Genes and Junk in Plant Mitochondria—Repair Mechanisms and Selection

PubMed Central

Christensen, Alan C.

2014-01-01

Plant mitochondrial genomes have very low mutation rates. In contrast, they also rearrange and expand frequently. This is easily understood if DNA repair in genes is accomplished by accurate mechanisms, whereas less accurate mechanisms including nonhomologous end joining or break-induced replication are used in nongenes. An important question is how different mechanisms of repair predominate in coding and noncoding DNA, although one possible mechanism is transcription-coupled repair (TCR). This work tests the predictions of TCR and finds no support for it. Examination of the mutation spectra and rates in genes and junk reveals what DNA repair mechanisms are available to plant mitochondria, and what selective forces act on the repair products. A model is proposed that mismatches and other DNA damages are repaired by converting them into double-strand breaks (DSBs). These can then be repaired by any of the DSB repair mechanisms, both accurate and inaccurate. Natural selection will eliminate coding regions repaired by inaccurate mechanisms, accounting for the low mutation rates in genes, whereas mutations, rearrangements, and expansions generated by inaccurate repair in noncoding regions will persist. Support for this model includes the structure of the mitochondrial mutS homolog in plants, which is fused to a double-strand endonuclease. The model proposes that plant mitochondria do not distinguish a damaged or mismatched DNA strand from the undamaged strand, they simply cut both strands and perform homology-based DSB repair. This plant-specific strategy for protecting future generations from mitochondrial DNA damage has the side effect of genome expansions and rearrangements. PMID:24904012

Multi-laboratory comparison of quantitative PCR assays for detection and quantification of Fusarium virguliforme from soybean roots and soil

USDA-ARS?s Scientific Manuscript database

Accurate identification and quantification of Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, within root tissue and soil are important tasks. Several quantitative PCR (qPCR) assays have been developed but there are no reports comparing their use in sensitive and specific...

SPECIES SPECIFIC DIETARY ARSENIC EXPOSURE ASSESSMENT: THE NEED TO ESTIMATE BIOACCESSIBILITY AND ASSESSING THE IMPLIED PRESYSTEMIC METABOLISM IMPLICATIONS

EPA Science Inventory

The chemical form specific toxicity of arsenic dictates the need for species specific quantification in order to accurately assess the risk from an exposure. The literature has begun to produce preliminary species specific databases for certain dietary sources, but a quantitativ...

Infectious helminth ova in wastewater and sludge: A review on public health issues and current quantification practices.

PubMed

Gyawali, P

2018-02-01

Raw and partially treated wastewater has been widely used to maintain the global water demand. Presence of viable helminth ova and larvae in the wastewater raised significant public health concern especially when used for agriculture and aquaculture. Depending on the prevalence of helminth infections in communities, up to 1.0 × 10 3 ova/larvae can be presented per litre of wastewater and 4 gm (dry weight) of sludge. Multi-barrier approaches including pathogen reduction, risk assessment, and exposure reduction have been suggested by health regulators to minimise the potential health risk. However, with a lack of a sensitive and specific method for the quantitative detection of viable helminth ova from wastewater, an accurate health risk assessment is difficult to achieve. As a result, helminth infections are difficult to control from the communities despite two decades of global effort (mass drug administration). Molecular methods can be more sensitive and specific than currently adapted culture-based and vital stain methods. The molecular methods, however, required more and thorough investigation for its ability with accurate quantification of viable helminth ova/larvae from wastewater and sludge samples. Understanding different cell stages and corresponding gene copy numbers is pivotal for accurate quantification of helminth ova/larvae in wastewater samples. Identifying specific genetic markers including protein, lipid, and metabolites using multiomics approach could be utilized for cheap, rapid, sensitive, specific and point of care detection tools for helminth ova and larva in the wastewater.

Background Signal as an in Situ Predictor of Dopamine Oxidation Potential: Improving Interpretation of Fast-Scan Cyclic Voltammetry Data.

PubMed

Meunier, Carl J; Roberts, James G; McCarty, Gregory S; Sombers, Leslie A

2017-02-15

Background-subtracted fast-scan cyclic voltammetry (FSCV) has emerged as a powerful analytical technique for monitoring subsecond molecular fluctuations in live brain tissue. Despite increasing utilization of FSCV, efforts to improve the accuracy of quantification have been limited due to the complexity of the technique and the dynamic recording environment. It is clear that variable electrode performance renders calibration necessary for accurate quantification; however, the nature of in vivo measurements can make conventional postcalibration difficult, or even impossible. Analyte-specific voltammograms and scaling factors that are critical for quantification can shift or fluctuate in vivo. This is largely due to impedance changes, and the effects of impedance on these measurements have not been characterized. We have previously reported that the background current can be used to predict electrode-specific scaling factors in situ. In this work, we employ model circuits to investigate the impact of impedance on FSCV measurements. Additionally, we take another step toward in situ electrode calibration by using the oxidation potential of quinones on the electrode surface to accurately predict the oxidation potential for dopamine at any point in an electrochemical experiment, as both are dependent on impedance. The model, validated both in adrenal slice and live brain tissue, enables information encoded in the shape of the background voltammogram to determine electrochemical parameters that are critical for accurate quantification. This improves data interpretation and provides a significant next step toward more automated methods for in vivo data analysis.

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.

PubMed

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.

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

PubMed

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.

Recommendations for Accurate Resolution of Gene and Isoform Allele-Specific Expression in RNA-Seq Data

PubMed Central

Wood, David L. A.; Nones, Katia; Steptoe, Anita; Christ, Angelika; Harliwong, Ivon; Newell, Felicity; Bruxner, Timothy J. C.; Miller, David; Cloonan, Nicole; Grimmond, Sean M.

2015-01-01

Genetic variation modulates gene expression transcriptionally or post-transcriptionally, and can profoundly alter an individual’s phenotype. Measuring allelic differential expression at heterozygous loci within an individual, a phenomenon called allele-specific expression (ASE), can assist in identifying such factors. Massively parallel DNA and RNA sequencing and advances in bioinformatic methodologies provide an outstanding opportunity to measure ASE genome-wide. In this study, matched DNA and RNA sequencing, genotyping arrays and computationally phased haplotypes were integrated to comprehensively and conservatively quantify ASE in a single human brain and liver tissue sample. We describe a methodological evaluation and assessment of common bioinformatic steps for ASE quantification, and recommend a robust approach to accurately measure SNP, gene and isoform ASE through the use of personalized haplotype genome alignment, strict alignment quality control and intragenic SNP aggregation. Our results indicate that accurate ASE quantification requires careful bioinformatic analyses and is adversely affected by sample specific alignment confounders and random sampling even at moderate sequence depths. We identified multiple known and several novel ASE genes in liver, including WDR72, DSP and UBD, as well as genes that contained ASE SNPs with imbalance direction discordant with haplotype phase, explainable by annotated transcript structure, suggesting isoform derived ASE. The methods evaluated in this study will be of use to researchers performing highly conservative quantification of ASE, and the genes and isoforms identified as ASE of interest to researchers studying those loci. PMID:25965996

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

PubMed

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.

Hairpin Bisulfite Sequencing: Synchronous Methylation Analysis on Complementary DNA Strands of Individual Chromosomes.

PubMed

Giehr, Pascal; Walter, Jörn

2018-01-01

The accurate and quantitative detection of 5-methylcytosine is of great importance in the field of epigenetics. The method of choice is usually bisulfite sequencing because of the high resolution and the possibility to combine it with next generation sequencing. Nevertheless, also this method has its limitations. Following the bisulfite treatment DNA strands are no longer complementary such that in a subsequent PCR amplification the DNA methylation patterns information of only one of the two DNA strand is preserved. Several years ago Hairpin Bisulfite sequencing was developed as a method to obtain the pattern information on complementary DNA strands. The method requires fragmentation (usually by enzymatic cleavage) of genomic DNA followed by a covalent linking of both DNA strands through ligation of a short DNA hairpin oligonucleotide to both strands. The ligated covalently linked dsDNA products are then subjected to a conventional bisulfite treatment during which all unmodified cytosines are converted to uracils. During the treatment the DNA is denatured forming noncomplementary ssDNA circles. These circles serve as a template for a locus specific PCR to amplify chromosomal patterns of the region of interest. As a result one ends up with a linearized product, which contains the methylation information of both complementary DNA strands.

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

PubMed

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.

Novel quantitative real-time LCR for the sensitive detection of SNP frequencies in pooled DNA: method development, evaluation and application.

PubMed

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-19

Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food.

Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application

PubMed Central

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-01

Background Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. Methods The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. Conclusions The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. Significance The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food. PMID:21283808

Carotenoid Extraction and Quantification from Capsicum annuum.

PubMed

Richins, Richard D; Kilcrease, James; Rodgriguez-Uribe, Laura; O'Connell, Mary A

2014-10-05

Carotenoids are ubiquitous pigments that play key roles in photosynthesis and also accumulate to high levels in fruit and flowers. Specific carotenoids play essential roles in human health as these compounds are precursors for Vitamin A; other specific carotenoids are important sources of macular pigments and all carotenoids are important anti-oxidants. Accurate determination of the composition and concentration of this complex set of natural products is therefore important in many different scientific areas. One of the richest sources of these compounds is the fruit of Capsicum ; these red, yellow and orange fruit accumulate multiple carotenes and xanthophylls. This report describes the detailed method for the extraction and quantification of specific carotenes and xanthophylls.

Transcriptome analysis by strand-specific sequencing of complementary DNA

PubMed Central

Parkhomchuk, Dmitri; Borodina, Tatiana; Amstislavskiy, Vyacheslav; Banaru, Maria; Hallen, Linda; Krobitsch, Sylvia; Lehrach, Hans; Soldatov, Alexey

2009-01-01

High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online. PMID:19620212

Transcriptome analysis by strand-specific sequencing of complementary DNA.

PubMed

Parkhomchuk, Dmitri; Borodina, Tatiana; Amstislavskiy, Vyacheslav; Banaru, Maria; Hallen, Linda; Krobitsch, Sylvia; Lehrach, Hans; Soldatov, Alexey

2009-10-01

High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online.

Fluorescent quantification of melanin.

PubMed

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.

STEM VQ Method, Using Scanning Transmission Electron Microscopy (STEM) for Accurate Virus Quantification

DTIC Science & Technology

2017-02-02

Corresponding Author Abstract Accurate virus quantification is sought, but a perfect method still eludes the scientific community. Electron...unlimited. UNCLASSIFIED 2 provides morphology data and counts all viral particles, including partial or noninfectious particles; however, EM methods ...consistent, reproducible virus quantification method called Scanning Transmission Electron Microscopy – Virus Quantification (STEM-VQ) which simplifies

Methods to Detect Nitric Oxide and its Metabolites in Biological Samples

PubMed Central

Bryan, Nathan S.; Grisham, Matthew B.

2007-01-01

Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. NO is involved in many physiological processes including regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification is critical to understanding health and disease. Due to the extremely short physiological half life of this gaseous free radical, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of NO metabolites in biological samples provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The methods described in this review is not an exhaustive or comprehensive discussion of all methods available for the detection of NO but rather a description of the most commonly used and practical methods which allow accurate and sensitive quantification of NO products/metabolites in multiple biological matrices under normal physiological conditions. PMID:17664129

Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR.

PubMed

Sidstedt, Maja; Hedman, Johannes; Romsos, Erica L; Waitara, Leticia; Wadsö, Lars; Steffen, Carolyn R; Vallone, Peter M; Rådström, Peter

2018-04-01

Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles. Graphical abstract PCR inhibition mechanisms of hemoglobin and immunoglobulin G (IgG). Cq quantification cycle, dsDNA double-stranded DNA, ssDNA single-stranded DNA.

In-Gel Stable-Isotope Labeling (ISIL): a strategy for mass spectrometry-based relative quantification.

PubMed

Asara, John M; Zhang, Xiang; Zheng, Bin; Christofk, Heather H; Wu, Ning; Cantley, Lewis C

2006-01-01

Most proteomics approaches for relative quantification of protein expression use a combination of stable-isotope labeling and mass spectrometry. Traditionally, researchers have used difference gel electrophoresis (DIGE) from stained 1D and 2D gels for relative quantification. While differences in protein staining intensity can often be visualized, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. A method is presented for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using In-gel Stable-Isotope Labeling (ISIL). Proteins extracted from any source (tissue, cell line, immunoprecipitate, etc.), treated under two experimental conditions, are resolved in separate lanes by gel electrophoresis. The regions of interest (visualized by staining) are reacted separately with light versus heavy isotope-labeled reagents, and the gel slices are then mixed and digested with proteases. The resulting peptides are then analyzed by LC-MS to determine relative abundance of light/heavy isotope pairs and analyzed by LC-MS/MS for identification of sequence and modifications. The strategy compares well with other relative quantification strategies, and in silico calculations reveal its effectiveness as a global relative quantification strategy. An advantage of ISIL is that visualization of gel differences can be used as a first quantification step followed by accurate and sensitive protein level stable-isotope labeling and mass spectrometry-based relative quantification.

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

PubMed

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.

Real-time PCR for differential quantification of CVI988 vaccine virus and virulent strains of Marek’s disease virus

PubMed Central

Baigent, Susan J.; Nair, Venugopal K.; Le Galludec, Hervé

2016-01-01

CVI988/Rispens vaccine, the ‘gold standard’ vaccine against Marek’s disease in poultry, is not easily distinguishable from virulent strains of Marek’s disease herpesvirus (MDV). Accurate differential measurement of CVI988 and virulent MDV is commercially important to confirm successful vaccination, to diagnose Marek’s disease, and to investigate causes of vaccine failure. A real-time quantitative PCR assay to distinguish CVI988 and virulent MDV based on a consistent single nucleotide polymorphism in the pp38 gene, was developed, optimised and validated using common primers to amplify both viruses, but differential detection of PCR products using two short probes specific for either CVI988 or virulent MDV. Both probes showed perfect specificity for three commercial preparations of CVI988 and 12 virulent MDV strains. Validation against BAC-sequence-specific and US2-sequence-specific q-PCR, on spleen samples from experimental chickens co-infected with BAC-cloned pCVI988 and wild-type virulent MDV, demonstrated that CVI988 and virulent MDV could be quantified very accurately. The assay was then used to follow kinetics of replication of commercial CVI988 and virulent MDV in feather tips and blood of vaccinated and challenged experimental chickens. The assay is a great improvement in enabling accurate differential quantification of CVI988 and virulent MDV over a biologically relevant range of virus levels. PMID:26973285

BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks.

PubMed

Yan, Winston X; Mirzazadeh, Reza; Garnerone, Silvano; Scott, David; Schneider, Martin W; Kallas, Tomasz; Custodio, Joaquin; Wernersson, Erik; Li, Yinqing; Gao, Linyi; Federova, Yana; Zetsche, Bernd; Zhang, Feng; Bienko, Magda; Crosetto, Nicola

2017-05-12

Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.

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

PubMed

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.

Quantification of transcription factor-DNA binding affinity in a living cell

PubMed Central

Belikov, Sergey; Berg, Otto G.; Wrange, Örjan

2016-01-01

The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [3H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element. PMID:26657626

Fully integrated graphene electronic biosensor for label-free detection of lead (II) ion based on G-quadruplex structure-switching.

PubMed

Li, Yijun; Wang, Cheng; Zhu, Yibo; Zhou, Xiaohong; Xiang, Yu; He, Miao; Zeng, Siyu

2017-03-15

This work presents a fully integrated graphene field-effect transistor (GFET) biosensor for the label-free detection of lead ions (Pb 2+ ) in aqueous-media, which first implements the G-quadruplex structure-switching biosensing principle in graphene nanoelectronics. We experimentally illustrate the biomolecular interplay that G-rich DNA single-strands with one-end confined on graphene surface can specifically interact with Pb 2+ ions and switch into G-quadruplex structures. Since the structure-switching of electrically charged DNA strands can disrupt the charge distribution in the vicinity of graphene surface, the carrier equilibrium in graphene sheet might be altered, and manifested by the conductivity variation of GFET. The experimental data and theoretical analysis show that our devices are capable of the label-free and specific quantification of Pb 2+ with a detection limit down to 163.7ng/L. These results first verify the signaling principle competency of G-quadruplex structure-switching in graphene electronic biosensors. Combining with the advantages of the compact device structure and convenient electrical signal, a label-free GFET biosensor for Pb 2+ monitoring is enabled with promising application potential. Copyright © 2016 Elsevier B.V. All rights reserved.

Simultaneous quantification of withanolides in Withania somnifera by a validated high-performance thin-layer chromatographic method.

PubMed

Srivastava, Pooja; Tiwari, Neerja; Yadav, Akhilesh K; Kumar, Vijendra; Shanker, Karuna; Verma, Ram K; Gupta, Madan M; Gupta, Anil K; Khanuja, Suman P S

2008-01-01

This paper describes a sensitive, selective, specific, robust, and validated densitometric high-performance thin-layer chromatographic (HPTLC) method for the simultaneous determination of 3 key withanolides, namely, withaferin-A, 12-deoxywithastramonolide, and withanolide-A, in Ashwagandha (Withania somnifera) plant samples. The separation was performed on aluminum-backed silica gel 60F254 HPTLC plates using dichloromethane-methanol-acetone-diethyl ether (15 + 1 + 1 + 1, v/v/v/v) as the mobile phase. The withanolides were quantified by densitometry in the reflection/absorption mode at 230 nm. Precise and accurate quantification could be performed in the linear working concentration range of 66-330 ng/band with good correlation (r2 = 0.997, 0.999, and 0.996, respectively). The method was validated for recovery, precision, accuracy, robustness, limit of detection, limit of quantitation, and specificity according to International Conference on Harmonization guidelines. Specificity of quantification was confirmed using retention factor (Rf) values, UV-Vis spectral correlation, and electrospray ionization mass spectra of marker compounds in sample tracks.

pyQms enables universal and accurate quantification of mass spectrometry data.

PubMed

Leufken, Johannes; Niehues, Anna; Sarin, L Peter; Wessel, Florian; Hippler, Michael; Leidel, Sebastian A; Fufezan, Christian

2017-10-01

Quantitative mass spectrometry (MS) is a key technique in many research areas (1), including proteomics, metabolomics, glycomics, and lipidomics. Because all of the corresponding molecules can be described by chemical formulas, universal quantification tools are highly desirable. Here, we present pyQms, an open-source software for accurate quantification of all types of molecules measurable by MS. pyQms uses isotope pattern matching that offers an accurate quality assessment of all quantifications and the ability to directly incorporate mass spectrometer accuracy. pyQms is, due to its universal design, applicable to every research field, labeling strategy, and acquisition technique. This opens ultimate flexibility for researchers to design experiments employing innovative and hitherto unexplored labeling strategies. Importantly, pyQms performs very well to accurately quantify partially labeled proteomes in large scale and high throughput, the most challenging task for a quantification algorithm. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed

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.

Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution

EPA Science Inventory

Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described cow feces-spec...

Ultraaccurate genome sequencing and haplotyping of single human cells.

PubMed

Chu, Wai Keung; Edge, Peter; Lee, Ho Suk; Bansal, Vikas; Bafna, Vineet; Huang, Xiaohua; Zhang, Kun

2017-11-21

Accurate detection of variants and long-range haplotypes in genomes of single human cells remains very challenging. Common approaches require extensive in vitro amplification of genomes of individual cells using DNA polymerases and high-throughput short-read DNA sequencing. These approaches have two notable drawbacks. First, polymerase replication errors could generate tens of thousands of false-positive calls per genome. Second, relatively short sequence reads contain little to no haplotype information. Here we report a method, which is dubbed SISSOR (single-stranded sequencing using microfluidic reactors), for accurate single-cell genome sequencing and haplotyping. A microfluidic processor is used to separate the Watson and Crick strands of the double-stranded chromosomal DNA in a single cell and to randomly partition megabase-size DNA strands into multiple nanoliter compartments for amplification and construction of barcoded libraries for sequencing. The separation and partitioning of large single-stranded DNA fragments of the homologous chromosome pairs allows for the independent sequencing of each of the complementary and homologous strands. This enables the assembly of long haplotypes and reduction of sequence errors by using the redundant sequence information and haplotype-based error removal. We demonstrated the ability to sequence single-cell genomes with error rates as low as 10 -8 and average 500-kb-long DNA fragments that can be assembled into haplotype contigs with N50 greater than 7 Mb. The performance could be further improved with more uniform amplification and more accurate sequence alignment. The ability to obtain accurate genome sequences and haplotype information from single cells will enable applications of genome sequencing for diverse clinical needs. Copyright © 2017 the Author(s). Published by PNAS.

Rapid, cost-effective and accurate quantification of Yucca schidigera Roezl. steroidal saponins using HPLC-ELSD method.

PubMed

Tenon, Mathieu; Feuillère, Nicolas; Roller, Marc; Birtić, Simona

2017-04-15

Yucca GRAS-labelled saponins have been and are increasingly used in food/feed, pharmaceutical or cosmetic industries. Existing techniques presently used for Yucca steroidal saponin quantification remain either inaccurate and misleading or accurate but time consuming and cost prohibitive. The method reported here addresses all of the above challenges. HPLC/ELSD technique is an accurate and reliable method that yields results of appropriate repeatability and reproducibility. This method does not over- or under-estimate levels of steroidal saponins. HPLC/ELSD method does not require each and every pure standard of saponins, to quantify the group of steroidal saponins. The method is a time- and cost-effective technique that is suitable for routine industrial analyses. HPLC/ELSD methods yield a saponin fingerprints specific to the plant species. As the method is capable of distinguishing saponin profiles from taxonomically distant species, it can unravel plant adulteration issues. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Structural Characterization and Absolute Quantification of Microcystin Peptides Using Collision-Induced and Ultraviolet Photo-Dissociation Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Attard, Troy J.; Carter, Melissa D.; Fang, Mengxuan; Johnson, Rudolph C.; Reid, Gavin E.

2018-05-01

Microcystin (MC) peptides produced by cyanobacteria pose a hepatotoxic threat to human health upon ingestion from contaminated drinking water. While rapid MC identification and quantification in contaminated body fluids or tissue samples is important for patient treatment and outcomes, conventional immunoassay-based measurement strategies typically lack the specificity required for unambiguous determination of specific MC variants, whose toxicity can significantly vary depending on their structures. Furthermore, the unambiguous identification and accurate quantitation of MC variants using tandem mass spectrometry (MS/MS)-based methods can be limited due to a current lack of appropriate stable isotope-labeled internal standards. To address these limitations, we have systematically examined here the sequence and charge state dependence to the formation and absolute abundance of both "global" and "variant-specific" product ions from representative MC-LR, MC-YR, MC-RR, and MC-LA peptides, using higher-energy collisional dissociation (HCD)-MS/MS, ion-trap collision-induced dissociation (CID)-MS/MS and CID-MS3, and 193 nm ultraviolet photodissociation (UPVD)-MS/MS. HCD-MS/MS was found to provide the greatest detection sensitivity for both global and variant-specific product ions in each of the MC variants, except for MC-YR where a variant-specific product uniquely formed via UPVD-MS/MS was observed with the greatest absolute abundance. A simple methodology for the preparation and characterization of 18O-stable isotope-labeled MC reference materials for use as internal standards was also developed. Finally, we have demonstrated the applicability of the methods developed herein for absolute quantification of MC-LR present in human urine samples, using capillary scale liquid chromatography coupled with ultra-high resolution / accurate mass spectrometry and HCD-MS/MS.

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

PubMed

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 < 1.5%). Third, 13 C-IS based lignin quantification was applied to four common poaceous biomass sources (wheat straw, barley straw, 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.

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

PubMed Central

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 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 13C 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, 12C and 13C lignin were isolated from nonlabeled and uniformly 13C 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, 13C-IS based lignin quantification by py-GC-SIM-MS was validated in reconstituted biomass model systems with known contents of the 12C lignin analogue and was shown to be extremely accurate (>99.9%, R2 > 0.999) and precise (RSD < 1.5%). Third, 13C-IS based lignin quantification was applied to four common poaceous biomass sources (wheat straw, barley straw, 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. PMID:28926698

Quantification of intestinal bacterial populations by real-time PCR with a universal primer set and minor groove binder probes: a global approach to the enteric flora.

PubMed

Ott, Stephan J; Musfeldt, Meike; Ullmann, Uwe; Hampe, Jochen; Schreiber, Stefan

2004-06-01

The composition of the human intestinal flora is important for the health status of the host. The global composition and the presence of specific pathogens are relevant to the effects of the flora. Therefore, accurate quantification of all major bacterial populations of the enteric flora is needed. A TaqMan real-time PCR-based method for the quantification of 20 dominant bacterial species and groups of the intestinal flora has been established on the basis of 16S ribosomal DNA taxonomy. A PCR with conserved primers was used for all reactions. In each real-time PCR, a universal probe for quantification of total bacteria and a specific probe for the species in question were included. PCR with conserved primers and the universal probe for total bacteria allowed relative and absolute quantification. Minor groove binder probes increased the sensitivity of the assays 10- to 100-fold. The method was evaluated by cross-reaction experiments and quantification of bacteria in complex clinical samples from healthy patients. A sensitivity of 10(1) to 10(3) bacterial cells per sample was achieved. No significant cross-reaction was observed. The real-time PCR assays presented may facilitate understanding of the intestinal bacterial flora through a normalized global estimation of the major contributing species.

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

PubMed

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.

Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze)

USDA-ARS?s Scientific Manuscript database

Quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used technique for measuring gene expression levels due to its simplicity, specificity, and sensitivity. Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a...

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

PubMed

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  < k). The improved analytical 'quantification of 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.

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

PubMed Central

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

Targeted Quantification of Isoforms of a Thylakoid-Bound Protein: MRM Method Development.

PubMed

Bru-Martínez, Roque; Martínez-Márquez, Ascensión; Morante-Carriel, Jaime; Sellés-Marchart, Susana; Martínez-Esteso, María José; Pineda-Lucas, José Luis; Luque, Ignacio

2018-01-01

Targeted mass spectrometric methods such as selected/multiple reaction monitoring (SRM/MRM) have found intense application in protein detection and quantification which competes with classical immunoaffinity techniques. It provides a universal procedure to develop a fast, highly specific, sensitive, accurate, and cheap methodology for targeted detection and quantification of proteins based on the direct analysis of their surrogate peptides typically generated by tryptic digestion. This methodology can be advantageously applied in the field of plant proteomics and particularly for non-model species since immunoreagents are scarcely available. Here, we describe the issues to take into consideration in order to develop a MRM method to detect and quantify isoforms of the thylakoid-bound protein polyphenol oxidase from the non-model and database underrepresented species Eriobotrya japonica Lindl.

DNA Meter: Energy Tunable, Quantitative Hybridization Assay

PubMed Central

Braunlin, William; Völker, Jens; Plum, G. Eric; Breslauer, Kenneth J.

2015-01-01

We describe a novel hybridization assay that employs a unique class of energy tunable, bulge loop-containing competitor strands (C*) that hybridize to a probe strand (P). Such initial “pre-binding” of a probe strand modulates its effective “availability” for hybridizing to a target site (T). More generally, the assay described here is based on competitive binding equilibria for a common probe strand (P) between such tunable competitor strands (C*) and a target strand (T). We demonstrate that loop variable, energy tunable families of C*P complexes exhibit enhanced discrimination between targets and mismatched targets, thereby reducing false positives/negatives. We refer to a C*P complex between a C* competitor single strand and the probe strand as a “tuning fork,” since the C* strand exhibits branch points (forks) at the duplex-bulge interfaces within the complex. By varying the loop to create families of such “tuning forks,” one can construct C*P “energy ladders” capable of resolving small differences within the target that may be of biological/functional consequence. The methodology further allows quantification of target strand concentrations, a determination heretofore not readily available by conventional hybridization assays. The dual ability of this tunable assay to discriminate and quantitate targets provides the basis for developing a technology we refer to as a “DNA Meter.” Here we present data that establish proof-of-principle for an in solution version of such a DNA Meter. We envision future applications of this tunable assay that incorporate surface bound/spatially resolved DNA arrays to yield enhanced discrimination and sensitivity. PMID:23529692

High-sensitivity MALDI-TOF MS quantification of anthrax lethal toxin for diagnostics and evaluation of medical countermeasures.

