information available to industry and government decision makers. Globally, stranded gas is pervasive, but large volumes in large accumulations are concentrated in only a few areas. The cost component of the paper focuses on stranded conventional gas accumulations in Africa and South America that have the potential to augment supplies to Europe. The methods described for the computation of extraction and transport costs are innovative in that they use information on the sizes and geographical distribution of the identified stranded gas fields. The costs are based on industry data specific to the country and geologic basin where the stranded gas is located. Gas supplies to Europe can be increased significantly at competitive costs by the development of stranded gas. Net extraction costs of producing the identified gas depend critically on the natural-gas-liquids (NGLs) content, the prevailing prices of liquids, the size of the gas accumulation, and the

Identification of cis-acting elements on positive-strand subgenomic mRNA required for the synthesis of negative-strand counterpart in bovine coronavirus.

Science.gov (United States)

Yeh, Po-Yuan; Wu, Hung-Yi

2014-07-30

It has been demonstrated that, in addition to genomic RNA, sgmRNA is able to serve as a template for the synthesis of the negative-strand [(-)-strand] complement. However, the cis-acting elements on the positive-strand [(+)-strand] sgmRNA required for (-)-strand sgmRNA synthesis have not yet been systematically identified. In this study, we employed real-time quantitative reverse transcription polymerase chain reaction to analyze the cis-acting elements on bovine coronavirus (BCoV) sgmRNA 7 required for the synthesis of its (-)-strand counterpart by deletion mutagenesis. The major findings are as follows. (1) Deletion of the 5'-terminal leader sequence on sgmRNA 7 decreased the synthesis of the (-)-strand sgmRNA complement. (2) Deletions of the 3' untranslated region (UTR) bulged stem-loop showed no effect on (-)-strand sgmRNA synthesis; however, deletion of the 3' UTR pseudoknot decreased the yield of (-)-strand sgmRNA. (3) Nucleotides positioned from -15 to -34 of the sgmRNA 7 3'-terminal region are required for efficient (-)-strand sgmRNA synthesis. (4) Nucleotide species at the 3'-most position (-1) of sgmRNA 7 is correlated to the efficiency of (-)-strand sgmRNA synthesis. These results together suggest, in principle, that the 5'- and 3'-terminal sequences on sgmRNA 7 harbor cis-acting elements are critical for efficient (-)-strand sgmRNA synthesis in BCoV.

Strand Tension Control in Anchor Span for Suspension Bridge Using Dynamic Balance Theory

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Da Wang

Full Text Available Abstract Strand tension control is essential in suspension bridge safety. However, few quantitative studies have examined the bending rigidity and boundary condition behavior of strands in the anchor span of suspension bridges because of their special structure and complex configuration. In this paper, a new calculation method for strand tension is explored by using dynamic balance theory to determine the effect of bending rigidity and boundary conditions. The accuracy and effectiveness of the proposed method are tested and confirmed with verification examples and application on Nanxi Yangtze Suspension Bridge in China. The results indicated that only low-order frequency calculation could be used to calculate the strand tension without considering the effect of bending rigidity to ensure control accuracy. The influence of bending rigidity on the control precision is related to the tension and the length of the strands, which is significantly determined by the specific value between the stress rigidity and the bending rigidity. The uncertain boundary conditions of the anchor span cable, which are fixed between consolidated and hinged, also have a major effect on the control accuracy. To improve the accuracy of strand tension control, the least squares method is proposed during the tension construction control of the anchor span. This approach can significantly improve the accuracy of the tension control of the main cable strand. Some recommendations for future bridge analysis are provided based on the results of this study.

Fair Exchange in Strand Spaces

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Joshua D. Guttman

2009-10-01

Full Text Available Many cryptographic protocols are intended to coordinate state changes among principals. Exchange protocols coordinate delivery of new values to the participants, e.g. additions to the set of values they possess. An exchange protocol is fair if it ensures that delivery of new values is balanced: If one participant obtains a new possession via the protocol, then all other participants will, too. Fair exchange requires progress assumptions, unlike some other protocol properties. The strand space model is a framework for design and verification of cryptographic protocols. A strand is a local behavior of a single principal in a single session of a protocol. A bundle is a partially ordered global execution built from protocol strands and adversary activities. The strand space model needs two additions for fair exchange protocols. First, we regard the state as a multiset of facts, and we allow strands to cause changes in this state via multiset rewriting. Second, progress assumptions stipulate that some channels are resilient-and guaranteed to deliver messages-and some principals are assumed not to stop at certain critical steps. This method leads to proofs of correctness that cleanly separate protocol properties, such as authentication and confidentiality, from invariants governing state evolution. G. Wang's recent fair exchange protocol illustrates the approach.

Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

Science.gov (United States)

Arts, Peer; van der Raadt, Jori; van Gestel, Sebastianus H.C.; Steehouwer, Marloes; Shendure, Jay; Hoischen, Alexander; Albers, Cornelis A.

2017-01-01

Whole-transcriptome or RNA sequencing (RNA-Seq) is a powerful and versatile tool for functional analysis of different types of RNA molecules, but sample reagent and sequencing cost can be prohibitive for hypothesis-driven studies where the aim is to quantify differential expression of a limited number of genes. Here we present an approach for quantification of differential mRNA expression by targeted resequencing of complementary DNA using single-molecule molecular inversion probes (cDNA-smMIPs) that enable highly multiplexed resequencing of cDNA target regions of ∼100 nucleotides and counting of individual molecules. We show that accurate estimates of differential expression can be obtained from molecule counts for hundreds of smMIPs per reaction and that smMIPs are also suitable for quantification of relative gene expression and allele-specific expression. Compared with low-coverage RNA-Seq and a hybridization-based targeted RNA-Seq method, cDNA-smMIPs are a cost-effective high-throughput tool for hypothesis-driven expression analysis in large numbers of genes (10 to 500) and samples (hundreds to thousands). PMID:28474677

Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates.

Science.gov (United States)

Stengel, Gudrun; Kuchta, Robert D

2011-01-01

The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not

Optimizing scoring function of protein-nucleic acid interactions with both affinity and specificity.

Directory of Open Access Journals (Sweden)

Zhiqiang Yan

Full Text Available Protein-nucleic acid (protein-DNA and protein-RNA recognition is fundamental to the regulation of gene expression. Determination of the structures of the protein-nucleic acid recognition and insight into their interactions at molecular level are vital to understanding the regulation function. Recently, quantitative computational approach has been becoming an alternative of experimental technique for predicting the structures and interactions of biomolecular recognition. However, the progress of protein-nucleic acid structure prediction, especially protein-RNA, is far behind that of the protein-ligand and protein-protein structure predictions due to the lack of reliable and accurate scoring function for quantifying the protein-nucleic acid interactions. In this work, we developed an accurate scoring function (named as SPA-PN, SPecificity and Affinity of the Protein-Nucleic acid interactions for protein-nucleic acid interactions by incorporating both the specificity and affinity into the optimization strategy. Specificity and affinity are two requirements of highly efficient and specific biomolecular recognition. Previous quantitative descriptions of the biomolecular interactions considered the affinity, but often ignored the specificity owing to the challenge of specificity quantification. We applied our concept of intrinsic specificity to connect the conventional specificity, which circumvents the challenge of specificity quantification. In addition to the affinity optimization, we incorporated the quantified intrinsic specificity into the optimization strategy of SPA-PN. The testing results and comparisons with other scoring functions validated that SPA-PN performs well on both the prediction of binding affinity and identification of native conformation. In terms of its performance, SPA-PN can be widely used to predict the protein-nucleic acid structures and quantify their interactions.

Molecular quantification of genes encoding for green-fluorescent proteins

DEFF Research Database (Denmark)

Felske, A; Vandieken, V; Pauling, B V

2003-01-01

A quantitative PCR approach is presented to analyze the amount of recombinant green fluorescent protein (gfp) genes in environmental DNA samples. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification...... PCR strategy is a highly specific and sensitive way to monitor recombinant DNA in environments like the efflux of a biotechnological plant....

Direct quantification of creatinine in human urine by using isotope dilution extractive electrospray ionization tandem mass spectrometry

International Nuclear Information System (INIS)

Li Xue; Fang Xiaowei; Yu Zhiqiang; Sheng Guoying; Wu Minghong; Fu Jiamo; Chen Huanwen

2012-01-01

Highlights: ► High throughput analysis of urinary creatinine is achieved by using ID-EESI–MS/MS. ► Urine sample is directly analyzed and no sample pre-treatment is required. ► Accurate quantification is accomplished with isotope dilution technique. - Abstract: Urinary creatinine (CRE) is an important biomarker of renal function. Fast and accurate quantification of CRE in human urine is required by clinical research. By using isotope dilution extractive electrospray ionization tandem mass spectrometry (EESI–MS/MS) a high throughput method for direct and accurate quantification of urinary CRE was developed in this study. Under optimized conditions, the method detection limit was lower than 50 μg L −1 . Over the concentration range investigated (0.05–10 mg L −1 ), the calibration curve was obtained with satisfactory linearity (R 2 = 0.9861), and the relative standard deviation (RSD) values for CRE and isotope-labeled CRE (CRE-d3) were 7.1–11.8% (n = 6) and 4.1–11.3% (n = 6), respectively. The isotope dilution EESI–MS/MS method was validated by analyzing six human urine samples, and the results were comparable with the conventional spectrophotometric method (based on the Jaffe reaction). Recoveries for individual urine samples were 85–111% and less than 0.3 min was taken for each measurement, indicating that the present isotope dilution EESI–MS/MS method is a promising strategy for the fast and accurate quantification of urinary CRE in clinical laboratories.

Site-Specific Modification Using the 2′-Methoxyethyl Group Improves the Specificity and Activity of siRNAs

Directory of Open Access Journals (Sweden)

Xinyun Song

2017-12-01

Full Text Available Rapid progress has been made toward small interfering RNA (siRNA-based therapy for human disorders, but rationally optimizing siRNAs for high specificity and potent silencing remains a challenge. In this study, we explored the effect of chemical modification at the cleavage site of siRNAs. We found that modifications at positions 9 and 10 markedly reduced the silencing potency of the unmodified strand of siRNAs but were well tolerated by the modified strand. Intriguingly, addition of the 2′-methoxyethyl (MOE group at the cleavage site improved both the specificity and silencing activity of siRNAs by facilitating the oriented RNA-induced silencing complex (RISC loading of the modified strand. Furthermore, we combined MOE modifications at positions 9 and 10 of one strand together with 2′-O-methylation (OMe at position 14 of the other strand and found a synergistic effect that improved the specificity of siRNAs. The surprisingly beneficial effect of the combined modification was validated using siRNA-targeting endogenous gene intercellular adhesion molecule 1 (ICAM1. We found that the combined modifications eliminated its off-target effects. In conclusion, we established effective strategies to optimize siRNAs using site-specific MOE modifications. The findings may allow the creation of superior siRNAs for therapy in terms of activity and specificity.

Protected DNA strand displacement for enhanced single nucleotide discrimination in double-stranded DNA.

Science.gov (United States)

Khodakov, Dmitriy A; Khodakova, Anastasia S; Huang, David M; Linacre, Adrian; Ellis, Amanda V

2015-03-04

Single nucleotide polymorphisms (SNPs) are a prime source of genetic diversity. Discriminating between different SNPs provides an enormous leap towards the better understanding of the uniqueness of biological systems. Here we report on a new approach for SNP discrimination using toehold-mediated DNA strand displacement. The distinctiveness of the approach is based on the combination of both 3- and 4-way branch migration mechanisms, which allows for reliable discrimination of SNPs within double-stranded DNA generated from real-life human mitochondrial DNA samples. Aside from the potential diagnostic value, the current study represents an additional way to control the strand displacement reaction rate without altering other reaction parameters and provides new insights into the influence of single nucleotide substitutions on 3- and 4-way branch migration efficiency and kinetics.

Single-strand DNA binding protein SSB1 facilitates TERT recruitment to telomeres and maintains telomere G-overhangs

Science.gov (United States)

Pandita, Raj K.; Chow, Tracy T.; Udayakumar, Durga; Bain, Amanda L.; Cubeddu, Liza; Hunt, Clayton R.; Shi, Wei; Horikoshi, Nobuo; Zhao, Yong; Wright, Woodring E.; Khanna, Kum Kum; Shay, Jerry W.; Pandita, Tej K.

2015-01-01

Proliferating mammalian stem and cancer cells express telomerase (TERT) in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA binding protein SSB1, which has a critical role in DNA double-strand break repair. Here we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacted with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduced TERT interaction with telomeres and lead to G-overhang loss. While SSB1 was recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1-TERT interaction relied upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. PMID:25589350

La quantification en Kabiye: une approche linguistique | Pali ...

African Journals Online (AJOL)

... which is denoted by lexical quantifiers. Quantification with specific reference is provided by different types of linguistic units (nouns, numerals, adjectives, adverbs, ideophones and verbs) in arguments/noun phrases and in the predicative phrase in the sense of Chomsky. Keywords: quantification, class, number, reference, ...

Liver iron concentration quantification by MRI: are recommended protocols accurate enough for clinical practice?

International Nuclear Information System (INIS)

Castiella, Agustin; Zapata, Eva Mia; Alustiza, Jose M.; Emparanza, Jose I.; Costero, Belen; Diez, Maria I.

2011-01-01

To assess the accuracy of quantification of liver iron concentration (LIC) by MRI using the Rennes University (URennes) algorithm. In the overall study period 1999-2006 the LIC in 171 patients was calculated with the URennes model and the results were compared with LIC measured by liver biopsy. The biopsy showed that 107 patients had no overload, 38 moderate overload and 26 high overload. The correlation between MRI and biopsy was r = 0.86. MRI correctly classified 105 patients according to the various levels of LIC. Diagnostic accuracy was 61.4%, with a tendency to overestimate overload: 43% of patients with no overload were diagnosed as having overload, and 44.7% of patients with moderate overload were diagnosed as having high overload. The sensitivity of the URennes method for high overload was 92.3%, and the specificity for the absence of overload was 57.0%. MRI values greater than 170 μmol Fe/g revealed a positive predictive value (PPV) for haemochromatosis of 100% (n = 18); concentrations below 60 μmol Fe/g had a negative predictive value (NPV) of 100% for haemochromatosis (n = 101). The diagnosis in 44 patients with intermediate values remained uncertain. The assessment of LIC with the URennes method was useful in 74.3% of the patients to rule out or to diagnose high iron overload. The method has a tendency to overestimate overload, which limits its diagnostic performance. (orig.)

Liver iron concentration quantification by MRI: are recommended protocols accurate enough for clinical practice?

Energy Technology Data Exchange (ETDEWEB)

Castiella, Agustin; Zapata, Eva M. [Mendaro Hospital, Gastroenterology Service, Mendaro (Spain); Alustiza, Jose M. [Osatek Donostia, Radiology Service, Donostia (Spain); Emparanza, Jose I. [Donostia Hospital CASPe, CIBER-ESP, Clinical Epidemiology Unit, Donostia (Spain); Costero, Belen [Principe de Asturias Hospital, Gastroenterology Service, Alcala de Henares (Spain); Diez, Maria I. [Principe de Asturias Hospital, Radiology Service, Alcala de Henares (Spain)

2011-01-15

To assess the accuracy of quantification of liver iron concentration (LIC) by MRI using the Rennes University (URennes) algorithm. In the overall study period 1999-2006 the LIC in 171 patients was calculated with the URennes model and the results were compared with LIC measured by liver biopsy. The biopsy showed that 107 patients had no overload, 38 moderate overload and 26 high overload. The correlation between MRI and biopsy was r = 0.86. MRI correctly classified 105 patients according to the various levels of LIC. Diagnostic accuracy was 61.4%, with a tendency to overestimate overload: 43% of patients with no overload were diagnosed as having overload, and 44.7% of patients with moderate overload were diagnosed as having high overload. The sensitivity of the URennes method for high overload was 92.3%, and the specificity for the absence of overload was 57.0%. MRI values greater than 170 {mu}mol Fe/g revealed a positive predictive value (PPV) for haemochromatosis of 100% (n = 18); concentrations below 60 {mu}mol Fe/g had a negative predictive value (NPV) of 100% for haemochromatosis (n = 101). The diagnosis in 44 patients with intermediate values remained uncertain. The assessment of LIC with the URennes method was useful in 74.3% of the patients to rule out or to diagnose high iron overload. The method has a tendency to overestimate overload, which limits its diagnostic performance. (orig.)

Identification of Cis-Acting Elements on Positive-Strand Subgenomic mRNA Required for the Synthesis of Negative-Strand Counterpart in Bovine Coronavirus

Directory of Open Access Journals (Sweden)

Po-Yuan Yeh

2014-07-01

Full Text Available It has been demonstrated that, in addition to genomic RNA, sgmRNA is able to serve as a template for the synthesis of the negative-strand [(−-strand] complement. However, the cis-acting elements on the positive-strand [(+-strand] sgmRNA required for (−-strand sgmRNA synthesis have not yet been systematically identified. In this study, we employed real-time quantitative reverse transcription polymerase chain reaction to analyze the cis-acting elements on bovine coronavirus (BCoV sgmRNA 7 required for the synthesis of its (−-strand counterpart by deletion mutagenesis. The major findings are as follows. (1 Deletion of the 5'-terminal leader sequence on sgmRNA 7 decreased the synthesis of the (−-strand sgmRNA complement. (2 Deletions of the 3' untranslated region (UTR bulged stem-loop showed no effect on (−-strand sgmRNA synthesis; however, deletion of the 3' UTR pseudoknot decreased the yield of (−-strand sgmRNA. (3 Nucleotides positioned from −15 to −34 of the sgmRNA 7 3'-terminal region are required for efficient (−-strand sgmRNA synthesis. (4 Nucleotide species at the 3'-most position (−1 of sgmRNA 7 is correlated to the efficiency of (−-strand sgmRNA synthesis. These results together suggest, in principle, that the 5'- and 3'-terminal sequences on sgmRNA 7 harbor cis-acting elements are critical for efficient (−-strand sgmRNA synthesis in BCoV.

MDA5 Detects the Double-Stranded RNA Replicative Form in Picornavirus-Infected Cells

Directory of Open Access Journals (Sweden)

Qian Feng

2012-11-01

Full Text Available RIG-I and MDA5 are cytosolic RNA sensors that play a critical role in innate antiviral responses. Major advances have been made in identifying RIG-I ligands, but our knowledge of the ligands for MDA5 remains restricted to data from transfection experiments mostly using poly(I:C, a synthetic dsRNA mimic. Here, we dissected the IFN-α/β-stimulatory activity of different viral RNA species produced during picornavirus infection, both by RNA transfection and in infected cells in which specific steps of viral RNA replication were inhibited. Our results show that the incoming genomic plus-strand RNA does not activate MDA5, but minus-strand RNA synthesis and production of the 7.5 kbp replicative form trigger a strong IFN-α/β response. IFN-α/β production does not rely on plus-strand RNA synthesis and thus generation of the partially double-stranded replicative intermediate. This study reports MDA5 activation by a natural RNA ligand under physiological conditions.

Dengue virus replicates and accumulates in Aedes aegypti salivary glands

Energy Technology Data Exchange (ETDEWEB)

Raquin, Vincent, E-mail: vincent.raquin@univ-lyon1.fr [Insect-Virus Interactions Group, Department of Genomes and Genetics, Institut Pasteur, 75015 Paris (France); Centre National de la Recherche Scientifique, Unité de Recherche Associée 3012, 75015 Paris (France); Lambrechts, Louis, E-mail: louis.lambrechts@pasteur.fr [Insect-Virus Interactions Group, Department of Genomes and Genetics, Institut Pasteur, 75015 Paris (France); Centre National de la Recherche Scientifique, Unité de Recherche Associée 3012, 75015 Paris (France)

2017-07-15

Dengue virus (DENV) is an RNA virus transmitted among humans by mosquito vectors, mainly Aedes aegypti. DENV transmission requires viral dissemination from the mosquito midgut to the salivary glands. During this process the virus undergoes several population bottlenecks, which are stochastic reductions in population size that restrict intra-host viral genetic diversity and limit the efficiency of natural selection. Despite the implications for virus transmission and evolution, DENV replication in salivary glands has not been directly demonstrated. Here, we used a strand-specific quantitative RT-PCR assay to demonstrate that negative-strand DENV RNA is produced in Ae. aegypti salivary glands, providing conclusive evidence that viral replication occurs in this tissue. Furthermore, we showed that the concentration of DENV genomic RNA in salivary glands increases significantly over time, indicating that active replication likely replenishes DENV genetic diversity prior to transmission. These findings improve our understanding of the biological determinants of DENV fitness and evolution. - Highlights: •Strand-specific RT-qPCR allows accurate quantification of DENV (-) RNA in mosquito tissues. •Detection of DENV (-) RNA in salivary glands provides evidence of viral replication in this tissue. •Viral replication in salivary glands likely replenishes DENV genetic diversity prior to transmission.

Dengue virus replicates and accumulates in Aedes aegypti salivary glands

International Nuclear Information System (INIS)

Raquin, Vincent; Lambrechts, Louis

2017-01-01

Dengue virus (DENV) is an RNA virus transmitted among humans by mosquito vectors, mainly Aedes aegypti. DENV transmission requires viral dissemination from the mosquito midgut to the salivary glands. During this process the virus undergoes several population bottlenecks, which are stochastic reductions in population size that restrict intra-host viral genetic diversity and limit the efficiency of natural selection. Despite the implications for virus transmission and evolution, DENV replication in salivary glands has not been directly demonstrated. Here, we used a strand-specific quantitative RT-PCR assay to demonstrate that negative-strand DENV RNA is produced in Ae. aegypti salivary glands, providing conclusive evidence that viral replication occurs in this tissue. Furthermore, we showed that the concentration of DENV genomic RNA in salivary glands increases significantly over time, indicating that active replication likely replenishes DENV genetic diversity prior to transmission. These findings improve our understanding of the biological determinants of DENV fitness and evolution. - Highlights: •Strand-specific RT-qPCR allows accurate quantification of DENV (-) RNA in mosquito tissues. •Detection of DENV (-) RNA in salivary glands provides evidence of viral replication in this tissue. •Viral replication in salivary glands likely replenishes DENV genetic diversity prior to transmission.

Site and strand specificity of UVB mutagenesis in the SUP4-o gene of yeast

International Nuclear Information System (INIS)

Armstrong, J.D.; Kunz, B.A.

1990-01-01

DNA sequencing was used to characterize 208 mutations induced in the SUP4-o tRNA gene of the yeast Saccharomyces cerevisiae by UVB (285-320 nm) radiation. The results were compared to those for an analysis of 211 SUP4-o mutations induced by 254-nm UVC light. In each case, greater than 90% of the mutations were single base-pair changes but G.C----A.T transitions predominated and accounted for more of the mutations induced by UVB than UVC. Double substitutions, single base-pair deletions, and more complex events were also recovered. However, UVB induced 3-fold more tandem substitutions than UVC and nontandem double events were detected only after irradiation with UVC. Virtually all induced substitutions occurred at sites where the pyrimidine of the base pair was part of a dipyrimidine sequence. Although the site specificities were consistent with roles for cyclobutane dimers and pyrimidine-pyrimidone(6-4) lesions in mutation induction, preliminary photoreactivation data implicated cyclobutane dimers as the major form of premutational DNA damage for both agents. Intriguingly, there was a preference for both UVB- and UVC-induced mutations to occur at sites where the dipyrimidine was on the transcribed strand

Development and UFLC-MS/MS Characterization of a Product-Specific Standard for Phenolic Quantification of Maple-Derived Foods.

Science.gov (United States)

Liu, Yongqiang; Ma, Hang; Seeram, Navindra P

2016-05-04

The phenolic contents of plant foods are commonly quantified by the Folin-Ciocalteu assay based on gallic acid equivalents (GAEs). However, this may lead to inaccuracies because gallic acid is not always representative of the structural heterogeneity of plant phenolics. Therefore, product-specific standards have been developed for the phenolic quantification of several foods. Currently, maple-derived foods (syrup, sugar, sap/water, and extracts) are quantified for phenolic contents based on GAEs. Because lignans are the predominant phenolics present in maple, herein, a maple phenolic lignan-enriched standard (MaPLES) was purified (by chromatography) and characterized (by UFLC-MS/MS with lignans previously isolated from maple syrup). Using MaPLES and secoisolariciresinol (a commercially available lignan), the phenolic contents of the maple-derived foods increased 3-fold compared to GAEs. Therefore, lignan-based standards are more appropriate for phenolic quantification of maple-derived foods versus GAEs. Also, MaPLES can be utilized for the authentication and detection of fake label claims on maple products.

Modeling active region transient brightenings observed with X-ray telescope as multi-stranded loops

Energy Technology Data Exchange (ETDEWEB)

Kobelski, Adam R.; McKenzie, David E. [Department of Physics, P.O. Box 173840, Montana State University, Bozeman, MT 59717-3840 (United States); Donachie, Martin, E-mail: kobelski@solar.physics.montana.edu [University of Glasgow, Glasgow, G128QQ, Scotland (United Kingdom)

2014-05-10

Strong evidence exists that coronal loops as observed in extreme ultraviolet and soft X-rays may not be monolithic isotropic structures, but can often be more accurately modeled as bundles of independent strands. Modeling the observed active region transient brightenings (ARTBs) within this framework allows for the exploration of the energetic ramifications and characteristics of these stratified structures. Here we present a simple method of detecting and modeling ARTBs observed with the Hinode X-Ray Telescope (XRT) as groups of zero-dimensional strands, which allows us to probe parameter space to better understand the spatial and temporal dependence of strand heating in impulsively heated loops. This partially automated method can be used to analyze a large number of observations to gain a statistical insight into the parameters of coronal structures, including the number of heating events required in a given model to fit the observations. In this article, we present the methodology and demonstrate its use in detecting and modeling ARTBs in a sample data set from Hinode/XRT. These initial results show that, in general, multiple heating events are necessary to reproduce observed ARTBs, but the spatial dependence of these heating events cannot yet be established.

Modeling active region transient brightenings observed with X-ray telescope as multi-stranded loops

International Nuclear Information System (INIS)

Kobelski, Adam R.; McKenzie, David E.; Donachie, Martin

2014-01-01

Strong evidence exists that coronal loops as observed in extreme ultraviolet and soft X-rays may not be monolithic isotropic structures, but can often be more accurately modeled as bundles of independent strands. Modeling the observed active region transient brightenings (ARTBs) within this framework allows for the exploration of the energetic ramifications and characteristics of these stratified structures. Here we present a simple method of detecting and modeling ARTBs observed with the Hinode X-Ray Telescope (XRT) as groups of zero-dimensional strands, which allows us to probe parameter space to better understand the spatial and temporal dependence of strand heating in impulsively heated loops. This partially automated method can be used to analyze a large number of observations to gain a statistical insight into the parameters of coronal structures, including the number of heating events required in a given model to fit the observations. In this article, we present the methodology and demonstrate its use in detecting and modeling ARTBs in a sample data set from Hinode/XRT. These initial results show that, in general, multiple heating events are necessary to reproduce observed ARTBs, but the spatial dependence of these heating events cannot yet be established.

Enhancement of Electroluminescence (EL) image measurements for failure quantification methods

DEFF Research Database (Denmark)

Parikh, Harsh; Spataru, Sergiu; Sera, Dezso

2018-01-01

Enhanced quality images are necessary for EL image analysis and failure quantification. A method is proposed which determines image quality in terms of more accurate failure detection of solar panels through electroluminescence (EL) imaging technique. The goal of the paper is to determine the most...

Strategies underlying RNA silencing suppression by negative strand RNA viruses

NARCIS (Netherlands)

Hemmes, J.C.

2007-01-01

The research described in this thesis focused on the strategies of negative strand RNA viruses to counteract antiviral RNA silencing. In plants and insects, RNA silencing has been shown to act as a sequence specific antiviral defence mechanism that is characterised by the processing of double

Strand-seq : A unifying tool for studies of chromosome segregation

NARCIS (Netherlands)

Falconer, Ester; Lansdorp, Peter M.

2013-01-01

Non random segregation of sister chromatids has been implicated to help specify daughter cell fate (the Silent Sister Hypothesis [1]) or to protect the genome of long-lived stem cells (the Immortal Strand Hypothesis [2]). The idea that sister chromatids are non-randomly segregated into specific

Accurately tracking single-cell movement trajectories in microfluidic cell sorting devices.

Science.gov (United States)

Jeong, Jenny; Frohberg, Nicholas J; Zhou, Enlu; Sulchek, Todd; Qiu, Peng

2018-01-01

Microfluidics are routinely used to study cellular properties, including the efficient quantification of single-cell biomechanics and label-free cell sorting based on the biomechanical properties, such as elasticity, viscosity, stiffness, and adhesion. Both quantification and sorting applications require optimal design of the microfluidic devices and mathematical modeling of the interactions between cells, fluid, and the channel of the device. As a first step toward building such a mathematical model, we collected video recordings of cells moving through a ridged microfluidic channel designed to compress and redirect cells according to cell biomechanics. We developed an efficient algorithm that automatically and accurately tracked the cell trajectories in the recordings. We tested the algorithm on recordings of cells with different stiffness, and showed the correlation between cell stiffness and the tracked trajectories. Moreover, the tracking algorithm successfully picked up subtle differences of cell motion when passing through consecutive ridges. The algorithm for accurately tracking cell trajectories paves the way for future efforts of modeling the flow, forces, and dynamics of cell properties in microfluidics applications.

Accurately tracking single-cell movement trajectories in microfluidic cell sorting devices.

Directory of Open Access Journals (Sweden)

Jenny Jeong

Full Text Available Microfluidics are routinely used to study cellular properties, including the efficient quantification of single-cell biomechanics and label-free cell sorting based on the biomechanical properties, such as elasticity, viscosity, stiffness, and adhesion. Both quantification and sorting applications require optimal design of the microfluidic devices and mathematical modeling of the interactions between cells, fluid, and the channel of the device. As a first step toward building such a mathematical model, we collected video recordings of cells moving through a ridged microfluidic channel designed to compress and redirect cells according to cell biomechanics. We developed an efficient algorithm that automatically and accurately tracked the cell trajectories in the recordings. We tested the algorithm on recordings of cells with different stiffness, and showed the correlation between cell stiffness and the tracked trajectories. Moreover, the tracking algorithm successfully picked up subtle differences of cell motion when passing through consecutive ridges. The algorithm for accurately tracking cell trajectories paves the way for future efforts of modeling the flow, forces, and dynamics of cell properties in microfluidics applications.

Quantification of Lignin and Its Structural Features in Plant Biomass Using

NARCIS (Netherlands)

Erven, Van Gijs; Visser, de Ries; Merkx, Donny W.H.; Strolenberg, Willem; Gijsel, de Peter; Gruppen, Harry; Kabel, Mirjam A.

2017-01-01

Understanding the mechanisms underlying plant biomass recalcitrance at the molecular level can only be achieved by accurate analyses of both the content and structural features of the molecules involved. Current quantification of lignin is, however, majorly based on unspecific gravimetric

A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load.

Science.gov (United States)

Malnati, Mauro S; Scarlatti, Gabriella; Gatto, Francesca; Salvatori, Francesca; Cassina, Giulia; Rutigliano, Teresa; Volpi, Rosy; Lusso, Paolo

2008-01-01

Quantification of human immunodeficiency virus type-1 (HIV-1) proviral DNA is increasingly used to measure the HIV-1 cellular reservoirs, a helpful marker to evaluate the efficacy of antiretroviral therapeutic regimens in HIV-1-infected individuals. Furthermore, the proviral DNA load represents a specific marker for the early diagnosis of perinatal HIV-1 infection and might be predictive of HIV-1 disease progression independently of plasma HIV-1 RNA levels and CD4(+) T-cell counts. The high degree of genetic variability of HIV-1 poses a serious challenge for the design of a universal quantitative assay capable of detecting all the genetic subtypes within the main (M) HIV-1 group with similar efficiency. Here, we describe a highly sensitive real-time PCR protocol that allows for the correct quantification of virtually all group-M HIV-1 strains with a higher degree of accuracy compared with other methods. The protocol involves three stages, namely DNA extraction/lysis, cellular DNA quantification and HIV-1 proviral load assessment. Owing to the robustness of the PCR design, this assay can be performed on crude cellular extracts, and therefore it may be suitable for the routine analysis of clinical samples even in developing countries. An accurate quantification of the HIV-1 proviral load can be achieved within 1 d from blood withdrawal.

Species-specific detection and quantification of common barnacle larvae from the Japanese coast using quantitative real-time PCR.

Science.gov (United States)

Endo, Noriyuki; Sato, Kana; Matsumura, Kiyotaka; Yoshimura, Erina; Odaka, Yukiko; Nogata, Yasuyuki

2010-11-01

Species-specific detection and quantification methods for barnacle larvae using quantitative real-time polymerase chain reaction (qPCR) were developed. Species-specific primers for qPCR were designed for 13 barnacle species in the mitochondrial 12S ribosomal RNA gene region. Primer specificity was examined by PCR using template DNA extracted from each of the 13 barnacle species, other unidentified barnacle species, and field collected zooplankton samples. The resulting PCR products comprised single bands following agarose gel electrophoresis when the templates corresponded to primers. The amplifications were highly species-specific even for the field plankton samples. The field plankton samples were subjected to qPCR assay. The calculated DNA contents for each barnacle species were closely correlated with the number of larvae measured by microscopic examination. The method could be applied to quantify barnacle larvae in natural plankton samples.

Correlation of superconductor strand, cable, and dipole critical currents in CBA magnets

International Nuclear Information System (INIS)

Tannenbaum, M.J.; Garber, M.; Sampson, W.B.