PubMed

Boyer, Anne E; Gallegos-Candela, Maribel; Quinn, Conrad P; Woolfitt, Adrian R; Brumlow, Judith O; Isbell, Katherine; Hoffmaster, Alex R; Lins, Renato C; Barr, John R

2015-04-01

Inhalation anthrax has a rapid progression and high fatality rate. Pathology and death from inhalation of Bacillus anthracis spores are attributed to the actions of secreted protein toxins. Protective antigen (PA) binds and imports the catalytic component lethal factor (LF), a zinc endoprotease, and edema factor (EF), an adenylyl cyclase, into susceptible cells. PA-LF is termed lethal toxin (LTx) and PA-EF, edema toxin. As the universal transporter for both toxins, PA is an important target for vaccination and immunotherapeutic intervention. However, its quantification has been limited to methods of relatively low analytic sensitivity. Quantification of LTx may be more clinically relevant than LF or PA alone because LTx is the toxic form that acts on cells. A method was developed for LTx-specific quantification in plasma using anti-PA IgG magnetic immunoprecipitation of PA and quantification of LF activity that co-purified with PA. The method was fast (<4 h total time to detection), sensitive at 0.033 ng/mL LTx in plasma for the fast analysis (0.0075 ng/mL LTx in plasma for an 18 h reaction), precise (6.3-9.9% coefficient of variation), and accurate (0.1-12.7%error; n ≥ 25). Diagnostic sensitivity was 100% (n = 27 animal/clinical cases). Diagnostic specificity was 100% (n = 141). LTx was detected post-antibiotic treatment in 6/6 treated rhesus macaques and 3/3 clinical cases of inhalation anthrax and as long as 8 days post-treatment. Over the course of infection in two rhesus macaques, LTx was first detected at 0.101 and 0.237 ng/mL at 36 h post-exposure and increased to 1147 and 12,107 ng/mL in late-stage anthrax. This demonstrated the importance of LTx as a diagnostic and therapeutic target. This method provides a sensitive, accurate tool for anthrax toxin detection and evaluation of PA-directed therapeutics.

Ultrasensitive liquid chromatography-tandem mass spectrometric methodologies for quantification of five HIV-1 integrase inhibitors in plasma for a microdose clinical trial.

PubMed

Sun, Li; Li, Hankun; Willson, Kenneth; Breidinger, Sheila; Rizk, Matthew L; Wenning, Larissa; Woolf, Eric J

2012-10-16

HIV-1 integrase strand transfer inhibitors are an important class of compounds targeted for the treatment of HIV-1 infection. Microdosing has emerged as an attractive tool to assist in drug candidate screening for clinical development, but necessitates extremely sensitive bioanalytical assays, typically in the pg/mL concentration range. Currently, accelerator mass spectrometry is the predominant tool for microdosing support, which requires a specialized facility and synthesis of radiolabeled compounds. There have been few studies attempted to comprehensively assess a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach in the context of microdosing applications. Herein, we describe the development of automated LC-MS/MS methods to quantify five integrase inhibitors in plasma with the limits of quantification at 1 pg/mL for raltegravir and 2 pg/mL for four proprietary compounds. The assays involved double extractions followed by UPLC coupled with negative ion electrospray MS/MS analysis. All methods were fully validated to the rigor of regulated bioanalysis requirements, with intraday precision between 1.20 and 14.1% and accuracy between 93.8 and 107% at the standard curve concentration range. These methods were successfully applied to a human microdose study and demonstrated to be accurate, reproducible, and cost-effective. Results of the study indicate that raltegravir displayed linear pharmacokinetics between a microdose and a pharmacologically active dose.

Gene expression profiling of human breast tissue samples using SAGE-Seq.

PubMed

Wu, Zhenhua Jeremy; Meyer, Clifford A; Choudhury, Sibgat; Shipitsin, Michail; Maruyama, Reo; Bessarabova, Marina; Nikolskaya, Tatiana; Sukumar, Saraswati; Schwartzman, Armin; Liu, Jun S; Polyak, Kornelia; Liu, X Shirley

2010-12-01

We present a powerful application of ultra high-throughput sequencing, SAGE-Seq, for the accurate quantification of normal and neoplastic mammary epithelial cell transcriptomes. We develop data analysis pipelines that allow the mapping of sense and antisense strands of mitochondrial and RefSeq genes, the normalization between libraries, and the identification of differentially expressed genes. We find that the diversity of cancer transcriptomes is significantly higher than that of normal cells. Our analysis indicates that transcript discovery plateaus at 10 million reads/sample, and suggests a minimum desired sequencing depth around five million reads. Comparison of SAGE-Seq and traditional SAGE on normal and cancerous breast tissues reveals higher sensitivity of SAGE-Seq to detect less-abundant genes, including those encoding for known breast cancer-related transcription factors and G protein-coupled receptors (GPCRs). SAGE-Seq is able to identify genes and pathways abnormally activated in breast cancer that traditional SAGE failed to call. SAGE-Seq is a powerful method for the identification of biomarkers and therapeutic targets in human disease.

Accurate Quantification of T Cells by Measuring Loss of Germline T-Cell Receptor Loci with Generic Single Duplex Droplet Digital PCR Assays.

PubMed

Zoutman, Willem H; Nell, Rogier J; Versluis, Mieke; van Steenderen, Debby; Lalai, Rajshri N; Out-Luiting, Jacoba J; de Lange, Mark J; Vermeer, Maarten H; Langerak, Anton W; van der Velden, Pieter A

2017-03-01

Quantifying T cells accurately in a variety of tissues of benign, inflammatory, or malignant origin can be of great importance in a variety of clinical applications. Flow cytometry and immunohistochemistry are considered to be gold-standard methods for T-cell quantification. However, these methods require fresh, frozen, or fixated cells and tissue of a certain quality. In addition, conventional and droplet digital PCR (ddPCR), whether followed by deep sequencing techniques, have been used to elucidate T-cell content by focusing on rearranged T-cell receptor (TCR) genes. These approaches typically target the whole TCR repertoire, thereby supplying additional information about TCR use. We alternatively developed and validated two novel generic single duplex ddPCR assays to quantify T cells accurately by measuring loss of specific germline TCR loci and compared them with flow cytometry-based quantification. These assays target sequences between the Dδ2 and Dδ3 genes (TRD locus) and Dβ1 and Jβ1.1 genes (TRB locus) that become deleted systematically early during lymphoid differentiation. Because these ddPCR assays require small amounts of DNA instead of freshly isolated, frozen, or fixated material, initially unanalyzable (scarce) specimens can be assayed from now on, supplying valuable information about T-cell content. Our ddPCR method provides a novel and sensitive way for quantifying T cells relatively fast, accurate, and independent of the cellular context. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Strand-Specific Analysis of DNA Synthesis and Proteins Association with DNA Replication Forks in Budding Yeast.

PubMed

Yu, Chuanhe; Gan, Haiyun; Zhang, Zhiguo

2018-01-01

DNA replication initiates at DNA replication origins after unwinding of double-strand DNA(dsDNA) by replicative helicase to generate single-stranded DNA (ssDNA) templates for the continuous synthesis of leading-strand and the discontinuous synthesis of lagging-strand. Therefore, methods capable of detecting strand-specific information will likely yield insight into the association of proteins at leading and lagging strand of DNA replication forks and the regulation of leading and lagging strand synthesis during DNA replication. The enrichment and Sequencing of Protein-Associated Nascent DNA (eSPAN), which measure the relative amounts of proteins at nascent leading and lagging strands of DNA replication forks, is a step-wise procedure involving the chromatin immunoprecipitation (ChIP) of a protein of interest followed by the enrichment of protein-associated nascent DNA through BrdU immunoprecipitation. The isolated ssDNA is then subjected to strand-specific sequencing. This method can detect whether a protein is enriched at leading or lagging strand of DNA replication forks. In addition to eSPAN, two other strand-specific methods, (ChIP-ssSeq), which detects potential protein-ssDNA binding and BrdU-IP-ssSeq, which can measure synthesis of both leading and lagging strand, were developed along the way. These methods can provide strand-specific and complementary information about the association of the target protein with DNA replication forks as well as synthesis of leading and lagging strands genome wide. Below, we describe the detailed eSPAN, ChIP-ssSeq, and BrdU-IP-ssSeq protocols.

Understanding the transmission dynamics of Leishmania donovani to provide robust evidence for interventions to eliminate visceral leishmaniasis in Bihar, India

USDA-ARS?s Scientific Manuscript database

Molecular tools enable the collection of accurate estimates of human blood index (HBI) in Phlebotomus argentipes. The refinement of a metacyclic-specific qPCR assay to identify L. donovani in P. argentipes would enable quantification of the entomological inoculation rate (EIR) for the first time. Li...

Understanding the transmission dynamics of Leishmania donovani to provide robust evidence for interventions to eliminate visceral leishmaniasis in Bihar, India

USDA-ARS?s Scientific Manuscript database

Molecular tools enable the collection of accurate estimates of human blood index (HBI) in P. argentipes. The refinement of a metacyclic-specific qPCR assay to identify L. donovani in P. argentipes would enable quantification of the entomological inoculation rate (EIR) for the first time. Likewise, a...

Resolution and quantification accuracy enhancement of functional delay and sum beamforming for three-dimensional acoustic source identification with solid spherical arrays

NASA Astrophysics Data System (ADS)

Chu, Zhigang; Yang, Yang; Shen, Linbang

2017-05-01

Functional delay and sum (FDAS) is a novel beamforming algorithm introduced for the three-dimensional (3D) acoustic source identification with solid spherical microphone arrays. Being capable of offering significantly attenuated sidelobes with a fast speed, the algorithm promises to play an important role in interior acoustic source identification. However, it presents some intrinsic imperfections, specifically poor spatial resolution and low quantification accuracy. This paper focuses on conquering these imperfections by ridge detection (RD) and deconvolution approach for the mapping of acoustic sources (DAMAS). The suggested methods are referred to as FDAS+RD and FDAS+RD+DAMAS. Both computer simulations and experiments are utilized to validate their effects. Several interesting conclusions have emerged: (1) FDAS+RD and FDAS+RD+DAMAS both can dramatically ameliorate FDAS's spatial resolution and at the same time inherit its advantages. (2) Compared to the conventional DAMAS, FDAS+RD+DAMAS enjoys the same super spatial resolution, stronger sidelobe attenuation capability and more than two hundred times faster speed. (3) FDAS+RD+DAMAS can effectively conquer FDAS's low quantification accuracy. Whether the focus distance is equal to the distance from the source to the array center or not, it can quantify the source average pressure contribution accurately. This study will be of great significance to the accurate and quick localization and quantification of acoustic sources in cabin environments.

Combining Metabolic ¹⁵N Labeling with Improved Tandem MOAC for Enhanced Probing of the Phosphoproteome.

PubMed

Thomas, Martin; Huck, Nicola; Hoehenwarter, Wolfgang; Conrath, Uwe; Beckers, Gerold J M

2015-01-01

In eukaryotic cells many diverse cellular functions are regulated by reversible protein phosphorylation. In recent years, phosphoproteomics has become a powerful tool for studying protein phosphorylation because it enables unbiased localization, and site-specific quantification of in vivo phosphorylation of hundreds of proteins in a single experiment. A common strategy for identifying phosphoproteins and their phosphorylation sites from complex biological samples is the enrichment of phosphopeptides from digested cellular lysates followed by mass spectrometry. However, despite high sensitivity of modern mass spectrometers the large dynamic range of protein abundance and the transient nature of protein phosphorylation remained major pitfalls in MS-based phosphoproteomics. This is particularly true for plants in which the presence of secondary metabolites and endogenous compounds, the overabundance of ribulose-1,5-bisphosphate carboxylase and other components of the photosynthetic apparatus, and the concurrent difficulties in protein extraction necessitate two-step phosphoprotein/phosphopeptide enrichment strategies (Nakagami et al., Plant Cell Physiol 53:118-124, 2012).Approaches for label-free peptide quantification are advantageous due to their low cost and experimental simplicity, but they lack precision. These drawbacks can be overcome by metabolic labeling of whole plants with heavy nitrogen ((15)N) which allows combining two samples very early in the phosphoprotein enrichment workflow. This avoids sample-to-sample variation introduced by the analytical procedures and it results in robust relative quantification values that need no further standardization. The integration of (15)N metabolic labeling into tandem metal-oxide affinity chromatography (MOAC) (Hoehenwarter et al., Mol Cell Proteomics 12:369-380, 2013) presents an improved and highly selective approach for the identification and accurate site-specific quantification of low-abundance phosphoproteins that is based on the successive enrichment of light and heavy nitrogen-labeled phosphoproteins and peptides. This improved strategy combines metabolic labeling of whole plants with the stable heavy nitrogen isotope ((15)N), protein extraction under denaturing conditions, phosphoprotein enrichment using Al(OH)3-based MOAC, and tryptic digest of enriched phosphoproteins followed by TiO2-based MOAC of phosphopeptides and quantitative phosphopeptide measurement by liquid chromatography (LC) and high-resolution accurate mass (HR/AM) mass spectrometry (MS). Thus, tandem MOAC effectively targets the phosphate moiety of phosphoproteins and phosphopeptides and allows probing of the phosphoproteome to unprecedented depth, while (15)N metabolic labeling enables accurate relative quantification of measured peptides and direct comparison between samples.

Simultaneous Quantification of Multiple Alternatively Spliced mRNA Transcripts Using Droplet Digital PCR.

PubMed

Sun, Bing; Zheng, Yun-Ling

2018-01-01

Currently there is no sensitive, precise, and reproducible method to quantitate alternative splicing of mRNA transcripts. Droplet digital™ PCR (ddPCR™) analysis allows for accurate digital counting for quantification of gene expression. Human telomerase reverse transcriptase (hTERT) is one of the essential components required for telomerase activity and for the maintenance of telomeres. Several alternatively spliced forms of hTERT mRNA in human primary and tumor cells have been reported in the literature. Using one pair of primers and two probes for hTERT, four alternatively spliced forms of hTERT (α-/β+, α+/β- single deletions, α-/β- double deletion, and nondeletion α+/β+) were accurately quantified through a novel analysis method via data collected from a single ddPCR reaction. In this chapter, we describe this ddPCR method that enables direct quantitative comparison of four alternatively spliced forms of the hTERT messenger RNA without the need for internal standards or multiple pairs of primers specific for each variant, eliminating the technical variation due to differential PCR amplification efficiency for different amplicons and the challenges of quantification using standard curves. This simple and straightforward method should have general utility for quantifying alternatively spliced gene transcripts.

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

PubMed

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.

Development of strand burner for solid propellant burning rate studies

NASA Astrophysics Data System (ADS)

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

2013-12-01

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.

7 CFR 1755.370 - RUS specification for seven wire galvanized steel strand.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 11 2012-01-01 2012-01-01 false RUS specification for seven wire galvanized steel... steel strand. (a) RUS incorporates by reference ASTM A475-78, Standard Specification for Zinc-Coated Steel Wire Strand, issued May 1978. All seven wire galvanized steel strand purchased after April 1, 1990...

7 CFR 1755.370 - RUS specification for seven wire galvanized steel strand.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 11 2014-01-01 2014-01-01 false RUS specification for seven wire galvanized steel... steel strand. (a) RUS incorporates by reference ASTM A475-78, Standard Specification for Zinc-Coated Steel Wire Strand, issued May 1978. All seven wire galvanized steel strand purchased after April 1, 1990...

7 CFR 1755.370 - RUS specification for seven wire galvanized steel strand.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 11 2013-01-01 2013-01-01 false RUS specification for seven wire galvanized steel... steel strand. (a) RUS incorporates by reference ASTM A475-78, Standard Specification for Zinc-Coated Steel Wire Strand, issued May 1978. All seven wire galvanized steel strand purchased after April 1, 1990...

RNA-Seq for Bacterial Gene Expression.

PubMed

Poulsen, Line Dahl; Vinther, Jeppe

2018-06-01

RNA sequencing (RNA-seq) has become the preferred method for global quantification of bacterial gene expression. With the continued improvements in sequencing technology and data analysis tools, the most labor-intensive and expensive part of an RNA-seq experiment is the preparation of sequencing libraries, which is also essential for the quality of the data obtained. Here, we present a straightforward and inexpensive basic protocol for preparation of strand-specific RNA-seq libraries from bacterial RNA as well as a computational pipeline for the data analysis of sequencing reads. The protocol is based on the Illumina platform and allows easy multiplexing of samples and the removal of sequencing reads that are PCR duplicates. © 2018 by John Wiley & Sons, Inc. © 2018 John Wiley & Sons, Inc.

Electrosynthesis and characterization of nanostructured polyquinone for use in detection and quantification of naturally occurring dsDNA.

PubMed

Hernández, Loreto A; Del Valle, María A; Armijo, Francisco

2016-05-15

The detection of naturally occurring desoxyribonucleic acid (DNA) has become a subject of study by the projections that would generate to be able to sense the genetic material for the detection of future diseases. Bearing this in mind, to provide new measuring strategies, in the current work the preparation of a low-cost electrode, modified with poly(1-amino-9,10-anthraquinone) nanowires using a SiO2 template, is carried out; the assembly is next modified by covalently attaching ssDNA strands. It must be noted that all this is accomplished by using solely electrochemical techniques, according to methodology developed for this purpose. SEM images of the modified surface show high order and homogeneity in the distribution of modified nanowires over the electrode surface. In turn, after the hybridization with its complementary strand, the voltammetric responses enable corroborating the linear relationship between hybridization at different DNA concentrations and normalized current response, obtaining a limit of detection (LOD) 5.7·10(-12)gL(-1) and limit of quantification (LOQ) 1.9·10(-11)gL(-1). The working dynamic range is between 1.4·10(-7) and 8.5·10(-9)gL(-1) with a correlation coefficient 0.9998. The successful obtaining of the modified electrode allows concluding that the high order reached by the nanostructures, guides the subsequent single strand of DNA (ssDNA) covalent attachment, which after hybridization with its complementary strand brings about a considerable current increase. This result allows foreseeing a guaranteed breakthrough with regard to the use of the biosensor in real samples. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed Central

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

PubMed

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.

Development of an optical biosensor based on surface-enhanced Raman scattering for DNA analysis

NASA Astrophysics Data System (ADS)

Yigit, Tugce; Akdogan, Ebru; Karagoz, Isık. Didem; Kahraman, Mehmet

2016-03-01

Rapid, accurate and sensitive DNA analysis is critically important for the diagnostic of genetic diseases. The most common method preferred in practice is fluorescence based microarrays to analyze the DNA. However, there exist some disadvantages related to the above-mentioned method such as the overlapping of the fluorescence emission wavelengths that can diminish in the performance of multiplexing, needed to obtain fluorescence spectra from each dye and photo degradation. In this study, a novel SERS based DNA analysis approach, which is Raman active dye-free and independent of SERS substrate properties, is developed. First, the single strand DNA probe is attached to the SERS substrate and half of the complimentary DNA is attached to gold nanoparticles, as well. We hypothesize that in the presence of target DNA, the complimentary DNA coupled colloids will bind to the SERS substrate surface via hybridization of single strand target DNA. To test this hypothesis, we used UV/Vis spectroscopy, atomic for microscopy (AFM) and dynamic light scattering (DLS). DNA analysis is demonstrated by a peak shift of the certain peak of the small molecules attached to the SERS substrate surface instead of SERS spectrum obtained in the presence of target DNA from the Raman reporter molecules. The degree of peak shifting will be used for the quantification of the target DNA in the sample. Plasmonic properties of SERS substrates and reproducibility issues will not be considerable due to the use of peak shifting instead of peak intensity for the qualitative analysis.

Droplet Digital™ PCR Next-Generation Sequencing Library QC Assay.

PubMed

Heredia, Nicholas J

2018-01-01

Digital PCR is a valuable tool to quantify next-generation sequencing (NGS) libraries precisely and accurately. Accurately quantifying NGS libraries enable accurate loading of the libraries on to the sequencer and thus improve sequencing performance by reducing under and overloading error. Accurate quantification also benefits users by enabling uniform loading of indexed/barcoded libraries which in turn greatly improves sequencing uniformity of the indexed/barcoded samples. The advantages gained by employing the Droplet Digital PCR (ddPCR™) library QC assay includes the precise and accurate quantification in addition to size quality assessment, enabling users to QC their sequencing libraries with confidence.

What controls the hybridization thermodynamics of spherical nucleic acids?

PubMed

Randeria, Pratik S; Jones, Matthew R; Kohlstedt, Kevin L; Banga, Resham J; Olvera de la Cruz, Monica; Schatz, George C; Mirkin, Chad A

2015-03-18

The hybridization of free oligonucleotides to densely packed, oriented arrays of DNA modifying the surfaces of spherical nucleic acid (SNA)-gold nanoparticle conjugates occurs with negative cooperativity; i.e., each binding event destabilizes subsequent binding events. DNA hybridization is thus an ever-changing function of the number of strands already hybridized to the particle. Thermodynamic quantification of this behavior reveals a 3 orders of magnitude decrease in the binding constant for the capture of a free oligonucleotide by an SNA conjugate as the fraction of pre-hybridized strands increases from 0 to ∼30%. Increasing the number of pre-hybridized strands imparts an increasing enthalpic penalty to hybridization that makes binding more difficult, while simultaneously decreasing the entropic penalty to hybridization, which makes binding more favorable. Hybridization of free DNA to an SNA is thus governed by both an electrostatic barrier as the SNA accumulates charge with additional binding events and an effect consistent with allostery, where hybridization at certain sites on an SNA modify the binding affinity at a distal site through conformational changes to the remaining single strands. Leveraging these insights allows for the design of conjugates that hybridize free strands with significantly higher efficiencies, some of which approach 100%.

A sensitive and accurate quantification method for the detection of hepatitis B virus covalently closed circular DNA by the application of a droplet digital polymerase chain reaction amplification system.

PubMed

Mu, Di; Yan, Liang; Tang, Hui; Liao, Yong

2015-10-01

To develop a sensitive and accurate assay system for the quantification of covalently closed circular HBV DNA (cccDNA) for future clinical monitoring of cccDNA fluctuation during antiviral therapy in the liver of infected patients. A droplet digital PCR (ddPCR)-based assay system detected template DNA input at the single copy level (or ~10(-5) pg of plasmid HBV DNA) by using serially diluted plasmid HBV DNA samples. Compared with the conventional quantitative PCR assay in the detection of cccDNA, which required at least 50 ng of template DNA input, a parallel experiment applying a ddPCR system demonstrates that the lowest detection limit of cccDNA from HepG2.215 cellular DNA samples is around 1 ng, which is equivalent to 0.54 ± 0.94 copies of cccDNA. In addition, we demonstrated that the addition of cccDNA-safe exonuclease and utilization of cccDNA-specific primers in the ddPCR assay system significantly improved the detection accuracy of HBV cccDNA from HepG2.215 cellular DNA samples. The ddPCR-based cccDNA detection system is a sensitive and accurate assay for the quantification of cccDNA in HBV-transfected HepG2.215 cellular DNA samples and may represent an important method for future application in monitoring cccDNA fluctuation during antiviral therapy.

Organ-specific SPECT activity calibration using 3D printed phantoms for molecular radiotherapy dosimetry.

PubMed

Robinson, Andrew P; Tipping, Jill; Cullen, David M; Hamilton, David; Brown, Richard; Flynn, Alex; Oldfield, Christopher; Page, Emma; Price, Emlyn; Smith, Andrew; Snee, Richard

2016-12-01

Patient-specific absorbed dose calculations for molecular radiotherapy require accurate activity quantification. This is commonly derived from Single-Photon Emission Computed Tomography (SPECT) imaging using a calibration factor relating detected counts to known activity in a phantom insert. A series of phantom inserts, based on the mathematical models underlying many clinical dosimetry calculations, have been produced using 3D printing techniques. SPECT/CT data for the phantom inserts has been used to calculate new organ-specific calibration factors for (99m) Tc and (177)Lu. The measured calibration factors are compared to predicted values from calculations using a Gaussian kernel. Measured SPECT calibration factors for 3D printed organs display a clear dependence on organ shape for (99m) Tc and (177)Lu. The observed variation in calibration factor is reproduced using Gaussian kernel-based calculation over two orders of magnitude change in insert volume for (99m) Tc and (177)Lu. These new organ-specific calibration factors show a 24, 11 and 8 % reduction in absorbed dose for the liver, spleen and kidneys, respectively. Non-spherical calibration factors from 3D printed phantom inserts can significantly improve the accuracy of whole organ activity quantification for molecular radiotherapy, providing a crucial step towards individualised activity quantification and patient-specific dosimetry. 3D printed inserts are found to provide a cost effective and efficient way for clinical centres to access more realistic phantom data.

Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli.

PubMed

Thomason, Lynn C; Costantino, Nina; Court, Donald L

2016-09-13

Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion. Bacteriophage homologous recombination systems are widely used for in vivo genetic engineering in bacteria. Single- or double-stranded linear DNA substrates containing short flanking homologies to chromosome targets are used to generate precise and accurate genetic modifications when introduced into bacteria expressing phage recombinases. Understanding the molecular mechanism of these recombination systems will facilitate improvements in the technology. Here, two phage-specific systems are shown to require exposure of complementary single-strand homologous targets for efficient recombination; these single-strand regions may be created during DNA replication or by single-strand exonuclease digestion of linear duplex DNA. Previously, in vitro studies reported that these recombinases promote the single-strand annealing of two complementary DNAs and also strand invasion of a single DNA strand into duplex DNA to create a three-stranded region. Here, in vivo experiments show that recombinase-mediated annealing of complementary single-stranded DNA is the predominant recombination pathway in E. coli. Copyright © 2016 Thomason et al.

Establishing Ion Ratio Thresholds Based on Absolute Peak Area for Absolute Protein Quantification using Protein Cleavage Isotope Dilution Mass Spectrometry

PubMed Central

Loziuk, Philip L.; Sederoff, Ronald R.; Chiang, Vincent L.; Muddiman, David C.

2014-01-01

Quantitative mass spectrometry has become central to the field of proteomics and metabolomics. Selected reaction monitoring is a widely used method for the absolute quantification of proteins and metabolites. This method renders high specificity using several product ions measured simultaneously. With growing interest in quantification of molecular species in complex biological samples, confident identification and quantitation has been of particular concern. A method to confirm purity or contamination of product ion spectra has become necessary for achieving accurate and precise quantification. Ion abundance ratio assessments were introduced to alleviate some of these issues. Ion abundance ratios are based on the consistent relative abundance (RA) of specific product ions with respect to the total abundance of all product ions. To date, no standardized method of implementing ion abundance ratios has been established. Thresholds by which product ion contamination is confirmed vary widely and are often arbitrary. This study sought to establish criteria by which the relative abundance of product ions can be evaluated in an absolute quantification experiment. These findings suggest that evaluation of the absolute ion abundance for any given transition is necessary in order to effectively implement RA thresholds. Overall, the variation of the RA value was observed to be relatively constant beyond an absolute threshold ion abundance. Finally, these RA values were observed to fluctuate significantly over a 3 year period, suggesting that these values should be assessed as close as possible to the time at which data is collected for quantification. PMID:25154770

Quantitative, non-invasive imaging of radiation-induced DNA double strand breaks in vivo

PubMed Central

Li, Wenrong; Li, Fang; Huang, Qian; Shen, Jingping; Wolf, Frank; He, Yujun; Liu, Xinjian; Hu, Y. Angela; Bedford, Joel. S.; Li, Chuan-Yuan

2011-01-01

DNA double strand breaks is a major form of DNA damage and a key mechanism through which radiotherapy and some chemotherapeutic agents kill cancer cells. Despite its importance, measuring DNA double strand breaks is still a tedious task that is normally carried out by gel electrophoresis or immunofluorescence staining. Here we report a novel approach to image and quantify DNA double strand breaks in live mammalian cells through bi-fragment luciferase reconstitution. N- and C- terminal fragments of firefly luciferase gene were fused with H2AX and MDC1 genes, respectively. Our strategy was based on the established fact that at the sites of DNA double strand breaks, H2AX protein is phosphoryated and physically associates with the MDC1 protein, thus bringing together N- and C- luciferase fragments and reconstituting luciferase activity. Our strategy allowed serial, non-invasive quantification of DNA double strand breaks in cells irradiated with x-rays and 56Fe ions. Furthermore, it allowed for the evaluation of DNA double strand breaks (DSBs) non-invasively in vivo in irradiated tumors over two weeks. Surprisingly, we detected a second wave of DSB induction in irradiated tumor cells days after radiation exposure in addition to the initial rapid induction of DSBs. We conclude that our new split-luciferase based method for imaging γ-H2AX-MDC1 interaction is a powerful new tool to study DNA double strand break repair kinetics in vivo with considerable advantage for experiments requiring observations over an extended period of time. PMID:21527553

Properties of targeted preamplification in DNA and cDNA quantification.

PubMed

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.