1982-01-01

A calibration between vendor critical current data for 0.0268'' diameter superconductor strand supplied to Fermilab, and the BNL 10 -12 Ωcm critical current specification is presented. Vendor critical current data for over 400 Fermilab type billets are shown, both as supplied by the vendor and converted to BNL units. Predictions of cable critical current are made using the sum of the critical currents of the 23 strands, where all strands from the same half billet are assigned the same critical current. The measured critical current shows excellent correlation to the predicted value and is approximately 14 +- 2 percent below it. Colliding Beam Accelerator (CBA) full length dipoles reach the conductor critical current limit, essentially without training. Magnet performance is predictable from the measured critical current of a short sample of cable to within 2%

Phase quantification in nanobainite via magnetic measurements and X-ray diffraction

Energy Technology Data Exchange (ETDEWEB)

Solano-Alvarez, W., E-mail: ws298@cam.ac.uk [Department of Materials Science and Metallurgy, University of Cambridge (United Kingdom); Abreu, H.F.G. [Departamento de Engenharia Metalúrgica e de Materiais, Universidade Federal do Ceará, Fortaleza (Brazil); Silva, M.R. da [Instituto de Física e Química, Universidade Federal de Itajubá, Itajubá, Minas Gerais (Brazil); Peet, M.J. [Department of Materials Science and Metallurgy, University of Cambridge (United Kingdom)

2015-03-15

Accurate phase quantification of nanostructured bainitic steel is of importance because of the nature of its percolating structure that controls many of its mechanical properties. X-ray diffraction is the technique of choice for such analysis, but magnetic methods can be more rapid and less sensitive to defect structures. In this study, the phase volume fractions measured using both of these techniques for the specific mixtures associated with nanostructured bainite have been compared and contrasted. An expression which relates the volume fraction and the saturation magnetization is obtained and its form is found to be consistent with previous work done on duplex stainless steels and TRIP steels. The fitting constants used in many of such analyses vary significantly so an attempt is made to rationalize the differences by considering the factors that determine the intrinsic saturation magnetization of ferrite. - Author-Highlights: • Magnetic phase quantification of nanobainite is presented for the first time. • Results are compared with x-ray diffraction. • Expression obtained that relates ferrite fraction and saturation magnetization. • Equation derived to calculate intrinsic saturation magnetization of ferrites. • These values agree with experimental data of the literature.

Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.

Science.gov (United States)

Vela, J; Vitorica, J; Ruano, D

2001-12-01

We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.

Development of a real-time PCR assay for Penicillium expansum quantification and patulin estimation in apples

International Nuclear Information System (INIS)

Atoui, A.; Tannous, J.; El Khoury, A.; Kantar, S.; Chdid, N.; Lteif, R.; Oswald, I.; Puel, O.

2015-01-01

Due to the occurrence and spread of the fungal contaminants in food and the difficulties to remove their resulting mycotoxins, rapid and accurate methods are needed for early detection of these mycotoxigenic fungi. The polymerase chain reaction and the real time PCR have been widely used for this purpose. Apples are suitable substrates for fungal colonization mostly caused by Penicillium expansum, which produces the mycotoxin patulin during fruit infection. This study describes the development of a realtime PCR assay incorporating an internal amplification control (IAC) to specifically detect and quantify P. expansum. A specific primer pair was designed from the patF gene, involved in patulin biosynthesis. The selected primer set showed a high specificity for P. expansum and was successfully employed in a standardized real-time PCR for the direct quantification of this fungus in apples. Using the developed system, twenty eight apples were analyzed for their DNA content. Apples were also analyzed for patulin content by HPLC. Interestingly, a positive correlation (R"2 = 0.701) was found between P. expansum DNA content and patulin concentration. This work offers an alternative to conventional methods of patulin quantification and mycological detection of P. expansum and could be very useful for the screening of patulin in fruits through the application of industrial quality control. (author)

Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

International Nuclear Information System (INIS)

Malik, Afshan N.; Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil

2011-01-01

Highlights: → Mitochondrial dysfunction is central to many diseases of oxidative stress. → 95% of the mitochondrial genome is duplicated in the nuclear genome. → Dilution of untreated genomic DNA leads to dilution bias. → Unique primers and template pretreatment are needed to accurately measure mitochondrial DNA content. -- Abstract: Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as β-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a 'dilution bias' when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

Subnuclear foci quantification using high-throughput 3D image cytometry

Science.gov (United States)

Wadduwage, Dushan N.; Parrish, Marcus; Choi, Heejin; Engelward, Bevin P.; Matsudaira, Paul; So, Peter T. C.

2015-07-01

Ionising radiation causes various types of DNA damages including double strand breaks (DSBs). DSBs are often recognized by DNA repair protein ATM which forms gamma-H2AX foci at the site of the DSBs that can be visualized using immunohistochemistry. However most of such experiments are of low throughput in terms of imaging and image analysis techniques. Most of the studies still use manual counting or classification. Hence they are limited to counting a low number of foci per cell (5 foci per nucleus) as the quantification process is extremely labour intensive. Therefore we have developed a high throughput instrumentation and computational pipeline specialized for gamma-H2AX foci quantification. A population of cells with highly clustered foci inside nuclei were imaged, in 3D with submicron resolution, using an in-house developed high throughput image cytometer. Imaging speeds as high as 800 cells/second in 3D were achieved by using HiLo wide-field depth resolved imaging and a remote z-scanning technique. Then the number of foci per cell nucleus were quantified using a 3D extended maxima transform based algorithm. Our results suggests that while most of the other 2D imaging and manual quantification studies can count only up to about 5 foci per nucleus our method is capable of counting more than 100. Moreover we show that 3D analysis is significantly superior compared to the 2D techniques.

Direct quantification of creatinine in human urine by using isotope dilution extractive electrospray ionization tandem mass spectrometry

Energy Technology Data Exchange (ETDEWEB)

Li Xue [Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444 (China); Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, Applied Chemistry Department, East China Institute of Technology, Nanchang 330013 (China); Fang Xiaowei [Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, Applied Chemistry Department, East China Institute of Technology, Nanchang 330013 (China); Yu Zhiqiang; Sheng Guoying [Guangdong Key Laboratory of Environmental Protection and Resource Utilization, State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640 (China); Wu Minghong [Shanghai Applied Radiation Institute, School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444 (China); Fu Jiamo [Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444 (China); Guangdong Key Laboratory of Environmental Protection and Resource Utilization, State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640 (China); Chen Huanwen, E-mail: chw8868@gmail.com [Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, Applied Chemistry Department, East China Institute of Technology, Nanchang 330013 (China)

2012-10-20

Highlights: Black-Right-Pointing-Pointer High throughput analysis of urinary creatinine is achieved by using ID-EESI-MS/MS. Black-Right-Pointing-Pointer Urine sample is directly analyzed and no sample pre-treatment is required. Black-Right-Pointing-Pointer Accurate quantification is accomplished with isotope dilution technique. - Abstract: Urinary creatinine (CRE) is an important biomarker of renal function. Fast and accurate quantification of CRE in human urine is required by clinical research. By using isotope dilution extractive electrospray ionization tandem mass spectrometry (EESI-MS/MS) a high throughput method for direct and accurate quantification of urinary CRE was developed in this study. Under optimized conditions, the method detection limit was lower than 50 {mu}g L{sup -1}. Over the concentration range investigated (0.05-10 mg L{sup -1}), the calibration curve was obtained with satisfactory linearity (R{sup 2} = 0.9861), and the relative standard deviation (RSD) values for CRE and isotope-labeled CRE (CRE-d3) were 7.1-11.8% (n = 6) and 4.1-11.3% (n = 6), respectively. The isotope dilution EESI-MS/MS method was validated by analyzing six human urine samples, and the results were comparable with the conventional spectrophotometric method (based on the Jaffe reaction). Recoveries for individual urine samples were 85-111% and less than 0.3 min was taken for each measurement, indicating that the present isotope dilution EESI-MS/MS method is a promising strategy for the fast and accurate quantification of urinary CRE in clinical laboratories.

Dynamic protein assembly by programmable DNA strand displacement

Science.gov (United States)

Chen, Rebecca P.; Blackstock, Daniel; Sun, Qing; Chen, Wilfred

2018-03-01

Inspired by the remarkable ability of natural protein switches to sense and respond to a wide range of environmental queues, here we report a strategy to engineer synthetic protein switches by using DNA strand displacement to dynamically organize proteins with highly diverse and complex logic gate architectures. We show that DNA strand displacement can be used to dynamically control the spatial proximity and the corresponding fluorescence resonance energy transfer between two fluorescent proteins. Performing Boolean logic operations enabled the explicit control of protein proximity using multi-input, reversible and amplification architectures. We further demonstrate the power of this technology beyond sensing by achieving dynamic control of an enzyme cascade. Finally, we establish the utility of the approach as a synthetic computing platform that drives the dynamic reconstitution of a split enzyme for targeted prodrug activation based on the sensing of cancer-specific miRNAs.

Automated selected reaction monitoring software for accurate label-free protein quantification.

Science.gov (United States)

Teleman, Johan; Karlsson, Christofer; Waldemarson, Sofia; Hansson, Karin; James, Peter; Malmström, Johan; Levander, Fredrik

2012-07-06

Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of Streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.5-19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology.

Gold nanoparticle enhanced fluorescence anisotropy for the assay of single nucleotide polymorphisms (SNPs) based on toehold-mediated strand-displacement reaction.

Science.gov (United States)

Wang, Xinyi; Zou, Mingjian; Huang, Hongduan; Ren, Yuqian; Li, Limei; Yang, Xiaoda; Li, Na

2013-03-15

We developed a highly differentiating, homogeneous gold nanoparticle (AuNP) enhanced fluorescence anisotropic method for single nucleotide polymorphism (SNP) detection at nanomolar level using toehold-mediated strand-displacement reaction. The template strand, containing a toehold domain with an allele-specific site, was immobilized on the surface of AuNPs, and the solution fluorescence anisotropy was markedly enhanced when the fluorescein-labeled blocking DNA was attached to the AuNP via hybridization. Strand-displacement by the target ssDNA strand resulted in detachment of fluorescein-labeled DNA from AuNPs, and thus decreased fluorescence anisotropy. The drastic kinetic difference in strand-displacement from toehold design was used to distinguish between the perfectly matched and the single-base mismatched strands. Free energy changes were calculated to elucidate the dependence of the differentiation ability on the mutation site in the toehold region. A solid negative signal change can be obtained for single-base mismatched strand in the dynamic range of the calibration curve, and a more than 10-fold signal difference can still be observed in a mixed solution containing 100 times the single-base mismatched strand, indicating the good specificity of the method. This proposed method can be performed with a standard spectrofluorimeter in a homogeneous and cost-effective manner, and has the potential to be extended to the application of fluorescence anisotropy method of SNP detection. Copyright © 2012 Elsevier B.V. All rights reserved.

Single-strand DNA-binding protein SSB1 facilitates TERT recruitment to telomeres and maintains telomere G-overhangs.

Science.gov (United States)

Pandita, Raj K; Chow, Tracy T; Udayakumar, Durga; Bain, Amanda L; Cubeddu, Liza; Hunt, Clayton R; Shi, Wei; Horikoshi, Nobuo; Zhao, Yong; Wright, Woodring E; Khanna, Kum Kum; Shay, Jerry W; Pandita, Tej K

2015-03-01

Proliferating mammalian stem and cancer cells express telomerase [telomerase reverse transcriptase (TERT)] in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA-binding protein SSB1, which has a critical role in DNA double-strand break (DSB) repair. Here, we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacts with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduces TERT interaction with telomeres and leads to G-overhang loss. Although SSB1 is recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1-TERT interaction relies upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. Cancer Res; 75(5); 858-69. ©2015 AACR. ©2015 American Association for Cancer Research.

A novel setup for the determination of absolute cross sections for low-energy electron induced strand breaks in oligonucleotides - The effect of the radiosensitizer 5-fluorouracil

International Nuclear Information System (INIS)

Rackwitz, J.; Rankovic, M.L.; Milosavljevic, A.R.; Bald, I.

2017-01-01

Low-energy electrons (LEEs) play an important role in DNA radiation damage. Here we present a method to quantify LEE induced strand breakage in well-defined oligonucleotide single strands in terms of absolute cross sections. An LEE irradiation setup covering electron energies <500 eV is constructed and optimized to irradiate DNA origami triangles carrying well-defined oligonucleotide target strands. Measurements are presented for 10.0 and 5.5 eV for different oligonucleotide targets. The determination of absolute strand break cross sections is performed by atomic force microscopy analysis. An accurate fluence determination ensures small margins of error of the determined absolute single strand break cross sections σ SSB . In this way, the influence of sequence modification with the radiosensitive 5-Fluorouracil ( 5F U) is studied using an absolute and relative data analysis. We demonstrate an increase in the strand break yields of 5F U containing oligonucleotides by a factor of 1.5 to 1.6 compared with non-modified oligonucleotide sequences when irradiated with 10 eV electrons. (authors)

Uncertainty Quantification for Large-Scale Ice Sheet Modeling

Energy Technology Data Exchange (ETDEWEB)

Ghattas, Omar [Univ. of Texas, Austin, TX (United States)

2016-02-05

This report summarizes our work to develop advanced forward and inverse solvers and uncertainty quantification capabilities for a nonlinear 3D full Stokes continental-scale ice sheet flow model. The components include: (1) forward solver: a new state-of-the-art parallel adaptive scalable high-order-accurate mass-conservative Newton-based 3D nonlinear full Stokes ice sheet flow simulator; (2) inverse solver: a new adjoint-based inexact Newton method for solution of deterministic inverse problems governed by the above 3D nonlinear full Stokes ice flow model; and (3) uncertainty quantification: a novel Hessian-based Bayesian method for quantifying uncertainties in the inverse ice sheet flow solution and propagating them forward into predictions of quantities of interest such as ice mass flux to the ocean.

PET optimization for improved assessment and accurate quantification of {sup 90}Y-microsphere biodistribution after radioembolization

Energy Technology Data Exchange (ETDEWEB)

Martí-Climent, Josep M., E-mail: jmmartic@unav.es; Prieto, Elena; Elosúa, César; Rodríguez-Fraile, Macarena; Domínguez-Prado, Inés; Vigil, Carmen; García-Velloso, María J.; Arbizu, Javier; Peñuelas, Iván; Richter, José A. [Nuclear Medicine Department, Clínica Universidad de Navarra, 36, Pío XII Avenue, 31008 Pamplona (Spain)

2014-09-15

Purpose: {sup 90}Y-microspheres are widely used for the radioembolization of metastatic liver cancer or hepatocellular carcinoma and there is a growing interest for imaging {sup 90}Y-microspheres with PET. The aim of this study is to evaluate the performance of a current generation PET/CT scanner for {sup 90}Y imaging and to optimize the PET protocol to improve the assessment and the quantification of {sup 90}Y-microsphere biodistribution after radioembolization. Methods: Data were acquired on a Biograph mCT-TrueV scanner with time of flight (TOF) and point spread function (PSF) modeling. Spatial resolution was measured with a{sup 90}Y point source. Sensitivity was evaluated using the NEMA 70 cm line source filled with {sup 90}Y. To evaluate the count rate performance, {sup 90}Y vials with activity ranging from 3.64 to 0.035 GBq were measured in the center of the field of view (CFOV). The energy spectrum was evaluated. Image quality with different reconstructions was studied using the Jaszczak phantom containing six hollow spheres (diameters: 31.3, 28.1, 21.8, 16.1, 13.3, and 10.5 mm), filled with a 207 kBq/ml {sup 90}Y concentration and a 5:1 sphere-to-background ratio. Acquisition time was adjusted to simulate the quality of a realistic clinical PET acquisition of a patient treated with SIR-Spheres{sup ®}. The developed methodology was applied to ten patients after SIR-Spheres{sup ®} treatment acquiring a 10 min per bed PET. Results: The energy spectrum showed the{sup 90}Y bremsstrahlung radiation. The {sup 90}Y transverse resolution, with filtered backprojection reconstruction, was 4.5 mm in the CFOV and degraded to 5.0 mm at 10 cm off-axis. {sup 90}Y absolute sensitivity was 0.40 kcps/MBq in the center of the field of view. Tendency of true and random rates as a function of the {sup 90}Y activity could be accurately described using linear and quadratic models, respectively. Phantom studies demonstrated that, due to low count statistics in {sup 90}Y PET

Dual-energy CT with iodine quantification in distinguishing between bland and neoplastic portal vein thrombosis in patients with hepatocellular carcinoma

International Nuclear Information System (INIS)

Ascenti, G.; Sofia, C.; Mazziotti, S.; Silipigni, S.; D'Angelo, T.; Pergolizzi, S.; Scribano, E.

2016-01-01

Aim: To investigate the diagnostic accuracy of dual-energy multidetector computed tomography (MDCT) with iodine quantification compared to conventional enhancement measurements in distinguishing bland from neoplastic portal vein thrombosis in patients with hepatocellular carcinoma. Material and methods: Thirty-four patients (26 men, eight women; mean age, 62 years) with hepatocellular carcinoma and portal vein thrombosis underwent contrast-enhanced dual-energy MDCT during the late hepatic arterial phase for the assessment of portal thrombosis (bland, n=21; neoplastic, n=13). Datasets were analysed separately by two different readers. Interobserver correlation and variability were calculated and compared with the Bland–Altman method. Diagnostic accuracy of conventional enhancement measurements and iodine quantification was calculated by setting either histopathology (n=7) or a reference standard based on MDCT imaging criteria and thrombus evolutionary characteristics compared to a previous MDCT examination (n=27). For iodine quantification threshold determination receiver operating characteristic (ROC) curves were drawn. p-Values <0.05 were considered significant. Results: For conventional enhancement measurements and iodine quantification interobserver correlation was 98% and 96%. Enhancement measurement resulted in a sensitivity of 92.3%, specificity of 85.7%, positive predictive value (PPV) of 80%, and negative predictive value (NPV) of 94.7%. An iodine concentration of 0.9 mg/ml optimised discrimination between neoplastic and bland thrombi (area under the ROC [AUC] 0.993) resulting in a sensitivity of 100%, specificity of 95.2%, PPV of 92.9%, and NPV of 100%. The overall diagnostic accuracy of iodine quantification (97%) was significantly better than conventional enhancement measurements (88.2%; p<0.001). Conclusion: Compared to conventional enhancement measurements, iodine quantification improves the characterisation of portal vein thrombi during the late

Evaluation of semi-automatic arterial stenosis quantification

International Nuclear Information System (INIS)

Hernandez Hoyos, M.; Universite Claude Bernard Lyon 1, 69 - Villeurbanne; Univ. de los Andes, Bogota; Serfaty, J.M.; Douek, P.C.; Universite Claude Bernard Lyon 1, 69 - Villeurbanne; Hopital Cardiovasculaire et Pneumologique L. Pradel, Bron; Maghiar, A.; Mansard, C.; Orkisz, M.; Magnin, I.; Universite Claude Bernard Lyon 1, 69 - Villeurbanne

2006-01-01

Object: To assess the accuracy and reproducibility of semi-automatic vessel axis extraction and stenosis quantification in 3D contrast-enhanced Magnetic Resonance Angiography (CE-MRA) of the carotid arteries (CA). Materials and methods: A total of 25 MRA datasets was used: 5 phantoms with known stenoses, and 20 patients (40 CAs) drawn from a multicenter trial database. Maracas software extracted vessel centerlines and quantified the stenoses, based on boundary detection in planes perpendicular to the centerline. Centerline accuracy was visually scored. Semi-automatic measurements were compared with: (1) theoretical phantom morphometric values, and (2) stenosis degrees evaluated by two independent radiologists. Results: Exploitable centerlines were obtained in 97% of CA and in all phantoms. In phantoms, the software achieved a better agreement with theoretic stenosis degrees (weighted kappa Κ W = 0.91) than the radiologists (Κ W = 0.69). In patients, agreement between software and radiologists varied from Κ W =0.67 to 0.90. In both, Maracas was substantially more reproducible than the readers. Mean operating time was within 1 min/ CA. Conclusion: Maracas software generates accurate 3D centerlines of vascular segments with minimum user intervention. Semi-automatic quantification of CA stenosis is also accurate, except in very severe stenoses that cannot be segmented. It substantially reduces the inter-observer variability. (orig.)

A phase quantification method based on EBSD data for a continuously cooled microalloyed steel

Energy Technology Data Exchange (ETDEWEB)

Zhao, H.; Wynne, B.P.; Palmiere, E.J., E-mail: e.j.palmiere@sheffield.ac.uk

2017-01-15

Mechanical properties of steels depend on the phase constitutions of the final microstructures which can be related to the processing parameters. Therefore, accurate quantification of different phases is necessary to investigate the relationships between processing parameters, final microstructures and mechanical properties. Point counting on micrographs observed by optical or scanning electron microscopy is widely used as a phase quantification method, and different phases are discriminated according to their morphological characteristics. However, it is difficult to differentiate some of the phase constituents with similar morphology. Differently, for EBSD based phase quantification methods, besides morphological characteristics, other parameters derived from the orientation information can also be used for discrimination. In this research, a phase quantification method based on EBSD data in the unit of grains was proposed to identify and quantify the complex phase constitutions of a microalloyed steel subjected to accelerated coolings. Characteristics of polygonal ferrite/quasi-polygonal ferrite, acicular ferrite and bainitic ferrite on grain averaged misorientation angles, aspect ratios, high angle grain boundary fractions and grain sizes were analysed and used to develop the identification criteria for each phase. Comparing the results obtained by this EBSD based method and point counting, it was found that this EBSD based method can provide accurate and reliable phase quantification results for microstructures with relatively slow cooling rates. - Highlights: •A phase quantification method based on EBSD data in the unit of grains was proposed. •The critical grain area above which GAM angles are valid parameters was obtained. •Grain size and grain boundary misorientation were used to identify acicular ferrite. •High cooling rates deteriorate the accuracy of this EBSD based method.

Activity quantification of phantom using dual-head SPECT with two-view planar image

International Nuclear Information System (INIS)

Guo Leiming; Chen Tao; Sun Xiaoguang; Huang Gang

2005-01-01

The absorbed radiation dose from internally deposited radionuclide is a major factor in assessing risk and therapeutic utility in nuclear medicine diagnosis or treatment. The quantification of absolute activity in vivo is necessary procedure of estimating the absorbed dose of organ or tissue. To understand accuracy in the determination of organ activity, the experiments on 99 Tc m activity quantification were made for a body phantom using dual-heat SPECT with the two-view counting technique. Accuracy in the activity quantification is credible and is not affected by depth of source organ in vivo. When diameter of the radiation source is ≤2 cm, the most accurate activity quantification result can be obtained on the basis of establishing the system calibration factor and transmission factor. The use of Buijs's method is preferable, especially at very low source-to-background activity concentration rations. (authors)

Summary of the performance of strand produced for the 1990 SSC dipole program

International Nuclear Information System (INIS)

Christopherson, D.; Capone, D. II; Seuntjens, J.; Pollock, D.; Hannaford, C.R.

1991-01-01

In 1990 and at the beginning of 1991, more than 4 million feet of wire was delivered to support the SSC Dipole Program. This wire was fabricated to meet specification SSC-MAG-M-4141, and test results and various statistics are compiled here. Certain strengths and weaknesses in the performance of the delivered strand are discussed, including analysis of strand breakage in certain billets. Test results of cable manufactured for 40 mm dipole magnets and 50 mm dipole magnets are reported, and a brief overview of the 1991-1992 Conductor Program is included

Ranking Fragment Ions Based on Outlier Detection for Improved Label-Free Quantification in Data-Independent Acquisition LC-MS/MS

Science.gov (United States)

Bilbao, Aivett; Zhang, Ying; Varesio, Emmanuel; Luban, Jeremy; Strambio-De-Castillia, Caterina; Lisacek, Frédérique; Hopfgartner, Gérard

2016-01-01

Data-independent acquisition LC-MS/MS techniques complement supervised methods for peptide quantification. However, due to the wide precursor isolation windows, these techniques are prone to interference at the fragment ion level, which in turn is detrimental for accurate quantification. The “non-outlier fragment ion” (NOFI) ranking algorithm has been developed to assign low priority to fragment ions affected by interference. By using the optimal subset of high priority fragment ions these interfered fragment ions are effectively excluded from quantification. NOFI represents each fragment ion as a vector of four dimensions related to chromatographic and MS fragmentation attributes and applies multivariate outlier detection techniques. Benchmarking conducted on a well-defined quantitative dataset (i.e. the SWATH Gold Standard), indicates that NOFI on average is able to accurately quantify 11-25% more peptides than the commonly used Top-N library intensity ranking method. The sum of the area of the Top3-5 NOFIs produces similar coefficients of variation as compared to the library intensity method but with more accurate quantification results. On a biologically relevant human dendritic cell digest dataset, NOFI properly assigns low priority ranks to 85% of annotated interferences, resulting in sensitivity values between 0.92 and 0.80 against 0.76 for the Spectronaut interference detection algorithm. PMID:26412574

Reservoir evaluation of thin-bedded turbidites and hydrocarbon pore thickness estimation for an accurate quantification of resource

Science.gov (United States)

Omoniyi, Bayonle; Stow, Dorrik

2016-04-01

One of the major challenges in the assessment of and production from turbidite reservoirs is to take full account of thin and medium-bedded turbidites (succession, they can go unnoticed by conventional analysis and so negatively impact on reserve estimation, particularly in fields producing from prolific thick-bedded turbidite reservoirs. Field development plans often take little note of such thin beds, which are therefore bypassed by mainstream production. In fact, the trapped and bypassed fluids can be vital where maximising field value and optimising production are key business drivers. We have studied in detail, a succession of thin-bedded turbidites associated with thicker-bedded reservoir facies in the North Brae Field, UKCS, using a combination of conventional logs and cores to assess the significance of thin-bedded turbidites in computing hydrocarbon pore thickness (HPT). This quantity, being an indirect measure of thickness, is critical for an accurate estimation of original-oil-in-place (OOIP). By using a combination of conventional and unconventional logging analysis techniques, we obtain three different results for the reservoir intervals studied. These results include estimated net sand thickness, average sand thickness, and their distribution trend within a 3D structural grid. The net sand thickness varies from 205 to 380 ft, and HPT ranges from 21.53 to 39.90 ft. We observe that an integrated approach (neutron-density cross plots conditioned to cores) to HPT quantification reduces the associated uncertainties significantly, resulting in estimation of 96% of actual HPT. Further work will focus on assessing the 3D dynamic connectivity of the low-pay sands with the surrounding thick-bedded turbidite facies.

Strand displacement amplification for ultrasensitive detection of human pluripotent stem cells.

Science.gov (United States)

Wu, Wei; Mao, Yiping; Zhao, Shiming; Lu, Xuewen; Liang, Xingguo; Zeng, Lingwen

2015-06-30

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide a powerful model system for studies of cellular identity and early mammalian development, which hold great promise for regenerative medicine. It is necessary to develop a convenient method to discriminate hPSCs from other cells in clinics and basic research. Herein, a simple and reliable biosensor for stem cell detection was established. In this biosensor system, stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4) were used to mark human pluripotent stem cells (hPSCs). Antibody specific for SSEA-3 was coated onto magnetic beads for hPSCs enrichment, and antibody specific for SSEA-4 was conjugated with carboxyl-modified tDNA sequence which was used as template for strand displacement amplification (SDA). The amplified single strand DNA (ssDNA) was detected with a lateral flow biosensor (LFB). This biosensor is capable of detecting a minimum of 19 human embryonic stem cells by a strip reader and 100 human embryonic stem cells by the naked eye within 80min. This approach has also shown excellent specificity to distinguish hPSCs from other types of cells, showing that it is promising for specific and handy detection of human pluripotent stem cells. Copyright © 2015 Elsevier B.V. All rights reserved.

DNA strand breaks, repair, and survival in x-irradiated mammalian cells

International Nuclear Information System (INIS)

Dugle, D.L.; Gillespie, C.J.; Chapman, J.D.

1976-01-01

The yields of unrepairable single- and double-strand breaks in the DNA of x-irradiated Chinese hamster cells were measured by low-speed neutral and alkaline sucrose density gradient sedimentation in order to investigate the relation between these lesions and reproductive death. After maximal single-strand rejoining, at all doses, the number of residual single-strand breaks was twice the number of residual double-strand breaks. Both double-strand and unrepairable single-strand breaks were proportional to the square of absorbed dose, in the range 10-50 krad. No rejoining of double-strand breaks was observed. These observations suggest that, in mammalian cells, most double-strand breaks are not repairable, while all single-strand breaks are repaired except those that are sufficiently close on complementary strands to constitute double-strand breaks. Comparison with cell survival measurements at much lower doses suggests that loss of reproductive capacity corresponds to induction of approximately one double-strand break

RPA Stabilization of Single-Stranded DNA Is Critical for Break-Induced Replication.

Science.gov (United States)

Ruff, Patrick; Donnianni, Roberto A; Glancy, Eleanor; Oh, Julyun; Symington, Lorraine S

2016-12-20

DNA double-strand breaks (DSBs) are cytotoxic lesions that must be accurately repaired to maintain genome stability. Replication protein A (RPA) plays an important role in homology-dependent repair of DSBs by protecting the single-stranded DNA (ssDNA) intermediates formed by end resection and by facilitating Rad51 loading. We found that hypomorphic mutants of RFA1 that support intra-chromosomal homologous recombination are profoundly defective for repair processes involving long tracts of DNA synthesis, in particular break-induced replication (BIR). The BIR defects of the rfa1 mutants could be partially suppressed by eliminating the Sgs1-Dna2 resection pathway, suggesting that Dna2 nuclease attacks the ssDNA formed during end resection when not fully protected by RPA. Overexpression of Rad51 was also found to suppress the rfa1 BIR defects. We suggest that Rad51 binding to the ssDNA formed by excessive end resection and during D-loop migration can partially compensate for dysfunctional RPA. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes

International Nuclear Information System (INIS)

Venema, J.; van Hoffen, A.; Karcagi, V.; Natarajan, A.T.; van Zeeland, A.A.; Mullenders, L.H.

1991-01-01

The authors have measured the removal of UV-induced pyrimidine dimers from DNA fragments of the adenosine deaminase (ADA) and dihydrofolate reductase (DHFR) genes in primary normal human and xeroderma pigmentosum complementation group C (XP-C) cells. Using strand-specific probes, we show that in normal cells, preferential repair of the 5' part of the ADA gene is due to the rapid and efficient repair of the transcribed strand. Within 8 h after irradiation with UV at 10 J m-2, 70% of the pyrimidine dimers in this strand are removed. The nontranscribed strand is repaired at a much slower rate, with 30% dimers removed after 8 h. Repair of the transcribed strand in XP-C cells occurs at a rate indistinguishable from that in normal cells, but the nontranscribed strand is not repaired significantly in these cells. Similar results were obtained for the DHFR gene. In the 3' part of the ADA gene, however, both normal and XP-C cells perform fast and efficient repair of either strand, which is likely to be caused by the presence of transcription units on both strands. The factor defective in XP-C cells is apparently involved in the processing of DNA damage in inactive parts of the genome, including nontranscribed strands of active genes. These findings have important implications for the understanding of the mechanism of UV-induced excision repair and mutagenesis in mammalian cells

Comparison of Suitability of the Most Common Ancient DNA Quantification Methods.

Science.gov (United States)

Brzobohatá, Kristýna; Drozdová, Eva; Smutný, Jiří; Zeman, Tomáš; Beňuš, Radoslav

2017-04-01

Ancient DNA (aDNA) extracted from historical bones is damaged and fragmented into short segments, present in low quantity, and usually copurified with microbial DNA. A wide range of DNA quantification methods are available. The aim of this study was to compare the five most common DNA quantification methods for aDNA. Quantification methods were tested on DNA extracted from skeletal material originating from an early medieval burial site. The tested methods included ultraviolet (UV) absorbance, real-time quantitative polymerase chain reaction (qPCR) based on SYBR ® green detection, real-time qPCR based on a forensic kit, quantification via fluorescent dyes bonded to DNA, and fragmentary analysis. Differences between groups were tested using a paired t-test. Methods that measure total DNA present in the sample (NanoDrop ™ UV spectrophotometer and Qubit ® fluorometer) showed the highest concentrations. Methods based on real-time qPCR underestimated the quantity of aDNA. The most accurate method of aDNA quantification was fragmentary analysis, which also allows DNA quantification of the desired length and is not affected by PCR inhibitors. Methods based on the quantification of the total amount of DNA in samples are unsuitable for ancient samples as they overestimate the amount of DNA presumably due to the presence of microbial DNA. Real-time qPCR methods give undervalued results due to DNA damage and the presence of PCR inhibitors. DNA quantification methods based on fragment analysis show not only the quantity of DNA but also fragment length.

Detection of bacteriophage phi 6 minus-strand RNA and novel mRNA isoconformers synthesized in vivo and in vitro, by strand-separating agarose gels

International Nuclear Information System (INIS)

Pagratis, N.; Revel, H.R.

1990-01-01

Two urea-free agarose gel protocols that resolve the six individual strands of bacteriophage phi 6 dsRNA were developed and used to analyze phage RNA synthesis in vivo and in vitro. Citrate gels separate strands of the large and medium chromosomes while Tris-borate-EDTA (TBE) gels resolve the medium and small dsRNA segments. Minus strands migrate faster than plus strands on citrate gels but are retarded on TBE gels. A study of electrophoretic conditions showed that pH affects strand resolution on citrate gels, and that voltage gradient, agarose concentration, and ethidium bromide significantly alter strand migration on TBE gels. Analysis of native phi 6 RNA synthesized in vivo and in vitro showed that the large and medium message RNAs comigrate with the corresponding plus strands of denatured virion dsRNA. The small messenger RNA is exceptional. Native small mRNA was detected as three isoconformers in vivo and in vitro. The isoconformers were converted by heat denaturation to a single RNA species that comigrates with the virion s+ strand. Minus strands labeled in vivo were detected only after heat denaturation. Minus strand synthesis was detected also in heat-denatured samples from in vitro phi 6 nucleocapsid RNA polymerase reactions at pH values suboptimal for transcription

A validated Fourier transform infrared spectroscopy method for quantification of total lactones in Inula racemosa and Andrographis paniculata.

Science.gov (United States)

Shivali, Garg; Praful, Lahorkar; Vijay, Gadgil

2012-01-01

Fourier transform infrared (FT-IR) spectroscopy is a technique widely used for detection and quantification of various chemical moieties. This paper describes the use of the FT-IR spectroscopy technique for the quantification of total lactones present in Inula racemosa and Andrographis paniculata. To validate the FT-IR spectroscopy method for quantification of total lactones in I. racemosa and A. paniculata. Dried and powdered I. racemosa roots and A. paniculata plant were extracted with ethanol and dried to remove ethanol completely. The ethanol extract was analysed in a KBr pellet by FT-IR spectroscopy. The FT-IR spectroscopy method was validated and compared with a known spectrophotometric method for quantification of lactones in A. paniculata. By FT-IR spectroscopy, the amount of total lactones was found to be 2.12 ± 0.47% (n = 3) in I. racemosa and 8.65 ± 0.51% (n = 3) in A. paniculata. The method showed comparable results with a known spectrophotometric method used for quantification of such lactones: 8.42 ± 0.36% (n = 3) in A. paniculata. Limits of detection and quantification for isoallantolactone were 1 µg and 10 µg respectively; for andrographolide they were 1.5 µg and 15 µg respectively. Recoveries were over 98%, with good intra- and interday repeatability: RSD ≤ 2%. The FT-IR spectroscopy method proved linear, accurate, precise and specific, with low limits of detection and quantification, for estimation of total lactones, and is less tedious than the UV spectrophotometric method for the compounds tested. This validated FT-IR spectroscopy method is readily applicable for the quality control of I. racemosa and A. paniculata. Copyright © 2011 John Wiley & Sons, Ltd.

Dynamics of Leading-strand Lesion Skipping by the Replisome

Science.gov (United States)

Yeeles, Joseph T.P.; Marians, Kenneth J.

2013-01-01

SUMMARY The E. coli replisome stalls transiently when it encounters a lesion in the leading-strand template, skipping over the damage by reinitiating replication at a new primer synthesized downstream by the primase. We report here that template unwinding and lagging-strand synthesis continue downstream of the lesion at a reduced rate after replisome stalling, that one replisome is capable of skipping multiple lesions, and that the rate limiting steps of replication restart involve the synthesis and activation of the new primer downstream. We also find little support for the concept that polymerase uncoupling, where extensive lagging-strand synthesis proceeds downstream in the absence of leading-strand synthesis, involves physical separation of the leading-strand polymerase from the replisome. Instead, our data indicate that extensive uncoupled replication likely results from a failure of the leading-strand polymerase still associated with the DNA helicase and the lagging-strand polymerase that are proceeding downstream to reinitiate synthesis. PMID:24268579

Quantification by qPCR of Pathobionts in Chronic Periodontitis: Development of Predictive Models of Disease Severity at Site-Specific Level

OpenAIRE

Tomás, Inmaculada; Regueira-Iglesias, Alba; López, Maria; Arias-Bujanda, Nora; Novoa, Lourdes; Balsa-Castro, Carlos; Tomás, Maria

2017-01-01

Currently, there is little evidence available on the development of predictive models for the diagnosis or prognosis of chronic periodontitis based on the qPCR quantification of subgingival pathobionts. Our objectives were to: (1) analyze and internally validate pathobiont-based models that could be used to distinguish different periodontal conditions at site-specific level within the same patient with chronic periodontitis; (2) develop nomograms derived from predictive models. Subgingival pl...

Possible causes of a harbour porpoise mass stranding in Danish waters in 2005.

Directory of Open Access Journals (Sweden)

Andrew J Wright

Full Text Available An unprecedented 85 harbour porpoises stranded freshly dead along approximately 100 km of Danish coastline from 7-15 April, 2005. This total is considerably above the mean weekly stranding rate for the whole of Denmark, both for any time of year, 1.23 animals/week (ranging from 0 to 20 during 2003-2008, excluding April 2005, and specifically in April, 0.65 animals/week (0 to 4, same period. Bycatch was established as the cause of death for most of the individuals through typical indications of fisheries interactions, including net markings in the skin and around the flippers, and loss of tail flukes. Local fishermen confirmed unusually large porpoise bycatch in nets set for lumpfish (Cyclopterus lumpus and the strandings were attributed to an early lumpfish season. However, lumpfish catches for 2005 were not unusual in terms of season onset, peak or total catch, when compared to 2003-2008. Consequently, human activity was combined with environmental factors and the variation in Danish fisheries landings (determined through a principal component analysis in a two-part statistical model to assess the correlation of these factors with both the presence of fresh strandings and the numbers of strandings on the Danish west coast. The final statistical model (which was forward selected using Akaike information criterion; AIC indicated that naval presence is correlated with higher rates of porpoise strandings, particularly in combination with certain fisheries, although it is not correlated with the actual presence of strandings. Military vessels from various countries were confirmed in the area from the 7th April, en route to the largest naval exercise in Danish waters to date (Loyal Mariner 2005, 11-28 April. Although sonar usage cannot be confirmed, it is likely that ships were testing various equipment prior to the main exercise. Thus naval activity cannot be ruled out as a possible contributing factor.

Connecting localized DNA strand displacement reactions

Science.gov (United States)

Mullor Ruiz, Ismael; Arbona, Jean-Michel; Lad, Amitkumar; Mendoza, Oscar; Aimé, Jean-Pierre; Elezgaray, Juan

2015-07-01

Logic circuits based on DNA strand displacement reactions have been shown to be versatile enough to compute the square root of four-bit numbers. The implementation of these circuits as a set of bulk reactions faces difficulties which include leaky reactions and intrinsically slow, diffusion-limited reaction rates. In this paper, we consider simple examples of these circuits when they are attached to platforms (DNA origamis). As expected, constraining distances between DNA strands leads to faster reaction rates. However, it also induces side-effects that are not detectable in the solution-phase version of this circuitry. Appropriate design of the system, including protection and asymmetry between input and fuel strands, leads to a reproducible behaviour, at least one order of magnitude faster than the one observed under bulk conditions.Logic circuits based on DNA strand displacement reactions have been shown to be versatile enough to compute the square root of four-bit numbers. The implementation of these circuits as a set of bulk reactions faces difficulties which include leaky reactions and intrinsically slow, diffusion-limited reaction rates. In this paper, we consider simple examples of these circuits when they are attached to platforms (DNA origamis). As expected, constraining distances between DNA strands leads to faster reaction rates. However, it also induces side-effects that are not detectable in the solution-phase version of this circuitry. Appropriate design of the system, including protection and asymmetry between input and fuel strands, leads to a reproducible behaviour, at least one order of magnitude faster than the one observed under bulk conditions. Electronic supplementary information (ESI) available. See DOI: 10.1039/C5NR02434J

ChIP-seq Accurately Predicts Tissue-Specific Activity of Enhancers

Energy Technology Data Exchange (ETDEWEB)

Visel, Axel; Blow, Matthew J.; Li, Zirong; Zhang, Tao; Akiyama, Jennifer A.; Holt, Amy; Plajzer-Frick, Ingrid; Shoukry, Malak; Wright, Crystal; Chen, Feng; Afzal, Veena; Ren, Bing; Rubin, Edward M.; Pennacchio, Len A.

2009-02-01

A major yet unresolved quest in decoding the human genome is the identification of the regulatory sequences that control the spatial and temporal expression of genes. Distant-acting transcriptional enhancers are particularly challenging to uncover since they are scattered amongst the vast non-coding portion of the genome. Evolutionary sequence constraint can facilitate the discovery of enhancers, but fails to predict when and where they are active in vivo. Here, we performed chromatin immunoprecipitation with the enhancer-associated protein p300, followed by massively-parallel sequencing, to map several thousand in vivo binding sites of p300 in mouse embryonic forebrain, midbrain, and limb tissue. We tested 86 of these sequences in a transgenic mouse assay, which in nearly all cases revealed reproducible enhancer activity in those tissues predicted by p300 binding. Our results indicate that in vivo mapping of p300 binding is a highly accurate means for identifying enhancers and their associated activities and suggest that such datasets will be useful to study the role of tissue-specific enhancers in human biology and disease on a genome-wide scale.

Southeast US Historical Marine Mammal Stranding Database

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — Data on marine mammal strandings are collected by the Southeast Marine Mammal Stranding Network. Basic data on the location, species identification, animal...

Cytological studies of human meiosis: sex-specific differences in recombination originate at, or prior to, establishment of double-strand breaks.

Directory of Open Access Journals (Sweden)

Jennifer R Gruhn

Full Text Available Meiotic recombination is sexually dimorphic in most mammalian species, including humans, but the basis for the male:female differences remains unclear. In the present study, we used cytological methodology to directly compare recombination levels between human males and females, and to examine possible sex-specific differences in upstream events of double-strand break (DSB formation and synaptic initiation. Specifically, we utilized the DNA mismatch repair protein MLH1 as a marker of recombination events, the RecA homologue RAD51 as a surrogate for DSBs, and the synaptonemal complex proteins SYCP3 and/or SYCP1 to examine synapsis between homologs. Consistent with linkage studies, genome-wide recombination levels were higher in females than in males, and the placement of exchanges varied between the sexes. Subsequent analyses of DSBs and synaptic initiation sites indicated similar male:female differences, providing strong evidence that sex-specific differences in recombination rates are established at or before the formation of meiotic DSBs. We then asked whether these differences might be linked to variation in the organization of the meiotic axis and/or axis-associated DNA and, indeed, we observed striking male:female differences in synaptonemal complex (SC length and DNA loop size. Taken together, our observations suggest that sex specific differences in recombination in humans may derive from chromatin differences established prior to the onset of the recombination pathway.

The stranding anomaly as population indicator

DEFF Research Database (Denmark)

Peltier, Helene; Baagøe, Hans J.; Camphuysen, Kees C. J.

2013-01-01

Ecological indicators for monitoring strategies are expected to combine three major characteristics: ecological significance, statistical credibility, and cost-effectiveness. Strategies based on stranding networks rank highly in cost-effectiveness, but their ecological significance and statistica...... surveys, mostly SCANS surveys (1994 and 2005). This new indicator could be applied to cetacean populations across the world and more widely to marine megafauna....... credibility are disputed. Our present goal is to improve the value of stranding data as population indicator as part of monitoring strategies by constructing the spatial and temporal null hypothesis for strandings. The null hypothesis is defined as: small cetacean distribution and mortality are uniform...

Formation of double-strand breaks in DNA of γ-irradiated bacteria depending on the function of fast repair processes of DNA single-strand breaks

International Nuclear Information System (INIS)

Petrov, S.I.; Gaziev, A.I.

1980-01-01

The formation of double-strand breaks in DNA of γ-irradiated ( 60 Co)Ex coli bacteria depending on the function of fast repair processes of DNA single-strand breaks, is investigated. The profiles of sedimentation of DNA Ex coli cells, irradiated at 0-2 deg C in the salt medium and in EDTA-borate buffer, are presented. It is shown that when irradiating cells in EDTA-borate buffer, the output of single- and double strand breaks in DNA is much higher than in the case of their irradiation in the minimum salt medium. The dependence of output of single-strand and double-strand breaks depending on the radiatier doze of E coli cells in the salt medium and EDTA-borate buffer, is studied. The supposition is made on the presence of a regulative interaction between the accumulation of DNA single-breaks and their repair with the formation of double-strand breaks. The functionating of fast and superfast repair processes considerably affects the formation of double-strand breaks in DNA of a bacterium cell. A considerable amount of double-breaks registered immediately after irradiation forms due to a close position of single-strand breaks on the opposite DNA strands

Immortal DNA strand cosegregation requires p53/IMPDH-dependent asymmetric self-renewal associated with adult stem cells.

Science.gov (United States)

Rambhatla, Lakshmi; Ram-Mohan, Sumati; Cheng, Jennifer J; Sherley, James L

2005-04-15

Because they are long-lived and cycle continuously, adult stem cells (ASCs) are predicted as the most common precursor for cancers in adult mammalian tissues. Two unique attributes have been proposed to restrict the carcinogenic potential of ASCs. These are asymmetric self-renewal that limits their number and immortal DNA strand cosegregation that limits their accumulation of mutations due to DNA replication errors. Until recently, the molecular basis and regulation of these important ASC-specific functions were unknown. We developed engineered cultured cells that exhibit asymmetric self-renewal and immortal DNA strand cosegregation. These model cells were used to show that both ASC-specific functions are regulated by the p53 cancer gene. Previously, we proposed that IMP dehydrogenase (IMPDH) was an essential factor for p53-dependent asymmetric self-renewal. We now confirm this proposal and provide quantitative evidence that asymmetric self-renewal is acutely sensitive to even modest changes in IMPDH expression. These analyses reveal that immortal DNA strand cosegregation is also regulated by IMPDH and confirm the original implicit precept that immortal DNA strand cosegregation is specific to cells undergoing asymmetric self-renewal (i.e., ASCs). With IMPDH being the rate-determining enzyme for guanine ribonucleotide (rGNP) biosynthesis, its requirement implicates rGNPs as important regulators of ASC asymmetric self-renewal and immortal DNA strand cosegregation. An in silico analysis of global gene expression data from human cancer cell lines underscored the importance of p53-IMPDH-rGNP regulation for normal tissue cell kinetics, providing further support for the concept that ASCs are key targets for adult tissue carcinogenesis.

Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA.

Science.gov (United States)

Pavšič, Jernej; Devonshire, Alison; Blejec, Andrej; Foy, Carole A; Van Heuverswyn, Fran; Jones, Gerwyn M; Schimmel, Heinz; Žel, Jana; Huggett, Jim F; Redshaw, Nicholas; Karczmarczyk, Maria; Mozioğlu, Erkan; Akyürek, Sema; Akgöz, Müslüm; Milavec, Mojca

2017-04-01

Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework.

Quantification of catechin in the spray-dried extract of Pimenta pseudocaryophyllus

Directory of Open Access Journals (Sweden)

Leonardo C. Silva

Full Text Available Abstract This paper describes the quantification of catechin in the spray-dried extract of Pimenta pseudocaryophyllus (Gomes Landrum, Myrtaceae, citral chemotype using a validated HPLC-PDA method. The method employs a RP-18 column with acetonitrile:water-orthophosphoric acid 0.05% (gradient system and UV detection at 210 nm. The method was demonstrated to be simple, sensitive, specific, linear, precise, accurate and robust. The response was linear over a range of 5–200 µg/ml (r > 0.999. The range of recoveries was 92.27–102.54%. The relative standard deviation values for intra- and inter-day precision studies were 4.30 and 3.78%, respectively. This assay can be readily utilized as quality control method for catechin in the dried extract of P. pseudocaryophyllus.

Round and Extracted Nb3Sn Strand Tests for LARP Magnet R and D

International Nuclear Information System (INIS)

Barzi, Emanuela; Bossert, Rodger; Caspi, Shlomo; Dietderich, Dan; Ferracin, Paolo; Ghosh, Arup; Turrioni, Daniele; Yamada, Ryuji; Zlobin, Alexander V.

2006-01-01

The first step in the magnet R and D of the U.S. LHC Accelerator Research Program (LARP) is fabrication of technology quadrupoles TQS01 and TQC01. These are two-layer magnets which use cables of same geometry made of 0.7 mm MJR Nb 3 Sn. Through strand billet qualification and tests of strands extracted from the cables, predictions of magnet performance are made. Measurements included the critical current, I c , using the voltage-current (VI) method at constant field, the stability current, I S , as the minimal quench current obtained with the voltage-field (VH) method at constant current in the sample, and RRR. Magnetization was measured at low and high fields to determine the effective filament size and to detect flux jumps. Effects of heat treatment duration and temperature on I c and I S were also studied. The Nb 3 Sn strand and cable samples, the equipment, measurement procedures, and results are described. Based on these results, strand specifications were formulated for next LARP quadrupole models

Site-specific quantification of lysine acetylation in the N-terminal tail of histone H4 using a double-labelling, targeted UHPLC MS/MS approach

NARCIS (Netherlands)

D'Urzo, Annalisa; Boichenko, Alexander P.; van den Bosch, Thea; Hermans, Jos; Dekker, Frank; Andrisano, Vincenza; Bischoff, Rainer

We developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 by combining chemical derivatization at the protein and peptide levels with digestion using chymotrypsin and

The importance of becoming double-stranded: Innate immunity and the kinetic model of HIV-1 central plus strand synthesis

International Nuclear Information System (INIS)

Poeschla, Eric

2013-01-01

Central initiation of plus strand synthesis is a conserved feature of lentiviruses and certain other retroelements. This complication of the standard reverse transcription mechanism produces a transient “central DNA flap” in the viral cDNA, which has been proposed to mediate its subsequent nuclear import. This model has assumed that the important feature is the flapped DNA structure itself rather than the process that produces it. Recently, an alternative kinetic model was proposed. It posits that central plus strand synthesis functions to accelerate conversion to the double-stranded state, thereby helping HIV-1 to evade single-strand DNA-targeting antiviral restrictions such as APOBEC3 proteins, and perhaps to avoid innate immune sensor mechanisms. The model is consistent with evidence that lentiviruses must often synthesize their cDNAs when dNTP concentrations are limiting and with data linking reverse transcription and uncoating. There may be additional kinetic advantages for the artificial genomes of lentiviral gene therapy vectors. - Highlights: • Two main functional models for HIV central plus strand synthesis have been proposed. • In one, a transient central DNA flap in the viral cDNA mediates HIV-1 nuclear import. • In the other, multiple kinetic consequences are emphasized. • One is defense against APOBEC3G, which deaminates single-stranded DNA. • Future questions pertain to antiviral restriction, uncoating and nuclear import

Error correction in multi-fidelity molecular dynamics simulations using functional uncertainty quantification

Energy Technology Data Exchange (ETDEWEB)

Reeve, Samuel Temple; Strachan, Alejandro, E-mail: strachan@purdue.edu

2017-04-01

We use functional, Fréchet, derivatives to quantify how thermodynamic outputs of a molecular dynamics (MD) simulation depend on the potential used to compute atomic interactions. Our approach quantifies the sensitivity of the quantities of interest with respect to the input functions as opposed to its parameters as is done in typical uncertainty quantification methods. We show that the functional sensitivity of the average potential energy and pressure in isothermal, isochoric MD simulations using Lennard–Jones two-body interactions can be used to accurately predict those properties for other interatomic potentials (with different functional forms) without re-running the simulations. This is demonstrated under three different thermodynamic conditions, namely a crystal at room temperature, a liquid at ambient pressure, and a high pressure liquid. The method provides accurate predictions as long as the change in potential can be reasonably described to first order and does not significantly affect the region in phase space explored by the simulation. The functional uncertainty quantification approach can be used to estimate the uncertainties associated with constitutive models used in the simulation and to correct predictions if a more accurate representation becomes available.

Summary of the performance of strand produced for the 1990 SSC Dipole Program

International Nuclear Information System (INIS)

Christopherson, D.; Capone, D.; Seuntjens, J.; Pollock, D.; Hannaford, C.R.

1991-03-01

In 1990 and at the beginning of 1991, more than 4 million feet of wire was delivered to support the SSC Dipole Program. This wire was fabricated to meet specification SSC-MAG-M-4141, and test results and various statistics are compiled here. Certain strengths and weaknesses in the performance of the delivered strand are discussed, including analysis of strand breakage in certain billets. Test results of cable manufactured for 40 mm dipole magnets and 50 mm dipole magnets are reported, and a brief overview of the 1991--1992 Conductor Program is included. 4 refs., 5 figs., 4 tabs

Possible causes of a harbour porpoise mass stranding in Danish waters in 2005

DEFF Research Database (Denmark)

Wright, Andrew J.; Maar, Marie; Mohn, Christian

2013-01-01

An unprecedented 85 harbour porpoises stranded freshly dead along approximately 100 km of Danish coastline from 7-15 April, 2005. This total is considerably above the mean weekly stranding rate for the whole of Denmark, both for any time of year, 1.23 animals/week (ranging from 0 to 20 during 2003......-2008, excluding April 2005), and specifically in April, 0.65 animals/week (0 to 4, same period). Bycatch was established as the cause of death for most of the individuals through typical indications of fisheries interactions, including net markings in the skin and around the flippers, and loss of tail flukes...

Quantification of Iodine-123-FP-CIT SPECT with a resolution-independent method

International Nuclear Information System (INIS)

Dobbeleir, A.A.; Ham, H.R.; Hambye, A.E.; Vervaet, A.M.

2005-01-01

Accurate quantification of small-sized objects by SPECT is hampered by the partial volume effect. The present work evaluates the magnitude of this phenomenon with Iodine- 123 in phantom studies, and presents a resolution- independent method to quantify striatal I-123 FP-CIT uptake in patients. At first five syringes with internal diameters varying between 9 and 29mm and an anthropomorphic striatal phantom were filled with known concentrations of Iodine-123 and imaged by SPECT using different collimators and radii of rotation. Data were processed with and without scatter correction. From the measured activities, calibration factors were calculated for each specific collimator. Then a resolution-independent method for FP-CIT quantification using large regions of interest was developed and validated in 34 human studies (controls and patients) acquired in 2 different hospitals, by comparing its results to those obtained by a semi- quantitative striatal-to-occipital analysis. Taking the injected activity and decay into account, the measured counts/volume could be converted into absolute tracer concentrations. For the fan-beam, high resolution and medium energy collimators, the measured maximum activity in comparison to the 29 mm-diameter syringe was respectively 38%, 16% and 9% for the 9 mm-diameter syringe and 82%, 80% and 30% for the 16 mm syringe, and not significantly modified after scatter correction. For the anthropomorphic phantom, the error in measurement in % of the true concentration ranged between 0.3-9.5% and was collimator dependent. Medium energy collimators yielded the most homogeneous results. In the human studies, inter- observer variability was 11.4% for the striatal-to-occipital ratio and 3.1% for the resolution-independent method, with correlation coefficients >0.8 between both. The resolution- independent method was 89%-sensitive and 100%-specific to separate the patients without and with abnormal FP-CIT uptake (accuracy: 94%). Also the

Low-residue euthanasia of stranded mysticetes.

Science.gov (United States)

Harms, Craig A; McLellan, William A; Moore, Michael J; Barco, Susan G; Clarke, Elsburgh O; Thayer, Victoria G; Rowles, Teresa K

2014-01-01

Euthanasia of stranded large whales poses logistic, safety, pharmaceutical, delivery, public relations, and disposal challenges. Reasonable arguments may be made for allowing a stranded whale to expire naturally. However, slow cardiovascular collapse from gravitational effects outside of neutral buoyancy, often combined with severely debilitating conditions, motivate humane efforts to end the animal's suffering. The size of the animal and prevailing environmental conditions often pose safety concerns for stranding personnel, which take priority over other considerations. When considering chemical euthanasia, the size of the animal also necessitates large quantities of euthanasia agents. Drug residues are a concern for relay toxicity to scavengers, particularly for pentobarbital-containing euthanasia solutions. Pentobarbital is also an environmental concern because of its stability and long persistence in aquatic environments. We describe a euthanasia technique for stranded mysticetes using readily available, relatively inexpensive, preanesthetic and anesthetic drugs (midazolam, acepromazine, xylazine) followed by saturated KCl delivered via custom-made needles and a low-cost, basic, pressurized canister. This method provides effective euthanasia while moderating personnel exposure to hazardous situations and minimizing drug residues of concern for relay toxicity.

CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.

Science.gov (United States)

Langston, Lance D; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E; Finkelstein, Jeff; Yao, Nina Y; Indiani, Chiara; O'Donnell, Mike E

2014-10-28

DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

Stranded Specimen Data

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — The Marine Mammal and Turtle Division collects data about individual cetaceans and sea turtles that come ashore, or strand on the beach. The date and location of...

Patient-specific distal radius locking plate for fixation and accurate 3D positioning in corrective osteotomy.

Science.gov (United States)

Dobbe, J G G; Vroemen, J C; Strackee, S D; Streekstra, G J

2014-11-01

Preoperative three-dimensional planning methods have been described extensively. However, transferring the virtual plan to the patient is often challenging. In this report, we describe the management of a severely malunited distal radius fracture using a patient-specific plate for accurate spatial positioning and fixation. Twenty months postoperatively the patient shows almost painless reconstruction and a nearly normal range of motion.

SU-G-IeP1-15: Towards Accurate Cerebral Blood Flow Quantification with Distortion- Corrected Pseudo-Continuous Arterial Spin Labeling

Energy Technology Data Exchange (ETDEWEB)

Hoff, M; Rane-Levandovsky, S; Andre, J [University of Washington, Seattle, WA (United States)

2016-06-15

Purpose: Traditional arterial spin labeling (ASL) acquisitions with echo planar imaging (EPI) readouts suffer from image distortion due to susceptibility effects, compromising ASL’s ability to accurately quantify cerebral blood flow (CBF) and assess disease-specific patterns associated with CBF abnormalities. Phase labeling for additional coordinate encoding (PLACE) can remove image distortion; our goal is to apply PLACE to improve the quantitative accuracy of ASL CBF in humans. Methods: Four subjects were imaged on a 3T Philips Ingenia scanner using a 16-channel receive coil with a 21/21/10cm (frequency/phase/slice direction) field-of-view. An ASL sequence with a pseudo-continuous ASL (pCASL) labeling scheme was employed to acquire thirty dynamics of single-shot EPI data, with control and label datasets for all dynamics, and PLACE gradients applied on odd dynamics. Parameters included a post-labeling delay = 2s, label duration = 1.8s, flip angle = 90°, TR/TE = 5000/23.5ms, and 2.9/2.9/5.0mm (frequency/phase/slice direction) voxel size. “M0” EPI-reference images and T1-weighted spin-echo images with 0.8/1.0/3.3mm (frequency/phase/slice directions) voxel size were also acquired. Complex conjugate image products of pCASL odd and even dynamics were formed, a linear phase ramp applied, and data expanded and smoothed. Data phase was extracted to map control, label, and M0 magnitude image pixels to their undistorted locations, and images were rebinned to original size. All images were corrected for motion artifacts in FSL 5.0. pCASL images were registered to M0 images, and control and label images were subtracted to compute quantitative CBF maps. Results: pCASL image and CBF map distortions were removed by PLACE in all subjects. Corrected images conformed well to the anatomical T1-weighted reference image, and deviations in corrected CBF maps were evident. Conclusion: Eliminating pCASL distortion with PLACE can improve CBF quantification accuracy using minimal

SU-G-IeP1-15: Towards Accurate Cerebral Blood Flow Quantification with Distortion- Corrected Pseudo-Continuous Arterial Spin Labeling

International Nuclear Information System (INIS)

Hoff, M; Rane-Levandovsky, S; Andre, J

2016-01-01

Purpose: Traditional arterial spin labeling (ASL) acquisitions with echo planar imaging (EPI) readouts suffer from image distortion due to susceptibility effects, compromising ASL’s ability to accurately quantify cerebral blood flow (CBF) and assess disease-specific patterns associated with CBF abnormalities. Phase labeling for additional coordinate encoding (PLACE) can remove image distortion; our goal is to apply PLACE to improve the quantitative accuracy of ASL CBF in humans. Methods: Four subjects were imaged on a 3T Philips Ingenia scanner using a 16-channel receive coil with a 21/21/10cm (frequency/phase/slice direction) field-of-view. An ASL sequence with a pseudo-continuous ASL (pCASL) labeling scheme was employed to acquire thirty dynamics of single-shot EPI data, with control and label datasets for all dynamics, and PLACE gradients applied on odd dynamics. Parameters included a post-labeling delay = 2s, label duration = 1.8s, flip angle = 90°, TR/TE = 5000/23.5ms, and 2.9/2.9/5.0mm (frequency/phase/slice direction) voxel size. “M0” EPI-reference images and T1-weighted spin-echo images with 0.8/1.0/3.3mm (frequency/phase/slice directions) voxel size were also acquired. Complex conjugate image products of pCASL odd and even dynamics were formed, a linear phase ramp applied, and data expanded and smoothed. Data phase was extracted to map control, label, and M0 magnitude image pixels to their undistorted locations, and images were rebinned to original size. All images were corrected for motion artifacts in FSL 5.0. pCASL images were registered to M0 images, and control and label images were subtracted to compute quantitative CBF maps. Results: pCASL image and CBF map distortions were removed by PLACE in all subjects. Corrected images conformed well to the anatomical T1-weighted reference image, and deviations in corrected CBF maps were evident. Conclusion: Eliminating pCASL distortion with PLACE can improve CBF quantification accuracy using minimal

SU-D-218-05: Material Quantification in Spectral X-Ray Imaging: Optimization and Validation.

Science.gov (United States)

Nik, S J; Thing, R S; Watts, R; Meyer, J

2012-06-01

To develop and validate a multivariate statistical method to optimize scanning parameters for material quantification in spectral x-rayimaging. An optimization metric was constructed by extensively sampling the thickness space for the expected number of counts for m (two or three) materials. This resulted in an m-dimensional confidence region ofmaterial quantities, e.g. thicknesses. Minimization of the ellipsoidal confidence region leads to the optimization of energy bins. For the given spectrum, the minimum counts required for effective material separation can be determined by predicting the signal-to-noise ratio (SNR) of the quantification. A Monte Carlo (MC) simulation framework using BEAM was developed to validate the metric. Projection data of the m-materials was generated and material decomposition was performed for combinations of iodine, calcium and water by minimizing the z-score between the expected spectrum and binned measurements. The mean square error (MSE) and variance were calculated to measure the accuracy and precision of this approach, respectively. The minimum MSE corresponds to the optimal energy bins in the BEAM simulations. In the optimization metric, this is equivalent to the smallest confidence region. The SNR of the simulated images was also compared to the predictions from the metric. TheMSE was dominated by the variance for the given material combinations,which demonstrates accurate material quantifications. The BEAMsimulations revealed that the optimization of energy bins was accurate to within 1keV. The SNRs predicted by the optimization metric yielded satisfactory agreement but were expectedly higher for the BEAM simulations due to the inclusion of scattered radiation. The validation showed that the multivariate statistical method provides accurate material quantification, correct location of optimal energy bins and adequateprediction of image SNR. The BEAM code system is suitable for generating spectral x- ray imaging simulations. �

An external standard method for quantification of human cytomegalovirus by PCR

International Nuclear Information System (INIS)

Rongsen, Shen; Liren, Ma; Fengqi, Zhou; Qingliang, Luo

1997-01-01

An external standard method for PCR quantification of HCMV was reported. [α- 32 P]dATP was used as a tracer. 32 P-labelled specific amplification product was separated by agarose gel electrophoresis. A gel piece containing the specific product band was excised and counted in a plastic scintillation counter. Distribution of [α- 32 P]dATP in the electrophoretic gel plate and effect of separation between the 32 P-labelled specific product and free [α- 32 P]dATP were observed. A standard curve for quantification of HCMV by PCR was established and detective results of quality control templets were presented. The external standard method and the electrophoresis separation effect were appraised. The results showed that the method could be used for relative quantification of HCMV. (author)

Heat Treatment Optimization Studies on PIT Nb$_3$Sn Strand for the NED Project

CERN Document Server

Boutboul, T; den Ouden, A; Pedrini, D; Seeber, B; Volpini, G

2009-01-01

For the Next European Dipole (NED) program, a Powder-In-Tube (PIT) strand was successfully developed by SMI. This high-performance Nb$_{3}$Sn strand presents a non-copper critical current density of ~ 2500 A/mm2 at 12 T applied field and 4.2 K and a filament diameter around 50 µm. Extensive heat treatment optimization studies were performed in order to maximize both critical current and RRR, with a plateau temperature down to 625 oC and duration up to 400 hours. It appears that a critical current enhancement of ~ 10 % can be achieved for a reaction schedule of 320 hours at 625 oC with non-copper critical current density respectively exceeding 2700 and 1500 A/mm2 at 12 and 15 T (4.2 K). Thanks to this modified heat treatment, this strand completely fulfils the NED stringent specification.

Current redistribution in cables made of insulated, soldered, or oxidized strands

International Nuclear Information System (INIS)

Turck, B.

1979-07-01

Current redistributions are compared in cables made of insulated strands, soldered, or oxidized strands and insulated strands with periodic joints. After discussing the different current redistributions in the cases of a rapidly changing current and a dc current, several particular situations are investigated: what happens if a strand is broken, or if a local normal zone appears that does not affect all the strands equally, the detection of this normal zone, and the influence of short circuits between strands

Lamb Wave Damage Quantification Using GA-Based LS-SVM

Directory of Open Access Journals (Sweden)

Fuqiang Sun

2017-06-01

Full Text Available Lamb waves have been reported to be an efficient tool for non-destructive evaluations (NDE for various application scenarios. However, accurate and reliable damage quantification using the Lamb wave method is still a practical challenge, due to the complex underlying mechanism of Lamb wave propagation and damage detection. This paper presents a Lamb wave damage quantification method using a least square support vector machine (LS-SVM and a genetic algorithm (GA. Three damage sensitive features, namely, normalized amplitude, phase change, and correlation coefficient, were proposed to describe changes of Lamb wave characteristics caused by damage. In view of commonly used data-driven methods, the GA-based LS-SVM model using the proposed three damage sensitive features was implemented to evaluate the crack size. The GA method was adopted to optimize the model parameters. The results of GA-based LS-SVM were validated using coupon test data and lap joint component test data with naturally developed fatigue cracks. Cases of different loading and manufacturer were also included to further verify the robustness of the proposed method for crack quantification.

Lamb Wave Damage Quantification Using GA-Based LS-SVM.

Science.gov (United States)

Sun, Fuqiang; Wang, Ning; He, Jingjing; Guan, Xuefei; Yang, Jinsong

2017-06-12

Lamb waves have been reported to be an efficient tool for non-destructive evaluations (NDE) for various application scenarios. However, accurate and reliable damage quantification using the Lamb wave method is still a practical challenge, due to the complex underlying mechanism of Lamb wave propagation and damage detection. This paper presents a Lamb wave damage quantification method using a least square support vector machine (LS-SVM) and a genetic algorithm (GA). Three damage sensitive features, namely, normalized amplitude, phase change, and correlation coefficient, were proposed to describe changes of Lamb wave characteristics caused by damage. In view of commonly used data-driven methods, the GA-based LS-SVM model using the proposed three damage sensitive features was implemented to evaluate the crack size. The GA method was adopted to optimize the model parameters. The results of GA-based LS-SVM were validated using coupon test data and lap joint component test data with naturally developed fatigue cracks. Cases of different loading and manufacturer were also included to further verify the robustness of the proposed method for crack quantification.