Design of a Sensitive and Selective Electrochemical Aptasensor for the Determination of the Complementary cDNA of miRNA-145 Based on the Intercalation and Electrochemical Reduction of Doxorubicin.

PubMed

Mohamadi, Maryam; Mostafavi, Ali; Torkzadeh-Mahani, Masoud

2017-11-01

The aim of this research was the determination of a microRNA (miRNA) using a DNA electrochemical aptasensor. In this biosensor, the complementary complementary DNA (cDNA) of miRNA-145 (a sense RNA transcript) was the target strand and the cDNA of miRNA-145 was the probe strand. Both cDNAs can be the product of the reverse transcriptase-polymerase chain reaction of miRNA. The proposed aptasensor's function was based on the hybridization of target strands with probes immobilized on the surface of a working electrode and the subsequent intercalation of doxorubicin (DOX) molecules functioning as the electroactive indicators of any double strands that formed. Electrochemical transduction was performed by measuring the cathodic current resulting from the electrochemical reduction of the intercalated molecules at the electrode surface. In the experiment, because many DOX molecules accumulated on each target strand on the electrode surface, amplification was inherently easy, without a need for enzymatic or complicated amplification strategies. The proposed aptasensor also had the excellent ability to regenerate as a result of the melting of the DNA duplex. Moreover, the use of DNA probe strands obviated the challenges of working with an RNA probe, such as sensitivity to RNase enzyme. In addition to the linear relationship between the electrochemical signal and the concentration of the target strands that ranged from 2.0 to 80.0 nM with an LOD of 0.27 nM, the proposed biosensor was clearly capable of distinguishing between complementary (target strand) and noncomplementary sequences. The presented biosensor was successfully applied for the quantification of DNA strands corresponding to miRNA-145 in human serum samples.

A rapid and accurate quantification method for real-time dynamic analysis of cellular lipids during microalgal fermentation processes in Chlorella protothecoides with low field nuclear magnetic resonance.

PubMed

Wang, Tao; Liu, Tingting; Wang, Zejian; Tian, Xiwei; Yang, Yi; Guo, Meijin; Chu, Ju; Zhuang, Yingping

2016-05-01

The rapid and real-time lipid determination can provide valuable information on process regulation and optimization in the algal lipid mass production. In this study, a rapid, accurate and precise quantification method of in vivo cellular lipids of Chlorella protothecoides using low field nuclear magnetic resonance (LF-NMR) was newly developed. LF-NMR was extremely sensitive to the algal lipids with the limits of the detection (LOD) of 0.0026g and 0.32g/L in dry lipid samples and algal broth, respectively, as well as limits of quantification (LOQ) of 0.0093g and 1.18g/L. Moreover, the LF-NMR signal was specifically proportional to the cellular lipids of C. protothecoides, thus the superior regression curves existing in a wide detection range from 0.02 to 0.42g for dry lipids and from 1.12 to 8.97gL(-1) of lipid concentration for in vivo lipid quantification were obtained with all R(2) higher than 0.99, irrespective of the lipid content and fatty acids profile variations. The accuracy of this novel method was further verified to be reliable by comparing lipid quantification results to those obtained by GC-MS. And the relative standard deviation (RSD) of LF-NMR results were smaller than 2%, suggesting the precision of this method. Finally, this method was successfully used in the on-line lipid monitoring during the algal lipid fermentation processes, making it possible for better understanding of the lipid accumulation mechanism and dynamic bioprocess control. Copyright © 2016 Elsevier B.V. All rights reserved.

Development and application of a multi-targeting reference plasmid as calibrator for analysis of five genetically modified soybean events.

PubMed

Pi, Liqun; Li, Xiang; Cao, Yiwei; Wang, Canhua; Pan, Liangwen; Yang, Litao

2015-04-01

Reference materials are important in accurate analysis of genetically modified organism (GMO) contents in food/feeds, and development of novel reference plasmid is a new trend in the research of GMO reference materials. Herein, we constructed a novel multi-targeting plasmid, pSOY, which contained seven event-specific sequences of five GM soybeans (MON89788-5', A2704-12-3', A5547-127-3', DP356043-5', DP305423-3', A2704-12-5', and A5547-127-5') and sequence of soybean endogenous reference gene Lectin. We evaluated the specificity, limit of detection and quantification, and applicability of pSOY in both qualitative and quantitative PCR analyses. The limit of detection (LOD) was as low as 20 copies in qualitative PCR, and the limit of quantification (LOQ) in quantitative PCR was 10 copies. In quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and Lectin assays were higher than 90%, and the squared regression coefficients (R(2)) were more than 0.999. The quantification bias varied from 0.21% to 19.29%, and the relative standard deviations were from 1.08% to 9.84% in simulated samples analysis. All the results demonstrated that the developed multi-targeting plasmid, pSOY, was a credible substitute of matrix reference materials, and could be used as a reliable reference calibrator in the identification and quantification of multiple GM soybean events.

GMO quantification: valuable experience and insights for the future.

PubMed

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.

A purified transcription factor (TIF-IB) binds to essential sequences of the mouse rDNA promoter.

PubMed Central

Clos, J; Buttgereit, D; Grummt, I

1986-01-01

A transcription factor that is specific for mouse rDNA has been partially purified from Ehrlich ascites cells. This factor [designated transcription initiation factor (TIF)-IB] is required for accurate in vitro synthesis of mouse rRNA in addition to RNA polymerase I and another regulatory factor, TIF-IA. TIF-IB activity is present in extracts both from growing and nongrowing cells in comparable amounts. Prebinding competition experiments with wild-type and mutant templates suggest that TIF-IB interacts with the core control element of the rDNA promoter, which is located immediately upstream of the initiation site. The specific binding of TIF-IB to the RNA polymerase I promoter is demonstrated by exonuclease III protection experiments. The 3' border of the sequences protected by TIF-IB is shown to be on the coding strand at position -21 and on the noncoding strand at position -7. The results suggest that direct binding of TIF-IB to sequences in the core promoter element is the mechanism by which this factor imparts promoter selectivity to RNA polymerase I. Images PMID:3456157

A fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp.

PubMed

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.

Spectral performance of a whole-body research photon counting detector CT: quantitative accuracy in derived image sets

NASA Astrophysics Data System (ADS)

Leng, Shuai; Zhou, Wei; Yu, Zhicong; Halaweish, Ahmed; Krauss, Bernhard; Schmidt, Bernhard; Yu, Lifeng; Kappler, Steffen; McCollough, Cynthia

2017-09-01

Photon-counting computed tomography (PCCT) uses a photon counting detector to count individual photons and allocate them to specific energy bins by comparing photon energy to preset thresholds. This enables simultaneous multi-energy CT with a single source and detector. Phantom studies were performed to assess the spectral performance of a research PCCT scanner by assessing the accuracy of derived images sets. Specifically, we assessed the accuracy of iodine quantification in iodine map images and of CT number accuracy in virtual monoenergetic images (VMI). Vials containing iodine with five known concentrations were scanned on the PCCT scanner after being placed in phantoms representing the attenuation of different size patients. For comparison, the same vials and phantoms were also scanned on 2nd and 3rd generation dual-source, dual-energy scanners. After material decomposition, iodine maps were generated, from which iodine concentration was measured for each vial and phantom size and compared with the known concentration. Additionally, VMIs were generated and CT number accuracy was compared to the reference standard, which was calculated based on known iodine concentration and attenuation coefficients at each keV obtained from the U.S. National Institute of Standards and Technology (NIST). Results showed accurate iodine quantification (root mean square error of 0.5 mgI/cc) and accurate CT number of VMIs (percentage error of 8.9%) using the PCCT scanner. The overall performance of the PCCT scanner, in terms of iodine quantification and VMI CT number accuracy, was comparable to that of EID-based dual-source, dual-energy scanners.

Normalized Quantitative Western Blotting Based on Standardized Fluorescent Labeling.

PubMed

Faden, Frederik; Eschen-Lippold, Lennart; Dissmeyer, Nico

2016-01-01

Western blot (WB) analysis is the most widely used method to monitor expression of proteins of interest in protein extracts of high complexity derived from diverse experimental setups. WB allows the rapid and specific detection of a target protein, such as non-tagged endogenous proteins as well as protein-epitope tag fusions depending on the availability of specific antibodies. To generate quantitative data from independent samples within one experiment and to allow accurate inter-experimental quantification, a reliable and reproducible method to standardize and normalize WB data is indispensable. To date, it is a standard procedure to normalize individual bands of immunodetected proteins of interest from a WB lane to other individual bands of so-called housekeeping proteins of the same sample lane. These are usually detected by an independent antibody or colorimetric detection and do not reflect the real total protein of a sample. Housekeeping proteins-assumed to be constitutively expressed mostly independent of developmental and environmental states-can greatly differ in their expression under these various conditions. Therefore, they actually do not represent a reliable reference to normalize the target protein's abundance to the total amount of protein contained in each lane of a blot.Here, we demonstrate the Smart Protein Layers (SPL) technology, a combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer via WB. SPL allows a rapid and highly sensitive protein visualization and quantification with a sensitivity comparable to conventional silver staining with a 1000-fold higher dynamic range. For normalization, standardization and quantification of protein gels and WBs, a sample-dependent bi-fluorescent standard reagent is applied and, for accurate quantification of data derived from different experiments, a second calibration standard is used. Together, the precise quantification of protein expression by lane-to-lane, gel-to-gel, and blot-to-blot comparisons is facilitated especially with respect to experiments in the area of proteostasis dealing with highly variable protein levels and involving protein degradation mutants and treatments modulating protein abundance.

DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication.

PubMed Central

Saunders, K; Lucy, A; Stanley, J

1991-01-01

We have analysed DNA from African cassava mosaic virus (ACMV)-infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot-hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND-cellulose and their ability to hybridise to strand-specific and double-stranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virus-sense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2). Images PMID:2041773

Quantitative real-time PCR approaches for microbial community studies in wastewater treatment systems: applications and considerations.

PubMed

Kim, Jaai; Lim, Juntaek; Lee, Changsoo

2013-12-01

Quantitative real-time PCR (qPCR) has been widely used in recent environmental microbial ecology studies as a tool for detecting and quantifying microorganisms of interest, which aids in better understandings of the complexity of wastewater microbial communities. Although qPCR can be used to provide more specific and accurate quantification than other molecular techniques, it does have limitations that must be considered when applying it in practice. This article reviews the principle of qPCR quantification and its applications to microbial ecology studies in various wastewater treatment environments. Here we also address several limitations of qPCR-based approaches that can affect the validity of quantification data: template nucleic acid quality, nucleic acid extraction efficiency, specificity of group-specific primers and probes, amplification of nonviable DNA, gene copy number variation, and limited number of sequences in the database. Even with such limitations, qPCR is reportedly among the best methods for quantitatively investigating environmental microbial communities. The application of qPCR is and will continue to be increasingly common in studies of wastewater treatment systems. To obtain reliable analyses, however, the limitations that have often been overlooked must be carefully considered when interpreting the results. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

PubMed

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.

A new background subtraction method for Western blot densitometry band quantification through image analysis software.

PubMed

Gallo-Oller, Gabriel; Ordoñez, Raquel; Dotor, Javier

2018-06-01

Since its first description, Western blot has been widely used in molecular labs. It constitutes a multistep method that allows the detection and/or quantification of proteins from simple to complex protein mixtures. Western blot quantification method constitutes a critical step in order to obtain accurate and reproducible results. Due to the technical knowledge required for densitometry analysis together with the resources availability, standard office scanners are often used for the imaging acquisition of developed Western blot films. Furthermore, the use of semi-quantitative software as ImageJ (Java-based image-processing and analysis software) is clearly increasing in different scientific fields. In this work, we describe the use of office scanner coupled with the ImageJ software together with a new image background subtraction method for accurate Western blot quantification. The proposed method represents an affordable, accurate and reproducible approximation that could be used in the presence of limited resources availability. Copyright © 2018 Elsevier B.V. All rights reserved.

Classifying Saturn's F Ring Strands

NASA Astrophysics Data System (ADS)

Albers, Nicole; Sremcevic, M.; Esposito, L. W.; Colwell, J. E.

2009-09-01

The Cassini Ultraviolet Imaging Spectrograph (UVIS) High Speed Photometer (HSP) has recorded more than 113 stellar occultations by Saturn's F ring providing measurements with ring plane resolutions of a few dozen meters and better. Inner and outer F ring strands have been seen throughout the Cassini mission where they revealed themselves as non-continuous, azimuthally and temporally highly variable structures. In the light of a more accurate orbit description of the F ring core we find evidence for a ring that becomes dynamically more active as the system approaches anti-apse alignment with Prometheus. This is consistent with the observed increased strand activity. A recent strand that morphologically resembles the core is the strongest seen to date and points to the intricate relation between core and strands indicating the strands' violent creation. Using more than 150 identifications of various strands, we trace their kinematics and infer dynamical timescales and photometric properties. Implications for the dynamical evolution of the F ring will be discussed. This research was supported by the Cassini Project.

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

PubMed

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.

Nuclear Proximity of Mtr4 with RNA exosome restricts DNA mutational asymmetry

PubMed Central

Lim, Junghyun; Giri, Pankaj Kumar; Kazadi, David; Laffleur, Brice; Zhang, Wanwei; Grinstein, Veronika; Pefanis, Evangelos; Brown, Lewis M.; Ladewig, Erik; Martin, Ophélie; Chen, Yuling; Rabadan, Raul; Boyer, François; Rothschild, Gerson; Cogné, Michel; Pinaud, Eric; Deng, Haiteng; Basu, Uttiya

2017-01-01

SUMMARY The distribution of sense and antisense strand DNA mutations on transcribed duplex DNA contributes to the development of immune and neural systems along with the progression of cancer. Because developmentally matured B cells undergo biologically programmed strand-specific DNA mutagenesis at focal DNA/RNA hybrid structures, they make a convenient system to investigate strand-specific mutagenesis mechanisms. We demonstrate that the sense and antisense strand DNA mutagenesis at the immunoglobulin heavy chain locus and some other regions of the B cell genome depends upon localized RNA processing protein complex formation in the nucleus. Both the physical proximity and coupled activities of RNA helicase Mtr4 (and Senataxin) with the noncoding RNA processing function of RNA exosome determine the strand specific distribution of DNA mutations. Our study suggests that strand-specific DNA mutagenesis-associated mechanisms will play major roles in other undiscovered aspects of organismic development. PMID:28431250

7 CFR 1755.370 - RUS specification for seven wire galvanized steel strand.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 11 2011-01-01 2011-01-01 false RUS specification for seven wire galvanized steel..., ACCEPTABLE MATERIALS, AND STANDARD CONTRACT FORMS § 1755.370 RUS specification for seven wire galvanized... Steel Wire Strand, issued May 1978. All seven wire galvanized steel strand purchased after April 1, 1990...

7 CFR 1755.370 - RUS specification for seven wire galvanized steel strand.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 11 2010-01-01 2010-01-01 false RUS specification for seven wire galvanized steel..., ACCEPTABLE MATERIALS, AND STANDARD CONTRACT FORMS § 1755.370 RUS specification for seven wire galvanized... Steel Wire Strand, issued May 1978. All seven wire galvanized steel strand purchased after April 1, 1990...

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

PubMed Central

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

2016-01-01

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

Reverse transcription strand invasion based amplification (RT-SIBA): a method for rapid detection of influenza A and B.

PubMed

Eboigbodin, Kevin; Filén, Sanna; Ojalehto, Tuomas; Brummer, Mirko; Elf, Sonja; Pousi, Kirsi; Hoser, Mark

2016-06-01

Rapid and accurate diagnosis of influenza viruses plays an important role in infection control, as well as in preventing the misuse of antibiotics. Isothermal nucleic acid amplification methods offer significant advantages over the polymerase chain reaction (PCR), since they are more rapid and do not require the sophisticated instruments needed for thermal cycling. We previously described a novel isothermal nucleic acid amplification method, 'Strand Invasion Based Amplification' (SIBA®), with high analytical sensitivity and specificity, for the detection of DNA. In this study, we describe the development of a variant of the SIBA method, namely, reverse transcription SIBA (RT-SIBA), for the rapid detection of viral RNA targets. The RT-SIBA method includes a reverse transcriptase enzyme that allows one-step reverse transcription of RNA to complementary DNA (cDNA) and simultaneous amplification and detection of the cDNA by SIBA under isothermal reaction conditions. The RT-SIBA method was found to be more sensitive than PCR for the detection of influenza A and B and could detect 100 copies of influenza RNA within 15 min. The development of RT-SIBA will enable rapid and accurate diagnosis of viral RNA targets within point-of-care or central laboratory settings.

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

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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.

[Progress of genome engineering technology via clustered regularly interspaced short palindromic repeats--a review].

PubMed

Li, Hao; Qiu, Shaofu; Song, Hongbin

2013-10-04

In survival competition with phage, bacteria and archaea gradually evolved the acquired immune system--Clustered regularly interspaced short palindromic repeats (CRISPR), presenting the trait of transcribing the crRNA and the CRISPR-associated protein (Cas) to silence or cleaving the foreign double-stranded DNA specifically. In recent years, strong interest arises in prokaryotes primitive immune system and many in-depth researches are going on. Recently, researchers successfully repurposed CRISPR as an RNA-guided platform for sequence-specific gene expression, which provides a simple approach for selectively perturbing gene expression on a genome-wide scale. It will undoubtedly bring genome engineering into a more convenient and accurate new era.

Specific and quantitative detection of human polyomaviruses BKV, JCV, and SV40 by real time PCR.

PubMed

McNees, Adrienne L; White, Zoe S; Zanwar, Preeti; Vilchez, Regis A; Butel, Janet S

2005-09-01

The polyomaviruses that infect humans, BK virus (BKV), JC virus (JCV), and simian virus 40 (SV40), typically establish subclinical persistent infections. However, reactivation of these viruses in immunocompromised hosts is associated with renal nephropathy and hemorrhagic cystitis (HC) caused by BKV and with progressive multifocal leukoencephalopathy (PML) caused by JCV. Additionally, SV40 is associated with several types of human cancers including primary brain and bone cancers, mesotheliomas, and non-Hodgkin's lymphoma. Advancements in detection of these viruses may contribute to improved diagnosis and treatment of affected patients. To develop sensitive and specific real time quantitative polymerase chain reaction (RQ-PCR) assays for the detection of T-antigen DNA sequences of the human polyomaviruses BKV, JCV, and SV40 using the ABI Prism 7000 Sequence Detection System. Assays for absolute quantification of the viral T-ag sequences were designed and the sensitivity and specificity were evaluated. A quantitative assay to measure the single copy human RNAse P gene was also developed and evaluated in order to normalize viral gene copy numbers to cell numbers. Quantification of the target genes is sensitive and specific over a 7 log dynamic range. Ten copies each of the viral and cellular genes are reproducibly and accurately detected. The sensitivity of detection of the RQ-PCR assays is increased 10- to 100-fold compared to conventional PCR and agarose gel protocols. The primers and probes used to detect the viral genes are specific for each virus and there is no cross reactivity within the dynamic range of the standard dilutions. The sensitivity of detection for these assays is not reduced in human cellular extracts; however, different DNA extraction protocols may affect quantification. These assays provide a technique for rapid and specific quantification of polyomavirus genomes per cell in human samples.

Simple and accurate temperature correction for moisture pin calibrations in oriented strand board

Treesearch

Charles Boardman; Samuel V. Glass; Patricia K. Lebow

2017-01-01

Oriented strand board (OSB) is commonly used in the residential construction market in North America and its moisture-related durability is a critical consideration for building envelope design. Measurement of OSB moisture content (MC), a key determinant of durability, is often done using moisture pins and relies on a correlation between MC and the electrical...

An accurate bacterial DNA quantification assay for HTS library preparation of human biological samples.

PubMed

Seashols-Williams, Sarah; Green, Raquel; Wohlfahrt, Denise; Brand, Angela; Tan-Torres, Antonio Limjuco; Nogales, Francy; Brooks, J Paul; Singh, Baneshwar

2018-05-17

Sequencing and classification of microbial taxa within forensically relevant biological fluids has the potential for applications in the forensic science and biomedical fields. The quantity of bacterial DNA from human samples is currently estimated based on quantity of total DNA isolated. This method can miscalculate bacterial DNA quantity due to the mixed nature of the sample, and consequently library preparation is often unreliable. We developed an assay that can accurately and specifically quantify bacterial DNA within a mixed sample for reliable 16S ribosomal DNA (16S rDNA) library preparation and high throughput sequencing (HTS). A qPCR method was optimized using universal 16S rDNA primers, and a commercially available bacterial community DNA standard was used to develop a precise standard curve. Following qPCR optimization, 16S rDNA libraries from saliva, vaginal and menstrual secretions, urine, and fecal matter were amplified and evaluated at various DNA concentrations; successful HTS data were generated with as low as 20 pg of bacterial DNA. Changes in bacterial DNA quantity did not impact observed relative abundances of major bacterial taxa, but relative abundance changes of minor taxa were observed. Accurate quantification of microbial DNA resulted in consistent, successful library preparations for HTS analysis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Smart DNA Tweezer for Detection of Human Telomerase Activity.

PubMed

Xu, Xiaowen; Wang, Lei; Li, Kan; Huang, Qihong; Jiang, Wei

2018-03-06

Reliable and accurate detection of telomerase activity is crucial to better understand its role in cancer cells and to further explore its function in cancer diagnosis and treatment. Here, we construct a smart DNA tweezer (DT) for detection of telomerase activity. The DT is assembled by three specially designed single-stranded oligonucleotides: a central strand dually labeled with donor/acceptor fluorophores and two arm strands containing overhangs complementary to telomerase reaction products (TRPs). It can get closed through hybridization with TRPs and get reopen through strand displacement reaction by TRPs' complementary sequences. First, under the action of telomerase, telomerase binding substrates (TS) are elongated to generate TRPs ended with telomeric repeats (TTAGGG) n . TRPs hybridize with the two arm overhangs cooperatively and strain DT to closed state, inducing an increased fluorescence resonance energy transfer (FRET) efficiency, which is utilized for telomerase activity detection. Second, upon introduction of a removal strand (RS) complementary to TRPs, the closed DT is relaxed to open state via the toehold-mediated strand displacement, inducing a decreased FRET efficiency, which is utilized for determination of TRP length distribution. The detection limit of telomerase activity is equivalent to 141 cells/μL for HeLa cells, and telomerase-active cellular extracts can be differentiated from telomerase-inactive cellular extracts. Furthermore, TRPs owning 1, 2, 3, 4, and ≥5 telomeric repeats are identified to account for 25.6%, 20.5%, 15.7%, 12.5%, and 25.7%, respectively. The proposed strategy will offer a new approach for reliable, accurate detection of telomerase activity and product length distribution for deeper studying its role and function in cancer.

IAOseq: inferring abundance of overlapping genes using RNA-seq data.

PubMed

Sun, Hong; Yang, Shuang; Tun, Liangliang; Li, Yixue

2015-01-01

Overlapping transcription constitutes a common mechanism for regulating gene expression. A major limitation of the overlapping transcription assays is the lack of high throughput expression data. We developed a new tool (IAOseq) that is based on reads distributions along the transcribed regions to identify the expression levels of overlapping genes from standard RNA-seq data. Compared with five commonly used quantification methods, IAOseq showed better performance in the estimation accuracy of overlapping transcription levels. For the same strand overlapping transcription, currently existing high-throughput methods are rarely available to distinguish which strand was present in the original mRNA template. The IAOseq results showed that the commonly used methods gave an average of 1.6 fold overestimation of the expression levels of same strand overlapping genes. This work provides a useful tool for mining overlapping transcription levels from standard RNA-seq libraries. IAOseq could be used to help us understand the complex regulatory mechanism mediated by overlapping transcripts. IAOseq is freely available at http://lifecenter.sgst.cn/main/en/IAO_seq.jsp.

Separation of 1-23-kb complementary DNA strands by urea-agarose gel electrophoresis.

PubMed

Hegedüs, Eva; Kókai, Endre; Kotlyar, Alexander; Dombrádi, Viktor; Szabó, Gábor

2009-09-01

Double-stranded (ds), as well as denatured, single-stranded (ss) DNA samples can be analyzed on urea-agarose gels. Here we report that after denaturation by heat in the presence of 8 M urea, the two strands of the same ds DNA fragment of approximately 1-20-kb size migrate differently in 1 M urea containing agarose gels. The two strands are readily distinguished on Southern blots by ss-specific probes. The different migration of the two strands could be attributed to their different, base composition-dependent conformation impinging on the electrophoretic mobility of the ss molecules. This phenomenon can be exploited for the efficient preparation of strand-specific probes and for the separation of the complementary DNA strands for subsequent analysis, offering a new tool for various cell biological research areas.

From Agrobacterium to viral vectors: genome modification of plant cells by rare cutting restriction enzymes.

PubMed

Marton, Ira; Honig, Arik; Omid, Ayelet; De Costa, Noam; Marhevka, Elena; Cohen, Barry; Zuker, Amir; Vainstein, Alexander

2013-01-01

Researchers and biotechnologists require methods to accurately modify the genome of higher eukaryotic cells. Such modifications include, but are not limited to, site-specific mutagenesis, site-specific insertion of foreign DNA, and replacement and deletion of native sequences. Accurate genome modifications in plant species have been rather limited, with only a handful of plant species and genes being modified through the use of early genome-editing techniques. The development of rare-cutting restriction enzymes as a tool for the induction of site-specific genomic double-strand breaks and their introduction as a reliable tool for genome modification in animals, animal cells and human cell lines have paved the way for the adaptation of rare-cutting restriction enzymes to genome editing in plant cells. Indeed, the number of plant species and genes which have been successfully edited using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and engineered homing endonucleases is on the rise. In our review, we discuss the basics of rare-cutting restriction enzyme-mediated genome-editing technology with an emphasis on its application in plant species.

Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR.

PubMed

Witte, Anna Kristina; Fister, Susanne; Mester, Patrick; Schoder, Dagmar; Rossmanith, Peter

2016-11-01

Fast and reliable pathogen detection is an important issue for human health. Since conventional microbiological methods are rather slow, there is growing interest in detection and quantification using molecular methods. The droplet digital polymerase chain reaction (ddPCR) is a relatively new PCR method for absolute and accurate quantification without external standards. Using the Listeria monocytogenes specific prfA assay, we focused on the questions of whether the assay was directly transferable to ddPCR and whether ddPCR was suitable for samples derived from heterogeneous matrices, such as foodstuffs that often included inhibitors and a non-target bacterial background flora. Although the prfA assay showed suboptimal cluster formation, use of ddPCR for quantification of L. monocytogenes from pure bacterial cultures, artificially contaminated cheese, and naturally contaminated foodstuff was satisfactory over a relatively broad dynamic range. Moreover, results demonstrated the outstanding detection limit of one copy. However, while poorer DNA quality, such as resulting from longer storage, can impair ddPCR, internal amplification control (IAC) of prfA by ddPCR, that is integrated in the genome of L. monocytogenes ΔprfA, showed even slightly better quantification over a broader dynamic range. Graphical Abstract Evaluating the absolute quantification potential of ddPCR targeting Listeria monocytogenes prfA.

Normal Databases for the Relative Quantification of Myocardial Perfusion

PubMed Central

Rubeaux, Mathieu; Xu, Yuan; Germano, Guido; Berman, Daniel S.; Slomka, Piotr J.

2016-01-01

Purpose of review Myocardial perfusion imaging (MPI) with SPECT is performed clinically worldwide to detect and monitor coronary artery disease (CAD). MPI allows an objective quantification of myocardial perfusion at stress and rest. This established technique relies on normal databases to compare patient scans against reference normal limits. In this review, we aim to introduce the process of MPI quantification with normal databases and describe the associated perfusion quantitative measures that are used. Recent findings New equipment and new software reconstruction algorithms have been introduced which require the development of new normal limits. The appearance and regional count variations of normal MPI scan may differ between these new scanners and standard Anger cameras. Therefore, these new systems may require the determination of new normal limits to achieve optimal accuracy in relative myocardial perfusion quantification. Accurate diagnostic and prognostic results rivaling those obtained by expert readers can be obtained by this widely used technique. Summary Throughout this review, we emphasize the importance of the different normal databases and the need for specific databases relative to distinct imaging procedures. use of appropriate normal limits allows optimal quantification of MPI by taking into account subtle image differences due to the hardware and software used, and the population studied. PMID:28138354

A validated method for measurement of serum total, serum free, and salivary cortisol, using high-performance liquid chromatography coupled with high-resolution ESI-TOF mass spectrometry.