Measurement of intracellular DNA double-strand break induction and rejoining along the track of carbon and neon particle beams in water

International Nuclear Information System (INIS)

Heilmann, Johannes; Taucher-Scholz, Gisela; Haberer, Thomas; Scholz, Michael; Kraft, Gerhard

1996-01-01

Purpose: The study was aimed at the measurement of effect-depth distributions of intracellularly induced DNA damage in water as tissue equivalent after heavy ion irradiation with therapy particle beams. Methods and Materials: An assay involving embedding of Chinese hamster ovary (CHO-K1) cells in large agarose plugs and electrophoretic elution of radiation induced DNA fragments by constant field gel electrophoresis was developed. Double-strand break production was quantified by densitometric analysis of DNA-fluorescence after staining with ethidium-bromide and determination of the fraction of DNA eluted out of the agarose plugs. Intracellular double-strand break induction and the effect of a 3 h rejoining incubation were investigated following irradiation with 250 kV x-rays and 190 MeV/u carbon- and 295 MeV/u neon-ions. Results and Conclusion: While the DNA damage induced by x-irradiation decreased continuously with penetration depth, a steady increase in the yield of double-strand breaks was observed for particle radiation, reaching distinct maxima at the position of the physical Bragg peaks. Beyond this, the extent of radiation damage dropped drastically. From comparison of DNA damage and calculated dose profiles, relative biological efficiencies (RBEs) for both double-strand break induction and unrejoined strand breaks after 3 h were determined. While RBE for the induction of DNA double-strand breaks decreased continuously with penetration depth, RBE maxima greater than unity were found with carbon- and neon-ions for double-strand break rejoining near the maximum range of the particles. The method presented here allows for a fast and accurate determination of depth profiles of relevant radiobiological effects for mixed particle fields in tissue equivalent

Strand SPA & Konverentsikeskus

Index Scriptorium Estoniae

2008-01-01

Strand SPA & Konverentsikeskus on Pärnu suurim äri- ja konverentsiklientidele suunatud hotell, mis klientide seas on hinnatud just selle kompleksuse tõttu, kuna kõik, mida külaline vajab ja soovib, on olemas ühe katuse all

Physical and biological parameters affecting DNA double strand break misrejoining in mammalian cells

International Nuclear Information System (INIS)

Kuehne, M.; Rothkamm, K.; Loebrich, M.

2002-01-01

In an attempt to investigate the effect of radiation quality, dose and specific repair pathways on correct and erroneous rejoining of DNA double strand breaks (DSBs), an assay was applied that allows the identification and quantification of incorrectly rejoined DSB ends produced by ionising radiation. While substantial misrejoining occurs in mammalian cells after high acute irradiation doses, decreasing misrejoining frequencies were observed in dose fractionation experiments with X rays. In line with this finding, continuous irradiation with gamma rays at low dose rate leads to non detectable misrejoining. This indicates that the probability for a DSB to be misrejoined decreases drastically when DSBs are separated in time and space. The same dose fractionation approach was applied to determine DSB misrejoining after a particle exposure. In contrast to the results with X rays, there was no significant decrease in DSB misrejoining with increasing fractionation. This suggests that DSB misrejoining after a irradiation is not significantly affected by a separation of particle tracks. To identify the enzymatic pathways that are involved in DSB misrejoining, cell lines deficient in non-homologous end-joining (NHEJ) were examined. After high X ray doses, DSB misrejoining is considerable reduced in NHEJ mutants. Low dose rate experiments show elevated DSB misrejoining in NHEJ mutants compared with wild-type cells. The authors propose that NHEJ serves as an efficient pathway for rejoining correct break ends in situations of separated breaks but generates genomic rearrangements if DSBs are close in time and space. (author)

A Major Role of DNA Polymerase δ in Replication of Both the Leading and Lagging DNA Strands.

Science.gov (United States)

Johnson, Robert E; Klassen, Roland; Prakash, Louise; Prakash, Satya

2015-07-16

Genetic studies with S. cerevisiae Polδ (pol3-L612M) and Polε (pol2-M644G) mutant alleles, each of which display a higher rate for the generation of a specific mismatch, have led to the conclusion that Polε is the primary leading strand replicase and that Polδ is restricted to replicating the lagging strand template. Contrary to this widely accepted view, here we show that Polδ plays a major role in the replication of both DNA strands, and that the paucity of pol3-L612M-generated errors on the leading strand results from their more proficient removal. Thus, the apparent lack of Polδ contribution to leading strand replication is due to differential mismatch removal rather than differential mismatch generation. Altogether, our genetic studies with Pol3 and Pol2 mutator alleles support the conclusion that Polδ, and not Polε, is the major DNA polymerase for carrying out both leading and lagging DNA synthesis. Copyright © 2015 Elsevier Inc. All rights reserved.

Simulation of 125I-induced DNA strand breaks in a CAP-DNA complex

International Nuclear Information System (INIS)

Li, W.; Friedland, W.; Jacob, P.

2000-01-01

DNA strand breakage induced by decay of 125 I incorporated into the pyrimidine of a small piece of DNA with a specific base pair sequence has been investigated theoretically and experimentally (Lobachevsky and Martin 2000a, 2000b; Nikjoo et al., 1996; Pomplun and Terrissol, 1994; Charlton and Humm, 1988). Recently an attempt was made to analyse the DNA kinks in a CAP-DNA complex with 125 I induced DNA strand breakage (Karamychev et al., 1999). This method could be used as a so called radioprobing for such DNa distortions like other chemical and biological assays, provided that it has been tested and confirmed in a corresponding theoretical simulation. In the measurement, the distribution of the first breaks on the DNA strands starting from their labeled end can be determined. Based on such first breakage distributions, the simulation calculation could then be used to derive information on the structure of a given DNA-protein complex. The biophysical model PARTRAC has been applied successfully in simulating DNA damage induced by irradiation (Friedland et al., 1998; 1999). In the present study PARTRAC is adapted to a DNA-protein complex in which a specific sequence of 30 base pairs of DNA is connected with the catabolite gene activator protein (CAP). This report presents the first step of the analysis in which the CAP-DNA model used in NIH is overlaid with electron track structures in liquid water and the strand breaks due to direct ionization and due to radical attack are simulated. The second step will be to take into account the neutralization of the heavily charged tellurium and the protective effect of the CAP protein against radical attack. (orig.)

Strand specific RNA-sequencing and membrane lipid profiling reveals growth phase-dependent cold stress response mechanisms in Listeria monocytogenes

Science.gov (United States)

Hingston, Patricia; Chen, Jessica; Allen, Kevin; Truelstrup Hansen, Lisbeth

2017-01-01

The human pathogen Listeria monocytogenes continues to pose a challenge in the food industry, where it is known to contaminate ready-to-eat foods and grow during refrigerated storage. Increased knowledge of the cold-stress response of this pathogen will enhance the ability to control it in the food-supply-chain. This study utilized strand-specific RNA sequencing and whole cell fatty acid (FA) profiling to characterize the bacterium’s cold stress response. RNA and FAs were extracted from a cold-tolerant strain at five time points between early lag phase and late stationary-phase, both at 4°C and 20°C. Overall, more genes (1.3×) were suppressed than induced at 4°C. Late stationary-phase cells exhibited the greatest number (n = 1,431) and magnitude (>1,000-fold) of differentially expressed genes (>2-fold, pmonocytogenes, the growth-phase dependency of its cold-stress regulon, and the active roles of antisense transcripts in regulating its cold stress response. PMID:28662112

Strand specific RNA-sequencing and membrane lipid profiling reveals growth phase-dependent cold stress response mechanisms in Listeria monocytogenes

DEFF Research Database (Denmark)

Hingston, Patricia; Chen, Jessica; Allen, Kevin

2017-01-01

The human pathogen Listeria monocytogenes continues to pose a challenge in the food industry, where it is known to contaminate ready-to-eat foods and grow during refrigerated storage. Increased knowledge of the cold-stress response of this pathogen will enhance the ability to control it in the food-supply-chain....... This study utilized strand-specific RNA sequencing and whole cell fatty acid (FA) profiling to characterize the bacterium’s cold stress response. RNA and FAs were extracted from a cold-tolerant strain at five time points between early lag phase and late stationary-phase, both at 4°C and 20°C. Overall, more...... genes (1.3×) were suppressed than induced at 4°C. Late stationary-phase cells exhibited the greatest number (n = 1,431) and magnitude (>1,000-fold) of differentially expressed genes (>2-fold, pcold. A core set of 22 genes was upregulated at all growth phases, including nine genes...

qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

International Nuclear Information System (INIS)

Jackson, Christopher B.; Gallati, Sabina; Schaller, André

2012-01-01

Highlights: ► Serial qPCR accurately determines fragmentation state of any given DNA sample. ► Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. ► Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. ► Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze–thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λ nDNA ) and mtDNA (λ mtDNA ) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two

qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

Energy Technology Data Exchange (ETDEWEB)

Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Gallati, Sabina, E-mail: sabina.gallati@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Schaller, Andre, E-mail: andre.schaller@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)

2012-07-06

Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct measurements in

Double-Stranded Peptide Nucleic Acids

DEFF Research Database (Denmark)

2001-01-01

A novel class of compounds, known as peptide nucleic acids, form double-stranded structures with one another and with ssDNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker.......A novel class of compounds, known as peptide nucleic acids, form double-stranded structures with one another and with ssDNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

The (not so) immortal strand hypothesis.

Science.gov (United States)

Tomasetti, Cristian; Bozic, Ivana

2015-03-01

Non-random segregation of DNA strands during stem cell replication has been proposed as a mechanism to minimize accumulated genetic errors in stem cells of rapidly dividing tissues. According to this hypothesis, an "immortal" DNA strand is passed to the stem cell daughter and not the more differentiated cell, keeping the stem cell lineage replication error-free. After it was introduced, experimental evidence both in favor and against the hypothesis has been presented. Using a novel methodology that utilizes cancer sequencing data we are able to estimate the rate of accumulation of mutations in healthy stem cells of the colon, blood and head and neck tissues. We find that in these tissues mutations in stem cells accumulate at rates strikingly similar to those expected without the protection from the immortal strand mechanism. Utilizing an approach that is fundamentally different from previous efforts to confirm or refute the immortal strand hypothesis, we provide evidence against non-random segregation of DNA during stem cell replication. Our results strongly suggest that parental DNA is passed randomly to stem cell daughters and provides new insight into the mechanism of DNA replication in stem cells. Copyright © 2015. Published by Elsevier B.V.

Prediction of guide strand of microRNAs from its sequence and secondary structure

Directory of Open Access Journals (Sweden)

Ahmed Firoz

2009-04-01

Full Text Available Abstract Background MicroRNAs (miRNAs are produced by the sequential processing of a long hairpin RNA transcript by Drosha and Dicer, an RNase III enzymes, and form transitory small RNA duplexes. One strand of the duplex, which incorporates into RNA-induced silencing complex (RISC and silences the gene expression is called guide strand, or miRNA; while the other strand of duplex is degraded and called the passenger strand, or miRNA*. Predicting the guide strand of miRNA is important for better understanding the RNA interference pathways. Results This paper describes support vector machine (SVM models developed for predicting the guide strands of miRNAs. All models were trained and tested on a dataset consisting of 329 miRNA and 329 miRNA* pairs using five fold cross validation technique. Firstly, models were developed using mono-, di-, and tri-nucleotide composition of miRNA strands and achieved the highest accuracies of 0.588, 0.638 and 0.596 respectively. Secondly, models were developed using split nucleotide composition and achieved maximum accuracies of 0.553, 0.641 and 0.602 for mono-, di-, and tri-nucleotide respectively. Thirdly, models were developed using binary pattern and achieved the highest accuracy of 0.708. Furthermore, when integrating the secondary structure features with binary pattern, an accuracy of 0.719 was seen. Finally, hybrid models were developed by combining various features and achieved maximum accuracy of 0.799 with sensitivity 0.781 and specificity 0.818. Moreover, the performance of this model was tested on an independent dataset that achieved an accuracy of 0.80. In addition, we also compared the performance of our method with various siRNA-designing methods on miRNA and siRNA datasets. Conclusion In this study, first time a method has been developed to predict guide miRNA strands, of miRNA duplex. This study demonstrates that guide and passenger strand of miRNA precursors can be distinguished using their

$Nb_{3}Sn macrostructure, microstructure, and property comparisons for bronze and internal Sn process strands

CERN Document Server

Lee, P J; Larbalestier, D C

2000-01-01

The variation in irreversibility field, B*(T), with temperature has been measured for Nb/sub 3/Sn superconducting strands manufactured for ITER using vibrating sample and SQUID magnetometers. The high performance strands were developed for both high transport critical current density, J/sub c/, and low hysteresis loss. Despite a wide variety of designs and components, the strands could be split into two distinctive groups, based on the extrapolated irreversibility fields, which lie about 10% lower than the upper critical field. "Bronze-process" strands exhibited consistently higher B*(T) (28 T to 31 T) compared with "internal Sn" process (24 T to 26 T) conductors. The intrinsic critical current density of the superconductor, J/sub c (sc)/, and the specific pinning force of the grain boundaries, Q/sub gb/, were evaluated using the measured J/sub c/, and image analysis of the macro- and micro-structures. A bronze-processed Nb(-Ta)/sub 3 /Sn was found to have a higher J/sub c(sc)/ but lower Q/sub gb/ than Nb/sub...

DNA strand breakage by 125I-decay in oligoDNA

International Nuclear Information System (INIS)

Lobachevsky, P.; Martin, R.F.

1996-01-01

Full text: A double-stranded oligodeoxynucleotide containing 125 I-dC in a defined location, with 5'- or 3'- 32 P-end-labelling of either strand, was used to investigate DNA strand breakage resulting from 125 I decay. Samples of the 32 P-end-labelled and 125 I-dC containing oligoDNA were incubated in 20 mM phosphate buffer (PB), or PB + 2 M dimethylsulphoxide (DMSO) at 4 deg during 18-20 days. The 32 P-end-labelled DNA fragments produced by 125 I decays were separated on denaturing polyacrylamide gels, and the 3P activity in each fragment was determined by scintillation counting after elution from the gel. The fragment size distribution was then converted to a distribution of single stranded break probabilities at each nucleotide position. The results indicate that each 125 I decay event produces at least one break in the 125 I-dC containing strand, and causes breakage of the opposite strand in 75-80% of events. Thus, the double stranded break is produced by 125 I decay with probability ∼0.8. Most of single stranded breaks (around 90%) occurred within 5-6 nucleotides of the 125 I-dC, however DNA breaks were detected up to 18-20 nucleotides from the decay site. The average numbers of single stranded breaks per decay are 3.7 (PB) and 3.3 (PB+DMSO) in 125 I-dC containing strand, and 1.5 (PB) and 1.3 (PB+DMSO) in the opposite strand. Deconvolution of strand break probabilities as a function of separation from the 125 I, in terms of both distance (to target deoxyribosyl carbon atoms, in B-DNA) and nucleotide number, show that the latter is an important parameter for the shorter-range damage. This could indicate a role for attenuation/dissipation of damage through the stacked bases. In summary, the results represent a much more extensive set of data than available from earlier experiments on DNA breakage from l25 I-decay, and may provide new mechanistic insights

Development of superconducting strand and cable with improved properties for use in SSC [Superconducting Super Collider] magnets

International Nuclear Information System (INIS)

Scanlan, R.M.

1989-02-01

The critical current requirement for the NbTi superconductor strand was set at 2750 A/mm 2 (5 T, 4.2 K) in the SSC Conceptual Design, compared with a value of 1800 A/mm 2 which was specified for the strand used in the Tevatron dipoles. In addition, a filament diameter of 5 μm, instead of the 9 μm diameter used in the Tevatron, was chosen to reduce field distortion at injection. In order to meet the requirements for field homogeneity, the dimensional requirements for both strand and cable were also tightened. The technical solutions employed to achieve these improved properties and the resulting specifications will be discussed. 9 refs

Southeast Region Level A Marine Mammal Stranding Data

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — Data on marine mammal strandings are collected by the Southeast Marine Mammal Stranding Network. Basic data on the location, species identification, animal...

On ISSM and leveraging the Cloud towards faster quantification of the uncertainty in ice-sheet mass balance projections

Science.gov (United States)

Larour, E.; Schlegel, N.

2016-11-01

With the Amazon EC2 Cloud becoming available as a viable platform for parallel computing, Earth System Models are increasingly interested in leveraging its capabilities towards improving climate projections. In particular, faced with long wait periods on high-end clusters, the elasticity of the Cloud presents a unique opportunity of potentially "infinite" availability of small-sized clusters running on high-performance instances. Among specific applications of this new paradigm, we show here how uncertainty quantification in climate projections of polar ice sheets (Antarctica and Greenland) can be significantly accelerated using the Cloud. Indeed, small-sized clusters are very efficient at delivering sensitivity and sampling analysis, core tools of uncertainty quantification. We demonstrate how this approach was used to carry out an extensive analysis of ice-flow projections on one of the largest basins in Greenland, the North-East Greenland Glacier, using the Ice Sheet System Model, the public-domain NASA-funded ice-flow modeling software. We show how errors in the projections were accurately quantified using Monte-Carlo sampling analysis on the EC2 Cloud, and how a judicious mix of high-end parallel computing and Cloud use can best leverage existing infrastructures, and significantly accelerate delivery of potentially ground-breaking climate projections, and in particular, enable uncertainty quantification that were previously impossible to achieve.

Thioflavin T binds dimeric parallel-stranded GA-containing non-G-quadruplex DNAs: a general approach to lighting up double-stranded scaffolds.

Science.gov (United States)

Liu, Shuangna; Peng, Pai; Wang, Huihui; Shi, Lili; Li, Tao

2017-12-01

A molecular rotor thioflavin T (ThT) is usually used as a fluorescent ligand specific for G-quadruplexes. Here, we demonstrate that ThT can tightly bind non-G-quadruplex DNAs with several GA motifs and dimerize them in a parallel double-stranded mode, accompanied by over 100-fold enhancement in the fluorescence emission of ThT. The introduction of reverse Watson-Crick T-A base pairs into these dimeric parallel-stranded DNA systems remarkably favors the binding of ThT into the pocket between G•G and A•A base pairs, where ThT is encapsulated thereby restricting its two rotary aromatic rings in the excited state. A similar mechanism is also demonstrated in antiparallel DNA duplexes where several motifs of two consecutive G•G wobble base pairs are incorporated and serve as the active pockets for ThT binding. The insight into the interactions of ThT with non-G-quadruplex DNAs allows us to introduce a new concept for constructing DNA-based sensors and devices. As proof-of-concept experiments, we design a DNA triplex containing GA motifs in its Hoogsteen hydrogen-bonded two parallel strands as a pH-driven nanoswitch and two GA-containing parallel duplexes as novel metal sensing platforms where C-C and T-T mismatches are included. This work may find further applications in biological systems (e.g. disease gene detection) where parallel duplex or triplex stretches are involved. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Reverse transcription-quantitative polymerase chain reaction: description of a RIN-based algorithm for accurate data normalization

Directory of Open Access Journals (Sweden)

Boissière-Michot Florence

2009-04-01

Full Text Available Abstract Background Reverse transcription-quantitative polymerase chain reaction (RT-qPCR is the gold standard technique for mRNA quantification, but appropriate normalization is required to obtain reliable data. Normalization to accurately quantitated RNA has been proposed as the most reliable method for in vivo biopsies. However, this approach does not correct differences in RNA integrity. Results In this study, we evaluated the effect of RNA degradation on the quantification of the relative expression of nine genes (18S, ACTB, ATUB, B2M, GAPDH, HPRT, POLR2L, PSMB6 and RPLP0 that cover a wide expression spectrum. Our results show that RNA degradation could introduce up to 100% error in gene expression measurements when RT-qPCR data were normalized to total RNA. To achieve greater resolution of small differences in transcript levels in degraded samples, we improved this normalization method by developing a corrective algorithm that compensates for the loss of RNA integrity. This approach allowed us to achieve higher accuracy, since the average error for quantitative measurements was reduced to 8%. Finally, we applied our normalization strategy to the quantification of EGFR, HER2 and HER3 in 104 rectal cancer biopsies. Taken together, our data show that normalization of gene expression measurements by taking into account also RNA degradation allows much more reliable sample comparison. Conclusion We developed a new normalization method of RT-qPCR data that compensates for loss of RNA integrity and therefore allows accurate gene expression quantification in human biopsies.

Low-Energy Electron-Induced Strand Breaks in Telomere-Derived DNA Sequences-Influence of DNA Sequence and Topology.

Science.gov (United States)

Rackwitz, Jenny; Bald, Ilko

2018-03-26

During cancer radiation therapy high-energy radiation is used to reduce tumour tissue. The irradiation produces a shower of secondary low-energy (DNA very efficiently by dissociative electron attachment. Recently, it was suggested that low-energy electron-induced DNA strand breaks strongly depend on the specific DNA sequence with a high sensitivity of G-rich sequences. Here, we use DNA origami platforms to expose G-rich telomere sequences to low-energy (8.8 eV) electrons to determine absolute cross sections for strand breakage and to study the influence of sequence modifications and topology of telomeric DNA on the strand breakage. We find that the telomeric DNA 5'-(TTA GGG) 2 is more sensitive to low-energy electrons than an intermixed sequence 5'-(TGT GTG A) 2 confirming the unique electronic properties resulting from G-stacking. With increasing length of the oligonucleotide (i.e., going from 5'-(GGG ATT) 2 to 5'-(GGG ATT) 4 ), both the variety of topology and the electron-induced strand break cross sections increase. Addition of K + ions decreases the strand break cross section for all sequences that are able to fold G-quadruplexes or G-intermediates, whereas the strand break cross section for the intermixed sequence remains unchanged. These results indicate that telomeric DNA is rather sensitive towards low-energy electron-induced strand breakage suggesting significant telomere shortening that can also occur during cancer radiation therapy. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antibiotic Resistome: Improving Detection and Quantification Accuracy for Comparative Metagenomics.

Science.gov (United States)

Elbehery, Ali H A; Aziz, Ramy K; Siam, Rania

2016-04-01

The unprecedented rise of life-threatening antibiotic resistance (AR), combined with the unparalleled advances in DNA sequencing of genomes and metagenomes, has pushed the need for in silico detection of the resistance potential of clinical and environmental metagenomic samples through the quantification of AR genes (i.e., genes conferring antibiotic resistance). Therefore, determining an optimal methodology to quantitatively and accurately assess AR genes in a given environment is pivotal. Here, we optimized and improved existing AR detection methodologies from metagenomic datasets to properly consider AR-generating mutations in antibiotic target genes. Through comparative metagenomic analysis of previously published AR gene abundance in three publicly available metagenomes, we illustrate how mutation-generated resistance genes are either falsely assigned or neglected, which alters the detection and quantitation of the antibiotic resistome. In addition, we inspected factors influencing the outcome of AR gene quantification using metagenome simulation experiments, and identified that genome size, AR gene length, total number of metagenomics reads and selected sequencing platforms had pronounced effects on the level of detected AR. In conclusion, our proposed improvements in the current methodologies for accurate AR detection and resistome assessment show reliable results when tested on real and simulated metagenomic datasets.

Structure-spectrophotometric selectivity relationship in interactions of quercetin related flavonoids with double stranded and single stranded RNA

Science.gov (United States)

Piantanida, Ivo; Mašić, Lozika; Rusak, Gordana

2009-04-01

Interactions of five flavonoids with dsRNA and single stranded ssRNA were studied by UV/vis titrations. The results obtained supported the intercalative binding mode as a dominant interaction of studied flavonoids with dsRNA as well as major interaction with ssRNA. Furthermore, changes of the UV/vis spectra of flavonoids induced by addition of poly G or poly C, respectively, are significantly stronger than changes induced by double stranded poly G-poly C, pointing to essential role of the free poly G or poly C sequence (not hydrogen bonded in double helix). Exclusively poly G caused significant batochromic shift of the UV/vis maxima of all studied flavonoids, whereby the intensity of batochromic shift is nicely correlated to the number of OH groups of flavonoid. Unlikely to poly G, addition of poly A and poly U induced measurable changes only in the UV/vis spectra of flavonoids characterised by no OH (galangin) or three OH groups (myricetin) on the phenyl part of the molecule. Consequently, flavonoids with one- or two-OH groups on the phenyl part of the molecule (luteolin, fisetin, kaempferol) specifically differentiate between poly A, poly U (negligible changes in the UV/Vis spectra) and poly G (strong changes in the UV/Vis spectra) as well as poly C (moderate changes in the UV/Vis spectra).

Effect of Twist Pitch in the Strands on the Saturation and Losses in the Nb3Sn Strands for the ITER TF CICC

International Nuclear Information System (INIS)

Martovetsky, N.N.

2007-01-01

ITER TF coils will see a significant longitudinal magnetic field in the event of the plasma disruption. This abrupt change of magnetic fields results in the appearance of an additional electrical field in the strands. The mechanism of this electrical field is the induced currents that expel the flux from the strands. This effect was known since the late 1970's [1-3] and most of the details necessary for the analyses given in this report are presented in [4]. Let's assume for simplicity a zero transport current in the strand. When a longitudinal pulsed field is applied, the outer filaments will carry an induced current repelling the change of flux. The current density of this current is 'critical' in the simplification of Bean's critical state model, where superconducting transition is represented as j=j c at any non-zero electrical field and zero where the electrical field has not penetrated. In reality, since the current density is roughly logarithmic with the electrical field, E=E c *exp[(j-j c )/jo], Bean's model is just a simplification, and current density is slightly nonuniform in the outer filament and more so for the interior strands. The inner portion of the filaments will carry a current of the opposite sign. Even in the Bean's model it is not uniform, but the assumption that it is uniform and less than critical simplifies mathematics significantly and does not deviate far from the real current density distribution. In certain circumstances, the average electrical field in the strands will be high enough to exceed the take-off electrical field averaged across the cross section. In this case, the multifilamentary strand will become unstable and will experience transition to the normal state. With zero transport current, it will eventually recover, of course. This phenomenon is analogous to the flux jump. If the strand carries a transport current, the situation becomes more complicated. If it goes unstable and the transport current is higher than the

In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication.

Science.gov (United States)

Schauer, Grant; Finkelstein, Jeff; O'Donnell, Mike

2017-09-20

The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other proteins. We have previously established the mechanisms by which the polymerases ε and δ are targeted to their 'correct' strands, as well as quality control mechanisms that evict polymerases when they associate with an 'incorrect' strand. Here, we provide a practical guide to differentially assay leading and lagging strand replication in vitro using pure proteins.

Ct shift: A novel and accurate real-time PCR quantification model for direct comparison of different nucleic acid sequences and its application for transposon quantifications.

Science.gov (United States)

Kolacsek, Orsolya; Pergel, Enikő; Varga, Nóra; Apáti, Ágota; Orbán, Tamás I

2017-01-20

There are numerous applications of quantitative PCR for both diagnostic and basic research. As in many other techniques the basis of quantification is that comparisons are made between different (unknown and known or reference) specimens of the same entity. When the aim is to compare real quantities of different species in samples, one cannot escape their separate precise absolute quantification. We have established a simple and reliable method for this purpose (Ct shift method) which combines the absolute and the relative approach. It requires a plasmid standard containing both sequences of amplicons to be compared (e.g. the target of interest and the endogenous control). It can serve as a reference sample with equal copies of templates for both targets. Using the ΔΔCt formula we can quantify the exact ratio of the two templates in each unknown sample. The Ct shift method has been successfully applied for transposon gene copy measurements, as well as for comparison of different mRNAs in cDNA samples. This study provides the proof of concept and introduces some potential applications of the method; the absolute nature of results even without the need for real reference samples can contribute to the universality of the method and comparability of different studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantification of methionine and selenomethionine in biological samples using multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS).

Science.gov (United States)

Vu, Dai Long; Ranglová, Karolína; Hájek, Jan; Hrouzek, Pavel

2018-05-01

Quantification of selenated amino-acids currently relies on methods employing inductively coupled plasma mass spectrometry (ICP-MS). Although very accurate, these methods do not allow the simultaneous determination of standard amino-acids, hampering the comparison of the content of selenated versus non-selenated species such as methionine (Met) and selenomethionine (SeMet). This paper reports two approaches for the simultaneous quantification of Met and SeMet. In the first approach, standard enzymatic hydrolysis employing Protease XIV was applied for the preparation of samples. The second approach utilized methanesulfonic acid (MA) for the hydrolysis of samples, either in a reflux system or in a microwave oven, followed by derivatization with diethyl ethoxymethylenemalonate. The prepared samples were then analyzed by multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS). Both approaches provided platforms for the accurate determination of selenium/sulfur substitution rate in Met. Moreover the second approach also provided accurate simultaneous quantification of Met and SeMet with a low limit of detection, low limit of quantification and wide linearity range, comparable to the commonly used gas chromatography mass spectrometry (GC-MS) method or ICP-MS. The novel method was validated using certified reference material in conjunction with the GC-MS reference method. Copyright © 2018. Published by Elsevier B.V.

Effects of DNA double-strand and single-strand breaks on intrachromosomal recombination events in cell-cycle-arrested yeast cells

International Nuclear Information System (INIS)

Galli, A.; Schiestl, R.H.

1998-01-01

Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events. We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of initiating these events. Two genetic systems were constructed in Saccharomyces cerevisiae that select for DEL recombination events between duplicated alleles of CDC28 and TUB2. We determined effects of double-strand breaks (DSBs) and single-strand breaks (SSBs) between the duplicated alleles on DEL recombination when induced in dividing cells or cells arrested in G1 or G2. Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gIIp), respectively. I-Sce I-induced DSBs caused an increase in DEL recombination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DSB repair. In contrast, gIIp-induced SSBs caused an increase in DEL recombination frequency only in dividing cells. To further examine these phenomena we used both γ-irradiation, inducing DSBs as its most relevant lesion, and UV, inducing other forms of DNA damage. UV irradiation did not increase DEL recombination frequencies in G1 or G2, whereas γ-rays increased DEL recombination frequencies in both phases. Both forms of radiation, however, induced DEL recombination in dividing cells. The results suggest that DSBsbut not SSBs induce DEL recombination, probably via the single-strand annealing pathway. Further, DSBs in dividing cells may result from the replication of a UV or SSB-damaged template. Alternatively, UV induced events may occur by replication slippage after DNA polymerase pausing in front of the damage. (author)

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells.

Science.gov (United States)

Holton, Nathaniel W; Andrews, Joel F; Gassman, Natalie R

2017-09-05

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Manufacture of Strand Board Bonded with Disposal Expanded Polystyrene as Binder

OpenAIRE

Hermawan, Andi; Ohuchi, Takeshi; Fujimoto, Noboru; 大内, 毅; 藤本, 登留

2010-01-01

The objective of this study was to evaluate the physical and mechanical properties of strand board bonded with disposal expanded polystyrene (EPS) as binder. The strand board was manufactured using strand made from Douglas-fir beams selected from construction scrap wood. The strands were oriented, and two types of three-layer (face-core-face) strand board were manufactured: one in which the board bounded with only disposal EPS (P board), and the other in which the board bonded with disposal...

Quantification analysis of CT for aphasic patients

International Nuclear Information System (INIS)

Watanabe, Shunzo; Ooyama, Hiroshi; Hojo, Kei; Tasaki, Hiroichi; Hanazono, Toshihide; Sato, Tokijiro; Metoki, Hirobumi; Totsuka, Motokichi; Oosumi, Noboru.

1987-01-01

Using a microcomputer, the locus and extent of the lesions, as demonstrated by computed tomography, for 44 aphasic patients with various types of aphasia were superimposed onto standardized matrices, composed of 10 slices with 3000 points (50 by 60). The relationships between the foci of the lesions and types of aphasia were investigated on the slices numbered 3, 4, 5, and 6 using a quantification theory, Type 3 (pattern analysis). Some types of regularities were observed on Slices 3, 4, 5, and 6. The group of patients with Broca's aphasia and the group with Wernicke's aphasia were generally separated on the 1st component and the 2nd component of the quantification theory, Type 3. On the other hand, the group with global aphasia existed between the group with Broca's aphasia and that with Wernicke's aphasia. The group of patients with amnestic aphasia had no specific findings, and the group with conduction aphasia existed near those with Wernicke's aphasia. The above results serve to establish the quantification theory, Type 2 (discrimination analysis) and the quantification theory, Type 1 (regression analysis). (author)

Four-Strand Core Suture Improves Flexor Tendon Repair Compared to Two-Strand Technique in a Rabbit Model

Directory of Open Access Journals (Sweden)

Alice Wichelhaus

2016-01-01

Full Text Available Introduction. This study was designed to investigate the influence of the amount of suture material on the formation of peritendinous adhesions of intrasynovial flexor tendon repairs. Materials and Methods. In 14 rabbits, the flexor tendons of the third and the fourth digit of the right hind leg were cut and repaired using a 2- or 4-strand core suture technique. The repaired tendons were harvested after three and eight weeks. The range of motion of the affected toes was measured and the tendons were processed histologically. The distance between the transected tendon ends, the changes in the peritendinous space, and cellular and extracellular inflammatory reaction were quantified by different staining. Results. A 4-strand core suture resulted in significantly less gap formation. The 2-strand core suture showed a tendency to less adhesion formation. Doubling of the intratendinous suture material was accompanied by an initial increase in leukocyte infiltration and showed a greater amount of formation of myofibroblasts. From the third to the eighth week after flexor tendon repair, both the cellular and the extracellular inflammation decreased significantly. Conclusion. A 4-strand core suture repair leads to a significantly better tendon healing process with less diastasis between the sutured tendon ends despite initially pronounced inflammatory response.

Control of DNA strand displacement kinetics using toehold exchange.

Science.gov (United States)

Zhang, David Yu; Winfree, Erik

2009-12-02

DNA is increasingly being used as the engineering material of choice for the construction of nanoscale circuits, structures, and motors. Many of these enzyme-free constructions function by DNA strand displacement reactions. The kinetics of strand displacement can be modulated by toeholds, short single-stranded segments of DNA that colocalize reactant DNA molecules. Recently, the toehold exchange process was introduced as a method for designing fast and reversible strand displacement reactions. Here, we characterize the kinetics of DNA toehold exchange and model it as a three-step process. This model is simple and quantitatively predicts the kinetics of 85 different strand displacement reactions from the DNA sequences. Furthermore, we use toehold exchange to construct a simple catalytic reaction. This work improves the understanding of the kinetics of nucleic acid reactions and will be useful in the rational design of dynamic DNA and RNA circuits and nanodevices.

Electro-mechanical behaviors of composite superconducting strand with filament breakage

International Nuclear Information System (INIS)

Wang, Xu; Gao, Yuanwen; Zhou, Youhe

2016-01-01

Highlights: • The electromechanical behaviors of the superconducting (SC) strand are investigated. • A 3D FEM model for bending behaviors and electric properties of strand is developed. • The influence of breakage of filaments on the critical current of SC strand is calculated. • The impact of current transfer length on the electric properties of SC strand is discussed. - Abstract: The bending behaviors of superconducting strand with typical multi-filament twist configuration are investigated based on a three-dimensional finite element method (FEM) model, named as the Multi-filament twist model, of the strand. In this 3D FEM model, the impacts of initial thermal residual stress, filament-breakage and its evaluation are taken into accounts. The mechanical responses of the strand under bending load are studied with the factors taken into consideration one by one. The distribution of the damage of the filaments and its evolution and the movement of the neutral axis caused by it are studied and displayed in detail. Besides, taking the advantages of the Multi-filament twist model, the normalized critical current of the strand under bending load is also calculated based on the invariant temperature and field strain functions. In addition, the non-negligible influences of the pitch length of the filaments on both the mechanical behaviors and the normalized critical current are discussed. The stress-strain characteristics of the strand under tensile load and the normalized critical current of it under axial and bending loads resulting from the Multi-filament twist model show good agreement with the experimental data.

Electro-mechanical behaviors of composite superconducting strand with filament breakage

Energy Technology Data Exchange (ETDEWEB)

Wang, Xu [Key Laboratory of Mechanics on Environment and Disaster in Western China, The Ministry of Education of China, Lanzhou, Gansu 730000 (China); Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou, Gansu 730000 (China); Gao, Yuanwen, E-mail: ywgao@lzu.edu.cn [Key Laboratory of Mechanics on Environment and Disaster in Western China, The Ministry of Education of China, Lanzhou, Gansu 730000 (China); Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou, Gansu 730000 (China); Zhou, Youhe [Key Laboratory of Mechanics on Environment and Disaster in Western China, The Ministry of Education of China, Lanzhou, Gansu 730000 (China); Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou, Gansu 730000 (China)

2016-10-15

Highlights: • The electromechanical behaviors of the superconducting (SC) strand are investigated. • A 3D FEM model for bending behaviors and electric properties of strand is developed. • The influence of breakage of filaments on the critical current of SC strand is calculated. • The impact of current transfer length on the electric properties of SC strand is discussed. - Abstract: The bending behaviors of superconducting strand with typical multi-filament twist configuration are investigated based on a three-dimensional finite element method (FEM) model, named as the Multi-filament twist model, of the strand. In this 3D FEM model, the impacts of initial thermal residual stress, filament-breakage and its evaluation are taken into accounts. The mechanical responses of the strand under bending load are studied with the factors taken into consideration one by one. The distribution of the damage of the filaments and its evolution and the movement of the neutral axis caused by it are studied and displayed in detail. Besides, taking the advantages of the Multi-filament twist model, the normalized critical current of the strand under bending load is also calculated based on the invariant temperature and field strain functions. In addition, the non-negligible influences of the pitch length of the filaments on both the mechanical behaviors and the normalized critical current are discussed. The stress-strain characteristics of the strand under tensile load and the normalized critical current of it under axial and bending loads resulting from the Multi-filament twist model show good agreement with the experimental data.

Double-Strand DNA Break Repair in Mycobacteria.

Science.gov (United States)

Glickman, Michael S

2014-10-01

Discontinuity of both strands of the chromosome is a lethal event in all living organisms because it compromises chromosome replication. As such, a diversity of DNA repair systems has evolved to repair double-strand DNA breaks (DSBs). In part, this diversity of DSB repair systems has evolved to repair breaks that arise in diverse physiologic circumstances or sequence contexts, including cellular states of nonreplication or breaks that arise between repeats. Mycobacteria elaborate a set of three genetically distinct DNA repair pathways: homologous recombination, nonhomologous end joining, and single-strand annealing. As such, mycobacterial DSB repair diverges substantially from the standard model of prokaryotic DSB repair and represents an attractive new model system. In addition, the presence in mycobacteria of a DSB repair system that can repair DSBs in nonreplicating cells (nonhomologous end joining) or when DSBs arise between repeats (single-strand annealing) has clear potential relevance to Mycobacterium tuberculosis pathogenesis, although the exact role of these systems in M. tuberculosis pathogenesis is still being elucidated. In this article we will review the genetics of mycobacterial DSB repair systems, focusing on recent insights.

Planar imaging quantification using 3D attenuation correction data and Monte Carlo simulated buildup factors

International Nuclear Information System (INIS)

Miller, C.; Filipow, L.; Jackson, S.; Riauka, T.

1996-01-01

A new method to correct for attenuation and the buildup of scatter in planar imaging quantification is presented. The method is based on the combined use of 3D density information provided by computed tomography to correct for attenuation and the application of Monte Carlo simulated buildup factors to correct for buildup in the projection pixels. CT and nuclear medicine images were obtained for a purpose-built nonhomogeneous phantom that models the human anatomy in the thoracic and abdominal regions. The CT transverse slices of the phantom were converted to a set of consecutive density maps. An algorithm was developed that projects the 3D information contained in the set of density maps to create opposing pairs of accurate 2D correction maps that were subsequently applied to planar images acquired from a dual-head gamma camera. A comparison of results obtained by the new method and the geometric mean approach based on published techniques is presented for some of the source arrangements used. Excellent results were obtained for various source - phantom configurations used to evaluate the method. Activity quantification of a line source at most locations in the nonhomogeneous phantom produced errors of less than 2%. Additionally, knowledge of the actual source depth is not required for accurate activity quantification. Quantification of volume sources placed in foam, Perspex and aluminium produced errors of less than 7% for the abdominal and thoracic configurations of the phantom. (author)

Rapid quantification of biomarkers during kerogen microscale pyrolysis

Energy Technology Data Exchange (ETDEWEB)

Stott, A.W.; Abbott, G.D. [Fossil Fuels and Environmental Geochemistry NRG, The University, Newcastle-upon-Tyne (United Kingdom)

1995-02-01

A rapid, reproducible method incorporating closed system microscale pyrolysis and thermal desorption-gas chromatography/mass spectrometry has been developed and applied to the quantification of sterane biomarkers released during pyrolysis of the Messel oil shale kerogen under confined conditions. This method allows a substantial experimental concentration-time data set to be collected at accurately controlled temperatures, due to the low thermal inertia of the microscale borosilicate glass reaction vessels, which facilitates kinetic studies of biomarker reactions during kerogen microscale pyrolysis

Quantification of Azospirillum brasilense FP2 Bacteria in Wheat Roots by Strain-Specific Quantitative PCR.

Science.gov (United States)

Stets, Maria Isabel; Alqueres, Sylvia Maria Campbell; Souza, Emanuel Maltempi; Pedrosa, Fábio de Oliveira; Schmid, Michael; Hartmann, Anton; Cruz, Leonardo Magalhães

2015-10-01

Azospirillum is a rhizobacterial genus containing plant growth-promoting species associated with different crops worldwide. Azospirillum brasilense strains exhibit a growth-promoting effect by means of phytohormone production and possibly by N2 fixation. However, one of the most important factors for achieving an increase in crop yield by plant growth-promoting rhizobacteria is the survival of the inoculant in the rhizosphere, which is not always achieved. The objective of this study was to develop quantitative PCR protocols for the strain-specific quantification of A. brasilense FP2. A novel approach was applied to identify strain-specific DNA sequences based on a comparison of the genomic sequences within the same species. The draft genome sequences of A. brasilense FP2 and Sp245 were aligned, and FP2-specific regions were filtered and checked for other possible matches in public databases. Strain-specific regions were then selected to design and evaluate strain-specific primer pairs. The primer pairs AzoR2.1, AzoR2.2, AzoR5.1, AzoR5.2, and AzoR5.3 were specific for the A. brasilense FP2 strain. These primer pairs were used to monitor quantitatively the population of A. brasilense in wheat roots under sterile and nonsterile growth conditions. In addition, coinoculations with other plant growth-promoting bacteria in wheat were performed under nonsterile conditions. The results showed that A. brasilense FP2 inoculated into wheat roots is highly competitive and achieves high cell numbers (∼10(7) CFU/g [fresh weight] of root) in the rhizosphere even under nonsterile conditions and when coinoculated with other rhizobacteria, maintaining the population at rather stable levels for at least up to 13 days after inoculation. The strategy used here can be applied to other organisms whose genome sequences are available. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Real-time PCR for the quantification of fungi in planta.

Science.gov (United States)

Klosterman, Steven J

2012-01-01

Methods enabling quantification of fungi in planta can be useful for a variety of applications. In combination with information on plant disease severity, indirect quantification of fungi in planta offers an additional tool in the screening of plants that are resistant to fungal diseases. In this chapter, a method is described for the quantification of DNA from a fungus in plant leaves using real-time PCR (qPCR). Although the method described entails quantification of the fungus Verticillium dahliae in lettuce leaves, the methodology described would be useful for other pathosystems as well. The method utilizes primers that are specific for amplification of a β-tubulin sequence from V. dahliae and a lettuce actin gene sequence as a reference for normalization. This approach enabled quantification of V. dahliae in the amount of 2.5 fg/ng of lettuce leaf DNA at 21 days following plant inoculation.

The (not so) Immortal Strand Hypothesis

OpenAIRE

Tomasetti, Cristian; Bozic, Ivana

2015-01-01

Background: Non-random segregation of DNA strands during stem cell replication has been proposed as a mechanism to minimize accumulated genetic errors in stem cells of rapidly dividing tissues. According to this hypothesis, an “immortal” DNA strand is passed to the stem cell daughter and not the more differentiated cell, keeping the stem cell lineage replication error-free. After it was introduced, experimental evidence both in favor and against the hypothesis has been presented. Principal...

Quantification of steroid hormones in human serum by liquid chromatography-high resolution tandem mass spectrometry.

Science.gov (United States)

Matysik, Silke; Liebisch, Gerhard

2017-12-01

A limited specificity is inherent to immunoassays for steroid hormone analysis. To improve selectivity mass spectrometric analysis of steroid hormones by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been introduced in the clinical laboratory over the past years usually with low mass resolution triple-quadrupole instruments or more recently by high resolution mass spectrometry (HR-MS). Here we introduce liquid chromatography-high resolution tandem mass spectrometry (LC-MS/HR-MS) to further increase selectivity of steroid hormone quantification. Application of HR-MS demonstrates an enhanced selectivity compared to low mass resolution. Separation of isobaric interferences reduces background noise and avoids overestimation. Samples were prepared by automated liquid-liquid extraction with MTBE. The LC-MS/HR-MS method using a quadrupole-Orbitrap analyzer includes eight steroid hormones i.e. androstenedione, corticosterone, cortisol, cortisone, 11-deoxycortisol, 17-hydroxyprogesterone, progesterone, and testosterone. It has a run-time of 5.3min and was validated according to the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) guidelines. For most of the analytes coefficient of variation were 10% or lower and LOQs were determined significantly below 1ng/ml. Full product ion spectra including accurate masses substantiate compound identification by matching their masses and ratios with authentic standards. In summary, quantification of steroid hormones by LC-MS/HR-MS is applicable for clinical diagnostics and holds also promise for highly selective quantification of other small molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

A new cable-in-conduit conductor magnet with insulated strands

International Nuclear Information System (INIS)

Yamaguchi, Satarou; Yamamoto, Junya; Motojima, Osamu.

1995-09-01

Many studies have used cable-in-conduit conductor (CICC) coils in trying to develop an AC superconducting magnet because of its enormous potential if AC losses were low and insulation voltage was high. The strands in the most recent CICC magnets are coated with chromium or another metal with high electrical resistance to order to induce current re-distribution among the strands and to avoid a quench caused by a current imbalance. Current re-distribution is highly complex and very difficult to analyze because the conditions of the strand surfaces and the contact areas vary greatly with the operation of the conductor. If, however, the cable currents were well-balanced, insulating the strands would be the best way to reduce AC losses. We propose a new CICC magnet structure featuring a current lead that balances the strand currents via its resistance. Having calculated current balances, we find that strand currents are well within the present parameters for nuclear fusion experiments and superconducting magnet energy storages. (author)

Detection of hepatitis A virus by hybridization with single-stranded RNA probes

International Nuclear Information System (INIS)

Xi, J.; Estes, M.K.; Metcalf, T.G.

1987-01-01

An improved method of dot-blot hybridization to detect hepatitis A virus (HAV) was developed with single-stranded RNA (ssRNA) probes. Radioactive and nonradioactive ssRNA probes were generated by in vitro transcription of HAV templates inserted into the plasmid pGEM-1. 32 P-labeled ssRNA probes were at least eightfold more sensitive than the 32 P-labeled double-stranded cDNA counterparts, whereas biotin-labeled ssRNA probes showed a sensitivity comparable with that of the 32 P-labeled double-stranded cDNA counterparts. Hybridization of HAV with the ssRNA probes at high stringency revealed specific reactions with a high signal-to-noise ratio. The differential hybridization reactions seen with probes of positive and negative sense (compared with HAV genomic RNA) were used to detect HAV in clinical and field samples. A positive/negative ratio was introduced as an indicator that permitted an semiquantitative expression of a positive HAV reaction. Good agreement of this indicator was observed with normal stool samples and with HAV-seeded samples. By using this system, HAV was detected in estuarine and freshwater samples collected from a sewage-polluted bayou in Houston and a saltwater tributary of Galveston Bay

Powder X-ray diffraction method for the quantification of cocrystals in the crystallization mixture.

Science.gov (United States)

Padrela, Luis; de Azevedo, Edmundo Gomes; Velaga, Sitaram P

2012-08-01

The solid state purity of cocrystals critically affects their performance. Thus, it is important to accurately quantify the purity of cocrystals in the final crystallization product. The aim of this study was to develop a powder X-ray diffraction (PXRD) quantification method for investigating the purity of cocrystals. The method developed was employed to study the formation of indomethacin-saccharin (IND-SAC) cocrystals by mechanochemical methods. Pure IND-SAC cocrystals were geometrically mixed with 1:1 w/w mixture of indomethacin/saccharin in various proportions. An accurately measured amount (550 mg) of the mixture was used for the PXRD measurements. The most intense, non-overlapping, characteristic diffraction peak of IND-SAC was used to construct the calibration curve in the range 0-100% (w/w). This calibration model was validated and used to monitor the formation of IND-SAC cocrystals by liquid-assisted grinding (LAG). The IND-SAC cocrystal calibration curve showed excellent linearity (R(2) = 0.9996) over the entire concentration range, displaying limit of detection (LOD) and limit of quantification (LOQ) values of 1.23% (w/w) and 3.74% (w/w), respectively. Validation results showed excellent correlations between actual and predicted concentrations of IND-SAC cocrystals (R(2) = 0.9981). The accuracy and reliability of the PXRD quantification method depend on the methods of sample preparation and handling. The crystallinity of the IND-SAC cocrystals was higher when larger amounts of methanol were used in the LAG method. The PXRD quantification method is suitable and reliable for verifying the purity of cocrystals in the final crystallization product.

The risk of stranded assets for utilities in Canada

International Nuclear Information System (INIS)

Schroeder, W.

1998-01-01

The problems of dealing with stranded assets in Canada and the United States were discussed. Compared to the United States, the risk associated with stranded assets for utilities in Canada was considered to be relatively low because of the following factors: (1) low variable cost, (2) isolation, (3) lack of transmission interconnection capacity, (4) lack of tight synchronization in North America, (5) the likelihood of an increase in natural gas prices, (6) the absence of jurisdictional disputes such as FERC versus the states, (7) social considerations, (8) the learning curve, (9) politics, (10) weak balance sheets, (11) relatively low electricity prices, (12) the weak Canadian dollar, and (13) the possibility of refinancing at lower interest rates. Ontario Hydro, New Brunswick and Nova Scotia Power are the three Canadian utilities that may have stranded costs. For Ontario Hydro and New Brunswick Power the stranded costs would be related to nuclear generator problems, whereas for Nova Scotia Power, the stranded costs would be related to the thermal generating base, the threat from Sable Island Gas and the changing tax structure of the utility. Some other reasons why stranded assets could be created in Canada would include low variable costs and high fixed costs, over capacity of at least 30 per cent in generation, limited domestic energy growth, competitive threat from gas, reliability and safety of nuclear plants, and technology change. Five factors in terms of which stranded assets can be expressed are: (1) variable cost definition, (2) total cost definition, (3) operating profit definition, (4) wide geographic definition, and (5) free market definition. In calculating stranded assets, the number of years over which the assets are recovered and the discount rate are considered to be key factors. 26 tabs

The impact of reconstruction method on the quantification of DaTSCAN images

Energy Technology Data Exchange (ETDEWEB)

Dickson, John C.; Erlandsson, Kjell; Hutton, Brian F. [UCLH NHS Foundation Trust and University College London, Institute of Nuclear Medicine, London (United Kingdom); Tossici-Bolt, Livia [Southampton University Hospitals NHS Trust, Department of Medical Physics, Southampton (United Kingdom); Sera, Terez [University of Szeged, Department of Nuclear Medicine and Euromedic Szeged, Szeged (Hungary); Varrone, Andrea [Psychiatry Section and Stockholm Brain Institute, Karolinska Institute, Department of Clinical Neuroscience, Stockholm (Sweden); Tatsch, Klaus [EANM/European Network of Excellence for Brain Imaging, Vienna (Austria)

2010-01-15

Reconstruction of DaTSCAN brain studies using OS-EM iterative reconstruction offers better image quality and more accurate quantification than filtered back-projection. However, reconstruction must proceed for a sufficient number of iterations to achieve stable and accurate data. This study assessed the impact of the number of iterations on the image quantification, comparing the results of the iterative reconstruction with filtered back-projection data. A striatal phantom filled with {sup 123}I using striatal to background ratios between 2:1 and 10:1 was imaged on five different gamma camera systems. Data from each system were reconstructed using OS-EM (which included depth-independent resolution recovery) with various combinations of iterations and subsets to achieve up to 200 EM-equivalent iterations and with filtered back-projection. Using volume of interest analysis, the relationships between image reconstruction strategy and quantification of striatal uptake were assessed. For phantom filling ratios of 5:1 or less, significant convergence of measured ratios occurred close to 100 EM-equivalent iterations, whereas for higher filling ratios, measured uptake ratios did not display a convergence pattern. Assessment of the count concentrations used to derive the measured uptake ratio showed that nonconvergence of low background count concentrations caused peaking in higher measured uptake ratios. Compared to filtered back-projection, OS-EM displayed larger uptake ratios because of the resolution recovery applied in the iterative algorithm. The number of EM-equivalent iterations used in OS-EM reconstruction influences the quantification of DaTSCAN studies because of incomplete convergence and possible bias in areas of low activity due to the nonnegativity constraint in OS-EM reconstruction. Nevertheless, OS-EM using 100 EM-equivalent iterations provides the best linear discriminatory measure to quantify the uptake in DaTSCAN studies. (orig.)

Ultrasensitive multiplex optical quantification of bacteria in large samples of biofluids

Science.gov (United States)

Pazos-Perez, Nicolas; Pazos, Elena; Catala, Carme; Mir-Simon, Bernat; Gómez-de Pedro, Sara; Sagales, Juan; Villanueva, Carlos; Vila, Jordi; Soriano, Alex; García de Abajo, F. Javier; Alvarez-Puebla, Ramon A.

2016-01-01

Efficient treatments in bacterial infections require the fast and accurate recognition of pathogens, with concentrations as low as one per milliliter in the case of septicemia. Detecting and quantifying bacteria in such low concentrations is challenging and typically demands cultures of large samples of blood (~1 milliliter) extending over 24–72 hours. This delay seriously compromises the health of patients. Here we demonstrate a fast microorganism optical detection system for the exhaustive identification and quantification of pathogens in volumes of biofluids with clinical relevance (~1 milliliter) in minutes. We drive each type of bacteria to accumulate antibody functionalized SERS-labelled silver nanoparticles. Particle aggregation on the bacteria membranes renders dense arrays of inter-particle gaps in which the Raman signal is exponentially amplified by several orders of magnitude relative to the dispersed particles. This enables a multiplex identification of the microorganisms through the molecule-specific spectral fingerprints. PMID:27364357

A mass stranding of the squid martialia hyadesi Rochebrune and ...

African Journals Online (AJOL)

1997-02-11

Feb 11, 1997 ... All animals were immature, with females in lower stages of maturity than males. No predatory marine mammals were seen in the area during or after the stranding event. An interpretation of the stranding is presented with reference to historical reports of squid strandings worldwide. Evidence suggests some ...

Quantification of mixed chimerism by real time PCR on whole blood-impregnated FTA cards.

Science.gov (United States)

Pezzoli, N; Silvy, M; Woronko, A; Le Treut, T; Lévy-Mozziconacci, A; Reviron, D; Gabert, J; Picard, C

2007-09-01

This study has investigated quantification of chimerism in sex-mismatched transplantations by quantitative real time PCR (RQ-PCR) using FTA paper for blood sampling. First, we demonstrate that the quantification of DNA from EDTA-blood which has been deposit on FTA card is accurate and reproducible. Secondly, we show that fraction of recipient cells detected by RQ-PCR was concordant between the FTA and salting-out method, reference DNA extraction method. Furthermore, the sensitivity of detection of recipient cells is relatively similar with the two methods. Our results show that this innovative method can be used for MC assessment by RQ-PCR.

Radiation induced strand breaks and time scale for repair of broken strands in superinfecting phage lambda DNA in Escherichia coli lysogenic for lambda

International Nuclear Information System (INIS)

Johansen, I.; Boye, E.; Brustad, T.

1975-01-01

The production of the first radiation induced break in covalent lambda DNA molecules in pol + and pol A 1 lysogenic host cells was measured after exposure to electrons from a linear accelerator and transfer to alkaline detergent within 100 ms from the onset of irradiation. The results revealed the presence of an oxygen effect in DNA strand breakage. In both pol + and pol A 1 host cells the rate of production in nitrogen was 1.2x10 -12 DNA single strand breaks per rad per dalton as compared to 5x10 -12 in oxygen. The yields of strand breaks in lambda DNA in pol + host cells under oxygenated or anoxic conditions are independent of whether the cells are irradiated in buffer at room temperature, in buffer at ice temperature, or in growth medium at 37 0 C. These results indicate that enzymic repair of DNA strand breaks before analysis is insignificant in these experiments. The presence of an oxygen effect in DNA strand breakage under these conditions suggest that an actual difference exists between initial number of breaks produced in nitrogen and in oxygen. The kinetics of rejoining of broken molecules under optimal growth conditions was measured by incubating the irradiated host cells prior to lysis. In pol + host cells 50% of the lambda DNA molecules broken in presence of oxygen are rejoined within 10 to 20 seconds of incubation. A significantly lower recovery is seen in pol + host cells after irradiation in nitrogen. The rejoining of broken lambda DNA strands in pol A 1 host cells is impaired after irradiation in presence of oxygen as well as under anoxia. These results show that DNA polymerase I is needed for the rapid rejoining of radiation induced strand breaks in the DNA, and that oxygen promoted strand breaks are more easily rejoined than are those produced in nitrogen. (author)

Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks.

Science.gov (United States)

Bañuelos, C A; Banáth, J P; MacPhail, S H; Zhao, J; Eaves, C A; O'Connor, M D; Lansdorp, P M; Olive, P L

2008-09-01

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse, but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed strand breaks more rapidly. Consistent with more rapid dsb rejoining, H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs), an increase in dsb rejoining rate, and a decrease in Ku70/80. Unlike mouse ES, human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells, they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM, and they add to the growing list of differences in the way rodent and human cells deal with DNA damage.

LC-MS/MS quantification of free and Fab-bound colchicine in plasma, urine and organs following colchicine administration and colchicine-specific Fab fragments treatment in Göttingen minipigs.

Science.gov (United States)

Fabresse, Nicolas; Allard, Julien; Sardaby, Marine; Thompson, Adrian; Clutton, R Eddie; Eddleston, Michael; Alvarez, Jean-Claude

2017-08-15

Clinical evaluation of a colchicine specific antigen-binding fragment (Fab) in order to treat colchicine poisoning required the development of an accurate method allowing quantification of free and Fab-bound colchicine in plasma and urine, and free colchicine in tissues, to measure colchicine redistribution after Fab administration. Three methods have been developed for this purpose, and validated in plasma, urine and liver: total colchicine was determined after denaturation of Fab by dilution in water and heating; free colchicine was separated from Fab-bound colchicine by filtration with 30KDa micro-filters; tissues were homogenized in a tissue mixer. Deuterated colchicine was used as internal standard. Samples were extracted by liquid-liquid extraction and analyzed with a LC-MS/MS. LOQ were 0.5ng/mL in plasma and urine for free and total colchicine and 5pg/mg in tissues. The methods were linear in the 0.5-100ng/mL range in plasma and urine, and 5-300pg/mg in tissues with determination coefficients>0.99. Precision and accuracy of QC samples presented a CVFab fragments. Copyright © 2017 Elsevier B.V. All rights reserved.

Radiation induced DNA double-strand breaks in radiology; Strahleninduzierte DNA-Doppelstrangbrueche in der Radiologie

Energy Technology Data Exchange (ETDEWEB)

Kuefner, M.A. [Dornbirn Hospital (Austria). Dept. of Radiology; Brand, M.; Engert, C.; Uder, M. [Erlangen University Hospital (Germany). Dept. of Radiology; Schwab, S.A. [Radiologis, Oberasbach (Germany)

2015-10-15

Shortly after the discovery of X-rays, their damaging effect on biological tissues was observed. The determination of radiation exposure in diagnostic and interventional radiology is usually based on physical measurements or mathematical algorithms with standardized dose simulations. γ-H2AX immunofluorescence microscopy is a reliable and sensitive method for the quantification of radiation induced DNA double-strand breaks (DSB) in blood lymphocytes. The detectable amount of these DNA damages correlates well with the dose received. However, the biological radiation damage depends not only on dose but also on other individual factors like radiation sensitivity and DNA repair capacity. Iodinated contrast agents can enhance the x-ray induced DNA damage level. After their induction DSB are quickly repaired. A protective effect of antioxidants has been postulated in experimental studies. This review explains the principle of the γ-H2AX technique and provides an overview on studies evaluating DSB in radiologic examinations.

Induction of DNA strand breaks in 14C-labelled cells

International Nuclear Information System (INIS)

Sundell-Bergman, S.; Johanson, K.J.

1979-01-01

Chinese hamster cells grown in vitro were labelled with 14 C-thymidine for 18 hours and after 3 hours in non-radioactive medium they were stored at 0 0 C for various periods ( 1 to 12 hours). During this treatment a number of DNA strand breaks were induced by 14 C decay which were not repaired at 0 0 C. The number of DNA strand breaks was determined using the DNA unwinding technique. At 0.5-1 dpm per cell a detectable number of DNA strand breaks were found. Treatment for six hours (1 dpm per cell) reduced the percentage of double-stranded DNA from 80 to 70%, corresponding to about 750 DNA strand breaks per cell. The rejoining of DNA strand breaks was studied after treatment for 12 hours at 0 0 C followed by incubation of the cells for various periods at 37 0 C. Most of the DNA strand breaks induced by 14 C decay at 0 0 C were repaired after incubation at 37 0 C for 15 minutes. Assuming an absorbed dose of 1.8 mGy per 14 C decay to the cell nucleus an RBE value close to 1 was found for internal irradiation from 14 C decay as compared with 60 Co-gamma irradiation. (author)

Stabilization of Pt nanoparticles by single stranded DNA and the binary assembly of Au and Pt nanoparticles without hybridization

International Nuclear Information System (INIS)

Yang, J.; Lee, Jim Yang; Too, Heng-Phon; Chow, Gan-Moog; Gan, Leong M.

2006-01-01

The non-specific interaction between single stranded DNA (ssDNA) and 12 nm Pt nanoparticles is investigated in this work. The data show a strong and non-specific interaction between the two which can be exploited for the stabilization of Pt nanoparticles in aqueous solutions. Based on the experimental findings, a non-hybridization based protocol to assemble 17 nm Au and Pt nanoparticles (12 nm cubic and 3.6 nm spherical) by single-stranded DNA was developed. Transmission electron microscopy (TEM) and UV-visible spectroscopy confirmed that Au and Pt nanoparticles could be assembled by the non-specific interaction in an orderly manner. The experimental results also caution against the potential pitfalls in using DNA melting point analysis to infer metal nanoparticle assembly by DNA hybridization

A fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp.

Science.gov (United States)

Alasaad, Samer; Soriguer, Ramón C; Abu-Madi, Marawan; El Behairy, Ahmed; Baños, Pablo Díez; Píriz, Ana; Fickel, Joerns; Zhu, Xing-Quan

2011-06-01

The present study aimed to establish a fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp. Based on the sequences of the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA, we designed a set of genus-specific primers for the amplification of Fasciola ITS-2, with an estimated size of 140 bp. These primers were labelled by fluorescence dyes, and the PCR products were analyzed by capillary electrophoresis under non-denaturing conditions (F-PCR-SSCP). Capillary electrophoresis analysis of the fluorescence-labelled DNA fragments displayed three different peak profiles that allowed the accurate identification of Fasciola species: one single peak specific for either Fasciola hepatica or Fasciola gigantica and a doublet peak corresponding to the "intermediate" Fasciola. Validation of our novel method was performed using Fasciola specimens from different host animals from China, Spain, Nigeria, and Egypt. This F-PCR-SSCP assay provides a rapid, simple, and robust tool for the identification and differentiation between Fasciola spp.

Getting Frustrated: Modelling Emotion Contagion in Stranded Passengers

NARCIS (Netherlands)

van der Wal, C. Natalie; Couwenberg, Maik; Bosse, T.

2017-01-01

Train passengers can get stranded due to a variety of events, such as a delay, technical malfunctioning or a natural disaster. Stranded passengers can get frustrated, which could escalate in misbehaviours. Examples are verbal and physical violence or dangerous behaviours such as opening emergency

Accurate and sensitive quantification of protein-DNA binding affinity.

Science.gov (United States)

Rastogi, Chaitanya; Rube, H Tomas; Kribelbauer, Judith F; Crocker, Justin; Loker, Ryan E; Martini, Gabriella D; Laptenko, Oleg; Freed-Pastor, William A; Prives, Carol; Stern, David L; Mann, Richard S; Bussemaker, Harmen J

2018-04-17

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. Copyright © 2018 the Author(s). Published by PNAS.

Inter-strand resistance measurements in the termination of the ITER SULTAN samples

International Nuclear Information System (INIS)

Cau, F; Bruzzone, P

2009-01-01

In cabled conductors a perfect uniformity of the current among the strands is hardly reached, due to the non-homogeneity of the contact resistance distribution between the strands and the copper of the electrical terminations. In the case of large current unbalance, the overloaded strands hit the critical surface at high field early, developing a current sharing voltage, which drives the redistribution of the current, mainly in the electrical terminations where the inter-strand resistance is lower than in the high field conductor. If the inter-strand resistance in the termination is low, the voltage levels are sufficiently low to allow an effective redistribution of the current to the less loaded strands. The inter-strand resistance of three different termination layouts of ITER short length samples is measured to make a database available which can be used to qualify the layout of the joints and their capability of redistributing the current among the strands.

A flexible and accurate quantification algorithm for electron probe X-ray microanalysis based on thin-film element yields

International Nuclear Information System (INIS)

Schalm, O.; Janssens, K.

2003-01-01

Quantitative analysis by means of electron probe X-ray microanalysis (EPXMA) of low Z materials such as silicate glasses can be hampered by the fact that ice or other contaminants build up on the Si(Li) detector beryllium window or (in the case of a windowless detector) on the Si(Li) crystal itself. These layers act as an additional absorber in front of the detector crystal, decreasing the detection efficiency at low energies (<5 keV). Since the layer thickness gradually changes with time, also the detector efficiency in the low energy region is not constant. Using the normal ZAF approach to quantification of EPXMA data is cumbersome in these conditions, because spectra from reference materials and from unknown samples must be acquired within a fairly short period of time in order to avoid the effect of the change in efficiency. To avoid this problem, an alternative approach to quantification of EPXMA data is proposed, following a philosophy often employed in quantitative analysis of X-ray fluorescence (XRF) and proton-induced X-ray emission (PIXE) data. This approach is based on the (experimental) determination of thin-film element yields, rather than starting from infinitely thick and single element calibration standards. These thin-film sensitivity coefficients can also be interpolated to allow quantification of elements for which no suitable standards are available. The change in detector efficiency can be monitored by collecting an X-ray spectrum of one multi-element glass standard. This information is used to adapt the previously determined thin-film sensitivity coefficients to the actual detector efficiency conditions valid on the day that the experiments were carried out. The main advantage of this method is that spectra collected from the standards and from the unknown samples should not be acquired within a short period of time. This new approach is evaluated for glass and metal matrices and is compared with a standard ZAF method

Immunological detection and quantification of DNA components structurally modified by alkylating carcinogens, mutagens and chemotherapeutic agents

International Nuclear Information System (INIS)

Rajewsky, M.F.