PubMed

Montskó, Gergely; Tarjányi, Zita; Mezősi, Emese; Kovács, Gábor L

2014-04-01

Blood cortisol level is routinely analysed in laboratory medicine, but the immunoassays in widespread use have the disadvantage of cross-reactivity with some commonly used steroid drugs. Mass spectrometry has become a method of increasing importance for cortisol estimation. However, current methods do not offer the option of accurate mass identification. Our objective was to develop a mass spectrometry method to analyse salivary, serum total, and serum free cortisol via accurate mass identification. The analysis was performed on a Bruker micrOTOF high-resolution mass spectrometer. Sample preparation involved protein precipitation, serum ultrafiltration, and solid-phase extraction. Limit of quantification was 12.5 nmol L(-1) for total cortisol, 440 pmol L(-1) for serum ultrafiltrate, and 600 pmol L(-1) for saliva. Average intra-assay variation was 4.7%, and inter-assay variation was 6.6%. Mass accuracy was <2.5 ppm. Serum total cortisol levels were in the range 35.6-1088 nmol L(-1), and serum free cortisol levels were in the range 0.5-12.4 nmol L(-1). Salivary cortisol levels were in the range 0.7-10.4 nmol L(-1). Mass accuracy was equal to or below 2.5 ppm, resulting in a mass error less than 1 mDa and thus providing high specificity. We did not observe any interference with routinely used steroidal drugs. The method is capable of specific cortisol quantification in different matrices on the basis of accurate mass identification.

Simultaneous quantification of alternatively spliced transcripts in a single droplet digital PCR reaction.

PubMed

Sun, Bing; Tao, Lian; Zheng, Yun-Ling

2014-06-01

Human telomerase reverse transcriptase (hTERT) is an essential component required for telomerase activity and telomere maintenance. Several alternatively spliced forms of hTERT mRNA have been reported in human primary and tumor cells. Currently, however, there is no sensitive and accurate method for the simultaneous quantification of multiple alternatively spliced RNA transcripts, such as in the case of hTERT. Here we show droplet digital PCR (ddPCR) provides sensitive, simultaneous digital quantification in a single reaction of two alternatively spliced single deletion hTERT transcripts (α-/β+ and α+/β-) as well as the opportunity to manually quantify non-deletion (α+/β+) and double deletion (α-/β-) transcripts. Our ddPCR method enables direct comparison among four alternatively spliced mRNAs without the need for internal standards or multiple primer pairs specific for each variant as real-time PCR (qPCR) requires, thus eliminating potential variation due to differences in PCR amplification efficiency.

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

PubMed Central

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

2013-01-01

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. PMID:24082118

Sequence-Specific DNA Photosplitting of Crosslinked DNAs Containing the 3-Cyanovinylcarbazole Nucleoside by Using DNA Strand Displacement.

PubMed

Nakamura, Shigetaka; Kawabata, Hayato; Fujimoto, Kenzo

2016-08-17

An oligodeoxynucleotide (ODN) containing the ultrafast reversible 3-cyanovinylcarbazole ((CNV) K) photo-crosslinker was photo-crosslinked to a complementary strand upon exposure to 366 nm irradiation and photosplit by use of 312 nm irradiation. In this paper we report that the photoreaction of (CNV) K on irradiation at 366 nm involves a photostationary state and that its reaction can be controlled by temperature. Guided by this new insight, we proposed and have now demonstrated previously unknown photosplitting of (CNV) K aided by DNA strand displacement as an alternative to heating. The photo-crosslinked double-stranded DNA (dsDNA) underwent >80 % photosplitting aided by DNA strand displacement on irradiation at 366 nm without heating. In this photosplitting based on DNA strand displacement, the relative thermal stability of the invader strand with respect to the template strands plays an important role, and an invader strand/template strand system that is more stable than the passenger strand/template strand system induces photosplitting without heating. This new strand-displacement-aided photosplitting occurred in a sequence-specific manner through irradiation at 366 nm in the presence of an invader strand. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

Tannous, Joanna; Atoui, Ali; El Khoury, André; Kantar, Sally; Chdid, Nader; Oswald, Isabelle P; Puel, Olivier; Lteif, Roger

2015-09-01

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 real-time 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

Programmable energy landscapes for kinetic control of DNA strand displacement.

PubMed

Machinek, Robert R F; Ouldridge, Thomas E; Haley, Natalie E C; Bath, Jonathan; Turberfield, Andrew J

2014-11-10

DNA is used to construct synthetic systems that sense, actuate, move and compute. The operation of many dynamic DNA devices depends on toehold-mediated strand displacement, by which one DNA strand displaces another from a duplex. Kinetic control of strand displacement is particularly important in autonomous molecular machinery and molecular computation, in which non-equilibrium systems are controlled through rates of competing processes. Here, we introduce a new method based on the creation of mismatched base pairs as kinetic barriers to strand displacement. Reaction rate constants can be tuned across three orders of magnitude by altering the position of such a defect without significantly changing the stabilities of reactants or products. By modelling reaction free-energy landscapes, we explore the mechanistic basis of this control mechanism. We also demonstrate that oxDNA, a coarse-grained model of DNA, is capable of accurately predicting and explaining the impact of mismatches on displacement kinetics.

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

PubMed

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.

Liver-Targeted Anti-HBV Single-Stranded Oligonucleotides with Locked Nucleic Acid Potently Reduce HBV Gene Expression In Vivo.

PubMed

Javanbakht, Hassan; Mueller, Henrik; Walther, Johanna; Zhou, Xue; Lopez, Anaïs; Pattupara, Thushara; Blaising, Julie; Pedersen, Lykke; Albæk, Nanna; Jackerott, Malene; Shi, Tianlai; Ploix, Corinne; Driessen, Wouter; Persson, Robert; Ravn, Jacob; Young, John A T; Ottosen, Søren

2018-06-01

Chronic hepatitis B infection (CHB) is an area of high unmet medical need. Current standard-of-care therapies only rarely lead to a functional cure, defined as durable hepatitis B surface antigen (HBsAg) loss following treatment. The goal for next generation CHB therapies is to achieve a higher rate of functional cure with finite treatment duration. To address this urgent need, we are developing liver-targeted single-stranded oligonucleotide (SSO) therapeutics for CHB based on the locked nucleic acid (LNA) platform. These LNA-SSOs target hepatitis B virus (HBV) transcripts for RNase-H-mediated degradation. Here, we describe a HBV-specific LNA-SSO that effectively reduces intracellular viral mRNAs and viral antigens (HBsAg and HBeAg) over an extended time period in cultured human hepatoma cell lines that were infected with HBV with mean 50% effective concentration (EC 50 ) values ranging from 1.19 to 1.66 μM. To achieve liver-specific targeting and minimize kidney exposure, this LNA-SSO was conjugated to a cluster of three N-acetylgalactosamine (GalNAc) moieties that direct specific binding to the asialoglycoprotein receptor (ASGPR) expressed specifically on the surface of hepatocytes. The GalNAc-conjugated LNA-SSO showed a strikingly higher level of potency when tested in the AAV-HBV mouse model as compared with its non-conjugated counterpart. Remarkably, higher doses of GalNAc-conjugated LNA-SSO resulted in a rapid and long-lasting reduction of HBsAg to below the detection limit for quantification, i.e., by 3 log10 (p < 0.0003). This antiviral effect depended on a close match between the sequences of the LNA-SSO and its HBV target, indicating that the antiviral effect is not due to non-specific oligonucleotide-driven immune activation. These data support the development of LNA-SSO therapeutics for the treatment of CHB infection. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2016-03-01

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.

All-atom 3D structure prediction of transmembrane β-barrel proteins from sequences.

PubMed

Hayat, Sikander; Sander, Chris; Marks, Debora S; Elofsson, Arne

2015-04-28

Transmembrane β-barrels (TMBs) carry out major functions in substrate transport and protein biogenesis but experimental determination of their 3D structure is challenging. Encouraged by successful de novo 3D structure prediction of globular and α-helical membrane proteins from sequence alignments alone, we developed an approach to predict the 3D structure of TMBs. The approach combines the maximum-entropy evolutionary coupling method for predicting residue contacts (EVfold) with a machine-learning approach (boctopus2) for predicting β-strands in the barrel. In a blinded test for 19 TMB proteins of known structure that have a sufficient number of diverse homologous sequences available, this combined method (EVfold_bb) predicts hydrogen-bonded residue pairs between adjacent β-strands at an accuracy of ∼70%. This accuracy is sufficient for the generation of all-atom 3D models. In the transmembrane barrel region, the average 3D structure accuracy [template-modeling (TM) score] of top-ranked models is 0.54 (ranging from 0.36 to 0.85), with a higher (44%) number of residue pairs in correct strand-strand registration than in earlier methods (18%). Although the nonbarrel regions are predicted less accurately overall, the evolutionary couplings identify some highly constrained loop residues and, for FecA protein, the barrel including the structure of a plug domain can be accurately modeled (TM score = 0.68). Lower prediction accuracy tends to be associated with insufficient sequence information and we therefore expect increasing numbers of β-barrel families to become accessible to accurate 3D structure prediction as the number of available sequences increases.

Development and validation of high-performance liquid chromatography and high-performance thin-layer chromatography methods for the quantification of khellin in Ammi visnaga seed

PubMed Central

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

Cause-specific temporal and spatial trends in green sea turtle strandings in the Hawaiian Archipelago (1982-2003)

USGS Publications Warehouse

Chaloupka, Milani; Work, Thierry M.; Balazs, George H.; Murakawa, Shawn K. K.; Morris, Robert

2008-01-01

We investigated cause-specific temporal and spatial trends in sea turtle strandings in the Hawaiian Archipelago. Five species of sea turtle were recorded in 3,861 strandings over a 22-year period (1982–2003). Green turtles comprised 97% of these strandings with size and gender composition reflecting the demographic structure of the resident green turtle population and relative green turtle abundance in Hawaiian waters. The cause of strandings was determined by necropsy based on a complete gross external and internal examination. Totally 75% of the 3,732 green turtle strandings were from Oahu where strandings occur year-round. The most common known cause of the green turtle strandings was the tumour-forming disease, fibropapillomatosis (28%) followed by hook-and-line fishing gear-induced trauma (7%), gillnet fishing gear-induced trauma (5%), boat strike (2.5%), and shark attack (2.7%). Miscellaneous causes comprised 5.4% of strandings whereas 49% of green turtle strandings could not be attributed to any known cause. Green turtle strandings attributable to boat strike were more likely from Kauai and Oahu while fibropapilloma strandings were more likely from Oahu and Maui. Hook-and-line gear strandings were more likely from Oahu due to higher per capita inshore fishing effort. The specific mortality rate (conditional probability) for fibropapillomatosis was 88%, 69% for gillnet gear and 52% for hook-and-line gear. The probability of a dead green turtle stranding increased from 1982 but levelled off by the mid-1990s. The declining mortality risk was because the prevalence and severity of fibropapillomatosis has decreased recently and so has the mortality risk attributable to gillnet gear. Despite exposure to disease and inshore fishing gears, the Hawaiian green turtle stock continues to recover following protection since the late 1970s. Nevertheless, measures to reduce incidental capture of sea turtles in coastal Hawaiian fisheries would be prudent, especially since strandings attributable to hook-and-line fishing gear have increased steadily since 1982.

Validation and Application of a Real-time PCR Protocol for the Specific Detection and Quantification of Clavibacter michiganensis subsp. sepedonicus in Potato.

PubMed

Cho, Min Seok; Park, Duck Hwan; Namgung, Min; Ahn, Tae-Young; Park, Dong Suk

2015-06-01

Clavibacter michiganensis subsp. sepedonicus (Cms) multiplies very rapidly, passing through the vascular strands and into the stems and petioles of a diseased potato. Therefore, the rapid and specific detection of this pathogen is highly important for the effective control of the pathogen. Although several PCR assays have been developed for detection, they cannot afford specific detection of Cms. Therefore, in this study, a computational genome analysis was performed to compare the sequenced genomes of the C. michiganensis subspecies and to identify an appropriate gene for the development of a subspecies-specific PCR primer set (Cms89F/R). The specificity of the primer set based on the putative phage-related protein was evaluated using genomic DNA from seven isolates of Cms and 27 other reference strains. The Cms89F/R primer set was more specific and sensitive than the existing assays in detecting Cms in in vitro using Cms cells and its genomic DNA. This assay was also able to detect at least 1.47×10(2) copies/μl of cloned-amplified target DNA, 5 fg of DNA using genomic DNA or 10(-6) dilution point of 0.12 at OD600 units of cells per reaction using a calibrated cell suspension.

Validation and Application of a Real-time PCR Protocol for the Specific Detection and Quantification of Clavibacter michiganensis subsp. sepedonicus in Potato

PubMed Central

Cho, Min Seok; Park, Duck Hwan; Namgung, Min; Ahn, Tae-Young; Park, Dong Suk

2015-01-01

Clavibacter michiganensis subsp. sepedonicus (Cms) multiplies very rapidly, passing through the vascular strands and into the stems and petioles of a diseased potato. Therefore, the rapid and specific detection of this pathogen is highly important for the effective control of the pathogen. Although several PCR assays have been developed for detection, they cannot afford specific detection of Cms. Therefore, in this study, a computational genome analysis was performed to compare the sequenced genomes of the C. michiganensis subspecies and to identify an appropriate gene for the development of a subspecies-specific PCR primer set (Cms89F/R). The specificity of the primer set based on the putative phage-related protein was evaluated using genomic DNA from seven isolates of Cms and 27 other reference strains. The Cms89F/R primer set was more specific and sensitive than the existing assays in detecting Cms in in vitro using Cms cells and its genomic DNA. This assay was also able to detect at least 1.47×102 copies/μl of cloned-amplified target DNA, 5 fg of DNA using genomic DNA or 10−6 dilution point of 0.12 at OD600 units of cells per reaction using a calibrated cell suspension. PMID:26060431

Epicocconone, a sensitive and specific fluorescent dye for in situ quantification of extracellular proteins within bacterial biofilms.

PubMed

Randrianjatovo, I; Girbal-Neuhauser, E; Marcato-Romain, C-E

2015-06-01

Biofilms are ecosystems of closely associated bacteria encapsulated in an extracellular matrix mainly composed of polysaccharides and proteins. A novel approach was developed for in situ quantification of extracellular proteins (ePNs) in various bacterial biofilms using epicocconone, a natural, fluorescent compound that binds amine residues of proteins. Six commercial proteins were tested for their reaction with epicocconone, and bovine serum albumin (BSA) was selected for assay optimization. The optimized protocol, performed as a microassay, allowed protein amounts as low as 0.7 μg to as high as 50 μg per well to be detected. Addition of monosaccharides or polysaccharides (glucose, dextran or alginate) to the standard BSA solutions (0 to 250 μg ml(-1)) showed little or no sugar interference up to 2000 μg ml(-1), thus providing an assessment of the specificity of epicocconone for proteins. The optimized protocol was then applied to three different biofilms, and in situ quantification of ePN showed contrasted protein amounts of 22.1 ± 3.1, 38.3 ± 7.1 and 0.3 ± 0.1 μg equivalent BSA of proteins for 48-h biofilms of Pseudomonas aeruginosa, Bacillus licheniformis and Weissella confusa, respectively. Possible interference due to global matrix compounds on the in situ quantification of proteins was also investigated by applying the standard addition method (SAM). Low error percentages were obtained, indicating a correct quantification of both the ePN and the added proteins. For the first time, a specific and sensitive assay has been developed for in situ determination of ePN produced by bacterial cells. This advance should lead to an accurate, rapid tool for further protein labelling and microscopic observation of the extracellular matrix of biofilms.

Enzyme- and label-free electrochemical aptasensor for kanamycin detection based on double stir bar-assisted toehold-mediated strand displacement reaction for dual-signal amplification.

PubMed

Hong, Feng; Chen, Xixue; Cao, Yuting; Dong, Youren; Wu, Dazhen; Hu, Futao; Gan, Ning

2018-07-30

It is critically important to detect antibiotic residues for monitoring food safety. In this study, an enzyme- and label-free electrochemical aptasensor for antibiotics, with kanamycin (Kana) as a typical analyte, was developed based on a double stir bar-assisted toehold-mediated strand displacement reaction (dSB-TMSDR) for dual-signal amplification. First, we modified two gold electrodes (E-1 and E-2) with different DNA probes (S1/S2 hybrid probe in E-1 and DNA fuel strand S3 in E-2). In the presence of Kana, an S1/S2 probe can be disassembled from E-1 to form an S2/Kana complex in supernatant. The S2/Kana could react with S3 on E-2 to form S2/S3 hybrid and release Kana through TMSDR. After then, the target recycling was triggered. Subsequently, the formed S2/S3 hybrid can also trigger a hybridization chain reaction (HCR). Consequently, the dual-signal amplification strategy was established, which resulted in many long dsDNA chains on E-2. The chains can associate with methylene blue (MB) as redox probes to produce a current response for the quantification of Kana. The assay exhibited high sensitivity and specificity with a detection limit at 16 fM Kana due to the dual-signal amplification. The double stir bars system can both increase phase separation and prevent leakage of DNA fuel to reduce background interference. Moreover, it allows flexible sequence design of the TMSDR probes. The assay was successfully employed to detect Kana residues in food and showed potential application value in food safety detection. Copyright © 2018 Elsevier B.V. All rights reserved.

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

PubMed

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.

18O-labeled proteome reference as global internal standards for targeted quantification by selected reaction monitoring-mass spectrometry.

PubMed

Kim, Jong-Seo; Fillmore, Thomas L; Liu, Tao; Robinson, Errol; Hossain, Mahmud; Champion, Boyd L; Moore, Ronald J; Camp, David G; Smith, Richard D; Qian, Wei-Jun

2011-12-01

Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an (18)O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The (18)O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope ((18)O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope ((18)O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light ((16)O)/heavy ((18)O) peak area ratios. The utility of (18)O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of (18)O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM.

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

PubMed

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.

Real-time PCR for detection and quantification of the biocontrol agent Trichoderma atroviride strain SC1 in soil.

PubMed

Savazzini, Federica; Longa, Claudia Maria Oliveira; Pertot, Ilaria; Gessler, Cesare

2008-05-01

Trichoderma (Hypocreales, Ascomycota) is a widespread genus in nature and several Trichoderma species are used in industrial processes and as biocontrol agents against crop diseases. It is very important that the persistence and spread of microorganisms released on purpose into the environment are accurately monitored. Real-time PCR methods for genus/species/strain identification of microorganisms are currently being developed to overcome the difficulties of classical microbiological and enzymatic methods for monitoring these populations. The aim of the present study was to develop and validate a specific real-time PCR-based method for detecting Trichoderma atroviride SC1 in soil. We developed a primer and TaqMan probe set constructed on base mutations in an endochitinase gene. This tool is highly specific for the detection and quantification of the SC1 strain. The limits of detection and quantification calculated from the relative standard deviation were 6000 and 20,000 haploid genome copies per gram of soil. Together with the low throughput time associated with this procedure, which allows the evaluation of many soil samples within a short time period, these results suggest that this method could be successfully used to trace the fate of T. atroviride SC1 applied as an open-field biocontrol agent.

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

PubMed Central

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

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

PubMed

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.

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

PubMed

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.

Advances in targeted proteomics and applications to biomedical research

PubMed Central

Shi, Tujin; Song, Ehwang; Nie, Song; Rodland, Karin D.; Liu, Tao; Qian, Wei-Jun; Smith, Richard D.

2016-01-01

Targeted proteomics technique has emerged as a powerful protein quantification tool in systems biology, biomedical research, and increasing for clinical applications. The most widely used targeted proteomics approach, selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), can be used for quantification of cellular signaling networks and preclinical verification of candidate protein biomarkers. As an extension to our previous review on advances in SRM sensitivity herein we review recent advances in the method and technology for further enhancing SRM sensitivity (from 2012 to present), and highlighting its broad biomedical applications in human bodily fluids, tissue and cell lines. Furthermore, we also review two recently introduced targeted proteomics approaches, parallel reaction monitoring (PRM) and data-independent acquisition (DIA) with targeted data extraction on fast scanning high-resolution accurate-mass (HR/AM) instruments. Such HR/AM targeted quantification with monitoring all target product ions addresses SRM limitations effectively in specificity and multiplexing; whereas when compared to SRM, PRM and DIA are still in the infancy with a limited number of applications. Thus, for HR/AM targeted quantification we focus our discussion on method development, data processing and analysis, and its advantages and limitations in targeted proteomics. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large-scale quantification of hundreds of target proteins are discussed. PMID:27302376

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

PubMed

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.

Advances in targeted proteomics and applications to biomedical research

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

Shi, Tujin; Song, Ehwang; Nie, Song

Targeted proteomics technique has emerged as a powerful protein quantification tool in systems biology, biomedical research, and increasing for clinical applications. The most widely used targeted proteomics approach, selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), can be used for quantification of cellular signaling networks and preclinical verification of candidate protein biomarkers. As an extension to our previous review on advances in SRM sensitivity (Shi et al., Proteomics, 12, 1074–1092, 2012) herein we review recent advances in the method and technology for further enhancing SRM sensitivity (from 2012 to present), and highlighting its broad biomedical applications inmore » human bodily fluids, tissue and cell lines. Furthermore, we also review two recently introduced targeted proteomics approaches, parallel reaction monitoring (PRM) and data-independent acquisition (DIA) with targeted data extraction on fast scanning high-resolution accurate-mass (HR/AM) instruments. Such HR/AM targeted quantification with monitoring all target product ions addresses SRM limitations effectively in specificity and multiplexing; whereas when compared to SRM, PRM and DIA are still in the infancy with a limited number of applications. Thus, for HR/AM targeted quantification we focus our discussion on method development, data processing and analysis, and its advantages and limitations in targeted proteomics. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large-scale quantification of hundreds of target proteins are discussed.« less

Simultaneous quantification of neuroactive dopamine serotonin and kynurenine pathway metabolites in gender-specific youth urine by ultra performance liquid chromatography tandem high resolution mass spectrometry.

PubMed

Lu, Haihua; Yu, Jing; Wang, Jun; Wu, Linlin; Xiao, Hang; Gao, Rong

2016-04-15

Neuroactive metabolites in dopamine, serotonin and kynurenine metabolic pathways play key roles in several physiological processes and their imbalances have been implicated in the pathophysiology of a wide range of disorders. The association of these metabolites' alterations with various pathologies has raised interest in analytical methods for accurate quantification in biological fluids. However, simultaneous measurement of various neuroactive metabolites represents great challenges due to their trace level, high polarity and instability. In this study, an analytical method was developed and validated for accurately quantifying 12 neuroactive metabolites covering three metabolic pathways in youth urine by ultra performance liquid chromatography coupled to electrospray tandem high resolution mass spectrometry (UPLC-ESI-HRMS/MS). The strategy of dansyl chloride derivatization followed by solid phase extraction on C18 cartridges were employed to reduce matrix interference and improve the extraction efficiency. The reverse phase chromatographic separation was achieved with a gradient elution program in 20 min. The high resolution mass spectrometer (Q Exactive) was employed, with confirmation and quantification by Target-MS/MS scan mode. Youth urine samples collected from 100 healthy volunteers (Female:Male=1:1) were analyzed to explore the differences in metabolite profile and their turnover between genders. The results demonstrated that the UPLC-ESI-HRMS/MS method is sensitive and robust, suitable for monitoring a large panel of metabolites and for discovering new biomarkers in the medical fields. Copyright © 2016 Elsevier B.V. All rights reserved.

SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients

PubMed Central

Stadler, Julia; Eder, Johanna; Pratscher, Barbara; Brandt, Sabine; Schneller, Doris; Müllegger, Robert; Vogl, Claus; Trautinger, Franz; Brem, Gottfried; Burgstaller, Joerg P.

2015-01-01

Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent “gold standard”. Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution), at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients. PMID:26562020

Streptavidin-coated magnetic beads for DNA strand separation implicate a multitude of problems during cell-SELEX.

PubMed

Paul, Angela; Avci-Adali, Meltem; Ziemer, Gerhard; Wendel, Hans P

2009-09-01

Using whole living cells as a target for SELEX (systematic evolution of ligands by exponential enrichment) experiments represents a promising method to generate cell receptor-specific aptamers. These aptamers have a huge potential in diagnostics, therapeutics, imaging, regenerative medicine, and target validation. During the SELEX for selecting DNA aptamers, one important step is the separation of 2 DNA strands to yield one of the 2 strands as single-stranded DNA aptamer. This is being done routinely by biotin labeling of the complementary DNA strand to the desired aptamer and then separating the DNA strand by using streptavidin-coated magnetic beads. After immobilization of the double-stranded DNA on these magnetic beads and alkaline denaturation, the non-biotinylated strand is being eluted and the biotinylated strand is retarded. Using Western blot analysis, we demonstrated the detachment of covalent-bonded streptavidin from the bead surface after alkaline treatment. The eluates were also contaminated with undesired biotinylated strands. Furthermore, a streptavidin-induced aggregation of target cells was demonstrated by flow cytometry and microscopic methods. Cell-specific enrichment of aptamers was not possible due to clustering and patching effects triggered by streptavidin. Therefore, the use of streptavidin-coated magnetic beads for DNA strand separation should be examined thoroughly, especially for cell-SELEX applications.

Targeted, Site-specific quantitation of N- and O-glycopeptides using 18O-labeling and product ion based mass spectrometry.

PubMed

Srikanth, Jandhyam; Agalyadevi, Rathinasamy; Babu, Ponnusamy

2017-02-01

The site-specific quantitation of N- and O-glycosylation is vital to understanding the function(s) of different glycans expressed at a given site of a protein under physiological and disease conditions. Most commonly used precursor ion intensity based quantification method is less accurate and other labeled methods are expensive and require enrichment of glycopeptides. Here, we used glycopeptide product (y and Y0) ions and 18 O-labeling of C-terminal carboxyl group as a strategy to obtain quantitative information about fold-change and relative abundance of most of the glycoforms attached to the glycopeptides. As a proof of concept, the accuracy and robustness of this targeted, relative quantification LC-MS method was demonstrated using Rituximab. Furthermore, the N-glycopeptide quantification results were compared with a biosimilar of Rituximab and validated with quantitative data obtained from 2-AB-UHPLC-FL method. We further demonstrated the intensity fold-change and relative abundance of 46 unique N- and O-glycopeptides and aglycopeptides from innovator and biosimilar samples of Etanercept using both the normal-MS and product ion based quantitation. The results showed a very similar site-specific expression of N- and O-glycopeptides between the samples but with subtle differences. Interestingly, we have also been able to quantify macro-heterogeneity of all N- and O-glycopetides of Etanercept. In addition to applications in biotherapeutics, the developed method can also be used for site-specific quantitation of N- and O-glycopeptides and aglycopeptides of glycoproteins with known glycosylation pattern.

pH-Modulated Watson-Crick duplex-quadruplex equilibria of guanine-rich and cytosine-rich DNA sequences 140 base pairs upstream of the c-kit transcription initiation site.

PubMed

Bucek, Pavel; Jaumot, Joaquim; Aviñó, Anna; Eritja, Ramon; Gargallo, Raimundo

2009-11-23

Guanine-rich regions of DNA are sequences capable of forming G-quadruplex structures. The formation of a G-quadruplex structure in a region 140 base pairs (bp) upstream of the c-kit transcription initiation site was recently proposed (Fernando et al., Biochemistry, 2006, 45, 7854). In the present study, the acid-base equilibria and the thermally induced unfolding of the structures formed by a guanine-rich region and by its complementary cytosine-rich strand in c-kit were studied by means of circular dichroism and molecular absorption spectroscopies. In addition, competition between the Watson-Crick duplex and the isolated structures was studied as a function of pH value and temperature. Multivariate data analysis methods based on both hard and soft modeling were used to allow accurate quantification of the various acid-base species present in the mixtures. Results showed that the G-quadruplex and i-motif coexist with the Watson-Crick duplex over the pH range from 3.0 to 6.5, approximately, under the experimental conditions tested in this study. At pH 7.0, the duplex is practically the only species present.

Sensitive detection of point mutation using exponential strand displacement amplification-based surface enhanced Raman spectroscopy.

PubMed

Huang, Si-Qiang; Hu, Juan; Zhu, Guichi; Zhang, Chun-Yang

2015-03-15

Accurate identification of point mutation is particularly imperative in the field of biomedical research and clinical diagnosis. Here, we develop a sensitive and specific method for point mutation assay using exponential strand displacement amplification (SDA)-based surface enhanced Raman spectroscopy (SERS). In this method, a discriminating probe and a hairpin probe are designed to specifically recognize the sequence of human K-ras gene. In the presence of K-ras mutant target (C→T), the 3'-terminal of discriminating probe and the 5'-terminal of hairpin probe can be ligated to form a SDA template. Subsequently, the 3'-terminal of hairpin probe can function as a primer to initiate the SDA reaction, producing a large amount of triggers. The resultant triggers can further hybridize with the discriminating probes to initiate new rounds of SDA reaction, leading to an exponential amplification reaction. With the addition of capture probe-modified gold nanoparticles (AuNPs) and the Rox-labeled reporter probes, the amplified triggers can be assembled on the surface of AuNPs through the formation of sandwich hybrids of capture probe-trigger-reporter probe, generating a strong Raman signal. While in the presence of K-ras wild-type target (C), neither ligation nor SDA reaction can be initiated and no Raman signal is observed. The proposed method exhibits high sensitivity with a detection limit of 1.4pM and can accurately discriminate as low as 1% variant frequency from the mixture of mutant target and wild-type target. Importantly, this method can be further applied to analyze the mutant target in the spiked HEK293T cell lysate, holding great potential for genetic analysis and disease prognosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid development of Proteomic applications with the AIBench framework.

PubMed

López-Fernández, Hugo; Reboiro-Jato, Miguel; Glez-Peña, Daniel; Méndez Reboredo, José R; Santos, Hugo M; Carreira, Ricardo J; Capelo-Martínez, José L; Fdez-Riverola, Florentino

2011-09-15

In this paper we present two case studies of Proteomics applications development using the AIBench framework, a Java desktop application framework mainly focused in scientific software development. The applications presented in this work are Decision Peptide-Driven, for rapid and accurate protein quantification, and Bacterial Identification, for Tuberculosis biomarker search and diagnosis. Both tools work with mass spectrometry data, specifically with MALDI-TOF spectra, minimizing the time required to process and analyze the experimental data. Copyright 2011 The Author(s). Published by Journal of Integrative Bioinformatics.

Collagen Quantification in Tissue Specimens.

PubMed

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.

Detection of benzo[a]pyrene-guanine adducts in single-stranded DNA using the α-hemolysin nanopore

NASA Astrophysics Data System (ADS)

Perera, Rukshan T.; Fleming, Aaron M.; Johnson, Robert P.; Burrows, Cynthia J.; White, Henry S.

2015-02-01

The carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, is released into the environment through the incomplete combustion of hydrocarbons. Metabolism of BP in the human body yields a potent alkylating agent (benzo[a]pyrene diol epoxide, BPDE) that reacts with guanine (G) in DNA to form an adduct implicated in cancer initiation. We report that the α-hemolysin (αHL) nanopore platform can be used to detect a BPDE adduct to G in synthetic oligodeoxynucleotides. Translocation of a 41-mer poly-2‧-deoxycytidine strand with a centrally located BPDE adduct to G through αHL in 1 M KCl produces a unique multi-level current signature allowing the adduct to be detected. This readily distinguishable current modulation was observed when the BPDE-adducted DNA strand translocated from either the 5‧ or 3‧ directions. This study suggests that BPDE adducts and other large aromatic biomarkers can be detected with αHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples.

Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients.

PubMed

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.

Single Color Multiplexed ddPCR Copy Number Measurements and Single Nucleotide Variant Genotyping.

PubMed

Wood-Bouwens, Christina M; Ji, Hanlee P

2018-01-01

Droplet digital PCR (ddPCR) allows for accurate quantification of genetic events such as copy number variation and single nucleotide variants. Probe-based assays represent the current "gold-standard" for detection and quantification of these genetic events. Here, we introduce a cost-effective single color ddPCR assay that allows for single genome resolution quantification of copy number and single nucleotide variation.

Equilibrious Strand Exchange Promoted by DNA Conformational Switching

NASA Astrophysics Data System (ADS)

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.

Isolation and characterization of naturally occurring hairpin structures in single-stranded DNA of coliphage M13

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

Niyogi, S.K.; Mitra, S.

With precise conditions of digestion with single-strand-specific nucleases, namely, endonuclease S1 of Aspergillus oryzae and exonuclease I of Escherichia coli, nuclease-resistant DNA cores can be obtained reproducibly from single-stranded M13 DNA. The DNA cores are composed almost exclusively of two sizes (60 and 44 nucleotides long). These have high (G + C)-contents relative to that of intact M13 DNA, and arise from restricted regions of the M13 genome. The resistance of these fragments to single-strand-specific nucleases and their nondenaturability strongly suggest the presence of double-stranded segments in these core pieces. That the core pieces are only partially double-stranded is shownmore » by their lack of complete base complementarity and their pattern of elution from hydroxyapatite.« less

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

PubMed

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.

Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

PubMed Central

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

Accuracy of iodine quantification in dual-layer spectral CT: Influence of iterative reconstruction, patient habitus and tube parameters.

PubMed

Sauter, Andreas P; Kopp, Felix K; Münzel, Daniela; Dangelmaier, Julia; Renz, Martin; Renger, Bernhard; Braren, Rickmer; Fingerle, Alexander A; Rummeny, Ernst J; Noël, Peter B

2018-05-01

Evaluation of the influence of iterative reconstruction, tube settings and patient habitus on the accuracy of iodine quantification with dual-layer spectral CT (DL-CT). A CT abdomen phantom with different extension rings and four iodine inserts (1, 2, 5 and 10 mg/ml) was scanned on a DL-CT. The phantom was scanned with tube-voltages of 120 and 140 kVp and CTDI vol of 2.5, 5, 10 and 20 mGy. Reconstructions were performed for eight levels of iterative reconstruction (i0-i7). Diagnostic dose levels are classified depending on patient-size and radiation dose. Measurements of iodine concentration showed accurate and reliable results. Taking all CTDI vol -levels into account, the mean absolute percentage difference (MAPD) showed less accuracy for low CTDI vol -levels (2.5 mGy: 34.72%) than for high CTDI vol -levels (20 mGy: 5.89%). At diagnostic dose levels, accurate quantification of iodine was possible (MAPD 3.38%). Level of iterative reconstruction did not significantly influence iodine measurements. Iodine quantification worked more accurately at a tube voltage of 140 kVp. Phantom size had a considerable effect only at low-dose-levels; at diagnostic dose levels the effect of phantom size decreased (MAPD <5% for all phantom sizes). With DL-CT, even low iodine concentrations can be accurately quantified. Accuracies are higher when diagnostic radiation doses are employed. Copyright © 2018 Elsevier B.V. All rights reserved.

Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase

PubMed Central

Langston, Lance; O’Donnell, Mike

2017-01-01

Replicative helicases are ring-shaped hexamers that encircle DNA for duplex unwinding. The currently accepted view of hexameric helicase function is by steric exclusion, where the helicase encircles one DNA strand and excludes the other, acting as a wedge with an external DNA unwinding point during translocation. Accordingly, strand-specific blocks only affect these helicases when placed on the tracking strand, not the excluded strand. We examined the effect of blocks on the eukaryotic CMG and, contrary to expectations, blocks on either strand inhibit CMG unwinding. A recent cryoEM structure of yeast CMG shows that duplex DNA enters the helicase and unwinding occurs in the central channel. The results of this report inform important aspects of the structure, and we propose that CMG functions by a modified steric exclusion process in which both strands enter the helicase and the duplex unwinding point is internal, followed by exclusion of the non-tracking strand. DOI: http://dx.doi.org/10.7554/eLife.23449.001 PMID:28346143

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

DOT National Transportation Integrated Search

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...

Quantification of neutral human milk oligosaccharides by graphitic carbon HPLC with tandem mass spectrometry

PubMed Central

Bao, Yuanwu; Chen, Ceng; Newburg, David S.

2012-01-01

Defining the biologic roles of human milk oligosaccharides (HMOS) requires an efficient, simple, reliable, and robust analytical method for simultaneous quantification of oligosaccharide profiles from multiple samples. The HMOS fraction of milk is a complex mixture of polar, highly branched, isomeric structures that contain no intrinsic facile chromophore, making their resolution and quantification challenging. A liquid chromatography-mass spectrometry (LC-MS) method was devised to resolve and quantify 11 major neutral oligosaccharides of human milk simultaneously. Crude HMOS fractions are reduced, resolved by porous graphitic carbon HPLC with a water/acetonitrile gradient, detected by mass spectrometric specific ion monitoring, and quantified. The HPLC separates isomers of identical molecular weights allowing 11 peaks to be fully resolved and quantified by monitoring mass to charge (m/z) ratios of the deprotonated negative ions. The standard curves for each of the 11 oligosaccharides is linear from 0.078 or 0.156 to 20 μg/mL (R2 > 0.998). Precision (CV) ranges from 1% to 9%. Accuracy is from 86% to 104%. This analytical technique provides sensitive, precise, accurate quantification for each of the 11 milk oligosaccharides and allows measurement of differences in milk oligosaccharide patterns between individuals and at different stages of lactation. PMID:23068043

Overcoming biofluid protein complexity during targeted mass spectrometry detection and quantification of protein biomarkers by MRM cubed (MRM3).

PubMed

Jeudy, Jeremy; Salvador, Arnaud; Simon, Romain; Jaffuel, Aurore; Fonbonne, Catherine; Léonard, Jean-François; Gautier, Jean-Charles; Pasquier, Olivier; Lemoine, Jerome

2014-02-01

Targeted mass spectrometry in the so-called multiple reaction monitoring mode (MRM) is certainly a promising way for the precise, accurate, and multiplexed measurement of proteins and their genetic or posttranslationally modified isoforms. MRM carried out on a low-resolution triple quadrupole instrument faces a lack of specificity when addressing the quantification of weakly concentrated proteins. In this case, extensive sample fractionation or immunoenrichment alleviates signal contamination by interferences, but in turn decreases assay performance and throughput. Recently, MRM(3) was introduced as an alternative to MRM to improve the limit of quantification of weakly concentrated protein biomarkers. In the present work, we compare MRM and MRM(3) modes for the detection of biomarkers in plasma and urine. Calibration curves drawn with MRM and MRM(3) showed a similar range of linearity (R(2) > 0.99 for both methods) with protein concentrations above 1 μg/mL in plasma and a few nanogram per milliliter in urine. In contrast, optimized MRM(3) methods improve the limits of quantification by a factor of 2 to 4 depending on the targeted peptide. This gain arises from the additional MS(3) fragmentation step, which significantly removes or decreases interfering signals within the targeted transition channels.

DNA breathing dynamics distinguish binding from nonbinding consensus sites for transcription factor YY1 in cells.

PubMed

Alexandrov, Boian S; Fukuyo, Yayoi; Lange, Martin; Horikoshi, Nobuo; Gelev, Vladimir; Rasmussen, Kim Ø; Bishop, Alan R; Usheva, Anny

2012-11-01

The genome-wide mapping of the major gene expression regulators, the transcription factors (TFs) and their DNA binding sites, is of great importance for describing cellular behavior and phenotypic diversity. Presently, the methods for prediction of genomic TF binding produce a large number of false positives, most likely due to insufficient description of the physiochemical mechanisms of protein-DNA binding. Growing evidence suggests that, in the cell, the double-stranded DNA (dsDNA) is subject to local transient strands separations (breathing) that contribute to genomic functions. By using site-specific chromatin immunopecipitations, gel shifts, BIOBASE data, and our model that accurately describes the melting behavior and breathing dynamics of dsDNA we report a specific DNA breathing profile found at YY1 binding sites in cells. We find that the genomic flanking sequence variations and SNPs, may exert long-range effects on DNA dynamics and predetermine YY1 binding. The ubiquitous TF YY1 has a fundamental role in essential biological processes by activating, initiating or repressing transcription depending upon the sequence context it binds. We anticipate that consensus binding sequences together with the related DNA dynamics profile may significantly improve the accuracy of genomic TF binding sites and TF binding-related functional SNPs.

Method for producing labeled single-stranded nucleic acid probes

DOEpatents

Dunn, John J.; Quesada, Mark A.; Randesi, Matthew

1999-10-19

Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector, the cloning vector having an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.

A homogeneous nucleic acid hybridization assay based on strand displacement.

PubMed Central

Vary, C P

1987-01-01

A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is described. The assay is based on the concept of strand displacement. In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest. Hybridization of the target nucleic acid with the probe strand ultimately results in displacement of the RNA signal strand. Strand displacement, therefore, causes conversion of the RNA from double to single-stranded form. The single-strand specificity of polynucleotide phosphorylase (EC 2.7.7.8) allows discrimination between double-helical and single-stranded forms of the RNA signal strand. As displacement proceeds, free RNA signal strands are preferentially phosphorolyzed to component nucleoside diphosphates, including adenosine diphosphate. The latter nucleotide is converted to ATP by pyruvate kinase(EC 2.7.1.40). Luciferase catalyzed bioluminescence is employed to measure the ATP generated as a result of strand displacement. Images PMID:3309890

Quantification of in vivo short echo-time proton magnetic resonance spectra at 14.1 T using two different approaches of modelling the macromolecule spectrum

NASA Astrophysics Data System (ADS)

Cudalbu, C.; Mlynárik, V.; Xin, L.; Gruetter, Rolf

2009-10-01

Reliable quantification of the macromolecule signals in short echo-time 1H MRS spectra is particularly important at high magnetic fields for an accurate quantification of metabolite concentrations (the neurochemical profile) due to effectively increased spectral resolution of the macromolecule components. The purpose of the present study was to assess two approaches of quantification, which take the contribution of macromolecules into account in the quantification step. 1H spectra were acquired on a 14.1 T/26 cm horizontal scanner on five rats using the ultra-short echo-time SPECIAL (spin echo full intensity acquired localization) spectroscopy sequence. Metabolite concentrations were estimated using LCModel, combined with a simulated basis set of metabolites using published spectral parameters and either the spectrum of macromolecules measured in vivo, using an inversion recovery technique, or baseline simulated by the built-in spline function. The fitted spline function resulted in a smooth approximation of the in vivo macromolecules, but in accordance with previous studies using Subtract-QUEST could not reproduce completely all features of the in vivo spectrum of macromolecules at 14.1 T. As a consequence, the measured macromolecular 'baseline' led to a more accurate and reliable quantification at higher field strengths.

[Progress in application of targeting viral vector regulated by microRNA in gene therapy: a review].

PubMed

Zhang, Guohai; Wang, Qizhao; Zhang, Jinghong; Xu, Ruian

2010-06-01

A safe and effective targeting viral vector is the key factor for successful clinical gene therapy. microRNA, a class of small, single-stranded endogenous RNAs, act as post-transcriptional regulators of gene expression. The discovery of these kind regulatory elements provides a new approach to regulate gene expression more accurately. In this review, we elucidated the principle of microRNA in regulation of targeting viral vector. The applications of microRNA in the fields of elimination contamination from replication competent virus, reduction of transgene-specific immunity, promotion of cancer-targeted gene therapy and development of live attenuated vaccines were also discussed.

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

PubMed Central

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

2013-01-01

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. PMID:24019238

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

PubMed

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.

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

PubMed

Townsley, Brad T; Covington, Michael F; Ichihashi, Yasunori; Zumstein, Kristina; Sinha, Neelima R

2015-01-01

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 the terminal breathing 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.

Comparison of Polymerase Subunits from Double-Stranded RNA Bacteriophages

PubMed Central

Yang, Hongyan; Makeyev, Eugene V.; Bamford, Dennis H.

2001-01-01

The family Cystoviridae comprises several bacteriophages with double-stranded RNA (dsRNA) genomes. We have previously purified the catalytic polymerase subunit (Pol) of one of the Cystoviridae members, bacteriophage φ6, and shown that the protein can catalyze RNA synthesis in vitro. In this reaction, both bacteriophage-specific and heterologous RNAs can serve as templates, but those containing 3′ termini from the φ6 minus strands are favored. This provides a molecular basis for the observation that only plus strands, not minus strands, are transcribed from φ6 dsRNA segments in vivo. To test whether such a regulatory mechanism is also found in other dsRNA viruses, we purified recombinant Pol subunits from the φ6-related bacteriophages φ8 and φ13 and assayed their polymerase activities in vitro. The enzymes catalyze template-dependent RNA synthesis using both single-stranded-RNA (ssRNA) and dsRNA templates. However, they differ from each other as well as from φ6 Pol in certain biochemical properties. Notably, each polymerase demonstrates a distinct preference for ssRNAs bearing short 3′-terminal sequences from the virus-specific minus strands. This suggests that, in addition to other factors, RNA transcription in Cystoviridae is controlled by the template specificity of the polymerase subunit. PMID:11602748

High-throughput quantification of hydroxyproline for determination of collagen.

PubMed

Hofman, Kathleen; Hall, Bronwyn; Cleaver, Helen; Marshall, Susan

2011-10-15

An accurate and high-throughput assay for collagen is essential for collagen research and development of collagen products. Hydroxyproline is routinely assayed to provide a measurement for collagen quantification. The time required for sample preparation using acid hydrolysis and neutralization prior to assay is what limits the current method for determining hydroxyproline. This work describes the conditions of alkali hydrolysis that, when combined with the colorimetric assay defined by Woessner, provide a high-throughput, accurate method for the measurement of hydroxyproline. Copyright © 2011 Elsevier Inc. All rights reserved.

Amino acids 16-275 of minute virus of mice NS1 include a domain that specifically binds (ACCA)2-3-containing DNA.

PubMed

Mouw, M; Pintel, D J

1998-11-10

GST-NS1 purified from Escherichia coli and insect cells binds double-strand DNA in an (ACCA)2-3-dependent fashion under similar ionic conditions, independent of the presence of anti-NS1 antisera or exogenously supplied ATP and interacts with single-strand DNA and RNA in a sequence-independent manner. An amino-terminal domain (amino acids 1-275) of NS1 [GST-NS1(1-275)], representing 41% of the full-length NS1 molecule, includes a domain that binds double-strand DNA in a sequence-specific manner at levels comparable to full-length GST-NS1, as well as single-strand DNA and RNA in a sequence-independent manner. The deletion of 15 additional amino-terminal amino acids yielded a molecule [GST-NS1(1-275)] that maintained (ACCA)2-3-specific double-strand DNA binding; however, this molecule was more sensitive to increasing ionic conditions than full-length GST-NS1 and GST-NS1(1-275) and could not be demonstrated to bind single-strand nucleic acids. A quantitative filter binding assay showed that E. coli- and baculovirus-expressed GST-NS1 and E. coli GST-NS1(1-275) specifically bound double-strand DNA with similar equilibrium kinetics [as measured by their apparent equilibrium DNA binding constants (KD)], whereas GST-NS1(16-275) bound 4- to 8-fold less well. Copyright 1998 Academic Press.

MAMA User Guide v2.0.1

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

Gaschen, Brian Keith; Bloch, Jeffrey Joseph; Porter, Reid

Morphological signatures of bulk SNM materials have significant promise, but these potential signatures are not fully utilized. This document describes software tools, collectively called the MAMA (Morphological Analysis for Material Attribution) software that can help provide robust and accurate quantification of morphological features in bulk material microscopy images (Optical, SEM). Although many of the specific tools are not unique to Mama, the software package has been designed specifically for nuclear material morphological analysis, and is at a point where it can be easily adapted (by Los Alamos or by collaborators) in response to new, different, or changing forensics needs. Themore » current release of the MAMA software only includes the image quantification, descriptions, and annotation functionality. Only limited information on a sample, its pedigree, and its chemistry are recorded inside this part of the software. This was decision based on initial feedback and the fact that there are several analytical chemistry databases being developed within the community. Currently MAMA is a standalone program that can export quantification results in a basic text format that can be imported into other programs such as Excel and Access. There is also a basic report generating feature that produces HTML formatted pages of the same information. We will be working with collaborators to provide better integration of MAMA into their particular systems, databases and workflows.« less

In vitro selection of single-stranded DNA molecular recognition elements against S. aureus alpha toxin and sensitive detection in human serum.

PubMed

Hong, Ka L; Battistella, Luisa; Salva, Alysia D; Williams, Ryan M; Sooter, Letha J

2015-01-27

Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX) variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE) targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (Kd) of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA) was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved.

Strawberry: Fast and accurate genome-guided transcript reconstruction and quantification from RNA-Seq.

PubMed

Liu, Ruolin; Dickerson, Julie

2017-11-01

We propose a novel method and software tool, Strawberry, for transcript reconstruction and quantification from RNA-Seq data under the guidance of genome alignment and independent of gene annotation. Strawberry consists of two modules: assembly and quantification. The novelty of Strawberry is that the two modules use different optimization frameworks but utilize the same data graph structure, which allows a highly efficient, expandable and accurate algorithm for dealing large data. The assembly module parses aligned reads into splicing graphs, and uses network flow algorithms to select the most likely transcripts. The quantification module uses a latent class model to assign read counts from the nodes of splicing graphs to transcripts. Strawberry simultaneously estimates the transcript abundances and corrects for sequencing bias through an EM algorithm. Based on simulations, Strawberry outperforms Cufflinks and StringTie in terms of both assembly and quantification accuracies. Under the evaluation of a real data set, the estimated transcript expression by Strawberry has the highest correlation with Nanostring probe counts, an independent experiment measure for transcript expression. Strawberry is written in C++14, and is available as open source software at https://github.com/ruolin/strawberry under the MIT license.

Development and Optimization of a Dedicated, Hybrid Dual-Modality SPECT-CmT System for Improved Breast Lesion Diagnosis

DTIC Science & Technology

2010-01-01

throughout the entire 3D volume which made quantification of the different tissues in the breast possible. The p eaks representing glandular and fat in...coefficients. Keywords: tissue quantification , absolute attenuation coefficient, scatter correction, computed tomography, tomography... tissue types. 1-4 Accurate measurements of t he quantification and di fferentiation of numerous t issues can be useful to identify di sease from

Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems

PubMed Central

Caplen, Natasha J.; Parrish, Susan; Imani, Farhad; Fire, Andrew; Morgan, Richard A.

2001-01-01

Short interfering RNAs (siRNAs) are double-stranded RNAs of ≈21–25 nucleotides that have been shown to function as key intermediaries in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates. siRNAs have a characteristic structure, with 5′-phosphate/3′-hydroxyl ends and a 2-base 3′ overhang on each strand of the duplex. In this study, we present data that synthetic siRNAs can induce gene-specific inhibition of expression in Caenorhabditis elegans and in cell lines from humans and mice. In each case, the interference by siRNAs was superior to the inhibition of gene expression mediated by single-stranded antisense oligonucleotides. The siRNAs seem to avoid the well documented nonspecific effects triggered by longer double-stranded RNAs in mammalian cells. These observations may open a path toward the use of siRNAs as a reverse genetic and therapeutic tool in mammalian cells. PMID:11481446

Method for introducing unidirectional nested deletions

DOEpatents

Dunn, J.J.; Quesada, M.A.; Randesi, M.

1999-07-27

Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector. The cloning vector has an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe. 1 fig.

Method for introducing unidirectional nested deletions

DOEpatents

Dunn, John J.; Quesada, Mark A.; Randesi, Matthew

1999-07-27

Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector, the cloning vector having an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.

Repairing the sickle cell mutation. I. Specific covalent binding of a photoreactive third strand to the mutated base pair.

PubMed

Broitman, S; Amosova, O; Dolinnaya, N G; Fresco, J R

1999-07-30

A DNA third strand with a 3'-psoralen substituent was designed to form a triplex with the sequence downstream of the T.A mutant base pair of the human sickle cell beta-globin gene. Triplex-mediated psoralen modification of the mutant T residue was sought as an approach to gene repair. The 24-nucleotide purine-rich target sequence switches from one strand to the other and has four pyrimidine interruptions. Therefore, a third strand sequence favorable to two triplex motifs was used, one parallel and the other antiparallel to it. To cope with the pyrimidine interruptions, which weaken third strand binding, 5-methylcytosine and 5-propynyluracil were used in the third strand. Further, a six residue "hook" complementary to an overhang of a linear duplex target was added to the 5'-end of the third strand via a T(4) linker. In binding to the overhang by Watson-Crick pairing, the hook facilitates triplex formation. This third strand also binds specifically to the target within a supercoiled plasmid. The psoralen moiety at the 3'-end of the third strand forms photoadducts to the targeted T with high efficiency. Such monoadducts are known to preferentially trigger reversion of the mutation by DNA repair enzymes.

Ultrasensitive multiplex optical quantification of bacteria in large samples of biofluids

PubMed Central

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

Improved Quantification of Free and Ester-Bound Gallic Acid in Foods and Beverages by UHPLC-MS/MS.

PubMed

Newsome, Andrew G; Li, Yongchao; van Breemen, Richard B

2016-02-17

Hydrolyzable tannins are measured routinely during the characterization of food and beverage samples. Most methods for the determination of hydrolyzable tannins use hydrolysis or methanolysis to convert complex tannins to small molecules (gallic acid, methyl gallate, and ellagic acid) for quantification by HPLC-UV. Often unrecognized, analytical limitations and variability inherent in these approaches for the measurement of hydrolyzable tannins include the variable mass fraction (0-0.90) that is released as analyte, contributions of sources other than tannins to hydrolyzable gallate (can exceed >10 wt %/wt), the measurement of both free and total analyte, and lack of controls to account for degradation. An accurate, specific, sensitive, and higher-throughput approach for the determination of hydrolyzable gallate based on ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) that overcomes these limitations was developed.

Formation of template-switching artifacts by linear amplification.

PubMed

Chakravarti, Dhrubajyoti; Mailander, Paula C

2008-07-01

Linear amplification is a method of synthesizing single-stranded DNA from either a single-stranded DNA or one strand of a double-stranded DNA. In this protocol, molecules of a single primer DNA are extended by multiple rounds of DNA synthesis at high temperature using thermostable DNA polymerases. Although linear amplification generates the intended full-length single-stranded product, it is more efficient over single-stranded templates than double-stranded templates. We analyzed linear amplification over single- or double-stranded mouse H-ras DNA (exon 1-2 region). The single-stranded H-ras template yielded only the intended product. However, when the double-stranded template was used, additional artifact products were observed. Increasing the concentration of the double-stranded template produced relatively higher amounts of these artifact products. One of the artifact DNA bands could be mapped and analyzed by sequencing. It contained three template-switching products. These DNAs were formed by incomplete DNA strand extension over the template strand, followed by switching to the complementary strand at a specific Ade nucleotide within a putative hairpin sequence, from which DNA synthesis continued over the complementary strand.

DNA Clutch Probes for Circulating Tumor DNA Analysis.

PubMed

Das, Jagotamoy; Ivanov, Ivaylo; Sargent, Edward H; Kelley, Shana O

2016-08-31

Progress toward the development of minimally invasive liquid biopsies of disease is being bolstered by breakthroughs in the analysis of circulating tumor DNA (ctDNA): DNA released from cancer cells into the bloodstream. However, robust, sensitive, and specific methods of detecting this emerging analyte are lacking. ctDNA analysis has unique challenges, since it is imperative to distinguish circulating DNA from normal cells vs mutation-bearing sequences originating from tumors. Here we report the electrochemical detection of mutated ctDNA in samples collected from cancer patients. By developing a strategy relying on the use of DNA clutch probes (DCPs) that render specific sequences of ctDNA accessible, we were able to readout the presence of mutated ctDNA. DCPs prevent reassociation of denatured DNA strands: they make one of the two strands of a dsDNA accessible for hybridization to a probe, and they also deactivate other closely related sequences in solution. DCPs ensure thereby that only mutated sequences associate with chip-based sensors detecting hybridization events. The assay exhibits excellent sensitivity and specificity in the detection of mutated ctDNA: it detects 1 fg/μL of a target mutation in the presence of 100 pg/μL of wild-type DNA, corresponding to detecting mutations at a level of 0.01% relative to wild type. This approach allows accurate analysis of samples collected from lung cancer and melanoma patients. This work represents the first detection of ctDNA without enzymatic amplification.

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

PubMed

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.

Power Control and Monitoring Requirements for Thermal Vacuum/Thermal Balance Testing of the MAP Observatory

NASA Technical Reports Server (NTRS)

Johnson, Chris; Hinkle, R. Kenneth (Technical Monitor)

2002-01-01

The specific heater control requirements for the thermal vacuum and thermal balance testing of the Microwave Anisotropy Probe (MAP) Observatory at the Goddard Space Flight Center (GSFC) in Greenbelt, Maryland are described. The testing was conducted in the 10m wide x 18.3m high Space Environment Simulator (SES) Thermal Vacuum Facility. The MAP thermal testing required accurate quantification of spacecraft and fixture power levels while minimizing heater electrical emissions. The special requirements of the MAP test necessitated construction of five (5) new heater racks.

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

PubMed

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.