1983-01-01

The detection and quantification of defined reaction products of chemical mutagens and carcinogens (and of many cancer chemotherapeutic agents) with DNA require highly sensitive analytical techniques. The exceptional capability of immunoglobulins to recognize subtle alterations of molecular structure (especially when monoclonal antibodies are used to maximize specificity), outstanding sensitivity of immunoanalysis by high-affinity antibodies, and the fact that radioactively-labelled agents are not required suggest the utility of a radioimmunoassay to recognize and quantitate alkylated DNA products. We have recently developed a set of high-affinity monoclonal antibodies (secreted by mouse x mouse as well as by rat x rat hybridomas; antibody affinity constants, 10 9 to > 10 10 lmol) specifically directed against several DNA alkylation products with possible relevance in relation to both mutagenesis and malignant transformation of mammalian cells. These alkylation products include 0 6 -N-butyldeoxyguanosine, and 0 4 -ethyldeoxythymidine. When used in a radioimmunassay, an antibody specific for 0 6 -ethyldeoxyguanosine, for example, will detect this product at an 0 6 -ethyldeoxyguanosine/deoxyguanosine molar ratio of approx. 3 x 10 -7 in a hydrolysate of 100 ug of DNA. The limit of detection can be lowered further if the respective alkyldeoxynucleosides are separated by HPLC from the DNA hydrolysate prior to the RIA. The anti-alkyldeoxynucleoside monoclonal antibodies can also be used to visualize, by immunostaining and fluorescence microscopy combined with electronic image intensification, specific alkylation products in the nuclear DNA of individual cells, and to localize structurally modified bases in double-stranded DNA molecules by transmission electron microscopy

Highly sensitive real-time PCR for specific detection and quantification of Coxiella burnetii

Directory of Open Access Journals (Sweden)

Linke Sonja

2006-01-01

Full Text Available Abstract Background Coxiella burnetii, the bacterium causing Q fever, is an obligate intracellular biosafety level 3 agent. Detection and quantification of these bacteria with conventional methods is time consuming and dangerous. During the last years, several PCR based diagnostic assays were developed to detect C. burnetii DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular icd (isocitrate dehydrogenase gene and the transposase of the IS1111a element present in multiple copies in the C. burnetii genome. Results To evaluate the precision of the icd and IS1111 real-time PCR assays, we performed different PCR runs with independent DNA dilutions of the C. burnetii Nine Mile RSA493 strain. The results showed very low variability, indicating efficient reproducibility of both assays. Using probit analysis, we determined that the minimal number of genome equivalents per reaction that could be detected with a 95% probability was 10 for the icd marker and 6.5 for the IS marker. Plasmid standards with cloned icd and IS1111 fragments were used to establish standard curves which were linear over a range from 10 to 107 starting plasmid copy numbers. We were able to quantify cell numbers of a diluted, heat-inactivated Coxiella isolate with a detection limit of 17 C. burnetii particles per reaction. Real-time PCR targeting both markers was performed with DNA of 75 different C. burnetii isolates originating from all over the world. Using this approach, the number of IS1111 elements in the genome of the Nine Mile strain was determined to be 23, close to 20, the number revealed by genome sequencing. In other isolates, the number of IS1111 elements varied widely (between seven and 110 and seemed to be very high in some isolates. Conclusion We validated TaqMan-based real-time PCR assays targeting the icd and IS1111 markers of C. burnetii. The assays were shown to be specific, highly

RRP Nb3Sn strand studies for LARP

International Nuclear Information System (INIS)

Barzi, Emanuela; Bossert, Rodger; Caspi, Shlomo; Dietderich, Daniel R.; Ferracin, Paolo; Ghosh, Arup; Turrioni, Daniele

2006-01-01

The Nb 3 Sn strand chosen for the next step in the magnet R and D of the U.S. LHC Accelerator Research Program is the 54/61 sub-element Restacked Rod Process by Oxford Instruments, Superconducting Technology. To ensure that the 0.7 mm RRP strands to be used in the upcoming LARP magnets are suitable, extensive studies were performed. Measurements included the critical current, e , using the voltage-current (V-I) method, the stability current, I S , as the minimal quench current obtained with the voltage-field (V-H) method, and RRR. Magnetization was measured at low and high fields to determine the effective filament size and to detect flux jumps. Effects of heat treatment temperature and durations on I e and I S were also studied. Using strand billet qualification and tests of strands extracted from cables, the short sample limits of magnet performance were obtained. The details and the results of this investigation are herein described

RRP Nb3Sn Strand Studies for LARP

International Nuclear Information System (INIS)

Barzi, Emanuela; Bossert, Rodger; Caspi, Shlomo; Dietderich, Daniel R.; Ferracin, Paolo; Ghosh, Arup; Turrioni, Daniele

2007-01-01

The Nb 3 Sn strand chosen for the next step in the magnet R and D of the U.S. LHC Accelerator Research Program is the 54/61 sub-element Restacked Rod Process by Oxford Instruments, Superconducting Technology. To ensure that the 0.7 mm RRP strands to be used in the upcoming LARP magnets are suitable, extensive studies were performed. Measurements included the critical current, I c , using the voltage-current (V-I) method, the stability current, I S , as the minimal quench current obtained with the voltage-field (V-H) method, and RRR. Magnetization was measured at low and high fields to determine the effective filament size and to detect flux jumps. Effects of heat treatment temperature and durations on I c and I S were also studied. Using strand billet qualification and tests of strands extracted from cables, the short sample limits of magnet performance were obtained. The details and the results of this investigation are herein described

Flexural behavior of bonded post-tensioned concrete beams under strand corrosion

International Nuclear Information System (INIS)

Zhang, Xuhui; Wang, Lei; Zhang, Jianren; Ma, Yafei; Liu, Yongming

2017-01-01

Highlights: • Flexural behavior of bonded PT beams with strand corrosion is experimental tested. • Cracking, stiffness, ultimate strength, failure & ductility of beams are clarified. • A coefficient is proposed to measure incompatible strain between strand & concrete. - Abstract: An experimental test is performed to investigate the flexural behavior of bonded post-tensioned concrete beams under strand corrosion. Eight beams are designed and subjected to accelerated method to different corrosion levels. The initial stiffness of beams is observed by cyclic loading-unloading test during the corrosion procedure. Corrosion effects on concrete cracking, post-cracking stiffness, ultimate strength, failure mode and ductility are then clarified by the flexural test. And, a coefficient is introduced to quantify the incompatible strain between corroded strand and concrete. Results show that the prestress force loss of strand has almost the linear relation with corrosion loss. Strand corrosion affects slightly the initial stiffness of beam before flexural cracking, but degrades significantly the post-cracking stiffness of beam as the corrosion loss exceeds 27.0%. Slight corrosion of strand has little effects on beams flexural behavior. The severe corrosion, however, decreases the number of crack, changes the failure mode form the concrete crushing to strand rupture, degrades the ductility and the ultimate strength of beams, and leads to the incompatible strain between strand and concrete. In the present test, the incompatible strain decreases about 20% of the flexural strength as the corrosion loss exceeds 27.0%.

Flexural behavior of bonded post-tensioned concrete beams under strand corrosion

Energy Technology Data Exchange (ETDEWEB)

Zhang, Xuhui [College of Civil Engineering and Mechanics, Xiangtan University, 411105 Xiangtan (China); School of Civil Engineering and Architecture, Changsha University of Science & Technology, 410114 Changsha (China); Industry Key Laboratory of Traffic Infrastructure Security Risk Management (CSUST), 410114 Changsha (China); Wang, Lei, E-mail: leiwlei@hotmail.com [School of Civil Engineering and Architecture, Changsha University of Science & Technology, 410114 Changsha (China); Industry Key Laboratory of Traffic Infrastructure Security Risk Management (CSUST), 410114 Changsha (China); Zhang, Jianren; Ma, Yafei [School of Civil Engineering and Architecture, Changsha University of Science & Technology, 410114 Changsha (China); Industry Key Laboratory of Traffic Infrastructure Security Risk Management (CSUST), 410114 Changsha (China); Liu, Yongming [School for Engineering of Matter, Transport and Energy, Arizona State University, 85281 Tempe, AZ (United States)

2017-03-15

Highlights: • Flexural behavior of bonded PT beams with strand corrosion is experimental tested. • Cracking, stiffness, ultimate strength, failure & ductility of beams are clarified. • A coefficient is proposed to measure incompatible strain between strand & concrete. - Abstract: An experimental test is performed to investigate the flexural behavior of bonded post-tensioned concrete beams under strand corrosion. Eight beams are designed and subjected to accelerated method to different corrosion levels. The initial stiffness of beams is observed by cyclic loading-unloading test during the corrosion procedure. Corrosion effects on concrete cracking, post-cracking stiffness, ultimate strength, failure mode and ductility are then clarified by the flexural test. And, a coefficient is introduced to quantify the incompatible strain between corroded strand and concrete. Results show that the prestress force loss of strand has almost the linear relation with corrosion loss. Strand corrosion affects slightly the initial stiffness of beam before flexural cracking, but degrades significantly the post-cracking stiffness of beam as the corrosion loss exceeds 27.0%. Slight corrosion of strand has little effects on beams flexural behavior. The severe corrosion, however, decreases the number of crack, changes the failure mode form the concrete crushing to strand rupture, degrades the ductility and the ultimate strength of beams, and leads to the incompatible strain between strand and concrete. In the present test, the incompatible strain decreases about 20% of the flexural strength as the corrosion loss exceeds 27.0%.

LHC superconducting strand

CERN Multimedia

Patrice Loiez

1999-01-01

This cross-section through a strand of superconducting matieral as used in the LHC shows the 8000 Niobium-Titanium filaments embedded like a honeycomb in copper. When cooled to 1.9 degrees above absolute zero in the LHC accelerator, these filaments will have zero resistance and so will carry a high electric current with no energy loss.

A Stress Measurement Method for Steel Strands Based on LC Oscillation

Directory of Open Access Journals (Sweden)

Dongjun Chen

2018-01-01

Full Text Available The prestress loss is one of the main factors affecting the safety of prestressed concrete structure. While the detecting signals like sound and light are difficult to spread in steel strands, there is no effective method for prestress detection of the bonded prestressed steel strands in existing structures yet. In this paper, taking into consideration that the electromagnetic oscillation characteristic can make the signal propagate effectively on the bonded prestressed steel strands, a nondestructive prestress detection method based on the electromagnetic effect to detect oscillation frequency is proposed. In a detection circuit, the steel strands are simulated as an inductance component, in which an induced electromagnetic signal passes through the steel strands to form resonance. And then, a frequency meter is used to detect the oscillation frequency of the resonant circuit. The oscillation frequency is supposed to have relationship with the prestress loading on the steel strands. A section of steel strands with a length of 1.2 m is adopted to test the correlation of stress and oscillation frequency. Both the theoretical and experimental results show that the resonant frequency of the circuit decreases with the increase of the stress of the strand and is linear in a certain range.

Fault-weighted quantification method of fault detection coverage through fault mode and effect analysis in digital I&C systems

Energy Technology Data Exchange (ETDEWEB)

Cho, Jaehyun; Lee, Seung Jun, E-mail: sjlee420@unist.ac.kr; Jung, Wondea

2017-05-15

Highlights: • We developed the fault-weighted quantification method of fault detection coverage. • The method has been applied to specific digital reactor protection system. • The unavailability of the module had 20-times difference with the traditional method. • Several experimental tests will be effectively prioritized using this method. - Abstract: The one of the most outstanding features of a digital I&C system is the use of a fault-tolerant technique. With an awareness regarding the importance of thequantification of fault detection coverage of fault-tolerant techniques, several researches related to the fault injection method were developed and employed to quantify a fault detection coverage. In the fault injection method, each injected fault has a different importance because the frequency of realization of every injected fault is different. However, there have been no previous studies addressing the importance and weighting factor of each injected fault. In this work, a new method for allocating the weighting to each injected fault using the failure mode and effect analysis data was proposed. For application, the fault-weighted quantification method has also been applied to specific digital reactor protection system to quantify the fault detection coverage. One of the major findings in an application was that we may estimate the unavailability of the specific module in digital I&C systems about 20-times smaller than real value when we use a traditional method. The other finding was that we can also classify the importance of the experimental case. Therefore, this method is expected to not only suggest an accurate quantification procedure of fault-detection coverage by weighting the injected faults, but to also contribute to an effective fault injection experiment by sorting the importance of the failure categories.

Automatic Drusen Quantification and Risk Assessment of Age-related Macular Degeneration on Color Fundus Images

NARCIS (Netherlands)

Grinsven, M.J.J.P. van; Lechanteur, Y.T.E.; Ven, J.P.H. van de; Ginneken, B. van; Hoyng, C.B.; Theelen, T.; Sanchez, C.I.

2013-01-01

PURPOSE: To evaluate a machine learning algorithm that allows for computer aided diagnosis (CAD) of non-advanced age-related macular degeneration (AMD) by providing an accurate detection and quantification of drusen location, area and size. METHODS: Color fundus photographs of 407 eyes without AMD

LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis

Science.gov (United States)

Lehle, Simon; Hildebrand, Dominic G.; Merz, Britta; Malak, Peter N.; Becker, Michael S.; Schmezer, Peter; Essmann, Frank; Schulze-Osthoff, Klaus; Rothfuss, Oliver

2014-01-01

DNA damage is tightly associated with various biological and pathological processes, such as aging and tumorigenesis. Although detection of DNA damage is attracting increasing attention, only a limited number of methods are available to quantify DNA lesions, and these techniques are tedious or only detect global DNA damage. In this study, we present a high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) in both the mitochondrial and nuclear genome. While most conventional methods are of low-sensitivity or restricted to abundant mitochondrial DNA samples, we established a protocol that enables the accurate sequence-specific quantification of DNA damage in >3-kb probes for any mitochondrial or nuclear DNA sequence. In order to validate the sensitivity of this method, we compared LORD-Q with a previously published qPCR-based method and the standard single-cell gel electrophoresis assay, demonstrating a superior performance of LORD-Q. Exemplarily, we monitored induction of DNA damage and repair processes in human induced pluripotent stem cells and isogenic fibroblasts. Our results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications. PMID:24371283

Quantification analysis of CT for aphasic patients

Energy Technology Data Exchange (ETDEWEB)

Watanabe, S.; Ooyama, H.; Hojo, K.; Tasaki, H.; Hanazono, T.; Sato, T.; Metoki, H.; Totsuka, M.; Oosumi, N.

1987-02-01

Using a microcomputer, the locus and extent of the lesions, as demonstrated by computed tomography, for 44 aphasic patients with various types of aphasia were superimposed onto standardized matrices, composed of 10 slices with 3000 points (50 by 60). The relationships between the foci of the lesions and types of aphasia were investigated on the slices numbered 3, 4, 5, and 6 using a quantification theory, Type 3 (pattern analysis). Some types of regularities were observed on slices 3, 4, 5, and 6. The group of patients with Broca's aphasia and the group with Wernicke's aphasia were generally separated on the 1st component and the 2nd component of the quantification theory, Type 3. On the other hand, the group with global aphasia existed between the group with Broca's aphasia and that with Wernicke's aphasia. The group of patients with amnestic aphasia had no specific findings, and the group with conduction aphasia existed near those with Wernicke's aphasia. The above results serve to establish the quantification theory, Type 2 (discrimination analysis) and the quantification theory, Type 1 (regression analysis).

Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity

International Nuclear Information System (INIS)

Hamada, Fumika N.; Koshiyama, Akiyo; Namekawa, Satoshi H.; Ishii, Satomi; Iwabata, Kazuki; Sugawara, Hiroko; Nara, Takayuki Y.; Sakaguchi, Kengo; Sawado, Tomoyuki

2007-01-01

PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg 2+ ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis

PCR amplification of repetitive sequences as a possible approach in relative species quantification

DEFF Research Database (Denmark)

Ballin, Nicolai Zederkopff; Vogensen, Finn Kvist; Karlsson, Anders H

2012-01-01

Abstract Both relative and absolute quantifications are possible in species quantification when single copy genomic DNA is used. However, amplification of single copy genomic DNA does not allow a limit of detection as low as one obtained from amplification of repetitive sequences. Amplification...... of repetitive sequences is therefore frequently used in absolute quantification but problems occur in relative quantification as the number of repetitive sequences is unknown. A promising approach was developed where data from amplification of repetitive sequences were used in relative quantification of species...... to relatively quantify the amount of chicken DNA in a binary mixture of chicken DNA and pig DNA. However, the designed PCR primers lack the specificity required for regulatory species control....

Accurate quantification of sphingosine-1-phosphate in normal and Fabry disease plasma, cells and tissues by LC-MS/MS with (13)C-encoded natural S1P as internal standard.

Science.gov (United States)

Mirzaian, Mina; Wisse, Patrick; Ferraz, Maria J; Marques, André R A; Gabriel, Tanit L; van Roomen, Cindy P A A; Ottenhoff, Roelof; van Eijk, Marco; Codée, Jeroen D C; van der Marel, Gijsbert A; Overkleeft, Herman S; Aerts, Johannes M

2016-08-01

We developed a mass spectrometric procedure to quantify sphingosine-1-phosphate (S1P) in biological materials. The use of newly synthesized (13)C5 C18-S1P and commercial C17-S1P as internal standards rendered very similar results with respect to linearity, limit of detection and limit of quantitation. Caution is warranted with determination of plasma S1P levels. Earlier it was reported that S1P is elevated in plasma of Fabry disease patients. We investigated this with the improved quantification. No clear conclusion could be drawn for patient plasma samples given the lack of uniformity of blood collection and plasma preparation. To still obtain insight, plasma and tissues were identically collected from α-galactosidase A deficient Fabry mice and matched control animals. No significant difference was observed in plasma S1P levels. A significant 2.3 fold increase was observed in kidney of Fabry mice, but not in liver and heart. Comparative analysis of S1P in cultured fibroblasts from normal subjects and classically affected Fabry disease males revealed no significant difference. In conclusion, accurate quantification of S1P in biological materials is feasible by mass spectrometry using the internal standards (13)C5 C18-S1P or C17-S1P. Significant local increases of S1P in the kidney might occur in Fabry disease as suggested by the mouse model. Copyright © 2016 Elsevier B.V. All rights reserved.

A model capturing novel strand symmetries in bacterial DNA

International Nuclear Information System (INIS)

Sobottka, Marcelo; Hart, Andrew G.

2011-01-01

Highlights: → We propose a simple stochastic model to construct primitive DNA sequences. → The model provide an explanation for Chargaff's second parity rule in primitive DNA sequences. → The model is also used to predict a novel type of strand symmetry in primitive DNA sequences. → We extend the results for bacterial DNA sequences and compare distributional properties intrinsic to the model to statistical estimates from 1049 bacterial genomes. → We find out statistical evidences that the novel type of strand symmetry holds for bacterial DNA sequences. -- Abstract: Chargaff's second parity rule for short oligonucleotides states that the frequency of any short nucleotide sequence on a strand is approximately equal to the frequency of its reverse complement on the same strand. Recent studies have shown that, with the exception of organellar DNA, this parity rule generally holds for double-stranded DNA genomes and fails to hold for single-stranded genomes. While Chargaff's first parity rule is fully explained by the Watson-Crick pairing in the DNA double helix, a definitive explanation for the second parity rule has not yet been determined. In this work, we propose a model based on a hidden Markov process for approximating the distributional structure of primitive DNA sequences. Then, we use the model to provide another possible theoretical explanation for Chargaff's second parity rule, and to predict novel distributional aspects of bacterial DNA sequences.

Sub-ensemble monitoring of DNA strand displacement using multiparameter single-molecule FRET

OpenAIRE

Baltierra Jasso, Laura; Morten, Michael; Magennis, Steven William

2018-01-01

Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constan...

Stranding Events of Kogia Whales along the Brazilian Coast.

Directory of Open Access Journals (Sweden)

Jailson F Moura

Full Text Available The genus Kogia, which comprises only two extant species, Kogia sima and Kogia breviceps, represents one of the least known groups of cetaceans in the global ocean. In some coastal regions, however, stranding events of these species have been relatively common over the last decades. Stranding provides the opportunity to investigate the biology of these cetaceans and to explore the epidemiological aspects associated with the mortality of the organisms found on the beach. A number of disturbances (including pelagic fisheries, chemical pollution, boat strikes, and noise pollution have been confirmed to pose a particular threat to the Kogia species. However, no study has yet investigated potential relationships between environmental conditions and stranding events. Here we analyse how a collection of environmental, physical, and biological variables, such as wind, sea surface temperature (SST, water depth, and chlorophyll-a, correlate to Kogia stranding events along the Brazilian coast. The results of our statistical analyses suggest that K. sima is more likely found in warm tropical waters, which provide an explanation for the high frequency of stranding in northeastern Brazilian coast. In contrast, K. breviceps appears to have a preference for temperate and productive waters. Wind speed results to be also an important factor for predicting Kogia strandings in Brazilian coast. Additionally, literature information in combination with our own data and analyses of stomach contents confirms that oceanic cephalopods constitute the primary nutritional source of both Kogia species. By using the available information as a qualitative proxy for habitat preference and feeding ecology, our study provides a novel and comprehensive assessment of Kogia stranding data in relation to environmental conditions along the Brazilian coast.

Multiplex electrochemical DNA platform for femtomolar-level quantification of genetically modified soybean.

Science.gov (United States)

Manzanares-Palenzuela, C Lorena; de-los-Santos-Álvarez, Noemí; Lobo-Castañón, María Jesús; López-Ruiz, Beatriz

2015-06-15

Current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) with a minimum content of 0.9% would benefit from the availability of reliable and rapid methods to detect and quantify DNA sequences specific for GMOs. Different genosensors have been developed to this aim, mainly intended for GMO screening. A remaining challenge, however, is the development of genosensing platforms for GMO quantification, which should be expressed as the number of event-specific DNA sequences per taxon-specific sequences. Here we report a simple and sensitive multiplexed electrochemical approach for the quantification of Roundup-Ready Soybean (RRS). Two DNA sequences, taxon (lectin) and event-specific (RR), are targeted via hybridization onto magnetic beads. Both sequences are simultaneously detected by performing the immobilization, hybridization and labeling steps in a single tube and parallel electrochemical readout. Hybridization is performed in a sandwich format using signaling probes labeled with fluorescein isothiocyanate (FITC) or digoxigenin (Dig), followed by dual enzymatic labeling using Fab fragments of anti-Dig and anti-FITC conjugated to peroxidase or alkaline phosphatase, respectively. Electrochemical measurement of the enzyme activity is finally performed on screen-printed carbon electrodes. The assay gave a linear range of 2-250 pM for both targets, with LOD values of 650 fM (160 amol) and 190 fM (50 amol) for the event-specific and the taxon-specific targets, respectively. Results indicate that the method could be applied for GMO quantification below the European labeling threshold level (0.9%), offering a general approach for the rapid quantification of specific GMO events in foods. Copyright © 2015 Elsevier B.V. All rights reserved.

Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli

International Nuclear Information System (INIS)

Moreau, P.L.

1988-01-01

Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes. (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected. These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein. However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair. Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA

Molecular Approaches for High Throughput Detection and Quantification of Genetically Modified Crops: A Review

Directory of Open Access Journals (Sweden)

Ibrahim B. Salisu

2017-10-01

Full Text Available As long as the genetically modified crops are gaining attention globally, their proper approval and commercialization need accurate and reliable diagnostic methods for the transgenic content. These diagnostic techniques are mainly divided into two major groups, i.e., identification of transgenic (1 DNA and (2 proteins from GMOs and their products. Conventional methods such as PCR (polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA were routinely employed for DNA and protein based quantification respectively. Although, these Techniques (PCR and ELISA are considered as significantly convenient and productive, but there is need for more advance technologies that allow for high throughput detection and the quantification of GM event as the production of more complex GMO is increasing day by day. Therefore, recent approaches like microarray, capillary gel electrophoresis, digital PCR and next generation sequencing are more promising due to their accuracy and precise detection of transgenic contents. The present article is a brief comparative study of all such detection techniques on the basis of their advent, feasibility, accuracy, and cost effectiveness. However, these emerging technologies have a lot to do with detection of a specific event, contamination of different events and determination of fusion as well as stacked gene protein are the critical issues to be addressed in future.

Genetic and biochemical identification of a novel single-stranded DNA binding complex in Haloferax volcanii

Directory of Open Access Journals (Sweden)

Amy eStroud

2012-06-01

Full Text Available Single-stranded DNA binding proteins play an essential role in DNA replication and repair. They use oligosaccharide-binding folds, a five-stranded ß-sheet coiled into a closed barrel, to bind to single-stranded DNA thereby protecting and stabilizing the DNA. In eukaryotes the single-stranded DNA binding protein is known as replication protein A (RPA and consists of three distinct subunits that function as a heterotrimer. The bacterial homolog is termed single-stranded DNA-binding protein (SSB and functions as a homotetramer. In the archaeon Haloferax volcanii there are three genes encoding homologs of RPA. Two of the rpa genes (rpa1 and rpa3 exist in operons with a novel gene specific to Euryarchaeota, this gene encodes a protein that we have termed rpa-associated protein (RPAP. The rpap genes encode proteins belonging to COG3390 group and feature oligosaccharide-binding folds, suggesting that they might cooperate with RPA in binding to single-stranded DNA. Our genetic analysis showed that rpa1 and rpa3 deletion mutants have differing phenotypes; only ∆rpa3 strains are hypersensitive to DNA damaging agents. Deletion of the rpa3-associated gene rpap3 led to similar levels of DNA damage sensitivity, as did deletion of the rpa3 operon, suggesting that RPA3 and RPAP3 function in the same pathway. Protein pull-downs involving recombinant hexahistidine-tagged RPAs showed that RPA3 co-purifies with RPAP3, and RPA1 co-purifies with RPAP1. This indicates that the RPAs interact only with their respective associated proteins; this was corroborated by the inability to construct rpa1 rpap3 and rpa3 rpap1 double mutants. This is the first report investigating the individual function of the archaeal COG3390 RPA-associated proteins. We have shown genetically and biochemically that the RPAPs interact with their respective RPAs, and have uncovered a novel single-stranded DNA binding complex that is unique to Euryarchaeota.

Linking probe thermodynamics to microarray quantification

International Nuclear Information System (INIS)

Li, Shuzhao; Pozhitkov, Alexander; Brouwer, Marius

2010-01-01

Understanding the difference in probe properties holds the key to absolute quantification of DNA microarrays. So far, Langmuir-like models have failed to link sequence-specific properties to hybridization signals in the presence of a complex hybridization background. Data from washing experiments indicate that the post-hybridization washing has no major effect on the specifically bound targets, which give the final signals. Thus, the amount of specific targets bound to probes is likely determined before washing, by the competition against nonspecific binding. Our competitive hybridization model is a viable alternative to Langmuir-like models. (comment)

75 FR 36678 - Prestressed Concrete Steel Wire Strand From China; Determinations

Science.gov (United States)

2010-06-28

... prestressed concrete steel wire strand (PC strand), provided for in subheading 7312.10.30 of the Harmonized... Publication 4162 (June 2010), entitled Prestressed Concrete Steel Wire Strand from China: Investigation Nos... INTERNATIONAL TRADE COMMISSION [Investigation Nos. 701-TA-464 and 731-TA-1160 (Final)] Prestressed...

Characteristics of Cu stabilized Nb3Al strands with low Cu ratio

Energy Technology Data Exchange (ETDEWEB)

Kikuchi, A.; Yamada, R.; Barzi, E.; Kobayashi, M.; Lamm, M.; Nakagawa, K.; Sasaki, K.; Takeuchi, T.; Turrioni, D.; Zlobin, A.V.; /NIMC, Tsukuba /Fermilab /Hitachi, Tsuchiura Works /KEK, Tsukuba

2008-12-01

Characteristics of recently developed F4-Nb{sub 3}Al strand with low Cu ratio are described. The overall J{sub c} of the Nb{sub 3}Al strand could be easily increased by decreasing of the Cu ratio. Although the quench of a pulse-like voltage generation is usually observed in superconducting unstable conductor, the F4 strand with a low Cu ratio of 0.61 exhibited an ordinary critical transition of gradual voltage generation. The F4 strand does not have magnetic instabilities at 4.2 K because of the tantalum interfilament matrix. The overall J{sub c} of the F4 strand achieved was 80-85% of the RRP strand. In the large mechanical stress above 100 MPa, the overall J{sub c} of the F4 strand might be comparable to that of high J{sub c} RRP-Nb{sub 3}Sn strands. The Rutherford cable with a high packing factor of 86.5% has been fabricated using F4 strands. The small racetrack magnet, SR07, was also fabricated by a 14 m F4 cable. The quench current, I{sub q}, of SR07 were obtained 22.4 kA at 4.5 K and 25.2 kA at 2.2 K. The tantalum matrix Nb{sub 3}Al strands are promising for the application of super-cooled high-field magnets as well as 4.2 K operation magnets.

Characterization of a parallel-stranded DNA hairpin

International Nuclear Information System (INIS)

Germann, M.W.; Vogel, H.J.; Pon, R.T.; van de Sande, J.H.

1989-01-01

Recently, the authors have shown that synthetic DNA containing homooligomeric A-T base pairs can form a parallel-stranded intramolecular hairpin structure. In the present study, they have employed NMR and optical spectroscopy to investigate the structure of the parallel-stranded (PS) DNA hairpin 3'-d(T) 8 C 4 (A) 8 -3' and the related antiparallel (APS) hair 5'-d(T) 8 C 4 (A) 8 -3'. The parallel orientation of the strands in the PS oligonucleotide is achieved by introducing a 5'-5' phosphodiester linkage in the hairpin loop. Ultraviolet spectroscopic and fluorescence data of drug binding are consistent with the formation of PS and APS structures, respectively, in these two hairpins. Vacuum circular dichroism measurements in combination with theoretical CD calculations indicate that the PS structure forms a right-handed helix. 31 P NMR measurements indicate that the conformation of the phosphodiester backbone of the PS structure is not drastically different from that of the APS control. The presence of slowly exchanging imino protons at 14 ppm and the observation of nuclear Overhauser enhancement between imino protons and the AH-2 protons demonstrate that similar base pairing and base stacking between T and A residues occur in both hairpins. On the basis of NOESY measurements, they find that the orientation of the bases is in the anti region and that the sugar puckering is in the 2'-endo range. The results indicate a B-like conformation for each of the strands in the stem part of the PS hairpin and reverse Watson-Crick base pairing between the T and A residues. These data are consistent with a previously calculated structure for parallel-stranded DNA

Torsional regulation of hRPA-induced unwinding of double-stranded DNA

NARCIS (Netherlands)

De Vlaminck, I.; Vidic, I.; Van Loenhout, M.T.J.; Kanaar, R.; Lebbink, J.H.G.; Dekker, C.

2010-01-01

All cellular single-stranded (ss) DNA is rapidly bound and stabilized by single stranded DNA-binding proteins (SSBs). Replication protein A, the main eukaryotic SSB, is able to unwind double-stranded (ds) DNA by binding and stabilizing transiently forming bubbles of ssDNA. Here, we study the

Role of stranded gas in increasing global gas supplies

Science.gov (United States)

Attanasi, E.D.; Freeman, P.A.

2013-01-01

This report synthesizes the findings of three regional studies in order to evaluate, at the global scale, the contribution that stranded gas resources can make to global natural gas supplies. Stranded gas, as defined for this study, is natural gas in discovered conventional gas and oil fields that is currently not commercially producible for either physical or economic reasons. The regional studies evaluated the cost of bringing the large volumes of undeveloped gas in stranded gas fields to selected markets. In particular, stranded gas fields of selected Atlantic Basin countries, north Africa, Russia, and central Asia are screened to determine whether the volumes are sufficient to meet Europe’s increasing demand for gas imports. Stranded gas fields in Russia, central Asia, Southeast Asia, and Australia are also screened to estimate development, production, and transport costs and corresponding gas volumes that could be supplied to Asian markets in China, India, Japan, and South Korea. The data and cost analysis presented here suggest that for the European market and the markets examined in Asia, the development of stranded gas provides a way to meet projected gas import demands for the 2020-to-2040 period. Although this is a reconnaissance-type appraisal, it is based on volumes of gas that are associated with individual identified fields. Individual field data were carefully examined. Some fields were not evaluated because current technology was insufficient or it appeared the gas was likely to be held off the export market. Most of the evaluated stranded gas can be produced and delivered to markets at costs comparable to historical prices. Moreover, the associated volumes of gas are sufficient to provide an interim supply while additional technologies are developed to unlock gas diffused in shale and hydrates or while countries transition to making a greater use of renewable energy sources.

Photochemical Acceleration of DNA Strand Displacement by Using Ultrafast DNA Photo-crosslinking.

Science.gov (United States)

Nakamura, Shigetaka; Hashimoto, Hirokazu; Kobayashi, Satoshi; Fujimoto, Kenzo

2017-10-18

DNA strand displacement is an essential reaction in genetic recombination, biological processes, and DNA nanotechnology. In particular, various DNA nanodevices enable complicated calculations. However, it takes time before the output is obtained, so acceleration of DNA strand displacement is required for a rapid-response DNA nanodevice. Herein, DNA strand displacement by using DNA photo-crosslinking to accelerate this displacement is evaluated. The DNA photo-crosslinking of 3-cyanovinylcarbazole ( CNV K) was accelerated at least 20 times, showing a faster DNA strand displacement. The rate of photo-crosslinking is a key factor and the rate of DNA strand displacement is accelerated through ultrafast photo-crosslinking. The rate of DNA strand displacement was regulated by photoirradiation energy. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Corrosion characteristics of unprotected post-tensioning strands under stress.

Science.gov (United States)

2014-05-01

An investigation was conducted to determine the effect of stress condition : and environmental exposure on corrosion of post-tensioned strands during ungrouted periods. : Exposures for periods of up to 4 weeks of stressed, as-received strand placed i...

Integrated site-specific quantification of faecal bacteria and detection of DNA markers in faecal contamination source tracking as a microbial risk tracking tool in urban Lake ecosystems

Science.gov (United States)

Donde, Oscar Omondi; Tian, Cuicui; Xiao, Bangding

2017-11-01

The presence of feacal-derived pathogens in water is responsible for several infectious diseases and deaths worldwide. As a solution, sources of fecal pollution in waters must be accurately assessed, properly determined and strictly controlled. However, the exercise has remained challenging due to the existing overlapping characteristics by different members of faecal coliform bacteria and the inadequacy of information pertaining to the contribution of seasonality and weather condition on tracking the possible sources of pollution. There are continued efforts to improve the Faecal Contamination Source Tracking (FCST) techniques such as Microbial Source Tracking (MST). This study aimed to make contribution to MST by evaluating the efficacy of combining site specific quantification of faecal contamination indicator bacteria and detection of DNA markers while accounting for seasonality and weather conditions' effects in tracking the major sources of faecal contamination in a freshwater system (Donghu Lake, China). The results showed that the use of cyd gene in addition to lacZ and uidA genes differentiates E. coli from other closely related faecal bacteria. The use of selective media increases the pollution source tracking accuracy. BSA addition boosts PCR detection and increases FCST efficiency. Seasonality and weather variability also influence the detection limit for DNA markers.

Accurate, fully-automated NMR spectral profiling for metabolomics.