Quantification of the total amount of black cohosh cycloartanoids by integration of one specific 1H NMR signal.

PubMed

Çiçek, Serhat Sezai; Girreser, Ulrich; Zidorn, Christian

2018-06-05

Quantitative analysis is an important field in the quality control of medicinal plants, aiming to determine the amount of pharmacologically active constituents in complex matrices. Often biological effects of herbal drugs are not restricted to single compounds, but are rather caused by a number of often biogenetically related plant metabolites. Depending on the complexity of the analyzed plant extract, conflicts between accuracy, such as total content assays using photometric or colorimetric methods, and comprehensiveness, e.g. quantification of one or a few lead compounds can occur. In this study, we present a qHNMR approach determining the total amount of cycloartanoids in black cohosh (Actaea racemosa) rhizomes. Perdeuterated methanol containing 1,2,4,5-tetrachloro-3-nitrobenzene as an internal standard was used for extraction. Amounts of cycloartanoids were then measured by integrating 1 H NMR signals of all cycloartenoids' H-19 exo protons. Due to their unusually low chemical shifts, these signals are well separated from all remaining signals in crude extracts. Thus, accurate (recovery rates of 99.5-102.5%) and precise (relative standard deviations below 2.5%) quantification of cycloartanoids was accomplished. To the best of our knowledge, this is the first example of a quantification of the total amount of a pharmacologically relevant compound class by integration of one 1 H NMR signal characteristic for all members of this particular compound class. Additionally, we propose a new term and unit for the evaluation of medicinal plants and herbal medicinal products: the "specific partial amount of substance" of pharmacologically active constituents, indicated in mmol/g. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting the groEL Gene

PubMed Central

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

Separation, identification, quantification, and method validation of anthocyanins in botanical supplement raw materials by HPLC and HPLC-MS.

PubMed

Chandra, A; Rana, J; Li, Y

2001-08-01

A method has been established and validated for identification and quantification of individual, as well as total, anthocyanins by HPLC and LC/ES-MS in botanical raw materials used in the herbal supplement industry. The anthocyanins were separated and identified on the basis of their respective M(+) (cation) using LC/ES-MS. Separated anthocyanins were individually calculated against one commercially available anthocyanin external standard (cyanidin-3-glucoside chloride) and expressed as its equivalents. Amounts of each anthocyanin calculated as external standard equivalent were then multiplied by a molecular-weight correction factor to afford their specific quantities. Experimental procedures and use of a molecular-weight correction factors are substantiated and validated using Balaton tart cherry and elderberry as templates. Cyanidin-3-glucoside chloride has been widely used in the botanical industry to calculate total anthocyanins. In our studies on tart cherry and elderberry, its use as external standard followed by use of molecular-weight correction factors should provide relatively accurate results for total anthocyanins, because of the presence of cyanidin as their major anthocyanidin backbone. The method proposed here is simple and has a direct sample preparation procedure without any solid-phase extraction. It enables selection and use of commercially available anthocyanins as external standards for quantification of specific anthocyanins in the sample matrix irrespective of their commercial availability as analytical standards. It can be used as a template and applied for similar quantification in several anthocyanin-containing raw materials for routine quality control procedures, thus providing consistency in analytical testing of botanical raw materials used for manufacturing efficacious and true-to-the-label nutritional supplements.

Techniques to improve the direct ex vivo detection of low frequency antigen-specific CD8+ T cells with peptide-major histocompatibility complex class I tetramers

PubMed Central

Chattopadhyay, Pratip K.; Melenhorst, J. Joseph; Ladell, Kristin; Gostick, Emma; Scheinberg, Philip; Barrett, A. John; Wooldridge, Linda; Roederer, Mario; Sewell, Andrew K.; Price, David A.

2008-01-01

The ability to quantify and characterize antigen-specific CD8+ T cells irrespective of functional readouts using fluorochrome-conjugated tetrameric peptide-MHC class I (pMHCI) complexes in conjunction with flow cytometry has transformed our understanding of cellular immune responses over the past decade. In the case of prevalent CD8+ T cell populations that engage cognate pMHCI tetramers with high avidities, direct ex vivo identification and subsequent data interpretation is relatively straightforward. However, the accurate identification of low frequency antigen-specific CD8+ T cell populations can be complicated, especially in situations where TCR-mediated tetramer binding occurs at low avidities. Here, we highlight a few simple techniques that can be employed to improve the visual resolution, and hence the accurate quantification, of tetramer-binding CD8+ T cell populations by flow cytometry. These methodological modifications enhance signal intensity, especially in the case of specific CD8+ T cell populations that bind cognate antigen with low avidity, minimize background noise and enable improved discrimination of true pMHCI tetramer binding events from nonspecific uptake. PMID:18836993

ACQUA: Automated Cyanobacterial Quantification Algorithm for toxic filamentous genera using spline curves, pattern recognition and machine learning.

PubMed

Gandola, Emanuele; Antonioli, Manuela; Traficante, Alessio; Franceschini, Simone; Scardi, Michele; Congestri, Roberta

2016-05-01

Toxigenic cyanobacteria are one of the main health risks associated with water resources worldwide, as their toxins can affect humans and fauna exposed via drinking water, aquaculture and recreation. Microscopy monitoring of cyanobacteria in water bodies and massive growth systems is a routine operation for cell abundance and growth estimation. Here we present ACQUA (Automated Cyanobacterial Quantification Algorithm), a new fully automated image analysis method designed for filamentous genera in Bright field microscopy. A pre-processing algorithm has been developed to highlight filaments of interest from background signals due to other phytoplankton and dust. A spline-fitting algorithm has been designed to recombine interrupted and crossing filaments in order to perform accurate morphometric analysis and to extract the surface pattern information of highlighted objects. In addition, 17 specific pattern indicators have been developed and used as input data for a machine-learning algorithm dedicated to the recognition between five widespread toxic or potentially toxic filamentous genera in freshwater: Aphanizomenon, Cylindrospermopsis, Dolichospermum, Limnothrix and Planktothrix. The method was validated using freshwater samples from three Italian volcanic lakes comparing automated vs. manual results. ACQUA proved to be a fast and accurate tool to rapidly assess freshwater quality and to characterize cyanobacterial assemblages in aquatic environments. Copyright © 2016 Elsevier B.V. All rights reserved.

Armored DNA in recombinant Baculoviruses as controls in molecular genetic assays.

PubMed

Freystetter, Andrea; Paar, Christian; Stekel, Herbert; Berg, Jörg

2017-10-01

The widespread use of molecular PCR-based assays in analytical and clinical laboratories brings about the need for test-specific, stable, and reliable external controls (EC) as well as standards and internal amplification controls (IC), in order to arrive at consistent test results. In addition, there is also a growing need to produce and provide stable, well-characterized molecular controls for quality assurance programs. In this study, we describe a novel approach to generate armored double-stranded DNA controls, which are encapsulated in baculovirus (BV) particles of the species Autographa californica multiple nucleopolyhedrovirus. We used the well-known BacPAK™ Baculovirus Expression System (Takara-Clontech), removed the polyhedrin promoter used for protein expression, and generated recombinant BV-armored DNAs. The obtained BV-armored DNAs were readily extracted by standard clinical DNA extraction methods, showed favorable linearity and performance in our clinical PCR assays, were resistant to DNase I digestion, and exhibited marked stability in human plasma and serum. BV-armored DNA ought to be used as ECs, quantification standards, and ICs in molecular assays, with the latter application allowing for the entire monitoring of clinical molecular assays for sample adequacy. BV-armored DNA may also be used to produce double-stranded DNA reference materials for, e.g., quality assurance programs. The ease to produce BV-armored DNA should make this approach feasible for a broad spectrum of molecular applications. Finally, as BV-armored DNAs are non-infectious to mammals, they may be even more conveniently shipped than clinical specimen.

Quantification of Functionalised Gold Nanoparticle-Targeted Knockdown of Gene Expression in HeLa Cells

PubMed Central

Jiwaji, Meesbah; Sandison, Mairi E.; Reboud, Julien; Stevenson, Ross; Daly, Rónán; Barkess, Gráinne; Faulds, Karen; Kolch, Walter; Graham, Duncan; Girolami, Mark A.; Cooper, Jonathan M.; Pitt, Andrew R.

2014-01-01

Introduction Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein. PMID:24926959

Quantification of Parvovirus B19 DNA Using COBAS AmpliPrep Automated Sample Preparation and LightCycler Real-Time PCR

PubMed Central

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

Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site

PubMed Central

Watzinger, Franz; Hörth, Elfriede; Lion, Thomas

2001-01-01

Despite the recent introduction of real-time PCR methods, competitive PCR techniques continue to play an important role in nucleic acid quantification because of the significantly lower cost of equipment and consumables. Here we describe a shifted restriction-site competitive PCR (SRS-cPCR) assay based on a modified type of competitor. The competitor fragments are designed to contain a recognition site for a restriction endonuclease that is also present in the target sequence to be quantified, but in a different position. Upon completion of the PCR, the amplicons are digested in the same tube with a single restriction enzyme, without the need to purify PCR products. The generated competitor- and target-specific restriction fragments display different sizes, and can be readily separated by electrophoresis and quantified by image analysis. Suboptimal digestion affects competitor- and target-derived amplicons to the same extent, thus eliminating the problem of incorrect quantification as a result of incomplete digestion of PCR products. We have established optimized conditions for a panel of 20 common restriction endonucleases permitting efficient digestion in PCR buffer. It is possible, therefore, to find a suitable restriction site for competitive PCR in virtually any sequence of interest. The assay presented is inexpensive, widely applicable, and permits reliable and accurate quantification of nucleic acid targets. PMID:11376164

Sensitivity of Chemical Shift-Encoded Fat Quantification to Calibration of Fat MR Spectrum

PubMed Central

Wang, Xiaoke; Hernando, Diego; Reeder, Scott B.

2015-01-01

Purpose To evaluate the impact of different fat spectral models on proton density fat-fraction (PDFF) quantification using chemical shift-encoded (CSE) MRI. Material and Methods Simulations and in vivo imaging were performed. In a simulation study, spectral models of fat were compared pairwise. Comparison of magnitude fitting and mixed fitting was performed over a range of echo times and fat fractions. In vivo acquisitions from 41 patients were reconstructed using 7 published spectral models of fat. T2-corrected STEAM-MRS was used as reference. Results Simulations demonstrate that imperfectly calibrated spectral models of fat result in biases that depend on echo times and fat fraction. Mixed fitting is more robust against this bias than magnitude fitting. Multi-peak spectral models showed much smaller differences among themselves than when compared to the single-peak spectral model. In vivo studies show all multi-peak models agree better (for mixed fitting, slope ranged from 0.967–1.045 using linear regression) with reference standard than the single-peak model (for mixed fitting, slope=0.76). Conclusion It is essential to use a multi-peak fat model for accurate quantification of fat with CSE-MRI. Further, fat quantification techniques using multi-peak fat models are comparable and no specific choice of spectral model is shown to be superior to the rest. PMID:25845713

A strategy for absolute proteome quantification with mass spectrometry by hierarchical use of peptide-concatenated standards.

PubMed

Kito, Keiji; Okada, Mitsuhiro; Ishibashi, Yuko; Okada, Satoshi; Ito, Takashi

2016-05-01

The accurate and precise absolute abundance of proteins can be determined using mass spectrometry by spiking the sample with stable isotope-labeled standards. In this study, we developed a strategy of hierarchical use of peptide-concatenated standards (PCSs) to quantify more proteins over a wider dynamic range. Multiple primary PCSs were used for quantification of many target proteins. Unique "ID-tag peptides" were introduced into individual primary PCSs, allowing us to monitor the exact amounts of individual PCSs using a "secondary PCS" in which all "ID-tag peptides" were concatenated. Furthermore, we varied the copy number of the "ID-tag peptide" in each PCS according to a range of expression levels of target proteins. This strategy accomplished absolute quantification over a wider range than that of the measured ratios. The quantified abundance of budding yeast proteins showed a high reproducibility for replicate analyses and similar copy numbers per cell for ribosomal proteins, demonstrating the accuracy and precision of this strategy. A comparison with the absolute abundance of transcripts clearly indicated different post-transcriptional regulation of expression for specific functional groups. Thus, the approach presented here is a faithful method for the absolute quantification of proteomes and provides insights into biological mechanisms, including the regulation of expressed protein abundance. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Developing a real-time PCR assay for direct identification and quantification of Pratylenchus penetrans in soil

USDA-ARS?s Scientific Manuscript database

The root-lesion nematode Pratylenchus penetrans is a major pathogen of potato world-wide. Yield losses may be exacerbated by interaction with the fungus Verticillium dahliae in the Potato early dying disease complex. Accurate identification and quantification of P. penetrans prior to planting are es...

Quantification of Efficiency of Beneficiation of Lunar Regolith

NASA Technical Reports Server (NTRS)

Trigwell, Steve; Lane, John; Captain, James; Weis, Kyle; Quinn, Jacqueline; Watanabe, Fumiya

2011-01-01

Electrostatic beneficiation of lunar regolith is being researched at Kennedy Space Center to enhance the ilmenite concentration of the regolith for the production of oxygen in in-situ resource utilization on the lunar surface. Ilmenite enrichment of up to 200% was achieved using lunar simulants. For the most accurate quantification of the regolith particles, standard petrographic methods are typically followed, but in order to optimize the process, many hundreds of samples were generated in this study that made the standard analysis methods time prohibitive. In the current studies, X-ray photoelectron spectroscopy (XPS) and Secondary Electron microscopy/Energy Dispersive Spectroscopy (SEM/EDS) were used that could automatically, and quickly, analyze many separated fractions of lunar simulant. In order to test the accuracy of the quantification, test mixture samples of known quantities of ilmenite (2, 5, 10, and 20 wt%) in silica (pure quartz powder), were analyzed by XPS and EDS. The results showed that quantification for low concentrations of ilmenite in silica could be accurately achieved by both XPS and EDS, knowing the limitations of the techniques. 1

Evaluation of empirical rule of linearly correlated peptide selection (ERLPS) for proteotypic peptide-based quantitative proteomics.

PubMed

Liu, Kehui; Zhang, Jiyang; Fu, Bin; Xie, Hongwei; Wang, Yingchun; Qian, Xiaohong

2014-07-01

Precise protein quantification is essential in comparative proteomics. Currently, quantification bias is inevitable when using proteotypic peptide-based quantitative proteomics strategy for the differences in peptides measurability. To improve quantification accuracy, we proposed an "empirical rule for linearly correlated peptide selection (ERLPS)" in quantitative proteomics in our previous work. However, a systematic evaluation on general application of ERLPS in quantitative proteomics under diverse experimental conditions needs to be conducted. In this study, the practice workflow of ERLPS was explicitly illustrated; different experimental variables, such as, different MS systems, sample complexities, sample preparations, elution gradients, matrix effects, loading amounts, and other factors were comprehensively investigated to evaluate the applicability, reproducibility, and transferability of ERPLS. The results demonstrated that ERLPS was highly reproducible and transferable within appropriate loading amounts and linearly correlated response peptides should be selected for each specific experiment. ERLPS was used to proteome samples from yeast to mouse and human, and in quantitative methods from label-free to O18/O16-labeled and SILAC analysis, and enabled accurate measurements for all proteotypic peptide-based quantitative proteomics over a large dynamic range. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mechanism of transcription termination by RNA polymerase III utilizes a nontemplate-strand sequence-specific signal element

PubMed Central

Arimbasseri, Aneeshkumar G.; Maraia, Richard J.

2015-01-01

SUMMARY Understanding the mechanism of transcription termination by a eukaryotic RNA polymerase (RNAP) has been limited by lack of a characterizable intermediate that reflects transition from an elongation complex to a true termination event. While other multisubunit RNAPs require multipartite cis-signals and/or ancillary factors to mediate pausing and release of the nascent transcript from the clutches of these enzymes, RNAP III does so with precision and efficiency on a simple oligo(dT) tract, independent of other cis-elements or trans-factors. We report a RNAP III pre-termination complex that reveals termination mechanisms controlled by sequence-specific elements in the non-template strand. Furthermore, the TFIIF-like, RNAP III subunit, C37 is required for this function of the non-template strand signal. The results reveal the RNAP III terminator as an information-rich control element. While the template strand promotes destabilization via a weak oligo(rU:dA) hybrid, the non-template strand provides distinct sequence-specific destabilizing information through interactions with the C37 subunit. PMID:25959395

Development of novel decoy oligonucleotides: advantages of circular dumb-bell decoy.

PubMed

Tomita, Naruya; Tomita, Tetsuya; Yuyama, Kazuhiko; Tougan, Takahiro; Tajima, Tsuyoshi; Ogihara, Toshio; Morishita, Ryuichi

2003-04-01

The inhibition of specific transcription regulatory proteins is a novel approach to regulate gene expression. The transcriptional activities of DNA binding proteins can be inhibited by the use of double-stranded oligonucleotides (ODNs) that compete for binding to their specific target sequences in promoters and enhancers. Transfection of this cis-element double-stranded ODN, referred to as decoy ODN, has been reported to be a powerful tool that provides a new class of anti-gene strategies to gene therapy and permits examination of specific gene regulation. We have demonstrated the usefulness of this decoy ODN strategy in animal models of restenosis, myocardial infarction, glomerulonephritis and rheumatoid arthritis. However, one of the major limitations of decoy ODN technology is the rapid degradation of phosphodiester ODNs by intracellular nucleases. To date, several different types of double-stranded decoy ODNs have been developed to overcome this issue. Circular dumb-bell (CD) double-stranded decoy ODNs that were developed to resolve this issue have attracted a high level of interest. In this review, the applications of decoy ODN strategy and the advantages of modified CD double-stranded decoy ODNs will be discussed.

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

PubMed Central

Hwang, Beomsoo; Jeon, Doyoung

2015-01-01

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. PMID:25860074

Rapid identification and quantification of tumor cells using an electrocatalytic method based on gold nanoparticles.

PubMed

de la Escosura-Muñiz, Alfredo; Sánchez-Espinel, Christian; Díaz-Freitas, Belén; González-Fernández, Africa; Maltez-da Costa, Marisa; Merkoçi, Arben

2009-12-15

There is a high demand for simple, rapid, efficient, and user-friendly alternative methods for the detection of cells in general and, in particular, for the detection of cancer cells. A biosensor able to detect cells would be an all-in-one dream device for such applications. The successful integration of nanoparticles into cell detection assays could allow for the development of this novel class of cell sensors. Indeed, their application could well have a great future in diagnostics, as well as other fields. As an example of a novel biosensor, we report here an electrocatalytic device for the specific identification of tumor cells that quantifies gold nanoparticles (AuNPs) coupled with an electrotransducing platform/sensor. Proliferation and adherence of tumor cells are achieved on the electrotransducer/detector, which consists of a mass-produced screen-printed carbon electrode (SPCE). In situ identification/quantification of tumor cells is achieved with a detection limit of 4000 cells per 700 microL of suspension. This novel and selective cell-sensing device is based on the reaction of cell surface proteins with specific antibodies conjugated with AuNPs. Final detection requires only a couple of minutes, taking advantage of the catalytic properties of AuNPs on hydrogen evolution. The proposed detection method does not require the chemical agents used in most existing assays for the detection of AuNPs. It allows for the miniaturization of the system and is much cheaper than other expensive and sophisticated methods used for tumor cell detection. We envisage that this device could operate in a simple way as an immunosensor or DNA sensor. Moreover, it could be used, even by inexperienced staff, for the detection of protein molecules or DNA strands.

The effect of applied transducer force on acoustic radiation force impulse quantification within the left lobe of the liver.

PubMed

Porra, Luke; Swan, Hans; Ho, Chien

2015-08-01

Introduction: Acoustic Radiation Force Impulse (ARFI) Quantification measures shear wave velocities (SWVs) within the liver. It is a reliable method for predicting the severity of liver fibrosis and has the potential to assess fibrosis in any part of the liver, but previous research has found ARFI quantification in the right lobe more accurate than in the left lobe. A lack of standardised applied transducer force when performing ARFI quantification in the left lobe of the liver may account for some of this inaccuracy. The research hypothesis of this present study predicted that an increase in applied transducer force would result in an increase in SWVs measured. Methods: ARFI quantification within the left lobe of the liver was performed within a group of healthy volunteers (n = 28). During each examination, each participant was subjected to ARFI quantification at six different levels of transducer force applied to the epigastric abdominal wall. Results: A repeated measures ANOVA test showed that ARFI quantification was significantly affected by applied transducer force (p = 0.002). Significant pairwise comparisons using Bonferroni correction for multiple comparisons showed that with an increase in applied transducer force, there was a decrease in SWVs. Conclusion: Applied transducer force has a significant effect on SWVs within the left lobe of the liver and it may explain some of the less accurate and less reliable results in previous studies where transducer force was not taken into consideration. Future studies in the left lobe of the liver should take this into account and control for applied transducer force.

Stability of local secondary structure determines selectivity of viral RNA chaperones.

PubMed

Bravo, Jack P K; Borodavka, Alexander; Barth, Anders; Calabrese, Antonio N; Mojzes, Peter; Cockburn, Joseph J B; Lamb, Don C; Tuma, Roman

2018-05-18

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.

Quantification of Fibrosis and Osteosclerosis in Myeloproliferative Neoplasms: A Computer-Assisted Image Study

PubMed Central

Teman, Carolin J.; Wilson, Andrew R.; Perkins, Sherrie L.; Hickman, Kimberly; Prchal, Josef T.; Salama, Mohamed E.

2010-01-01

Evaluation of bone marrow fibrosis and osteosclerosis in myeloproliferative neoplasms (MPN) is subject to interobserver inconsistency. Performance data for currently utilized fibrosis grading systems are lacking, and classification scales for osteosclerosis do not exist. Digital imaging can serve as a quantification method for fibrosis and osteosclerosis. We used digital imaging techniques for trabecular area assessment and reticulin-fiber quantification. Patients with all Philadelphia negative MPN subtypes had higher trabecular volume than controls (p ≤0.0015). Results suggest that the degree of osteosclerosis helps differentiate primary myelofibrosis from other MPN. Numerical quantification of fibrosis highly correlated with subjective scores, and interobserver correlation was satisfactory. Digital imaging provides accurate quantification for osteosclerosis and fibrosis. PMID:20122729

Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection.

PubMed

Wu, Fengchi; Wu, Yuqiang; Niu, Zhongwei; Vollmer, Frank

2016-07-29

Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world's attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg(2+) ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species.

Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection

PubMed Central

Wu, Fengchi; Wu, Yuqiang; Niu, Zhongwei; Vollmer, Frank

2016-01-01

Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world’s attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg2+ ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species. PMID:27483277

A novel quantitative electrochemical method to monitor DNA double-strand breaks caused by a DNA cleavage agent at a DNA sensor.

PubMed

Banasiak, Anna; Cassidy, John; Colleran, John

2018-06-01

To date, DNA cleavage, caused by cleavage agents, has been monitored mainly by gel and capillary electrophoresis. However, these techniques are time-consuming, non-quantitative and require gel stains. In this work, a novel, simple and, importantly, a quantitative method for monitoring the DNA nuclease activity of potential anti-cancer drugs, at a DNA electrochemical sensor, is presented. The DNA sensors were prepared using thiol-modified oligonucleotides that self-assembled to create a DNA monolayer at gold electrode surfaces. The quantification of DNA double-strand breaks is based on calculating the DNA surface coverage, before and after exposure to a DNA cleavage agent. The nuclease properties of a model DNA cleavage agent, copper bis-phenanthroline ([Cu II (phen) 2 ] 2+ ), that can cleave DNA in a Fenton-type reaction, were quantified electrochemically. The DNA surface coverage decreased on average by 21% after subjecting the DNA sensor to a nuclease assay containing [Cu II (phen) 2 ] 2+ , a reductant and an oxidant. This percentage indicates that 6 base pairs were cleaved in the nuclease assay from the immobilised 30 base pair strands. The DNA cleavage can be also induced electrochemically in the absence of a chemical reductant. [Cu II (phen) 2 ] 2+ intercalates between DNA base pairs and, on application of a suitable potential, can be reduced to [Cu I (phen) 2 ] + , with dissolved oxygen acting as the required oxidant. This reduction process is facilitated through DNA strands via long-range electron transfer, resulting in DNA cleavage of 23%. The control measurements for both chemically and electrochemically induced cleavage revealed that DNA strand breaks did not occur under experimental conditions in the absence of [Cu II (phen) 2 ] 2+ . Copyright © 2018 Elsevier B.V. All rights reserved.

Secretion of Hepatitis C Virus Replication Intermediates Reduces Activation of Toll-Like Receptor 3 in Hepatocytes.

PubMed

Grünvogel, Oliver; Colasanti, Ombretta; Lee, Ji-Young; Klöss, Volker; Belouzard, Sandrine; Reustle, Anna; Esser-Nobis, Katharina; Hesebeck-Brinckmann, Jasper; Mutz, Pascal; Hoffmann, Katrin; Mehrabi, Arianeb; Koschny, Ronald; Vondran, Florian W R; Gotthardt, Daniel; Schnitzler, Paul; Neumann-Haefelin, Christoph; Thimme, Robert; Binder, Marco; Bartenschlager, Ralf; Dubuisson, Jean; Dalpke, Alexander H; Lohmann, Volker

2018-06-01

Hepatitis C virus (HCV) infections most often result in chronic outcomes, although the virus constantly produces replication intermediates, in particular double-stranded RNA (dsRNA), representing potent inducers of innate immunity. We aimed to characterize the fate of HCV dsRNA in hepatocyte cultures to identify mechanisms contributing to viral persistence in presence of an active innate immune response. We analyzed hepatocyte-based culture models for HCV for induction of innate immunity, secretion of virus positive- or negative-strand RNA, and viral replication using different quantification methods and microscopy techniques. Expression of pattern recognition receptors was reconstituted in hepatoma cells by lentiviral transduction. HCV-infected cells secrete substantial amounts of virus positive- and negative-strand RNAs in extracellular vesicles (EVs), toward the apical and basolateral domain of hepatocytes. Secretion of negative-strand RNA was independent from virus production, and viral RNA secreted in EVs contained higher relative amounts of negative-strands, indicating that mostly virus dsRNA is released. A substantial part of viral replication complexes and dsRNA was found in the endosomal compartment and multivesicular bodies, indicating that secretion of HCV replication intermediates is mediated by the exosomal pathway. Block of vesicle release in HCV-positive cells increased intracellular dsRNA levels and increased activation of toll-like receptor 3, inhibiting HCV replication. Using hepatocyte-based culture models for HCV, we found a portion of HCV dsRNA intermediates to be released from infected cells in EVs, which reduces activation of toll-like receptor 3. This represents a novel mechanism how HCV evades host immune responses, potentially contributing to viral persistence. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Comprehensive Panel of Real-Time TaqMan™ Polymerase Chain Reaction Assays for Detection and Absolute Quantification of Filoviruses, Arenaviruses, and New World Hantaviruses

PubMed Central

Trombley, Adrienne R.; Wachter, Leslie; Garrison, Jeffrey; Buckley-Beason, Valerie A.; Jahrling, Jordan; Hensley, Lisa E.; Schoepp, Randal J.; Norwood, David A.; Goba, Augustine; Fair, Joseph N.; Kulesh, David A.

2010-01-01

Viral hemorrhagic fever is caused by a diverse group of single-stranded, negative-sense or positive-sense RNA viruses belonging to the families Filoviridae (Ebola and Marburg), Arenaviridae (Lassa, Junin, Machupo, Sabia, and Guanarito), and Bunyaviridae (hantavirus). Disease characteristics in these families mark each with the potential to be used as a biological threat agent. Because other diseases have similar clinical symptoms, specific laboratory diagnostic tests are necessary to provide the differential diagnosis during outbreaks and for instituting acceptable quarantine procedures. We designed 48 TaqMan™-based polymerase chain reaction (PCR) assays for specific and absolute quantitative detection of multiple hemorrhagic fever viruses. Forty-six assays were determined to be virus-specific, and two were designated as pan assays for Marburg virus. The limit of detection for the assays ranged from 10 to 0.001 plaque-forming units (PFU)/PCR. Although these real-time hemorrhagic fever virus assays are qualitative (presence of target), they are also quantitative (measure a single DNA/RNA target sequence in an unknown sample and express the final results as an absolute value (e.g., viral load, PFUs, or copies/mL) on the basis of concentration of standard samples and can be used in viral load, vaccine, and antiviral drug studies. PMID:20439981

Stranded dolphin stomach contents represent the free-ranging population's diet

PubMed Central

Dunshea, Glenn; Barros, Nélio B.; Berens McCabe, Elizabeth J.; Gales, Nicholas J.; Hindell, Mark A.; Jarman, Simon N.; Wells, Randall S.