Directory of Open Access Journals (Sweden)

Siamak Ravanbakhsh

Full Text Available Many diseases cause significant changes to the concentrations of small molecules (a.k.a. metabolites that appear in a person's biofluids, which means such diseases can often be readily detected from a person's "metabolic profile"-i.e., the list of concentrations of those metabolites. This information can be extracted from a biofluids Nuclear Magnetic Resonance (NMR spectrum. However, due to its complexity, NMR spectral profiling has remained manual, resulting in slow, expensive and error-prone procedures that have hindered clinical and industrial adoption of metabolomics via NMR. This paper presents a system, BAYESIL, which can quickly, accurately, and autonomously produce a person's metabolic profile. Given a 1D 1H NMR spectrum of a complex biofluid (specifically serum or cerebrospinal fluid, BAYESIL can automatically determine the metabolic profile. This requires first performing several spectral processing steps, then matching the resulting spectrum against a reference compound library, which contains the "signatures" of each relevant metabolite. BAYESIL views spectral matching as an inference problem within a probabilistic graphical model that rapidly approximates the most probable metabolic profile. Our extensive studies on a diverse set of complex mixtures including real biological samples (serum and CSF, defined mixtures and realistic computer generated spectra; involving > 50 compounds, show that BAYESIL can autonomously find the concentration of NMR-detectable metabolites accurately (~ 90% correct identification and ~ 10% quantification error, in less than 5 minutes on a single CPU. These results demonstrate that BAYESIL is the first fully-automatic publicly-accessible system that provides quantitative NMR spectral profiling effectively-with an accuracy on these biofluids that meets or exceeds the performance of trained experts. We anticipate this tool will usher in high-throughput metabolomics and enable a wealth of new applications of

Strand-seq: A unifying tool for studies of chromosome segregation

OpenAIRE

Falconer, Ester; Lansdorp, Peter M.

2013-01-01

Non random segregation of sister chromatids has been implicated to help specify daughter cell fate (the Silent Sister Hypothesis [1]) or to protect the genome of long-lived stem cells (the Immortal Strand Hypothesis [2]). The idea that sister chromatids are non-randomly segregated into specific daughter cells is only marginally supported by data in sporadic and often contradictory studies. As a result, the field has moved forward rather slowly. The advent of being able to directly label and d...

Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand.

Science.gov (United States)

Reis, António M C; Mills, Wilbur K; Ramachandran, Ilangovan; Friedberg, Errol C; Thompson, David; Queimado, Lurdes

2012-01-01

Endogenous DNA damage is removed mainly via base excision repair (BER), however, whether there is preferential strand repair of endogenous DNA damage is still under intense debate. We developed a highly sensitive primer-anchored DNA damage detection assay (PADDA) to map and quantify in vivo endogenous DNA damage. Using PADDA, we documented significantly higher levels of endogenous damage in Saccharomyces cerevisiae cells in stationary phase than in exponential phase. We also documented that yeast BER-defective cells have significantly higher levels of endogenous DNA damage than isogenic wild-type cells at any phase of growth. PADDA provided detailed fingerprint analysis at the single-nucleotide level, documenting for the first time that persistent endogenous nucleotide damage in CAN1 co-localizes with previously reported spontaneous CAN1 mutations. To quickly and reliably quantify endogenous strand-specific DNA damage in the constitutively expressed CAN1 gene, we used PADDA on a real-time PCR setting. We demonstrate that wild-type cells repair endogenous damage preferentially on the CAN1 transcribed strand. In contrast, yeast BER-defective cells accumulate endogenous damage preferentially on the CAN1 transcribed strand. These data provide the first direct evidence for preferential strand repair of endogenous DNA damage and documents the major role of BER in this process.

Quantification of Parvovirus B19 DNA Using COBAS AmpliPrep Automated Sample Preparation and LightCycler Real-Time PCR

Science.gov (United States)

Schorling, Stefan; Schalasta, Gunnar; Enders, Gisela; Zauke, Michael

2004-01-01

The COBAS AmpliPrep instrument (Roche Diagnostics GmbH, D-68305 Mannheim, Germany) automates the entire sample preparation process of nucleic acid isolation from serum or plasma for polymerase chain reaction analysis. We report the analytical performance of the LightCycler Parvovirus B19 Quantification Kit (Roche Diagnostics) using nucleic acids isolated with the COBAS AmpliPrep instrument. Nucleic acids were extracted using the Total Nucleic Acid Isolation Kit (Roche Diagnostics) and amplified with the LightCycler Parvovirus B19 Quantification Kit. The kit combination processes 72 samples per 8-hour shift. The lower detection limit is 234 IU/ml at a 95% hit-rate, linear range approximately 104-1010 IU/ml, and overall precision 16 to 40%. Relative sensitivity and specificity in routine samples from pregnant women are 100% and 93%, respectively. Identification of a persistent parvovirus B19-infected individual by the polymerase chain reaction among 51 anti-parvovirus B19 IgM-negative samples underlines the importance of additional nucleic acid testing in pregnancy and its superiority to serology in identifying the risk of parvovirus B19 transmission via blood or blood products. Combination of the Total Nucleic Acid Isolation Kit on the COBAS AmpliPrep instrument with the LightCycler Parvovirus B19 Quantification Kit provides a reliable and time-saving tool for sensitive and accurate detection of parvovirus B19 DNA. PMID:14736825

Stranded cost recovery in electricity market reforms in the US

International Nuclear Information System (INIS)

Woo, C.K.; Lloyd, D.; Karimov, R.; Tishler, A.

2003-01-01

An important element of an electricity market reform is stranded cost recovery. This paper explains the cause of stranded costs, describes four recovery mechanisms, evaluates these mechanisms using the criteria of recovery certainty, economic efficiency and equity, reviews the financial performance of 12 utilities in the US in connection to stranded cost recovery, and shows why the mechanism used in California has contributed to the reform failure in that state. (Author)

Genetic recombination induced by DNA double-strand break in bacteriophage T4: nature of the left/right bias.

Science.gov (United States)

Shcherbakov, Victor P; Shcherbakova, Tamara; Plugina, Lidiya; Sizova, Svetlana; Kudryashova, Elena; Granovsky, Igor

2008-06-01

The experimental system combining double-strand breaks (DSBs), produced site-specifically by SegC endonuclease, with the famous advantages of the bacteriophage T4 rII mutant recombination analysis was used here to elucidate the origin of the recombination bias on two sides of the DSB, especially pronounced in gene 39 (topoisomerase II) and gene 59 (41-helicase loader) mutants. Three sources were found to contribute to the bias: (1) the SegC endonuclease may remain bound to the end of the broken DNA and thus protect it from exonuclease degradation; (2) in heteroduplex heterozygotes (HHs), arising as the recombinant products in the left-hand crosses, the transcribed strands are of rII mutant phenotype, so they, in contrast to the right-hand HHs, do not produce plaques on the lawn of the lambda-lysogenic host; and (3) the intrinsic polarity of T4 chromosome, reflected in transcription, may be a cause for discrimination of promoter-proximal and promoter-distal DNA sequences. It is shown that the apparent recombination bias does not imply one-sidedness of the DSB repair but just reflects a different depth of the end processing. It is inferred that the cause, underlying the "intrinsic" bias, might be interference between strand exchange and transcription. Topoisomerase and helicase functions are necessary to turn the process in favor of strand exchange. The idea is substantiated that the double-stranded to single-stranded DNA transition edge (not ss-DNA tip) serves as an actual recombinogenic element.

Modeling qRT-PCR dynamics with application to cancer biomarker quantification.

Science.gov (United States)

Chervoneva, Inna; Freydin, Boris; Hyslop, Terry; Waldman, Scott A

2017-01-01

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used for molecular diagnostics and evaluating prognosis in cancer. The utility of mRNA expression biomarkers relies heavily on the accuracy and precision of quantification, which is still challenging for low abundance transcripts. The critical step for quantification is accurate estimation of efficiency needed for computing a relative qRT-PCR expression. We propose a new approach to estimating qRT-PCR efficiency based on modeling dynamics of polymerase chain reaction amplification. In contrast, only models for fluorescence intensity as a function of polymerase chain reaction cycle have been used so far for quantification. The dynamics of qRT-PCR efficiency is modeled using an ordinary differential equation model, and the fitted ordinary differential equation model is used to obtain effective polymerase chain reaction efficiency estimates needed for efficiency-adjusted quantification. The proposed new qRT-PCR efficiency estimates were used to quantify GUCY2C (Guanylate Cyclase 2C) mRNA expression in the blood of colorectal cancer patients. Time to recurrence and GUCY2C expression ratios were analyzed in a joint model for survival and longitudinal outcomes. The joint model with GUCY2C quantified using the proposed polymerase chain reaction efficiency estimates provided clinically meaningful results for association between time to recurrence and longitudinal trends in GUCY2C expression.

A novel single fluorophore-labeled double-stranded oligonucleotide probe for fluorescence-enhanced nucleic acid detection based on the inherent quenching ability of deoxyguanosine bases and competitive strand-displacement reaction.

Science.gov (United States)

Zhang, Yingwei; Tian, Jingqi; Li, Hailong; Wang, Lei; Sun, Xuping

2012-01-01

We develop a novel single fluorophore-labeled double-stranded oligonucleotide (OND) probe for rapid, nanostructure-free, fluorescence-enhanced nucleic acid detection for the first time. We further demonstrate such probe is able to well discriminate single-base mutation in nucleic acid. The design takes advantage of an inherent quenching ability of guanine bases. The short strand of the probe is designed with an end-labeled fluorophore that is placed adjacent to two guanines as the quencher located on the long opposite strand, resulting in great quenching of dye fluorescence. In the presence of a target complementary to the long strand of the probe, a competitive strand-displacement reaction occurs and the long strand forms a more stable duplex with the target, resulting in the two strands of the probe being separated from each other. As a consequence of this displacement, the fluorophore and the quencher are no longer in close proximity and dye fluorescence increases, signaling the presence of target.

A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins.

Science.gov (United States)

Soriano, Brian D; Tam, Lei-Ting T; Lu, Hsieng S; Valladares, Violeta G

2012-01-01

Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein. Copyright © 2011. Published by Elsevier B.V.

Development of computational algorithms for quantification of pulmonary structures

International Nuclear Information System (INIS)

Oliveira, Marcela de; Alvarez, Matheus; Alves, Allan F.F.; Miranda, Jose R.A.; Pina, Diana R.

2012-01-01

The high-resolution computed tomography has become the imaging diagnostic exam most commonly used for the evaluation of the squeals of Paracoccidioidomycosis. The subjective evaluations the radiological abnormalities found on HRCT images do not provide an accurate quantification. The computer-aided diagnosis systems produce a more objective assessment of the abnormal patterns found in HRCT images. Thus, this research proposes the development of algorithms in MATLAB® computing environment can quantify semi-automatically pathologies such as pulmonary fibrosis and emphysema. The algorithm consists in selecting a region of interest (ROI), and by the use of masks, filter densities and morphological operators, to obtain a quantification of the injured area to the area of a healthy lung. The proposed method was tested on ten HRCT scans of patients with confirmed PCM. The results of semi-automatic measurements were compared with subjective evaluations performed by a specialist in radiology, falling to a coincidence of 80% for emphysema and 58% for fibrosis. (author)

Bioprinting Using Mechanically Robust Core-Shell Cell-Laden Hydrogel Strands.

Science.gov (United States)

Mistry, Pritesh; Aied, Ahmed; Alexander, Morgan; Shakesheff, Kevin; Bennett, Andrew; Yang, Jing

2017-06-01

The strand material in extrusion-based bioprinting determines the microenvironments of the embedded cells and the initial mechanical properties of the constructs. One unmet challenge is the combination of optimal biological and mechanical properties in bioprinted constructs. Here, a novel bioprinting method that utilizes core-shell cell-laden strands with a mechanically robust shell and an extracellular matrix-like core has been developed. Cells encapsulated in the strands demonstrate high cell viability and tissue-like functions during cultivation. This process of bioprinting using core-shell strands with optimal biochemical and biomechanical properties represents a new strategy for fabricating functional human tissues and organs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mechanical behaviors of multi-filament twist superconducting strand under tensile and cyclic loading

Science.gov (United States)

Wang, Xu; Li, Yingxu; Gao, Yuanwen

2016-01-01

The superconducting strand, serving as the basic unit cell of the cable-in-conduit-conductors (CICCs), is a typical multi-filament twist composite which is always subjected to a cyclic loading under the operating condition. Meanwhile, the superconducting material Nb3Sn in the strand is sensitive to strain frequently relating to the performance degradation of the superconductivity. Therefore, a comprehensive study on the mechanical behavior of the strand helps understanding the superconducting performance of the strained Nb3Sn strands. To address this issue, taking the LMI (internal tin) strand as an example, a three-dimensional structural finite element model, named as the Multi-filament twist model, of the strand with the real configuration of the LMI strand is built to study the influences of the plasticity of the component materials, the twist of the filament bundle, the initial thermal residual stress and the breakage and its evolution of the filaments on the mechanical behaviors of the strand. The effective properties of superconducting filament bundle with random filament breakage and its evolution versus strain are obtained based on the damage theory of fiber-reinforced composite materials proposed by Curtin and Zhou. From the calculation results of this model, we find that the occurrence of the hysteresis loop in the cyclic loading curve is determined by the reverse yielding of the elastic-plastic materials in the strand. Both the initial thermal residual stress in the strand and the pitch length of the filaments have significant impacts on the axial and hysteretic behaviors of the strand. The damage of the filaments also affects the axial mechanical behavior of the strand remarkably at large axial strain. The critical current of the strand is calculated by the scaling law with the results of the Multi-filament twist model. The predicted results of the Multi-filament twist model show an acceptable agreement with the experiment.

Molecular investigation of evaporation of biodroplets containing single-strand DNA on graphene surface.

Science.gov (United States)

Akbari, Fahimeh; Foroutan, Masumeh

2018-02-14

In this study, the water droplet behaviour of four different types of single-strand DNA with homogeneous base sequence on a graphene substrate during evaporation of the droplet was investigated using molecular dynamics (MD) simulation. The simulation results indicated that the evaporation depended on the DNA sequence. The observed changes can be divided into four parts: (i) vaporization mode, (ii) evaporation flux, (iii) mechanism of single-strand placement on the surface, and (iv) consideration of remaining single strands after evaporation. Our simulation observations indicated different evaporation modes for thymine biodroplets as compared to those for other biodroplets. The evaporation of the thymine biodroplets occurred with an increase in the contact angle, while that of the other biodroplets occur in a constant contact angle mode. Moreover, thymine biodroplets generate the lowest contact line compared to other single strands, and it is always placed far away from the centre of the droplets during evaporation. Investigating variations in the evaporation flux shows that thymine has the highest evaporation flux and guanine has the lowest. Moreover, during initial evaporation, the flux of evaporation increases at the triple point of the biodroplets containing thymine single strands, while it decreases in the other biodroplets. The following observation was obtained from the study of the placement of single strands on the substrate: guanine and thymine interacted slower than other single strands during evaporation with graphene, adenine single strand had a higher folding during evaporation, and guanine single strand showed the lowest end-to-end distance. The investigation of single-strand DNA after evaporation shows that adenine produces the most stable structure at the end of evaporation. In addition, cytosine is the most stretched single-strand DNA due to its lack of internal π-π stacking and hydrogen bonding. Therefore, cytosine single strand is more

Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR.

Science.gov (United States)

Furet, Jean-Pierre; Quénée, Pascal; Tailliez, Patrick

2004-12-15

Real-time quantitative PCR assays were developed for the absolute quantification of lactic acid bacteria (LAB) (Streptococcus thermophilus, Lactobacillus delbrueckii, L. casei, L. paracasei, L. rhamnosus, L. acidophilus and L. johnsonii) in fermented milk products. The results of molecular quantification and classic bacterial enumeration did not differ significantly with respect to S. thermophilus and the species of the L. casei group which were detected in the six commercial fermented products tested, thus showing that DNA extraction was efficient and that genomic DNA solutions were free of PCR inhibitors. For L. delbrueckii, the results of bacterial enumeration were generally lower by a factor 10 to 100 than those of PCR quantification, suggesting a loss of viability during storage of the dairy products at 1-8 degrees C for most of the strains in this species. Real-time quantitative assays enabled identification of the species of lactic acid bacterial strains initially present in commercial fermented milk products and their accurate quantification with a detection threshold of 10(3) cells per ml of product.

Sites of termination of in vitro DNA synthesis on psoralen phototreated single-stranded templates

International Nuclear Information System (INIS)

Piette, J.; Hearst, J.

1985-01-01

Single-stranded DNA has been photochemically induced to react with 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and used as substrate for DNA replication with E. coli DNA polymerase I large fragment. By using the dideoxy sequencing procedure, it is possible to map the termination sites on the template photoreacted with HMT. These sites occur at the nucleotides preceding each thymine residue (and a few cytosine residues), emphasizing the fact that in a single-stranded stretch of DNA, HMT reacts with each thymine residue without any specificity regarding the flanking base sequence of the thymine residues. In addition, termination of DNA synthesis due to psoralen-adducted thymine is not influenced by the efficiency of the 3'-5' exonuclease proof-reading activity of the DNA polymerase. (author)

Quantification of underivatised amino acids on dry blood spot, plasma, and urine by HPLC-ESI-MS/MS.

Science.gov (United States)

Giordano, Giuseppe; Di Gangi, Iole Maria; Gucciardi, Antonina; Naturale, Mauro

2012-01-01

Enzyme deficiencies in amino acid (AA) metabolism affecting the levels of amino acids and their derivatives in physiological fluids may serve as diagnostically significant biomarkers for one or a group of metabolic disorders. Therefore, it is important to monitor a wide range of free amino acids simultaneously and to quantify them. This is time consuming if we use the classical methods and more than ever now that many laboratories have introduced Newborn Screening Programs for the semiquantitative analysis, detection, and quantification of some amino acids needed to be performed in a short time to reduce the rate of false positives.We have modified the stable isotope dilution HPLC-electrospray ionization (ESI)-MS/MS method previously described by Qu et al. (Anal Chem 74: 2034-2040, 2002) for a more rapid, robust, sensitive, and specific detection and quantification of underivatised amino acids. The modified method reduces the time of analysis to 10 min with very good reproducibility of retention times and a better separation of the metabolites and their isomers.The omission of the derivatization step allowed us to achieve some important advantages: fast and simple sample preparation and exclusion of artefacts and interferences. The use of this technique is highly sensitive, specific, and allows monitoring of 40 underivatized amino acids, including the key isomers and quantification of some of them, in order to cover many diagnostically important intermediates of metabolic pathways.We propose this HPLC-ESI-MS/MS method for underivatized amino acids as a support for the Newborn Screening as secondary test using the same dried blood spots for a more accurate and specific examination in case of suspected metabolic diseases. In this way, we avoid plasma collection from the patient as it normally occurs, reducing anxiety for the parents and further costs for analysis.The same method was validated and applied also to plasma and urine samples with good reproducibility

Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients.

Science.gov (United States)

van Ginkel, Joost H; Huibers, Manon M H; van Es, Robert J J; de Bree, Remco; Willems, Stefan M

2017-06-19

During posttreatment surveillance of head and neck cancer patients, imaging is insufficiently accurate for the early detection of relapsing disease. Free circulating tumor DNA (ctDNA) may serve as a novel biomarker for monitoring tumor burden during posttreatment surveillance of these patients. In this exploratory study, we investigated whether low level ctDNA in plasma of head and neck cancer patients can be detected using Droplet Digital PCR (ddPCR). TP53 mutations were determined in surgically resected primary tumor samples from six patients with high stage (II-IV), moderate to poorly differentiated head and neck squamous cell carcinoma (HNSCC). Subsequently, mutation specific ddPCR assays were designed. Pretreatment plasma samples from these patients were examined on the presence of ctDNA by ddPCR using the mutation-specific assays. The ddPCR results were evaluated alongside clinicopathological data. In all cases, plasma samples were found positive for targeted TP53 mutations in varying degrees (absolute quantification of 2.2-422 mutational copies/ml plasma). Mutations were detected in wild-type TP53 background templates of 7667-156,667 copies/ml plasma, yielding fractional abundances of down to 0.01%. Our results show that detection of tumor specific TP53 mutations in low level ctDNA from HNSCC patients using ddPCR is technically feasible and provide ground for future research on ctDNA quantification for the use of diagnostic biomarkers in the posttreatment surveillance of HNSCC patients.

Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting the groEL Gene

Science.gov (United States)

Junick, Jana

2012-01-01

Quantitative real-time PCR assays targeting the groEL gene for the specific enumeration of 12 human fecal Bifidobacterium species were developed. The housekeeping gene groEL (HSP60 in eukaryotes) was used as a discriminative marker for the differentiation of Bifidobacterium adolescentis, B. angulatum, B. animalis, B. bifidum, B. breve, B. catenulatum, B. dentium, B. gallicum, B. longum, B. pseudocatenulatum, B. pseudolongum, and B. thermophilum. The bifidobacterial chromosome contains a single copy of the groEL gene, allowing the determination of the cell number by quantification of the groEL copy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a given Bifidobacterium species. Independent of the Bifidobacterium species tested, the proportion of groEL copies recovered from fecal samples spiked with 5 to 9 log10 cells/g feces was approximately 50%. The quantification limit was 5 to 6 log10 groEL copies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3 Bifidobacterium species and B. longum frequently detected. The predominant species in infant and adult fecal samples were B. breve and B. adolescentis, respectively. It was possible to distinguish B. catenulatum and B. pseudocatenulatum. We conclude that the groEL gene is a suitable molecular marker for the specific and accurate quantification of human fecal Bifidobacterium species by real-time PCR. PMID:22307308

RPA prevents G-rich structure formation at lagging-strand telomeres to allow maintenance of chromosome ends.

Science.gov (United States)

Audry, Julien; Maestroni, Laetitia; Delagoutte, Emmanuelle; Gauthier, Tiphaine; Nakamura, Toru M; Gachet, Yannick; Saintomé, Carole; Géli, Vincent; Coulon, Stéphane

2015-07-14

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1-D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging-strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52. The presence of these secondary DNA structures correlates with reduced association of shelterin subunits Pot1 and Ccq1 at telomeres. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G-quadruplex rescues all the telomeric defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G-quadruplex. We propose that RPA prevents the formation of G-quadruplex structures at lagging-strand telomeres to promote shelterin association and facilitate telomerase action at telomeres. © 2015 The Authors.

Towards quantitative viromics for both double-stranded and single-stranded DNA viruses

Directory of Open Access Journals (Sweden)

Simon Roux

2016-12-01

Full Text Available Background Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA viral genomes captured in quantitative viral metagenomes (viromes. This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation. Methods Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses. Results Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5% of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample. Discussion Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

Accurate LC peak boundary detection for ¹⁶O/¹⁸O labeled LC-MS data.

Science.gov (United States)

Cui, Jian; Petritis, Konstantinos; Tegeler, Tony; Petritis, Brianne; Ma, Xuepo; Jin, Yufang; Gao, Shou-Jiang S J; Zhang, Jianqiu Michelle

2013-01-01

In liquid chromatography-mass spectrometry (LC-MS), parts of LC peaks are often corrupted by their co-eluting peptides, which results in increased quantification variance. In this paper, we propose to apply accurate LC peak boundary detection to remove the corrupted part of LC peaks. Accurate LC peak boundary detection is achieved by checking the consistency of intensity patterns within peptide elution time ranges. In addition, we remove peptides with erroneous mass assignment through model fitness check, which compares observed intensity patterns to theoretically constructed ones. The proposed algorithm can significantly improve the accuracy and precision of peptide ratio measurements.

Cetacean strandings along the coast of Izmir Bay, Turkey

NARCIS (Netherlands)

Guclusoy, H.; Veryeri, N.; Cirik, S.

2004-01-01

The present paper provides information on the stranding of cetaceans in Izmir Bay, Aegean Sea, between 1992 and 2004. The data were collected opportunistically during sightings and stranding data collection for Monk Seals. A total of 12 cetaceans, namely Bottle-nosed Dolphin, Tursiops truncatus

DNA turnover and strand breaks in Escherichia coli

International Nuclear Information System (INIS)

Hanawalt, P.; Grivell, A.; Nakayama, H.

1975-01-01

The extent of DNA turnover has been measured in a dnaB mutant of Escherichia coli, temperature sensitive for semiconservative DNA replication. At the nonpermissive temperature about 0.02 percent of the deoxynucleotides in DNA are exchanged per generation period. This turnover rate is markedly depressed in the presence of rifampicin. During thymine starvation strand breaks accumulate in the DNA of E. coli strains that are susceptible to thymineless death. Rifampicin suppresses the appearance of these breaks, consistent with our hypothesis that transcription may be accompanied by repairable single-strand breaks in DNA. DNA turnover is enhanced severalfold in strands containing 5-bromodeoxyuridine in place of thymidine, possibly because the analog (or the deoxyuridine, following debromination) is sometimes recognized and excised

Residual Resistivity Ratio (RRR) Measurements of LHC Superconducting NbTi Cable Strands

CERN Document Server

Charifoulline, Z

2006-01-01

The Rutherford-type superconducting NbTi cables of the LHC accelerator are currently manufactured by six industrial companies. As a part of the acceptance tests, the Residual Resistivity Ratio (RRR) of superconducting strands is systematically measured on virgin strands to qualify the strands before cabling and on extracted strands to qualify the cables and to check the final heat treatment (controlled oxidation to control interstrand resistance). More than 12000 samples of virgin and extracted strands have been measured during last five years. Results show good correlation with the measurements done by the companies and reflect well the technological process of cable production (strand annealing, cabling, cable heat treatment). This paper presents a description of the RRR-test station and the measurement procedure, the summary of the results over all suppliers and finally the correlation between RRR-values of the cables and the magnets.

Strategy study of quantification harmonization of SUV in PET/CT images; Estudo da estrategia de harmonizacao da quantificacao do SUV em imagens de PET/CT

Energy Technology Data Exchange (ETDEWEB)

Fischer, Andreia Caroline Fischer da Silveira

2014-07-01

In clinical practice, PET/CT images are often analyzed qualitatively by visual comparison of tumor lesions and normal tissues uptake; and semi-quantitatively by means of a parameter called SUV (Standardized Uptake Value). To ensure that longitudinal studies acquired on different scanners are interchangeable, and information of quantification is comparable, it is necessary to establish a strategy to harmonize the quantification of SUV. The aim of this study is to evaluate the strategy to harmonize the quantification of PET/CT images, performed with different scanner models and manufacturers. For this purpose, a survey of the technical characteristics of equipment and acquisition protocols of clinical images of different services of PET/CT in the state of Rio Grande do Sul was conducted. For each scanner, the accuracy of SUV quantification, and the Recovery Coefficient (RC) curves were determined, using the reconstruction parameters clinically relevant and available. From these data, harmonized performance specifications among the evaluated scanners were identified, as well as the algorithm that produces, for each one, the most accurate quantification. Finally, the most appropriate reconstruction parameters to harmonize the SUV quantification in each scanner, either regionally or internationally were identified. It was found that the RC values of the analyzed scanners proved to be overestimated by up to 38%, particularly for objects larger than 17mm. These results demonstrate the need for further optimization, through the reconstruction parameters modification, and even the change of the reconstruction algorithm used in each scanner. It was observed that there is a decoupling between the best image for PET/CT qualitative analysis and the best image for quantification studies. Thus, the choice of reconstruction method should be tied to the purpose of the PET/CT study in question, since the same reconstruction algorithm is not adequate, in one scanner, for qualitative

The 3'-terminal 55 nucleotides of bovine coronavirus defective interfering RNA harbor cis-acting elements required for both negative- and positive-strand RNA synthesis.

Directory of Open Access Journals (Sweden)

Wei-Yu Liao

Full Text Available The synthesis of the negative-strand [(--strand] complement of the ∼30 kilobase, positive-strand [(+-strand] coronaviral genome is a necessary early step for genome replication. The identification of cis-acting elements required for (--strand RNA synthesis in coronaviruses, however, has been hampered due to insufficiencies in the techniques used to detect the (--strand RNA species. Here, we employed a method of head-to-tail ligation and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR to detect and quantitate the synthesis of bovine coronavirus (BCoV defective interfering (DI RNA (- strands. Furthermore, using the aforementioned techniques along with Northern blot assay, we specifically defined the cis-acting RNA elements within the 3'-terminal 55 nucleotides (nts which function in the synthesis of (-- or (+-strand BCoV DI RNA. The major findings are as follows: (i nts from -5 to -39 within the 3'-terminal 55 nts are the cis-acting elements responsible for (--strand BCoV DI RNA synthesis, (ii nts from -3 to -34 within the 3'-terminal 55 nts are cis-acting elements required for (+-strand BCoV DI RNA synthesis, and (iii the nucleotide species at the 3'-most position (-1 is important, but not critical, for both (-- and (+-strand BCoV DI RNA synthesis. These results demonstrate that the 3'-terminal 55 nts in BCoV DI RNA harbor cis-acting RNA elements required for both (-- and (+-strand DI RNA synthesis and extend our knowledge on the mechanisms of coronavirus replication. The method of head-to-tail ligation and qRT-PCR employed in the study may also be applied to identify other cis-acting elements required for (--strand RNA synthesis in coronaviruses.

Accurate quantification of microorganisms in PCR-inhibiting environmental DNA extracts by a novel Internal Amplification Control approach using Biotrove OpenArrays

NARCIS (Netherlands)

Van Doorn, R.; Klerks, M.; van Gent-Pelzer, M.; Speksnijder, A.G.C.L.; Kowalchuk, G.A.; Schoen, C.D.

2009-01-01

PCR-based detection assays are prone to inhibition by substances present in environmental samples, thereby potentially leading to inaccurate target quantification or false-negative results. Internal amplification controls (IACs) have been developed to help alleviate this problem but are generally

Statistically accurate low-order models for uncertainty quantification in turbulent dynamical systems.

Science.gov (United States)

Sapsis, Themistoklis P; Majda, Andrew J

2013-08-20

A framework for low-order predictive statistical modeling and uncertainty quantification in turbulent dynamical systems is developed here. These reduced-order, modified quasilinear Gaussian (ROMQG) algorithms apply to turbulent dynamical systems in which there is significant linear instability or linear nonnormal dynamics in the unperturbed system and energy-conserving nonlinear interactions that transfer energy from the unstable modes to the stable modes where dissipation occurs, resulting in a statistical steady state; such turbulent dynamical systems are ubiquitous in geophysical and engineering turbulence. The ROMQG method involves constructing a low-order, nonlinear, dynamical system for the mean and covariance statistics in the reduced subspace that has the unperturbed statistics as a stable fixed point and optimally incorporates the indirect effect of non-Gaussian third-order statistics for the unperturbed system in a systematic calibration stage. This calibration procedure is achieved through information involving only the mean and covariance statistics for the unperturbed equilibrium. The performance of the ROMQG algorithm is assessed on two stringent test cases: the 40-mode Lorenz 96 model mimicking midlatitude atmospheric turbulence and two-layer baroclinic models for high-latitude ocean turbulence with over 125,000 degrees of freedom. In the Lorenz 96 model, the ROMQG algorithm with just a single mode captures the transient response to random or deterministic forcing. For the baroclinic ocean turbulence models, the inexpensive ROMQG algorithm with 252 modes, less than 0.2% of the total, captures the nonlinear response of the energy, the heat flux, and even the one-dimensional energy and heat flux spectra.

Tensile and dimensional properties of wood strands made from plantation southern pine lumber

Science.gov (United States)

Qinglin Wu; Zhiyong Cai; Jong N. Lee

2005-01-01

Working stresses and performance of strand composite lumber largely depend upon the properties of each individual strand. Southern pine strands from plantation lumber grown in southern Louisiana were investigated in this study in order to understand strand behaviors. The effects of hot-pressing and resin application on tensile modulus, strength, and dimensional...

Accurate and rapid modeling of iron–bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis

OpenAIRE

Harsch, Andreas; Marzilli, Lisa A.; Bunt, Richard C.; Stubbe, Joanne; Vouros, Paul

2000-01-01

Bleomycin B2 (BLM) in the presence of iron [Fe(II)] and O2 catalyzes single-stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe–BLM was incorporated into each an...

Global Sensitivity Analysis and Estimation of Model Error, Toward Uncertainty Quantification in Scramjet Computations

Science.gov (United States)

Huan, Xun; Safta, Cosmin; Sargsyan, Khachik; Geraci, Gianluca; Eldred, Michael S.; Vane, Zachary P.; Lacaze, Guilhem; Oefelein, Joseph C.; Najm, Habib N.

2018-03-01

The development of scramjet engines is an important research area for advancing hypersonic and orbital flights. Progress toward optimal engine designs requires accurate flow simulations together with uncertainty quantification. However, performing uncertainty quantification for scramjet simulations is challenging due to the large number of uncertain parameters involved and the high computational cost of flow simulations. These difficulties are addressed in this paper by developing practical uncertainty quantification algorithms and computational methods, and deploying them in the current study to large-eddy simulations of a jet in crossflow inside a simplified HIFiRE Direct Connect Rig scramjet combustor. First, global sensitivity analysis is conducted to identify influential uncertain input parameters, which can help reduce the systems stochastic dimension. Second, because models of different fidelity are used in the overall uncertainty quantification assessment, a framework for quantifying and propagating the uncertainty due to model error is presented. These methods are demonstrated on a nonreacting jet-in-crossflow test problem in a simplified scramjet geometry, with parameter space up to 24 dimensions, using static and dynamic treatments of the turbulence subgrid model, and with two-dimensional and three-dimensional geometries.

Global Sensitivity Analysis and Estimation of Model Error, Toward Uncertainty Quantification in Scramjet Computations

Energy Technology Data Exchange (ETDEWEB)

Huan, Xun [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Safta, Cosmin [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Sargsyan, Khachik [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Geraci, Gianluca [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Eldred, Michael S. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Vane, Zachary P. [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Lacaze, Guilhem [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Oefelein, Joseph C. [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Najm, Habib N. [Sandia National Lab. (SNL-CA), Livermore, CA (United States)

2018-02-09

The development of scramjet engines is an important research area for advancing hypersonic and orbital flights. Progress toward optimal engine designs requires accurate flow simulations together with uncertainty quantification. However, performing uncertainty quantification for scramjet simulations is challenging due to the large number of uncertain parameters involved and the high computational cost of flow simulations. These difficulties are addressed in this paper by developing practical uncertainty quantification algorithms and computational methods, and deploying them in the current study to large-eddy simulations of a jet in crossflow inside a simplified HIFiRE Direct Connect Rig scramjet combustor. First, global sensitivity analysis is conducted to identify influential uncertain input parameters, which can help reduce the system’s stochastic dimension. Second, because models of different fidelity are used in the overall uncertainty quantification assessment, a framework for quantifying and propagating the uncertainty due to model error is presented. Finally, these methods are demonstrated on a nonreacting jet-in-crossflow test problem in a simplified scramjet geometry, with parameter space up to 24 dimensions, using static and dynamic treatments of the turbulence subgrid model, and with two-dimensional and three-dimensional geometries.