2013-01-01

Diet is a fundamental aspect of animal ecology. Cetacean prey species are generally identified by examining stomach contents of stranded individuals. Critical uncertainty in these studies is whether samples from stranded animals are representative of the diet of free-ranging animals. Over two summers, we collected faecal and gastric samples from healthy free-ranging individuals of an extensively studied bottlenose dolphin population. These samples were analysed by molecular prey detection and these data compared with stomach contents data derived from stranded dolphins from the same population collected over 22 years. There was a remarkable consistency in the prey species composition and relative amounts between the two datasets. The conclusions of past stomach contents studies regarding dolphin habitat associations, prey selection and proposed foraging mechanisms are supported by molecular data from live animals and the combined dataset. This is the first explicit test of the validity of stomach contents analysis for accurate population-scale diet determination of an inshore cetacean. PMID:23637389

Precise assignment of the heavy-strand promoter of mouse mitochondrial DNA: cognate start sites are not required for transcriptional initiation.

PubMed Central

Chang, D D; Clayton, D A

1986-01-01

Transcription of the heavy strand of mouse mitochondrial DNA starts from two closely spaced, distinct sites located in the displacement loop region of the genome. We report here an analysis of regulatory sequences required for faithful transcription from these two sites. Data obtained from in vitro assays demonstrated that a 51-base-pair region, encompassing nucleotides -40 to +11 of the downstream start site, contains sufficient information for accurate transcription from both start sites. Deletion of the 3' flanking sequences, including one or both start sites to -17, resulted in the initiation of transcription by the mitochondrial RNA polymerase from alternative sites within vector DNA sequences. This feature places the mouse heavy-strand promoter uniquely among other known mitochondrial promoters, all of which absolutely require cognate start sites for transcription. Comparison of the heavy-strand promoter with those of other vertebrate mitochondrial DNAs revealed a remarkably high rate of sequence divergence among species. Images PMID:3785226

Development and validation of a rapid and simple LC-MS/MS method for quantification of vemurafenib in human plasma: application to a human pharmacokinetic study.

PubMed

Bihan, Kevin; Sauzay, Chloé; Goldwirt, Lauriane; Charbonnier-Beaupel, Fanny; Hulot, Jean-Sebastien; Funck-Brentano, Christian; Zahr, Noël

2015-02-01

Vemurafenib (Zelboraf) is a new tyrosine kinase inhibitor that selectively targets activated BRAF V600E gene and is indicated for the treatment of advanced BRAF mutation-positive melanoma. We developed a simple method for vemurafenib quantification using liquid chromatography-tandem mass spectrometry. A stability study of vemurafenib in human plasma was also performed. (13)C(6)-vemurafenib was used as the internal standard. A single-step protein precipitation was used for plasma sample preparation. Chromatography was performed on an Acquity UPLC system (Waters) with chromatographic separation by the use of an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7-mm particle size; Waters). Quantification was performed using the monitoring of multiple reactions of following transitions: m/z 488.2 → 381.0 for vemurafenib and m/z 494.2 → 387.0 for internal standard. This method was linear over the range from 1.0 to 100.0 mcg/mL. The lower limit of quantification was 0.1 mcg/mL for vemurafenib in plasma. Vemurafenib remained stable for 1 month at all levels tested, when stored indifferently at room temperature (20 °C), at +4 °C, or at -20 °C. This method was used successfully to perform a plasma pharmacokinetic study of vemurafenib in a patient after oral administration at a steady state. This liquid chromatography-tandem mass spectrometry method for vemurafenib quantification in human plasma is simple, rapid, specific, sensitive, accurate, precise, and reliable.

3-dimensional examination of the adult mouse subventricular zone reveals lineage-specific microdomains.

PubMed

Azim, Kasum; Fiorelli, Roberto; Zweifel, Stefan; Hurtado-Chong, Anahi; Yoshikawa, Kazuaki; Slomianka, Lutz; Raineteau, Olivier

2012-01-01

Recent studies suggest that the subventricular zone (SVZ) of the lateral ventricle is populated by heterogeneous populations of stem and progenitor cells that, depending on their exact location, are biased to acquire specific neuronal fates. This newly described heterogeneity of SVZ stem and progenitor cells underlines the necessity to develop methods for the accurate quantification of SVZ stem and progenitor subpopulations. In this study, we provide 3-dimensional topographical maps of slow cycling "stem" cells and progenitors based on their unique cell cycle properties. These maps revealed that both cell populations are present throughout the lateral ventricle wall as well as in discrete regions of the dorsal wall. Immunodetection of transcription factors expressed in defined progenitor populations further reveals that divergent lineages have clear regional enrichments in the rostro-caudal as well as in the dorso-ventral span of the lateral ventricle. Thus, progenitors expressing Tbr2 and Dlx2 were confined to dorsal and dorso-lateral regions of the lateral ventricle, respectively, while Mash1+ progenitors were more homogeneously distributed. All cell populations were enriched in the rostral-most region of the lateral ventricle. This diversity and uneven distribution greatly impede the accurate quantification of SVZ progenitor populations. This is illustrated by measuring the coefficient of error of estimates obtained by using increasing section sampling interval. Based on our empirical data, we provide such estimates for all progenitor populations investigated in this study. These can be used in future studies as guidelines to judge if the precision obtained with a sampling scheme is sufficient to detect statistically significant differences between experimental groups if a biological effect is present. Altogether, our study underlines the need to consider the SVZ of the lateral ventricle as a complex 3D structure and define methods to accurately assess neural stem cells or progenitor diversity and population sizes in physiological or experimental paradigms.

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

PubMed

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

2017-09-21

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 ([Formula: see text]) 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 [Formula: see text] was found to be a good substitute for the measured CTAC map in brain PET/MR when used with a Gaussian smoothing kernel of [Formula: see text] 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.

Near-infrared spectral tomography integrated with digital breast tomosynthesis: Effects of tissue scattering on optical data acquisition design

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

Michaelsen, Kelly; Krishnaswamy, Venkat; Pogue, Brian W.

2012-07-15

Purpose: Design optimization and phantom validation of an integrated digital breast tomosynthesis (DBT) and near-infrared spectral tomography (NIRST) system targeting improvement in sensitivity and specificity of breast cancer detection is presented. Factors affecting instrumentation design include minimization of cost, complexity, and examination time while maintaining high fidelity NIRST measurements with sufficient information to recover accurate optical property maps. Methods: Reconstructed DBT slices from eight patients with abnormal mammograms provided anatomical information for the NIRST simulations. A limited frequency domain (FD) and extensive continuous wave (CW) NIRST system was modeled. The FD components provided tissue scattering estimations used in the reconstructionmore » of the CW data. Scattering estimates were perturbed to study the effects on hemoglobin recovery. Breast mimicking agar phantoms with inclusions were imaged using the combined DBT/NIRST system for comparison with simulation results. Results: Patient simulations derived from DBT images show successful reconstruction of both normal and malignant lesions in the breast. They also demonstrate the importance of accurately quantifying tissue scattering. Specifically, 20% errors in optical scattering resulted in 22.6% or 35.1% error in quantification of total hemoglobin concentrations, depending on whether scattering was over- or underestimated, respectively. Limited frequency-domain optical signal sampling provided two regions scattering estimates (for fat and fibroglandular tissues) that led to hemoglobin concentrations that reduced the error in the tumor region by 31% relative to when a single estimate of optical scattering was used throughout the breast volume of interest. Acquiring frequency-domain data with six wavelengths instead of three did not significantly improve the hemoglobin concentration estimates. Simulation results were confirmed through experiments in two-region breast mimicking gelatin phantoms. Conclusions: Accurate characterization of scattering is necessary for quantification of hemoglobin. Based on this study, a system design is described to optimally combine breast tomosynthesis with NIRST.« less

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

NASA Astrophysics Data System (ADS)

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.

Correlation between energy deposition and molecular damage from Auger electrons: A case study of ultra-low energy (5-18 eV) electron interactions with DNA.

PubMed

Rezaee, Mohammad; Hunting, Darel J; Sanche, Léon

2014-07-01

The present study introduces a new method to establish a direct correlation between biologically related physical parameters (i.e., stopping and damaging cross sections, respectively) for an Auger-electron emitting radionuclide decaying within a target molecule (e.g., DNA), so as to evaluate the efficacy of the radionuclide at the molecular level. These parameters can be applied to the dosimetry of Auger electrons and the quantification of their biological effects, which are the main criteria to assess the therapeutic efficacy of Auger-electron emitting radionuclides. Absorbed dose and stopping cross section for the Auger electrons of 5-18 eV emitted by(125)I within DNA were determined by developing a nanodosimetric model. The molecular damages induced by these Auger electrons were investigated by measuring damaging cross section, including that for the formation of DNA single- and double-strand breaks. Nanoscale films of pure plasmid DNA were prepared via the freeze-drying technique and subsequently irradiated with low-energy electrons at various fluences. The damaging cross sections were determined by employing a molecular survival model to the measured exposure-response curves for induction of DNA strand breaks. For a single decay of(125)I within DNA, the Auger electrons of 5-18 eV deposit the energies of 12.1 and 9.1 eV within a 4.2-nm(3) volume of a hydrated or dry DNA, which results in the absorbed doses of 270 and 210 kGy, respectively. DNA bases have a major contribution to the deposited energies. Ten-electronvolt and high linear energy transfer 100-eV electrons have a similar cross section for the formation of DNA double-strand break, while 100-eV electrons are twice as efficient as 10 eV in the induction of single-strand break. Ultra-low-energy electrons (<18 eV) substantially contribute to the absorbed dose and to the molecular damage from Auger-electron emitting radionuclides; hence, they should be considered in the dosimetry calculation of such radionuclides. Moreover, absorbed dose is not an appropriate physical parameter for nanodosimetry. Instead, stopping cross section, which describes the probability of energy deposition in a target molecule can be an appropriate nanodosimetric parameter. The stopping cross section is correlated with a damaging cross section (e.g., cross section for the double-strand break formation) to quantify the number of each specific lesion in a target molecule for each nuclear decay of a single Auger-electron emitting radionuclide.

Recent advances in stable isotope labeling based techniques for proteome relative quantification.

PubMed

Zhou, Yuan; Shan, Yichu; Zhang, Lihua; Zhang, Yukui

2014-10-24

The large scale relative quantification of all proteins expressed in biological samples under different states is of great importance for discovering proteins with important biological functions, as well as screening disease related biomarkers and drug targets. Therefore, the accurate quantification of proteins at proteome level has become one of the key issues in protein science. Herein, the recent advances in stable isotope labeling based techniques for proteome relative quantification were reviewed, from the aspects of metabolic labeling, chemical labeling and enzyme-catalyzed labeling. Furthermore, the future research direction in this field was prospected. Copyright © 2014 Elsevier B.V. All rights reserved.

New Views on Strand Asymmetry in Insect Mitochondrial Genomes

PubMed Central

Wei, Shu-Jun; Shi, Min; Chen, Xue-Xin; Sharkey, Michael J.; van Achterberg, Cornelis; Ye, Gong-Yin; He, Jun-Hua

2010-01-01

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. PMID:20856815

High-throughput illumina strand-specific RNA sequencing library preparation

USDA-ARS?s Scientific Manuscript database

Conventional Illumina RNA-Seq does not have the resolution to decode the complex eukaryote transcriptome due to the lack of RNA polarity information. Strand-specific RNA sequencing (ssRNA-Seq) can overcome these limitations and as such is better suited for genome annotation, de novo transcriptome as...

Exploring the bacterial gut microbiota of supralittoral talitrid amphipods.

PubMed

Abdelrhman, Khaled F A; Bacci, Giovanni; Marras, Barbara; Nistri, Annamaria; Schintu, Marco; Ugolini, Alberto; Mengoni, Alessio

2017-01-01

Talitrid amphipods (sandhoppers and beach fleas) are typical of the supralittoral zone. They are known to thrive on stranded materials, including detrital marine angiosperms and macroalgae, as well as occasional dead animals. In this work, the gut microbiota of five species of talitrid amphipods (Talitrus saltator, Talorchestia ugolinii, Sardorchestia pelecaniformis, Orchestia montagui and Orchestia stephenseni) collected in Sardinia (Italy) has been investigated through: i) metabarcoding analysis of the amplified 16S rRNA V4 region; and ii) quantification of family 48 glycosyl hydrolase genes (GHF48), involved in cellulose degradation. Results indicate that, though talitrid gut biodiversity is not directly related to taxon or sampling locality, the animals' digestive tracts may host species-specific bacterial communities. In particular, gut microbiota of O. montagui, an inhabitant of Posidonia banquettes and macro-algae mat, showed the greatest differences in taxonomic composition and the highest proportion of GHF48 genes with respect to 16S rRNA genes. These results suggest that the different talitrid species may host species-specific bacterial communities whose function could partially reflect the different microhabitats and food preferences of their host. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Cryptic MCAT enhancer regulation in fibroblasts and smooth muscle cells. Suppression of TEF-1 mediated activation by the single-stranded DNA-binding proteins, Pur alpha, Pur beta, and MSY1.

PubMed

Carlini, Leslie E; Getz, Michael J; Strauch, Arthur R; Kelm, Robert J

2002-03-08

An asymmetric polypurine-polypyrimidine cis-element located in the 5' region of the mouse vascular smooth muscle alpha-actin gene serves as a binding site for multiple proteins with specific affinity for either single- or double-stranded DNA. Here, we test the hypothesis that single-stranded DNA-binding proteins are responsible for preventing a cryptic MCAT enhancer centered within this element from cooperating with a nearby serum response factor-interacting CArG motif to trans-activate the minimal promoter in fibroblasts and smooth muscle cells. DNA binding studies revealed that the core MCAT sequence mediates binding of transcription enhancer factor-1 to the double-stranded polypurine-polypyrimidine element while flanking nucleotides account for interaction of Pur alpha and Pur beta with the purine-rich strand and MSY1 with the complementary pyrimidine-rich strand. Mutations that selectively impaired high affinity single-stranded DNA binding by fibroblast or smooth muscle cell-derived Pur alpha, Pur beta, and MSY1 in vitro, released the cryptic MCAT enhancer from repression in transfected cells. Additional experiments indicated that Pur alpha, Pur beta, and MSY1 also interact specifically, albeit weakly, with double-stranded DNA and with transcription enhancer factor-1. These results are consistent with two plausible models of cryptic MCAT enhancer regulation by Pur alpha, Pur beta, and MSY1 involving either competitive single-stranded DNA binding or masking of MCAT-bound transcription enhancer factor-1.

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

PubMed

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.

Accurate quantification of fluorescent targets within turbid media based on a decoupled fluorescence Monte Carlo model.

PubMed

Deng, Yong; Luo, Zhaoyang; Jiang, Xu; Xie, Wenhao; Luo, Qingming

2015-07-01

We propose a method based on a decoupled fluorescence Monte Carlo model for constructing fluorescence Jacobians to enable accurate quantification of fluorescence targets within turbid media. The effectiveness of the proposed method is validated using two cylindrical phantoms enclosing fluorescent targets within homogeneous and heterogeneous background media. The results demonstrate that our method can recover relative concentrations of the fluorescent targets with higher accuracy than the perturbation fluorescence Monte Carlo method. This suggests that our method is suitable for quantitative fluorescence diffuse optical tomography, especially for in vivo imaging of fluorophore targets for diagnosis of different diseases and abnormalities.

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

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

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

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 undermore » 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.« less

Precise montaging and metric quantification of retinal surface area from ultra-widefield fundus photography and fluorescein angiography.

PubMed

Croft, Daniel E; van Hemert, Jano; Wykoff, Charles C; Clifton, David; Verhoek, Michael; Fleming, Alan; Brown, David M

2014-01-01

Accurate quantification of retinal surface area from ultra-widefield (UWF) images is challenging due to warping produced when the retina is projected onto a two-dimensional plane for analysis. By accounting for this, the authors sought to precisely montage and accurately quantify retinal surface area in square millimeters. Montages were created using Optos 200Tx (Optos, Dunfermline, U.K.) images taken at different gaze angles. A transformation projected the images to their correct location on a three-dimensional model. Area was quantified with spherical trigonometry. Warping, precision, and accuracy were assessed. Uncorrected, posterior pixels represented up to 79% greater surface area than peripheral pixels. Assessing precision, a standard region was quantified across 10 montages of the same eye (RSD: 0.7%; mean: 408.97 mm(2); range: 405.34-413.87 mm(2)). Assessing accuracy, 50 patients' disc areas were quantified (mean: 2.21 mm(2); SE: 0.06 mm(2)), and the results fell within the normative range. By accounting for warping inherent in UWF images, precise montaging and accurate quantification of retinal surface area in square millimeters were achieved. Copyright 2014, SLACK Incorporated.

COSMOS: accurate detection of somatic structural variations through asymmetric comparison between tumor and normal samples

PubMed Central

Yamagata, Koichi; Yamanishi, Ayako; Kokubu, Chikara; Takeda, Junji; Sese, Jun

2016-01-01

An important challenge in cancer genomics is precise detection of structural variations (SVs) by high-throughput short-read sequencing, which is hampered by the high false discovery rates of existing analysis tools. Here, we propose an accurate SV detection method named COSMOS, which compares the statistics of the mapped read pairs in tumor samples with isogenic normal control samples in a distinct asymmetric manner. COSMOS also prioritizes the candidate SVs using strand-specific read-depth information. Performance tests on modeled tumor genomes revealed that COSMOS outperformed existing methods in terms of F-measure. We also applied COSMOS to an experimental mouse cell-based model, in which SVs were induced by genome engineering and gamma-ray irradiation, followed by polymerase chain reaction-based confirmation. The precision of COSMOS was 84.5%, while the next best existing method was 70.4%. Moreover, the sensitivity of COSMOS was the highest, indicating that COSMOS has great potential for cancer genome analysis. PMID:26833260

Multiplex Droplet Digital PCR Protocols for Quantification of GM Maize Events.

PubMed

Dobnik, David; Spilsberg, Bjørn; Bogožalec Košir, Alexandra; Štebih, Dejan; Morisset, Dany; Holst-Jensen, Arne; Žel, Jana

2018-01-01

The standard-curve based simplex quantitative polymerase chain reaction (qPCR) has been the gold standard for DNA target quantification for more than a decade. The large and growing number of individual analyses needed to test for genetically modified organisms (GMOs) is reducing the cost-effectiveness of qPCR. Droplet digital PCR (ddPCR) enables absolute quantification without standard curves, avoids the amplification efficiency bias observed with qPCR, allows more accurate estimations at low target copy numbers and, in combination with multiplexing, significantly improves cost efficiency. Here we describe two protocols for multiplex quantification of GM maize events: (1) nondiscriminating, with multiplex quantification of targets as a group (12 GM maize lines) and (2) discriminating, with multiplex quantification of individual targets (events). The first enables the quantification of twelve European Union authorized GM maize events as a group with only two assays, but does not permit determination of the individual events present. The second protocol enables the quantification of four individual targets (three GM events and one endogene) in a single reaction. Both protocols can be modified for quantification of any other DNA target.

Establishment and evaluation of a bead-based luminex assay allowing simultaneous quantification of equine IL-12 and IFN-γ.

PubMed

Duran, Maria Carolina; Willenbrock, Saskia; Müller, Jessika-M V; Nolte, Ingo; Feige, Karsten; Murua Escobar, Hugo

2013-04-01

Interleukin-12 (IL-12) and interferon gamma (IFN-γ) are key cytokines in immunemediated equine melanoma therapy. Currently, a method for accurate simultaneous quantification of these equine cytokines is lacking. Therefore, we sought to establish an assay that allows for accurate and simultaneous quantification of equine IL-12 (eIL-12) and IFN-γ (eIFN-γ). Several antibodies were evaluated for cross-reactivity to eIL-12 and eIFN-γ and were used to establish a bead-based Luminex assay, which was subsequently applied to quantify cytokine concentrations in biological samples. Cytokine detection ranged from 31.5-5,000 pg/ml and 15-10,000 pg/ml for eIL-12 and eIFN-γ, respectively. eIL-12 was detected in supernatants of stimulated peripheral blood mononuclear cells (PBMCs) and supernatants/cell lysates of eIL-12 expression plasmid-transfected cells. Low or undetectable cytokine concentrations were measured in negative controls. In equine serum samples, the mean measured eIL-12 concentration was 1,374 ± 8 pg/ml. The bead-based assay and ELISA for eIFN-γ used to measure eIFN-γ concentrations, showed similar concentrations. Results demonstrate, to our knowledge for the first time, that cross-reactive antibody pairs to eIL-12 and eIFN-γ and Luminex bead-based technology allow for accurate, simultaneous and multiplexed quantification of these key cytokines in biological samples.

Melanin fate in the human epidermis: a reassessment of how best to detect and analyse histologically.

PubMed

Joly-Tonetti, Nicolas; Wibawa, Judata I D; Bell, Mike; Tobin, Desmond

2016-07-01

Melanin is the predominant pigment responsible for skin colour and is synthesized by the melanocyte in the basal layer of the epidermis and then transferred to surrounding keratinocytes. Despite its optical properties, melanin is barely detectable in unstained sections of human epidermis. However, identification and localization of melanin is of importance for the study of skin pigmentation in health and disease. Current methods for the histologic quantification of melanin are suboptimal and are associated with significant risk of misinterpretation. The aim of this study was to reassess the existing literature and to develop a more effective histological method of melanin quantification in human skin. Moreover, we confirm that Warthin-Starry (WS) stain provides a much more sensitive and more specific melanin detection method than the commonplace Fontana-Masson (FM) stain. For example, WS staining sensitivity allowed the visualization of melanin even in very pale Caucasian skin that was missed by FM or Von Kossa (VK) stains. From our reassessment of the histology-related literature, we conclude that so-called melanin dust is most likely an artifact of discoloration due to non-specific silver deposition in the stratum corneum. Unlike FM and VK, WS was not associated with this non-specific stratum corneum darkening, misinterpreted previously as 'degraded' melanin. Finally, WS melanin particle counts were largely similar to previously reported manual counts by transmission electron microscopy, in contrast to both FM and VK. Together these findings allow us to propose a new histology/Image J-informed method for the accurate and precise quantification of epidermal melanin in skin. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Novel Real-Time PCR Assay of microRNAs Using S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer with Excellent Sensitivity and Specificity

PubMed Central

Kang, Kang; Zhang, Xiaoying; Liu, Hongtao; Wang, Zhiwei; Zhong, Jiasheng; Huang, Zhenting; Peng, Xiao; Zeng, Yan; Wang, Yuna; Yang, Yi; Luo, Jun; Gou, Deming

2012-01-01

Background MicroRNAs (miRNAs) are small, non-coding RNAs capable of postranscriptionally regulating gene expression. Accurate expression profiling is crucial for understanding the biological roles of miRNAs, and exploring them as biomarkers of diseases. Methodology/Principal Findings A novel, highly sensitive, and reliable miRNA quantification approach,termed S-Poly(T) miRNA assay, is designed. In this assay, miRNAs are subjected to polyadenylation and reverse transcription with a S-Poly(T) primer that contains a universal reverse primer, a universal Taqman probe, an oligo(dT)11 sequence and six miRNA-specific bases. Individual miRNAs are then amplified by a specific forward primer and a universal reverse primer, and the PCR products are detected by a universal Taqman probe. The S-Poly(T) assay showed a minimum of 4-fold increase in sensitivity as compared with the stem-loop or poly(A)-based methods. A remarkable specificity in discriminating among miRNAs with high sequence similarity was also obtained with this approach. Using this method, we profiled miRNAs in human pulmonary arterial smooth muscle cells (HPASMC) and identified 9 differentially expressed miRNAs associated with hypoxia treatment. Due to its outstanding sensitivity, the number of circulating miRNAs from normal human serum was significantly expanded from 368 to 518. Conclusions/Significance With excellent sensitivity, specificity, and high-throughput, the S-Poly(T) method provides a powerful tool for miRNAs quantification and identification of tissue- or disease-specific miRNA biomarkers. PMID:23152780

Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system.

PubMed

Herz, Katia; Becker, Alexandra; Shi, Chenyue; Ema, Masatsugo; Takahashi, Satoru; Potente, Michael; Hesse, Michael; Fleischmann, Bernd K; Wenzel, Daniela

2018-05-01

Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP + signals specifically in Ki67 + /PECAM + endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.

Detection of malondialdehyde in processed meat products without interference from the ingredients.

PubMed

Jung, Samooel; Nam, Ki Chang; Jo, Cheorun

2016-10-15

Our aim was to develop a method for accurate quantification of malondialdehyde (MDA) in meat products. MDA content of uncured ground pork (Control); ground pork cured with sodium nitrite (Nitrite); and ground pork cured with sodium nitrite, sodium chloride, sodium pyrophosphate, maltodextrin, and a sausage seasoning (Mix) was measured by the 2-thiobarbituric acid (TBA) assay with MDA extraction by trichloroacetic acid (method A) and two high-performance liquid chromatography (HPLC) methods: i) HPLC separation of the MDA-dinitrophenyl hydrazine adduct (method B) and ii) HPLC separation of MDA (method C) after MDA extraction with acetonitrile. Methods A and B could not quantify MDA accurately in groups Nitrite and Mix. Nevertheless, MDA in groups Control, Nitrite, and Mix was accurately quantified by method C with good recovery. Therefore, direct MDA quantification by HPLC after MDA extraction with acetonitrile (method C) is useful for accurate measurement of MDA content in processed meat products. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

PubMed

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.

DNA Photo Lithography with Cinnamate-based Photo-Bio-Nano-Glue

NASA Astrophysics Data System (ADS)

Feng, Lang; Li, Minfeng; Romulus, Joy; Sha, Ruojie; Royer, John; Wu, Kun-Ta; Xu, Qin; Seeman, Nadrian; Weck, Marcus; Chaikin, Paul

2013-03-01

We present a technique to make patterned functional surfaces, using a cinnamate photo cross-linker and photolithography. We have designed and modified a complementary set of single DNA strands to incorporate a pair of opposing cinnamate molecules. On exposure to 360nm UV, the cinnamate makes a highly specific covalent bond permanently linking only the complementary strands containing the cinnamates. We have studied this specific and efficient crosslinking with cinnamate-containing DNA in solution and on particles. UV addressability allows us to pattern surfaces functionally. The entire surface is coated with a DNA sequence A incorporating cinnamate. DNA strands A'B with one end containing a complementary cinnamated sequence A' attached to another sequence B, are then hybridized to the surface. UV photolithography is used to bind the A'B strand in a specific pattern. The system is heated and the unbound DNA is washed away. The pattern is then observed by thermo-reversibly hybridizing either fluorescently dyed B' strands complementary to B, or colloids coated with B' strands. Our techniques can be used to reversibly and/or permanently bind, via DNA linkers, an assortment of molecules, proteins and nanostructures. Potential applications range from advanced self-assembly, such as templated self-replication schemes recently reported, to designed physical and chemical patterns, to high-resolution multi-functional DNA surfaces for genetic detection or DNA computing.

Phenomenological Partial Specific Volumes for G-Quadruplex DNAs

PubMed Central

Hellman, Lance M.; Rodgers, David W.; Fried, Michael G.