Double-stranded DNA-dependent ATPase Irc3p is directly involved in mitochondrial genome maintenance.

Science.gov (United States)

Sedman, Tiina; Gaidutšik, Ilja; Villemson, Karin; Hou, YingJian; Sedman, Juhan

2014-12-01

Nucleic acid-dependent ATPases are involved in nearly all aspects of DNA and RNA metabolism. Previous studies have described a number of mitochondrial helicases. However, double-stranded DNA-dependent ATPases, including translocases or enzymes remodeling DNA-protein complexes, have not been identified in mitochondria of the yeast Saccharomyces cerevisae. Here, we demonstrate that Irc3p is a mitochondrial double-stranded DNA-dependent ATPase of the Superfamily II. In contrast to the other mitochondrial Superfamily II enzymes Mss116p, Suv3p and Mrh4p, which are RNA helicases, Irc3p has a direct role in mitochondrial DNA (mtDNA) maintenance. Specific Irc3p-dependent mtDNA metabolic intermediates can be detected, including high levels of double-stranded DNA breaks that accumulate in irc3Δ mutants. irc3Δ-related topology changes in rho- mtDNA can be reversed by the deletion of mitochondrial RNA polymerase RPO41, suggesting that Irc3p counterbalances adverse effects of transcription on mitochondrial genome stability. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Improving strand quality of upland oaks for use in oriented strand board

Science.gov (United States)

David B. DeValliance; Jody D. Gray; Shawn T. Grushecky

2013-01-01

Past research estimates that more than 1 million tons of oak logging residues go unused in West Virginia each year. Much research has been done investigating potential products and markets for this underutilized resource. West Virginia is home to an oriented strand board (OSB) producer that consumes large volumes of small diameter, low quality round wood. However, the...

Quantification of complex modular architecture in plants.

Science.gov (United States)

Reeb, Catherine; Kaandorp, Jaap; Jansson, Fredrik; Puillandre, Nicolas; Dubuisson, Jean-Yves; Cornette, Raphaël; Jabbour, Florian; Coudert, Yoan; Patiño, Jairo; Flot, Jean-François; Vanderpoorten, Alain

2018-04-01

Morphometrics, the assignment of quantities to biological shapes, is a powerful tool to address taxonomic, evolutionary, functional and developmental questions. We propose a novel method for shape quantification of complex modular architecture in thalloid plants, whose extremely reduced morphologies, combined with the lack of a formal framework for thallus description, have long rendered taxonomic and evolutionary studies extremely challenging. Using graph theory, thalli are described as hierarchical series of nodes and edges, allowing for accurate, homologous and repeatable measurements of widths, lengths and angles. The computer program MorphoSnake was developed to extract the skeleton and contours of a thallus and automatically acquire, at each level of organization, width, length, angle and sinuosity measurements. Through the quantification of leaf architecture in Hymenophyllum ferns (Polypodiopsida) and a fully worked example of integrative taxonomy in the taxonomically challenging thalloid liverwort genus Riccardia, we show that MorphoSnake is applicable to all ramified plants. This new possibility of acquiring large numbers of quantitative traits in plants with complex modular architectures opens new perspectives of applications, from the development of rapid species identification tools to evolutionary analyses of adaptive plasticity. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

PET Quantification of the Norepinephrine Transporter in Human Brain with (S,S)-18F-FMeNER-D2.

Science.gov (United States)

Moriguchi, Sho; Kimura, Yasuyuki; Ichise, Masanori; Arakawa, Ryosuke; Takano, Harumasa; Seki, Chie; Ikoma, Yoko; Takahata, Keisuke; Nagashima, Tomohisa; Yamada, Makiko; Mimura, Masaru; Suhara, Tetsuya

2017-07-01

Norepinephrine transporter (NET) in the brain plays important roles in human cognition and the pathophysiology of psychiatric disorders. Two radioligands, ( S , S )- 11 C-MRB and ( S , S )- 18 F-FMeNER-D 2 , have been used for imaging NETs in the thalamus and midbrain (including locus coeruleus) using PET in humans. However, NET density in the equally important cerebral cortex has not been well quantified because of unfavorable kinetics with ( S , S )- 11 C-MRB and defluorination with ( S , S )- 18 F-FMeNER-D 2 , which can complicate NET quantification in the cerebral cortex adjacent to the skull containing defluorinated 18 F radioactivity. In this study, we have established analysis methods of quantification of NET density in the brain including the cerebral cortex using ( S , S )- 18 F-FMeNER-D 2 PET. Methods: We analyzed our previous ( S , S )- 18 F-FMeNER-D 2 PET data of 10 healthy volunteers dynamically acquired for 240 min with arterial blood sampling. The effects of defluorination on the NET quantification in the superficial cerebral cortex was evaluated by establishing a time stability of NET density estimations with an arterial input 2-tissue-compartment model, which guided the less-invasive reference tissue model and area under the time-activity curve methods to accurately quantify NET density in all brain regions including the cerebral cortex. Results: Defluorination of ( S , S )- 18 F-FMeNER-D 2 became prominent toward the latter half of the 240-min scan. Total distribution volumes in the superficial cerebral cortex increased with the scan duration beyond 120 min. We verified that 90-min dynamic scans provided a sufficient amount of data for quantification of NET density unaffected by defluorination. Reference tissue model binding potential values from the 90-min scan data and area under the time-activity curve ratios of 70- to 90-min data allowed for the accurate quantification of NET density in the cerebral cortex. Conclusion: We have established

TALEN-Induced Double-Strand Break Repair of CTG Trinucleotide Repeats

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Valentine Mosbach

2018-02-01

Full Text Available Trinucleotide repeat expansions involving CTG/CAG triplets are responsible for several neurodegenerative disorders, including myotonic dystrophy and Huntington’s disease. Because expansions trigger the disease, contracting repeat length could be a possible approach to gene therapy for these disorders. Here, we show that a TALEN-induced double-strand break was very efficient at contracting expanded CTG repeats in yeast. We show that RAD51, POL32, and DNL4 are dispensable for double-strand break repair within CTG repeats, the only required genes being RAD50, SAE2, and RAD52. Resection was totally abolished in the absence of RAD50 on both sides of the break, whereas it was reduced in a sae2Δ mutant on the side of the break containing the longest repeat tract, suggesting that secondary structures at double-strand break ends must be removed by the Mre11-Rad50 complex and Sae2. Following the TALEN double-strand break, single-strand annealing occurred between both sides of the repeat tract, leading to repeat contraction.

Programmable autonomous synthesis of single-stranded DNA

Science.gov (United States)

Kishi, Jocelyn Y.; Schaus, Thomas E.; Gopalkrishnan, Nikhil; Xuan, Feng; Yin, Peng

2018-02-01

DNA performs diverse functional roles in biology, nanotechnology and biotechnology, but current methods for autonomously synthesizing arbitrary single-stranded DNA are limited. Here, we introduce the concept of primer exchange reaction (PER) cascades, which grow nascent single-stranded DNA with user-specified sequences following prescribed reaction pathways. PER synthesis happens in a programmable, autonomous, in situ and environmentally responsive fashion, providing a platform for engineering molecular circuits and devices with a wide range of sensing, monitoring, recording, signal-processing and actuation capabilities. We experimentally demonstrate a nanodevice that transduces the detection of a trigger RNA into the production of a DNAzyme that degrades an independent RNA substrate, a signal amplifier that conditionally synthesizes long fluorescent strands only in the presence of a particular RNA signal, molecular computing circuits that evaluate logic (AND, OR, NOT) combinations of RNA inputs, and a temporal molecular event recorder that records in the PER transcript the order in which distinct RNA inputs are sequentially detected.

Feasibility and accuracy of dual-layer spectral detector computed tomography for quantification of gadolinium: a phantom study.

Science.gov (United States)

van Hamersvelt, Robbert W; Willemink, Martin J; de Jong, Pim A; Milles, Julien; Vlassenbroek, Alain; Schilham, Arnold M R; Leiner, Tim

2017-09-01

The aim of this study was to evaluate the feasibility and accuracy of dual-layer spectral detector CT (SDCT) for the quantification of clinically encountered gadolinium concentrations. The cardiac chamber of an anthropomorphic thoracic phantom was equipped with 14 tubular inserts containing different gadolinium concentrations, ranging from 0 to 26.3 mg/mL (0.0, 0.1, 0.2, 0.4, 0.5, 1.0, 2.0, 3.0, 4.0, 5.1, 10.6, 15.7, 20.7 and 26.3 mg/mL). Images were acquired using a novel 64-detector row SDCT system at 120 and 140 kVp. Acquisitions were repeated five times to assess reproducibility. Regions of interest (ROIs) were drawn on three slices per insert. A spectral plot was extracted for every ROI and mean attenuation profiles were fitted to known attenuation profiles of water and pure gadolinium using in-house-developed software to calculate gadolinium concentrations. At both 120 and 140 kVp, excellent correlations between scan repetitions and true and measured gadolinium concentrations were found (R > 0.99, P  0.99, CI 0.99-1.00). Relative mean measurement errors stayed below 10% down to 2.0 mg/mL true gadolinium concentration at 120 kVp and below 5% down to 1.0 mg/mL true gadolinium concentration at 140 kVp. SDCT allows for accurate quantification of gadolinium at both 120 and 140 kVp. Lowest measurement errors were found for 140 kVp acquisitions. • Gadolinium quantification may be useful in patients with contraindication to iodine. • Dual-layer spectral detector CT allows for overall accurate quantification of gadolinium. • Interscan variability of gadolinium quantification using SDCT material decomposition is excellent.

MR Spectroscopy: Real-Time Quantification of in-vivo MR Spectroscopic data

OpenAIRE

Massé, Kunal

2009-01-01

In the last two decades, magnetic resonance spectroscopy (MRS) has had an increasing success in biomedical research. This technique has the faculty of discerning several metabolites in human tissue non-invasively and thus offers a multitude of medical applications. In clinical routine, quantification plays a key role in the evaluation of the different chemical elements. The quantification of metabolites characterizing specific pathologies helps physicians establish the patient's diagnosis. E...

Critical assessment of digital PCR for the detection and quantification of genetically modified organisms.

Science.gov (United States)

Demeke, Tigst; Dobnik, David

2018-07-01

The number of genetically modified organisms (GMOs) on the market is steadily increasing. Because of regulation of cultivation and trade of GMOs in several countries, there is pressure for their accurate detection and quantification. Today, DNA-based approaches are more popular for this purpose than protein-based methods, and real-time quantitative PCR (qPCR) is still the gold standard in GMO analytics. However, digital PCR (dPCR) offers several advantages over qPCR, making this new technique appealing also for GMO analysis. This critical review focuses on the use of dPCR for the purpose of GMO quantification and addresses parameters which are important for achieving accurate and reliable results, such as the quality and purity of DNA and reaction optimization. Three critical factors are explored and discussed in more depth: correct classification of partitions as positive, correctly determined partition volume, and dilution factor. This review could serve as a guide for all laboratories implementing dPCR. Most of the parameters discussed are applicable to fields other than purely GMO testing. Graphical abstract There are generally three different options for absolute quantification of genetically modified organisms (GMOs) using digital PCR: droplet- or chamber-based and droplets in chambers. All have in common the distribution of reaction mixture into several partitions, which are all subjected to PCR and scored at the end-point as positive or negative. Based on these results GMO content can be calculated.

Development of a 10 m quasi-isotropic strand assembled from 2G wires

Science.gov (United States)

Kan, Changtao; Wang, Yinshun; Hou, Yanbing; Li, Yan; Zhang, Han; Fu, Yu; Jiang, Zhe

2018-03-01

Quasi-isotropic strands made of second generation (2G) high temperature superconducting (HTS) wires are attractive to applications of high-field magnets at low temperatures and power transmission cables at liquid nitrogen temperature in virtue of their high current carrying capability and well mechanical property. In this contribution, a 10 m length quasi-isotropic strand is manufactured and successfully tested in liquid nitrogen to verify the feasibility of an industrial scale production of the strand by the existing cabling technologies. The strand with copper sheath consists of 72 symmetrically assembled 2G wires. The uniformity of critical properties of long quasi-isotropic strands, including critical current and n-value, is very important for their using. Critical currents as well as n-values of the strand are measured every 1 m respectively and compared with the simulation results. Critical current and n-value of the strand are calculated basing on the self-consistent model solved by the finite element method (FEM). Effects of self-field on the critical current and n-value distributions in wires of the strand are analyzed in detail. The simulation results show good agreement with the experimental data and the 10 m quasi-isotropic strand has good critical properties uniformity.

Absolute Quantification of the Host-To-Parasite DNA Ratio in Theileria parva-Infected Lymphocyte Cell Lines.

Science.gov (United States)

Gotia, Hanzel T; Munro, James B; Knowles, Donald P; Daubenberger, Claudia A; Bishop, Richard P; Silva, Joana C

2016-01-01

Theileria parva is a tick-transmitted intracellular apicomplexan pathogen of cattle in sub-Saharan Africa that causes East Coast fever (ECF). ECF is an acute fatal disease that kills over one million cattle annually, imposing a tremendous burden on African small-holder cattle farmers. The pathology and level of T. parva infections in its wildlife host, African buffalo (Syncerus caffer), and in cattle are distinct. We have developed an absolute quantification method based on quantitative PCR (qPCR) in which recombinant plasmids containing single copy genes specific to the parasite (apical membrane antigen 1 gene, ama1) or the host (hypoxanthine phosphoribosyltransferase 1, hprt1) are used as the quantification reference standards. Our study shows that T. parva and bovine cells are present in similar numbers in T. parva-infected lymphocyte cell lines and that consequently, due to its much smaller genome size, T. parva DNA comprises between 0.9% and 3% of the total DNA samples extracted from these lines. This absolute quantification assay of parasite and host genome copy number in a sample provides a simple and reliable method of assessing T. parva load in infected bovine lymphocytes, and is accurate over a wide range of host-to-parasite DNA ratios. Knowledge of the proportion of target DNA in a sample, as enabled by this method, is essential for efficient high-throughput genome sequencing applications for a variety of intracellular pathogens. This assay will also be very useful in future studies of interactions of distinct host-T. parva stocks and to fully characterize the dynamics of ECF infection in the field.

Development and validation of high-performance liquid chromatography and high-performance thin-layer chromatography methods for the quantification of khellin in Ammi visnaga seed

Science.gov (United States)

Kamal, Abid; Khan, Washim; Ahmad, Sayeed; Ahmad, F. J.; Saleem, Kishwar

2015-01-01

Objective: The present study was used to design simple, accurate and sensitive reversed phase-high-performance liquid chromatography RP-HPLC and high-performance thin-layer chromatography (HPTLC) methods for the development of quantification of khellin present in the seeds of Ammi visnaga. Materials and Methods: RP-HPLC analysis was performed on a C18 column with methanol: Water (75: 25, v/v) as a mobile phase. The HPTLC method involved densitometric evaluation of khellin after resolving it on silica gel plate using ethyl acetate: Toluene: Formic acid (5.5:4.0:0.5, v/v/v) as a mobile phase. Results: The developed HPLC and HPTLC methods were validated for precision (interday, intraday and intersystem), robustness and accuracy, limit of detection and limit of quantification. The relationship between the concentration of standard solutions and the peak response was linear in both HPLC and HPTLC methods with the concentration range of 10–80 μg/mL in HPLC and 25–1,000 ng/spot in HPTLC for khellin. The % relative standard deviation values for method precision was found to be 0.63–1.97%, 0.62–2.05% in HPLC and HPTLC for khellin respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels and the average percentage recovery was found to be 100.53% in HPLC and 100.08% in HPTLC for khellin. Conclusions: The developed HPLC and HPTLC methods for the quantification of khellin were found simple, precise, specific, sensitive and accurate which can be used for routine analysis and quality control of A. visnaga and several formulations containing it as an ingredient. PMID:26681890

Quantification of in vivo oxidative damage in Caenorhabditis elegans during aging by endogenous F3-isoprostane measurement

NARCIS (Netherlands)

Labuschagne, C.F.; Stigter, E.C.; Hendriks, M.M.; Berger, R.; Rokach, J.; Korswagen, H.C.; Brenkman, A.B.

2013-01-01

Oxidative damage is thought to be a major cause in development of pathologies and aging. However, quantification of oxidative damage is methodologically difficult. Here, we present a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach for accurate, sensitive, and linear in vivo

Assessing reference genes for accurate transcript normalization using quantitative real-time PCR in pearl millet [Pennisetum glaucum (L. R. Br].

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Prasenjit Saha

Full Text Available Pearl millet [Pennisetum glaucum (L. R.Br.], a close relative of Panicoideae food crops and bioenergy grasses, offers an ideal system to perform functional genomics studies related to C4 photosynthesis and abiotic stress tolerance. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR provides a sensitive platform to conduct such gene expression analyses. However, the lack of suitable internal control reference genes for accurate transcript normalization during qRT-PCR analysis in pearl millet is the major limitation. Here, we conducted a comprehensive assessment of 18 reference genes on 234 samples which included an array of different developmental tissues, hormone treatments and abiotic stress conditions from three genotypes to determine appropriate reference genes for accurate normalization of qRT-PCR data. Analyses of Ct values using Stability Index, BestKeeper, ΔCt, Normfinder, geNorm and RefFinder programs ranked PP2A, TIP41, UBC2, UBQ5 and ACT as the most reliable reference genes for accurate transcript normalization under different experimental conditions. Furthermore, we validated the specificity of these genes for precise quantification of relative gene expression and provided evidence that a combination of the best reference genes are required to obtain optimal expression patterns for both endogeneous genes as well as transgenes in pearl millet.

A novel automatic quantification method for high-content screening analysis of DNA double strand-break response.

Science.gov (United States)

Feng, Jingwen; Lin, Jie; Zhang, Pengquan; Yang, Songnan; Sa, Yu; Feng, Yuanming

2017-08-29

High-content screening is commonly used in studies of the DNA damage response. The double-strand break (DSB) is one of the most harmful types of DNA damage lesions. The conventional method used to quantify DSBs is γH2AX foci counting, which requires manual adjustment and preset parameters and is usually regarded as imprecise, time-consuming, poorly reproducible, and inaccurate. Therefore, a robust automatic alternative method is highly desired. In this manuscript, we present a new method for quantifying DSBs which involves automatic image cropping, automatic foci-segmentation and fluorescent intensity measurement. Furthermore, an additional function was added for standardizing the measurement of DSB response inhibition based on co-localization analysis. We tested the method with a well-known inhibitor of DSB response. The new method requires only one preset parameter, which effectively minimizes operator-dependent variations. Compared with conventional methods, the new method detected a higher percentage difference of foci formation between different cells, which can improve measurement accuracy. The effects of the inhibitor on DSB response were successfully quantified with the new method (p = 0.000). The advantages of this method in terms of reliability, automation and simplicity show its potential in quantitative fluorescence imaging studies and high-content screening for compounds and factors involved in DSB response.

Direct quantification of lipopeptide biosurfactants in biological samples via HPLC and UPLC-MS requires sample modification with an organic solvent.

Science.gov (United States)

Biniarz, Piotr; Łukaszewicz, Marcin

2017-06-01

The rapid and accurate quantification of biosurfactants in biological samples is challenging. In contrast to the orcinol method for rhamnolipids, no simple biochemical method is available for the rapid quantification of lipopeptides. Various liquid chromatography (LC) methods are promising tools for relatively fast and exact quantification of lipopeptides. Here, we report strategies for the quantification of the lipopeptides pseudofactin and surfactin in bacterial cultures using different high- (HPLC) and ultra-performance liquid chromatography (UPLC) systems. We tested three strategies for sample pretreatment prior to LC analysis. In direct analysis (DA), bacterial cultures were injected directly and analyzed via LC. As a modification, we diluted the samples with methanol and detected an increase in lipopeptide recovery in the presence of methanol. Therefore, we suggest this simple modification as a tool for increasing the accuracy of LC methods. We also tested freeze-drying followed by solvent extraction (FDSE) as an alternative for the analysis of "heavy" samples. In FDSE, the bacterial cultures were freeze-dried, and the resulting powder was extracted with different solvents. Then, the organic extracts were analyzed via LC. Here, we determined the influence of the extracting solvent on lipopeptide recovery. HPLC methods allowed us to quantify pseudofactin and surfactin with run times of 15 and 20 min per sample, respectively, whereas UPLC quantification was as fast as 4 and 5.5 min per sample, respectively. Our methods provide highly accurate measurements and high recovery levels for lipopeptides. At the same time, UPLC-MS provides the possibility to identify lipopeptides and their structural isoforms.

Stability Indicating HPLC Method for Simultaneous Quantification of Trihexyphenidyl Hydrochloride, Trifluoperazine Hydrochloride and Chlorpromazine Hydrochloride from Tablet Formulation

Directory of Open Access Journals (Sweden)

P. Shetti

2010-01-01

Full Text Available A new, simple, precise, rapid, selective and stability indicating reversed-phase high performance liquid chromatographic (HPLC method has been developed and validated for simultaneous quantification of trihexyphenidyl hydrochloride, trifluoperazine hydrochloride and chlorpromazine hydrochloride from combined tablet formulation. The method is based on reverse-phase using C-18 (250×4.6 mm, 5 μm particle size column. The separation is achieved using isocratic elution by methanol and ammonium acetate buffer (1% w/v, pH 6.5 in the ratio of 85:15 v/v, pumped at flow rate 1.0 mL/min and UV detection at 215 nm. The column is maintained at 30 °C through out the analysis. This method gives baseline resolution. The total run time is 15 min. Stability indicating capability is established buy forced degradation experiment. The method is validated for specificity, accuracy, precision and linearity as per International conference of harmonisation (ICH. The method is accurate and linear for quantification of trihexyphenidyl hydrochloride, trifluoperazine hydrochloride and Chlorpromazine hydrochloride between 5 - 15 μg/mL, 12.5- 37.5 μg/mL and 62.5 - 187.5 μg/mL respectively.

Method for indirect quantification of CH4 production via H2O production using hydrogenotrophic methanogens

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Ruth-Sophie eTaubner

2016-04-01

Full Text Available ydrogenotrophic methanogens are an intriguing group of microorganisms from the domain Archaea. They exhibit extraordinary ecological, biochemical, physiological characteristics colorbox{yellow}{and have a huge biotechnological potential}. Yet, the only possibility to assess the methane (CH$_4$ production potential of hydrogenotrophic methanogens is to apply gas chromatographic quantification of CH$_4$.In order to be able to effectively screen pure cultures of hydrogenotrophic methanogens regarding their CH$_4$ production potential we developed a novel method for indirect quantification of colorbox{yellow}{the} volumetric CH$_4$ production rate by measuring colorbox{yellow}{the} volumetric water production rate. This colorbox{yellow}{ } method was established in serum bottles for cultivation of methanogens in closed batch cultivation mode. Water production was colorbox{yellow}{estimated} by determining the difference in mass increase in an isobaric setting.This novel CH$_4$ quantification method is an accurate and precise analytical technique, colorbox{yellow}{which can be used} to rapidly screen pure cultures of methanogens regarding colorbox{yellow}{their} volumetric CH$_{4}$ evolution rate. colorbox{yellow}{It} is a cost effective alternative colorbox{yellow}{determining} CH$_4$ production of methanogens over CH$_4$ quantification by using gas chromatography, especially if colorbox{yellow}{ } applied as a high throughput quantification method. colorbox{yellow}{Eventually, the} method can be universally applied for quantification of CH$_4$ production from psychrophilic, thermophilic and hyperthermophilic hydrogenotrophic methanogens.

Stem cell identity and template DNA strand segregation.

Science.gov (United States)

Tajbakhsh, Shahragim

2008-12-01

The quest for stem cell properties to distinguish their identity from that of committed daughters has led to a re-investigation of the notion that DNA strands are not equivalent, and 'immortal' DNA strands are retained in stem cells whereas newly replicated DNA strands segregate to the differentiating daughter cell during mitosis. Whether this process occurs only in stem cells, and also in all tissues, remains unclear. That individual chromosomes can be also partitioned non-randomly raises the question if this phenomenon is related to the immortal DNA hypothesis, and it underscores the need for high-resolution techniques to observe these events empirically. Although initially postulated as a mechanism to avoid DNA replication errors, alternative views including epigenetic regulation and sister chromatid silencing may provide insights into this process.

Regulation of Gene Expression with Double-Stranded Phosphorothioate Oligonucleotides

Science.gov (United States)

Bielinska, Anna; Shivdasani, Ramesh A.; Zhang, Liquan; Nabel, Gary J.

1990-11-01

Alteration of gene transcription by inhibition of specific transcriptional regulatory proteins is necessary for determining how these factors participate in cellular differentiation. The functions of these proteins can be antagonized by several methods, each with specific limitations. Inhibition of sequence-specific DNA-binding proteins was achieved with double-stranded (ds) phosphorothioate oligonucleotides that contained octamer or kappaB consensus sequences. The phosphorothioate oligonucleotides specifically bound either octamer transcription factor or nuclear factor (NF)-kappaB. The modified oligonucleotides accumulated in cells more effectively than standard ds oligonucleotides and modulated gene expression in a specific manner. Octamer-dependent activation of a reporter plasmid or NF-kappaB-dependent activation of the human immunodeficiency virus (HIV) enhancer was inhibited when the appropriate phosphorothioate oligonucleotide was added to a transiently transfected B cell line. Addition of phosphorothioate oligonucleotides that contained the octamer consensus to Jurkat T leukemia cells inhibited interleukin-2 (IL-2) secretion to a degree similar to that observed with a mutated octamer site in the IL-2 enhancer. The ds phosphorothioate oligonucleotides probably compete for binding of specific transcription factors and may provide anti-viral, immunosuppressive, or other therapeutic effects.

Mechanical behavior of Nb{sub 3}Sn strands under transverse electromagnetic loads

Energy Technology Data Exchange (ETDEWEB)

Ru, Yanyun; Yong, Huadong, E-mail: yonghd@lzu.edu.cn; Zhou, Youhe

2016-11-15

Highlights: • The contact forces between the strands under transverse electromagnetic load are considered. • The indentation depth for two-dimensional contact problem is calculated. • The strand damage for different layers is presented. • We discuss the effect of bending wavelength on the Nb{sub 3}Sn strand. - Abstract: The performance of Nb{sub 3}Sn cable-in-conduit-conductor (CICC) shows a significant degradation with increasing electromagnetic load in the International Thermonuclear Experimental Reactor (ITER). As the strand is under the compression caused by adjacent strands, the strand damage may occur for high contact force. In this paper, we present a 3D helix model based on the TEMLOP and FEMCAM to simulate the contact force among the strands under transverse load. The maximum linear strain and indentation depth induced by contact stress are calculated with two-dimensional contact model of cylinder. The numerical results are compared with the experimental values. Finally, the indentation depth is discussed for different layers and cross angles based on two models. With the increasing of layer number, the degradation of performance will occur. The short bending wavelength leads to small indentation depth.

Sub-Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single-Molecule FRET.

Science.gov (United States)

Baltierra-Jasso, Laura E; Morten, Michael J; Magennis, Steven W

2018-03-05

Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here, we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constant of 10 m -1  s -1 . We also followed the displacement from a DNA three-way junction (3WJ) by ssDNA. The presence of three internal mismatched bases in the middle of the invading strand did not prevent displacement from the 3WJ, but reduced the second-order rate constant by about 50 %. We attribute strand exchange in the dsDNA and 3WJ to a zero-toehold pathway from the blunt-ended duplex arms. The single-molecule approach demonstrated here will be useful for studying complex DNA networks. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Outcome quantification using SPHARM-PDM toolbox in orthognathic surgery

Science.gov (United States)

Cevidanes, Lucia; Zhu, HongTu; Styner, Martin

2011-01-01

Purpose Quantification of surgical outcomes in longitudinal studies has led to significant progress in the treatment of dentofacial deformity, both by offering options to patients who might not otherwise have been recommended for treatment and by clarifying the selection of appropriate treatment methods. Most existing surgical treatments have not been assessed in a systematic way. This paper presents the quantification of surgical outcomes in orthognathic surgery via our localized shape analysis framework. Methods In our setting, planning and surgical simulation is performed using the surgery planning software CMFapp. We then employ the SPHARM-PDM to measure the difference between pre-surgery and virtually simulated post-surgery models. This SPHARM-PDM shape framework is validated for use with craniofacial structures via simulating known 3D surgical changes within CMFapp. Results Our results show that SPHARM-PDM analysis accurately measures surgical displacements, compared with known displacement values. Visualization of color maps of virtually simulated surgical displacements describe corresponding surface distances that precisely describe location of changes, and difference vectors indicate directionality and magnitude of changes. Conclusions SPHARM-PDM-based quantification of surgical outcome is feasible. When compared to prior solutions, our method has the potential to make the surgical planning process more flexible, increase the level of detail and accuracy of the plan, yield higher operative precision and control and enhance the follow-up and documentation of clinical cases. PMID:21161693

Quantification of viral DNA during HIV-1 infection: A review of relevant clinical uses and laboratory methods.

Science.gov (United States)

Alidjinou, E K; Bocket, L; Hober, D

2015-02-01

Effective antiretroviral therapy usually leads to undetectable HIV-1 RNA in the plasma. However, the virus persists in some cells of infected patients as various DNA forms, both integrated and unintegrated. This reservoir represents the greatest challenge to the complete cure of HIV-1 infection and its characteristics highly impact the course of the disease. The quantification of HIV-1 DNA in blood samples constitutes currently the most practical approach to measure this residual infection. Real-time quantitative PCR (qPCR) is the most common method used for HIV-DNA quantification and many strategies have been developed to measure the different forms of HIV-1 DNA. In the literature, several "in-house" PCR methods have been used and there is a need for standardization to have comparable results. In addition, qPCR is limited for the precise quantification of low levels by background noise. Among new assays in development, digital PCR was shown to allow an accurate quantification of HIV-1 DNA. Total HIV-1 DNA is most commonly measured in clinical routine. The absolute quantification of proviruses and unintegrated forms is more often used for research purposes. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Sequence-specific capture of protein-DNA complexes for mass spectrometric protein identification.

Directory of Open Access Journals (Sweden)

Cheng-Hsien Wu

Full Text Available The regulation of gene transcription is fundamental to the existence of complex multicellular organisms such as humans. Although it is widely recognized that much of gene regulation is controlled by gene-specific protein-DNA interactions, there presently exists little in the way of tools to identify proteins that interact with the genome at locations of interest. We have developed a novel strategy to address this problem, which we refer to as GENECAPP, for Global ExoNuclease-based Enrichment of Chromatin-Associated Proteins for Proteomics. In this approach, formaldehyde cross-linking is employed to covalently link DNA to its associated proteins; subsequent fragmentation of the DNA, followed by exonuclease digestion, produces a single-stranded region of the DNA that enables sequence-specific hybridization capture of the protein-DNA complex on a solid support. Mass spectrometric (MS analysis of the captured proteins is then used for their identification and/or quantification. We show here the development and optimization of GENECAPP for an in vitro model system, comprised of the murine insulin-like growth factor-binding protein 1 (IGFBP1 promoter region and FoxO1, a member of the forkhead rhabdomyosarcoma (FoxO subfamily of transcription factors, which binds specifically to the IGFBP1 promoter. This novel strategy provides a powerful tool for studies of protein-DNA and protein-protein interactions.

Performance optimization of internal tin process multifilamentary Nb{sub 3}Sn strands

Energy Technology Data Exchange (ETDEWEB)

Zhang, Ke, E-mail: zhangke@c-wst.com [State Key Laboratory of Solidification Processing, Northwestern Polytechnical University, Xi’an, Shaanxi 710072 (China); Western Superconducting Technologies Co. Ltd, Xi’an, Shaanxi 710018 (China); State Engineering Lab. of Superconducting Material Preparation, Xi’an, Shaanxi 710018 (China); Zhang, Pingxiang [State Key Laboratory of Solidification Processing, Northwestern Polytechnical University, Xi’an, Shaanxi 710072 (China); Western Superconducting Technologies Co. Ltd, Xi’an, Shaanxi 710018 (China); State Engineering Lab. of Superconducting Material Preparation, Xi’an, Shaanxi 710018 (China); Northwest Institute for Non-ferrous Metal Research, Xi’an, Shaanxi 710016 (China); Shi, Yigong; Liu, Jianwei; Gao, Huixian; Li, Jianfeng; Liu, Xianghong; Feng, Yong [Western Superconducting Technologies Co. Ltd, Xi’an, Shaanxi 710018 (China); State Engineering Lab. of Superconducting Material Preparation, Xi’an, Shaanxi 710018 (China)

2016-11-01

Highlights: • J{sub c} increases by adding Sn cores in the interspaces, hysteresis loss increases at the same time. • Enlarger the number of filaments with the same Sn proportion will increase J{sub c} and hysteresis loss. • Cu split adopted in each sub-element leads to a low hysteresis loss. - Abstract: In this study, internal tin process Nb{sub 3}Sn strands for the International Thermo-nuclear Experimental Reactor are developed in Western Superconducting Technologies. Short cable-in-conduit conductors manufactured with these strands have been qualified. Mass production is accomplished, and all the performances of the strands meet the requirement of ITER project. Besides, more researches on the effect of strand design on critical current density and hysteresis loss for Nb{sub 3}Sn strands are carried out. It is found that critical current density can be enhanced by reducing filament diameter and increasing Sn content, and hysteresis loss of the strands can be significantly reduced through Cu split adopted in sub-element.

Metallographic investigation of fracture behavior in ITER-style Nb$_{3}$Sn superconducting strands

CERN Document Server

Jewell, M C; Larbalestier, D C; Nijhuis, A

2009-01-01

In this work we specify the extent to which fracture in two ITER-style Nb$_{3}$Sn composite strands occurs in a collective or individual manner, under mechanical tension and bending from the TARSIS apparatus at the University of Twente. A bronze-route strand from European Advanced Superconductors (EAS), which has very uniform, well-spaced filaments, has a widely distributed (200 μm) fracture field and exhibits a composite of individual and collective cracks. An internal tin strand from Oxford Instruments – Superconducting Technology (OST) demonstrates much more localized, collective fracture behavior. The filaments in this strand are about four times larger (in area) than the filaments in the EAS strand, and also agglomerate significantly during heat treatment upon conversion of the Nb to Nb$_{3}$Sn. These results demonstrate that the architecture of the strand can play a significant role in determining the mechanical toughness of the composite, and that strand design should incorporate mechanical consider...