2009-01-01

Accurate partial specific volume (ν̄) values are required for sedimentation velocity and sedimentation equilibrium analyses. For nucleic acids, the estimation of these values is complicated by the fact that ν̄ depends on base composition, secondary structure, solvation and the concentrations and identities of ions in the surrounding buffer. Here we describe sedimentation equilibrium measurements of the apparent isopotential partial specific volume φ′ for two G-quadruplex DNAs and a single-stranded DNA of similar molecular weight and base composition. The G-quadruplex DNAs are a 22 nucleotide fragment of the human telomere consensus sequence and a 27 nucleotide fragment from the human c-myc promoter. The single-stranded DNA is 26 nucleotides long and is designed to have low propensity to form secondary structures. Parallel measurements were made in buffers containing NaCl and in buffers containing KCl, spanning the range 0.09M ≤ [salt] ≤ 2.3M. Limiting values of φ′, extrapolated to [salt] = 0M, were: 22-mer (NaCl-form), 0.525 ± 0.004 mL/g; 22-mer (KCl-form), 0.531 ± 0.006 mL/g; 27-mer (NaCl-form), 0.548 ± 0.005 mL/g; 27-mer (KCl-form), 0.557 ± 0.006 mL/g; 26-mer (NaCl-form), 0.555 ± 0.004 mL/g; 26-mer (KCl-form), 0.564 ± 0.006 mL/g. Small changes in φ′ with [salt] suggest that large changes in counterion association or hydration are unlikely to take place over these concentration ranges. PMID:19238377

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

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

Zhao, H.; Wynne, B.P.; Palmiere, E.J., E-mail: e.j

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 orientationmore » 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.« less

Isolation and Expression Profile of the Ca2+-Activated Chloride Channel-like Membrane Protein 6 Gene in Xenopus laevis

PubMed Central

Lee, Ra Mi; Ryu, Rae Hyung; Jeong, Seong Won; Oh, Soo Jin; Huang, Hue; Han, Jin Soo; Lee, Chi Ho; Lee, C. Justin; Jan, Lily Yeh

2011-01-01

To clone the first anion channel from Xenopus laevis (X. laevis), we isolated a calcium-activated chloride channel (CLCA)-like membrane protein 6 gene (CMP6) in X. laevis. As a first step in gene isolation, an expressed sequence tags database was screened to find the partial cDNA fragment. A putative partial cDNA sequence was obtained by comparison with rat CLCAs identified in our laboratory. First stranded cDNA was synthesized by reverse transcription polymerase-chain reaction (RT-PCR) using a specific primer designed for the target cDNA. Repeating the 5' and 3' rapid amplification of cDNA ends, full-length cDNA was constructed from the cDNA pool. The full-length CMP6 cDNA completed via 5'- and 3'-RACE was 2,940 bp long and had an open reading frame (ORF) of 940 amino acids. The predicted 940 polypeptides have four major transmembrane domains and showed about 50% identity with that of rat brain CLCAs in our previously published data. Semi-quantification analysis revealed that CMP6 was most abundantly expressed in small intestine, colon and liver. However, all tissues except small intestine, colon and liver had undetectable levels. This result became more credible after we did real-time PCR quantification for the target gene. In view of all CLCA studies focused on human or murine channels, this finding suggests a hypothetical protein as an ion channel, an X. laevis CLCA. PMID:21826170

Programmable and multiparameter DNA-based logic platform for cancer recognition and targeted therapy.

PubMed

You, Mingxu; Zhu, Guizhi; Chen, Tao; Donovan, Michael J; Tan, Weihong

2015-01-21

The specific inventory of molecules on diseased cell surfaces (e.g., cancer cells) provides clinicians an opportunity for accurate diagnosis and intervention. With the discovery of panels of cancer markers, carrying out analyses of multiple cell-surface markers is conceivable. As a trial to accomplish this, we have recently designed a DNA-based device that is capable of performing autonomous logic-based analysis of two or three cancer cell-surface markers. Combining the specific target-recognition properties of DNA aptamers with toehold-mediated strand displacement reactions, multicellular marker-based cancer analysis can be realized based on modular AND, OR, and NOT Boolean logic gates. Specifically, we report here a general approach for assembling these modular logic gates to execute programmable and higher-order profiling of multiple coexisting cell-surface markers, including several found on cancer cells, with the capacity to report a diagnostic signal and/or deliver targeted photodynamic therapy. The success of this strategy demonstrates the potential of DNA nanotechnology in facilitating targeted disease diagnosis and effective therapy.

Development and Validation of Different Ultraviolet-Spectrophotometric Methods for the Estimation of Besifloxacin in Different Simulated Body Fluids.

PubMed

Singh, C L; Singh, A; Kumar, S; Kumar, M; Sharma, P K; Majumdar, D K

2015-01-01

In the present study a simple, accurate, precise, economical and specific UV-spectrophotometric method for estimation of besifloxacin in bulk and in different pharmaceutical formulation has been developed. The drug shows maximum λmax289 nm in distilled water, simulated tears and phosphate buffer saline. The linearity range of developed methods were in the range of 3-30 μg/ml of drug with a correlation coefficient (r(2)) 0.9992, 0.9989 and 0.9984 with respect to distilled water, simulated tears and phosphate buffer saline, respectively. Reproducibility by repeating methods as %RSD were found to be less than 2%. The limit of detection in different media was found to be 0.62, 0.72 and 0.88 μg/ml, respectively. The limit of quantification was found to be 1.88, 2.10, 2.60 μg/ml, respectively. The proposed method was validated statically according to International Conference on Harmonization guidelines with respect to specificity, linearity, range, accuracy, precision and robustness. The proposed methods of validation were found to be accurate and highly specific for the estimation of besifloxacin in different pharmaceutical formulations.

Phylogenetic Quantification of Intra-tumour Heterogeneity

PubMed Central

Schwarz, Roland F.; Trinh, Anne; Sipos, Botond; Brenton, James D.; Goldman, Nick; Markowetz, Florian

2014-01-01

Intra-tumour genetic heterogeneity is the result of ongoing evolutionary change within each cancer. The expansion of genetically distinct sub-clonal populations may explain the emergence of drug resistance, and if so, would have prognostic and predictive utility. However, methods for objectively quantifying tumour heterogeneity have been missing and are particularly difficult to establish in cancers where predominant copy number variation prevents accurate phylogenetic reconstruction owing to horizontal dependencies caused by long and cascading genomic rearrangements. To address these challenges, we present MEDICC, a method for phylogenetic reconstruction and heterogeneity quantification based on a Minimum Event Distance for Intra-tumour Copy-number Comparisons. Using a transducer-based pairwise comparison function, we determine optimal phasing of major and minor alleles, as well as evolutionary distances between samples, and are able to reconstruct ancestral genomes. Rigorous simulations and an extensive clinical study show the power of our method, which outperforms state-of-the-art competitors in reconstruction accuracy, and additionally allows unbiased numerical quantification of tumour heterogeneity. Accurate quantification and evolutionary inference are essential to understand the functional consequences of tumour heterogeneity. The MEDICC algorithms are independent of the experimental techniques used and are applicable to both next-generation sequencing and array CGH data. PMID:24743184

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

PubMed Central

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

Subnuclear foci quantification using high-throughput 3D image cytometry

NASA Astrophysics Data System (ADS)

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.

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

PubMed

Li, Xin; Wang, Xun; Song, Tao; Lu, Wei; Chen, Zhihua; Shi, Xiaolong

2015-01-01

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.

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

PubMed Central

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

A sensitive analytical procedure for monitoring acrylamide in environmental water samples by offline SPE-UPLC/MS/MS.

PubMed

Togola, Anne; Coureau, Charlotte; Guezennec, Anne-Gwenaëlle; Touzé, Solène

2015-05-01

The presence of acrylamide in natural systems is of concern from both environmental and health points of view. We developed an accurate and robust analytical procedure (offline solid phase extraction combined with UPLC/MS/MS) with a limit of quantification (20 ng L(-1)) compatible with toxicity threshold values. The optimized (considering the nature of extraction phases, sampling volumes, and solvent of elution) solid phase extraction (SPE) was validated according to ISO Standard ISO/IEC 17025 on groundwater, surface water, and industrial process water samples. Acrylamide is highly polar, which induces a high variability during the SPE step, therefore requiring the use of C(13)-labeled acrylamide as an internal standard to guarantee the accuracy and robustness of the method (uncertainty about 25 % (k = 2) at limit of quantification level). The specificity of the method and the stability of acrylamide were studied for these environmental media, and it was shown that the method is suitable for measuring acrylamide in environmental studies.

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

Bloomquist, R.G.

District heating and cooling (DHC) can provide multiple opportunities to reduce air emissions associated with space conditioning and electricity generation, which contribute 30% to 50% of all such emissions. When DHC is combined with cogeneration (CHP), maximum reductions in sulfur oxides (SO{sub x}), nitrogen oxides (NO{sub x}), carbon dioxide (CO{sub 2}), particulates, and ozone-depleting chlorofluorocarbon (CFC) refrigerants can most effectively be achieved. Although significant improvements in air quality have been documented in Europe and Scandinavia due to DHC and CHP implementation, accurately predicting such improvements has been difficult. Without acceptable quantification methods, regulatory bodies are reluctant to grant air emissionsmore » credits, and local community leaders are unwilling to invest in DHC and CHP as preferred methods of providing energy or strategies for air quality improvement. The recent development and release of a number of computer models designed specifically to provide quantification of air emissions that can result from DHC and CHP implementation should help provide local, state, and national policymakers with information vital to increasing support and investment in DHC development.« less

Writing and applications of fiber Bragg grating arrays

NASA Astrophysics Data System (ADS)

LaRochelle, Sophie; Cortes, Pierre-Yves; Fathallah, H.; Rusch, Leslie A.; Jaafar, H. B.

2000-12-01

Multiple Bragg gratings are written in a single fibre strand with accurate positioning to achieve predetermined time delays between optical channels. Applications of fibre Bragg grating arrays include encoders/decoders with series of identical gratings for optical code-division multiple access.

Detection of bacterial 16S rRNA using a molecular beacon-based X sensor

PubMed Central

Gerasimova, Yulia V.; Kolpashchikov, Dmitry M.

2012-01-01

We demonstrate how a long structurally constrained RNA can be analyzed in homogeneous solution at ambient temperatures with high specificity using a sophisticated biosensor. The sensor consists of a molecular beacon probe as a signal reporter and two DNA adaptor strands, which have fragments complementary to the reporter and to the analyzed RNA. One adaptor strand uses its long RNA-binding arm to unwind the RNA secondary structure. Second adaptor strand with a short RNA-binding arm hybridizes only to a fully complementary site, thus providing high recognition specificity. Overall the three-component sensor and the target RNA form a four-stranded DNA crossover (X) structure. Using this sensor, E.coli 16S rRNA was detected in real time with the detection limit of ~ 0.17 nM. The high specificity of the analysis was proven by differentiating B.subtilus from E.coli 16S rRNA sequences. The sensor responds to the presence of the analyte within seconds. PMID:23021850

Modulation of the Pyrococcus abyssi NucS Endonuclease Activity by Replication Clamp at Functional and Structural Levels*

PubMed Central

Creze, Christophe; Ligabue, Alessio; Laurent, Sébastien; Lestini, Roxane; Laptenok, Sergey P.; Khun, Joelle; Vos, Marten H.; Czjzek, Mirjam; Myllykallio, Hannu; Flament, Didier

2012-01-01

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5′ and 3′ flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction. PMID:22431731

Modulation of the Pyrococcus abyssi NucS endonuclease activity by replication clamp at functional and structural levels.

PubMed

Creze, Christophe; Ligabue, Alessio; Laurent, Sébastien; Lestini, Roxane; Laptenok, Sergey P; Khun, Joelle; Vos, Marten H; Czjzek, Mirjam; Myllykallio, Hannu; Flament, Didier

2012-05-04

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5' and 3' flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction.

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

PubMed Central

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

2006-01-01

Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was 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. PMID:16907967

A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins.

PubMed

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.

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

PubMed

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.

Determining orientation and direction of DNA sequences

DOEpatents

Goodwin, Edwin H.; Meyne, Julianne

2000-01-01

Determining orientation and direction of DNA sequences. A method by which fluorescence in situ hybridization can be made strand specific is described. Cell cultures are grown in a medium containing a halogenated nucleotide. The analog is partially incorporated in one DNA strand of each chromatid. This substitution takes place in opposite strands of the two sister chromatids. After staining with the fluorescent DNA-binding dye Hoechst 33258, cells are exposed to long-wavelength ultraviolet light which results in numerous strand nicks. These nicks enable the substituted strand to be denatured and solubilized by heat, treatment with high or low pH aqueous solutions, or by immersing the strands in 2.times.SSC (0.3M NaCl+0.03M sodium citrate), to name three procedures. It is unnecessary to enzymatically digest the strands using Exo III or another exonuclease in order to excise and solubilize nucleotides starting at the sites of the nicks. The denaturing/solubilizing process removes most of the substituted strand while leaving the prereplication strand largely intact. Hybridization of a single-stranded probe of a tandem repeat arranged in a head-to-tail orientation will result in hybridization only to the chromatid with the complementary strand present.

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

PubMed

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.

Accurate quantification of PGE2 in the polyposis in rat colon (Pirc) model by surrogate analyte-based UPLC-MS/MS.

PubMed

Yun, Changhong; Dashwood, Wan-Mohaiza; Kwong, Lawrence N; Gao, Song; Yin, Taijun; Ling, Qinglan; Singh, Rashim; Dashwood, Roderick H; Hu, Ming

2018-01-30

An accurate and reliable UPLC-MS/MS method is reported for the quantification of endogenous Prostaglandin E2 (PGE 2 ) in rat colonic mucosa and polyps. This method adopted the "surrogate analyte plus authentic bio-matrix" approach, using two different stable isotopic labeled analogs - PGE 2 -d9 as the surrogate analyte and PGE 2 -d4 as the internal standard. A quantitative standard curve was constructed with the surrogate analyte in colonic mucosa homogenate, and the method was successfully validated with the authentic bio-matrix. Concentrations of endogenous PGE 2 in both normal and inflammatory tissue homogenates were back-calculated based on the regression equation. Because of no endogenous interference on the surrogate analyte determination, the specificity was particularly good. By using authentic bio-matrix for validation, the matrix effect and exaction recovery are identically same for the quantitative standard curve and actual samples - this notably increased the assay accuracy. The method is easy, fast, robust and reliable for colon PGE 2 determination. This "surrogate analyte" approach was applied to measure the Pirc (an Apc-mutant rat kindred that models human FAP) mucosa and polyps PGE 2 , one of the strong biomarkers of colorectal cancer. A similar concept could be applied to endogenous biomarkers in other tissues. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantifying Motor Impairment in Movement Disorders.

PubMed

FitzGerald, James J; Lu, Zhongjiao; Jareonsettasin, Prem; Antoniades, Chrystalina A

2018-01-01

Until recently the assessment of many movement disorders has relied on clinical rating scales that despite careful design are inherently subjective and non-linear. This makes accurate and truly observer-independent quantification difficult and limits the use of sensitive parametric statistical methods. At last, devices capable of measuring neurological problems quantitatively are becoming readily available. Examples include the use of oculometers to measure eye movements and accelerometers to measure tremor. Many applications are being developed for use on smartphones. The benefits include not just more accurate disease quantification, but also consistency of data for longitudinal studies, accurate stratification of patients for entry into trials, and the possibility of automated data capture for remote follow-up. In this mini review, we will look at movement disorders with a particular focus on Parkinson's disease, describe some of the limitations of existing clinical evaluation tools, and illustrate the ways in which objective metrics have already been successful.

Day and night variations in the repair of ionizing-radiation-induced DNA damage in mouse splenocytes.

PubMed

Palombo, Philipp; Moreno-Villanueva, Maria; Mangerich, Aswin

2015-04-01

In mammals, biological rhythms synchronize physiological and behavioral processes to the 24-h light-dark (LD) cycle. At the molecular level, self-sustaining processes, such as oscillations of transcription-translation feedback loops, control the circadian clock, which in turn regulates a wide variety of cellular processes, including gene expression and cell cycle progression. Furthermore, previous studies reported circadian oscillations in the repair capacity of DNA lesions specifically repaired by nucleotide excision repair (NER). However, it is so far only poorly understood if DNA repair pathways other than NER are under circadian control, in particular base excision and DNA strand break repair. In the present study, we analyzed potential day and night variations in the repair of DNA lesions induced by ionizing radiation (i.e., mainly oxidative damage and DNA strand breaks) in living mouse splenocytes using a modified protocol of the automated FADU assay. Our results reveal that splenocytes isolated from mice during the light phase (ZT06) displayed higher DNA repair activity than those of the dark phase (ZT18). As analyzed by highly sensitive and accurate qPCR arrays, these alterations were accompanied by significant differences in expression profiles of genes involved in the circadian clock and DNA repair. Notably, the majority of the DNA repair genes were expressed at higher levels during the light phase (ZT06). This included genes of all major DNA repair pathways with the strongest differences observed for genes of base excision and DNA double strand break repair. In conclusion, here we provide novel evidence that mouse splenocytes exhibit significant differences in the repair of IR-induced DNA damage during the LD cycle, both on a functional and on a gene expression level. It will be interesting to test if these findings could be exploited for therapeutic purposes, e.g. time-of-the-day-specific application of DNA-damaging treatments used against blood malignancies. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of endogenous reference genes on digital PCR assessment of genetically engineered canola events.

PubMed

Demeke, Tigst; Eng, Monika

2018-05-01

Droplet digital PCR (ddPCR) has been used for absolute quantification of genetically engineered (GE) events. Absolute quantification of GE events by duplex ddPCR requires the use of appropriate primers and probes for target and reference gene sequences in order to accurately determine the amount of GE materials. Single copy reference genes are generally preferred for absolute quantification of GE events by ddPCR. Study has not been conducted on a comparison of reference genes for absolute quantification of GE canola events by ddPCR. The suitability of four endogenous reference sequences ( HMG-I/Y , FatA(A), CruA and Ccf) for absolute quantification of GE canola events by ddPCR was investigated. The effect of DNA extraction methods and DNA quality on the assessment of reference gene copy numbers was also investigated. ddPCR results were affected by the use of single vs. two copy reference genes. The single copy, FatA(A), reference gene was found to be stable and suitable for absolute quantification of GE canola events by ddPCR. For the copy numbers measured, the HMG-I/Y reference gene was less consistent than FatA(A) reference gene. The expected ddPCR values were underestimated when CruA and Ccf (two copy endogenous Cruciferin sequences) were used because of high number of copies. It is important to make an adjustment if two copy reference genes are used for ddPCR in order to obtain accurate results. On the other hand, real-time quantitative PCR results were not affected by the use of single vs. two copy reference genes.

Direct quantification of lipopeptide biosurfactants in biological samples via HPLC and UPLC-MS requires sample modification with an organic solvent.

PubMed

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.

21 CFR 866.5820 - Systemic lupus erythema-tosus immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... with cellular nuclear double-stranded deoxyribonucleic acid (DNA) or other nuclear constituents that are specifically diagnostic of SLE. Measurement of nuclear double-stranded DNA antibodies aids in the...

21 CFR 866.5820 - Systemic lupus erythema-tosus immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... with cellular nuclear double-stranded deoxyribonucleic acid (DNA) or other nuclear constituents that are specifically diagnostic of SLE. Measurement of nuclear double-stranded DNA antibodies aids in the...

21 CFR 866.5820 - Systemic lupus erythema-tosus immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... with cellular nuclear double-stranded deoxyribonucleic acid (DNA) or other nuclear constituents that are specifically diagnostic of SLE. Measurement of nuclear double-stranded DNA antibodies aids in the...

21 CFR 866.5820 - Systemic lupus erythema-tosus immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... with cellular nuclear double-stranded deoxyribonucleic acid (DNA) or other nuclear constituents that are specifically diagnostic of SLE. Measurement of nuclear double-stranded DNA antibodies aids in the...

Optical control of filamentation-induced damage to DNA by intense, ultrashort, near-infrared laser pulses

PubMed Central

Dharmadhikari, J. A.; Dharmadhikari, A. K.; Kasuba, K. C.; Bharambe, H.; D’Souza, J. S.; Rathod, K. D.; Mathur, D.

2016-01-01

We report on damage to DNA in an aqueous medium induced by ultrashort pulses of intense laser light of 800 nm wavelength. Focusing of such pulses, using lenses of various focal lengths, induces plasma formation within the aqueous medium. Such plasma can have a spatial extent that is far in excess of the Rayleigh range. In the case of water, the resulting ionization and dissociation gives rise to in situ generation of low-energy electrons and OH-radicals. Interactions of these with plasmid DNA produce nicks in the DNA backbone: single strand breaks (SSBs) are induced as are, at higher laser intensities, double strand breaks (DSBs). Under physiological conditions, the latter are not readily amenable to repair. Systematic quantification of SSBs and DSBs at different values of incident laser energy and under different external focusing conditions reveals that damage occurs in two distinct regimes. Numerical aperture is the experimental handle that delineates the two regimes, permitting simple optical control over the extent of DNA damage. PMID:27279565

Detection of proteins using a colorimetric bio-barcode assay.

PubMed

Nam, Jwa-Min; Jang, Kyung-Jin; Groves, Jay T

2007-01-01

The colorimetric bio-barcode assay is a red-to-blue color change-based protein detection method with ultrahigh sensitivity. This assay is based on both the bio-barcode amplification method that allows for detecting miniscule amount of targets with attomolar sensitivity and gold nanoparticle-based colorimetric DNA detection method that allows for a simple and straightforward detection of biomolecules of interest (here we detect interleukin-2, an important biomarker (cytokine) for many immunodeficiency-related diseases and cancers). The protocol is composed of the following steps: (i) conjugation of target capture molecules and barcode DNA strands onto silica microparticles, (ii) target capture with probes, (iii) separation and release of barcode DNA strands from the separated probes, (iv) detection of released barcode DNA using DNA-modified gold nanoparticle probes and (v) red-to-blue color change analysis with a graphic software. Actual target detection and quantification steps with premade probes take approximately 3 h (whole protocol including probe preparations takes approximately 3 days).

An UPLC-MS/MS method for separation and accurate quantification of tamoxifen and its metabolites isomers.

PubMed

Arellano, Cécile; Allal, Ben; Goubaa, Anwar; Roché, Henri; Chatelut, Etienne

2014-11-01

A selective and accurate analytical method is needed to quantify tamoxifen and its phase I metabolites in a prospective clinical protocol, for evaluation of pharmacokinetic parameters of tamoxifen and its metabolites in adjuvant treatment of breast cancer. The selectivity of the analytical method is a fundamental criteria to allow the quantification of the main active metabolites (Z)-isomers from (Z)'-isomers. An UPLC-MS/MS method was developed and validated for the quantification of (Z)-tamoxifen, (Z)-endoxifen, (E)-endoxifen, Z'-endoxifen, (Z)'-endoxifen, (Z)-4-hydroxytamoxifen, (Z)-4'-hydroxytamoxifen, N-desmethyl tamoxifen, and tamoxifen-N-oxide. The validation range was set between 0.5ng/mL and 125ng/mL for 4-hydroxytamoxifen and endoxifen isomers, and between 12.5ng/mL and 300ng/mL for tamoxifen, tamoxifen N-desmethyl and tamoxifen-N-oxide. The application to patient plasma samples was performed. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of genomic high-throughput sequencing data generated on Illumina HiSeq and Genome Analyzer systems

PubMed Central

2011-01-01

Background The generation and analysis of high-throughput sequencing data are becoming a major component of many studies in molecular biology and medical research. Illumina's Genome Analyzer (GA) and HiSeq instruments are currently the most widely used sequencing devices. Here, we comprehensively evaluate properties of genomic HiSeq and GAIIx data derived from two plant genomes and one virus, with read lengths of 95 to 150 bases. Results We provide quantifications and evidence for GC bias, error rates, error sequence context, effects of quality filtering, and the reliability of quality values. By combining different filtering criteria we reduced error rates 7-fold at the expense of discarding 12.5% of alignable bases. While overall error rates are low in HiSeq data we observed regions of accumulated wrong base calls. Only 3% of all error positions accounted for 24.7% of all substitution errors. Analyzing the forward and reverse strands separately revealed error rates of up to 18.7%. Insertions and deletions occurred at very low rates on average but increased to up to 2% in homopolymers. A positive correlation between read coverage and GC content was found depending on the GC content range. Conclusions The errors and biases we report have implications for the use and the interpretation of Illumina sequencing data. GAIIx and HiSeq data sets show slightly different error profiles. Quality filtering is essential to minimize downstream analysis artifacts. Supporting previous recommendations, the strand-specificity provides a criterion to distinguish sequencing errors from low abundance polymorphisms. PMID:22067484

Quantification of Neural Ethanol and Acetaldehyde Using Headspace GC-MS

PubMed Central

Heit, Claire; Eriksson, Peter; Thompson, David C; Fritz, Kristofer S; Vasiliou, Vasilis

2016-01-01

BACKGROUND There is controversy regarding the active agent responsible for alcohol addiction. The theory that ethanol itself was the agent in alcohol drinking behavior was widely accepted until acetaldehyde was found in the brain. The importance of acetaldehyde formation in the brain role is still subject to speculation due to the lack of a method to accurately assay the acetaldehyde levels directly. A highly sensitive GC-MS method to reliably determine acetaldehyde concentration with certainty is needed to address whether neural acetaldehyde is indeed responsible for increased alcohol consumption. METHODS A headspace gas chromatograph coupled to selected ion monitoring mass spectrometry was utilized to develop a quantitative assay for acetaldehyde and ethanol. Our GC-MS approach was carried out using a Bruker Scion 436-GC SQ MS. RESULTS Our approach yields limits of detection of acetaldehyde in the nanomolar range and limits of quantification in the low micromolar range. Our linear calibration includes 5 concentrations with a least square regression greater than 0.99 for both acetaldehyde and ethanol. Tissue analyses using this method revealed the capacity to quantify ethanol and acetaldehyde in blood, brain, and liver tissue from mice. CONCLUSIONS By allowing quantification of very low concentrations, this method may be used to examine the formation of ethanol metabolites, specifically acetaldehyde, in murine brain tissue in alcohol research. PMID:27501276

Molecular Approaches for High Throughput Detection and Quantification of Genetically Modified Crops: A Review

PubMed Central

Salisu, Ibrahim B.; Shahid, Ahmad A.; Yaqoob, Amina; Ali, Qurban; Bajwa, Kamran S.; Rao, Abdul Q.; Husnain, Tayyab

2017-01-01

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. PMID:29085378

UV-Visible Spectroscopy-Based Quantification of Unlabeled DNA Bound to Gold Nanoparticles.

PubMed

Baldock, Brandi L; Hutchison, James E

2016-12-20

DNA-functionalized gold nanoparticles have been increasingly applied as sensitive and selective analytical probes and biosensors. The DNA ligands bound to a nanoparticle dictate its reactivity, making it essential to know the type and number of DNA strands bound to the nanoparticle surface. Existing methods used to determine the number of DNA strands per gold nanoparticle (AuNP) require that the sequences be fluorophore-labeled, which may affect the DNA surface coverage and reactivity of the nanoparticle and/or require specialized equipment and other fluorophore-containing reagents. We report a UV-visible-based method to conveniently and inexpensively determine the number of DNA strands attached to AuNPs of different core sizes. When this method is used in tandem with a fluorescence dye assay, it is possible to determine the ratio of two unlabeled sequences of different lengths bound to AuNPs. Two sizes of citrate-stabilized AuNPs (5 and 12 nm) were functionalized with mixtures of short (5 base) and long (32 base) disulfide-terminated DNA sequences, and the ratios of sequences bound to the AuNPs were determined using the new method. The long DNA sequence was present as a lower proportion of the ligand shell than in the ligand exchange mixture, suggesting it had a lower propensity to bind the AuNPs than the short DNA sequence. The ratio of DNA sequences bound to the AuNPs was not the same for the large and small AuNPs, which suggests that the radius of curvature had a significant influence on the assembly of DNA strands onto the AuNPs.

PET Quantification of the Norepinephrine Transporter in Human Brain with (S,S)-18F-FMeNER-D2.

PubMed

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 methods of quantification of NET densities in the brain including the cerebral cortex unaffected by defluorination using ( S , S )- 18 F-FMeNER-D 2 These results suggest that we can accurately quantify NET density with a 90-min ( S , S )- 18 F-FMeNER-D 2 scan in broad brain areas. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Centrifuge: rapid and sensitive classification of metagenomic sequences

PubMed Central

Song, Li; Breitwieser, Florian P.

2016-01-01

Centrifuge is a novel microbial classification engine that enables rapid, accurate, and sensitive labeling of reads and quantification of species on desktop computers. The system uses an indexing scheme based on the Burrows-Wheeler transform (BWT) and the Ferragina-Manzini (FM) index, optimized specifically for the metagenomic classification problem. Centrifuge requires a relatively small index (4.2 GB for 4078 bacterial and 200 archaeal genomes) and classifies sequences at very high speed, allowing it to process the millions of reads from a typical high-throughput DNA sequencing run within a few minutes. Together, these advances enable timely and accurate analysis of large metagenomics data sets on conventional desktop computers. Because of its space-optimized indexing schemes, Centrifuge also makes it possible to index the entire NCBI nonredundant nucleotide sequence database (a total of 109 billion bases) with an index size of 69 GB, in contrast to k-mer-based indexing schemes, which require far more extensive space. PMID:27852649

Neutron-Encoded Protein Quantification by Peptide Carbamylation

NASA Astrophysics Data System (ADS)

Ulbrich, Arne; Merrill, Anna E.; Hebert, Alexander S.; Westphall, Michael S.; Keller, Mark P.; Attie, Alan D.; Coon, Joshua J.

2014-01-01

We describe a chemical tag for duplex proteome quantification using neutron encoding (NeuCode). The method utilizes the straightforward, efficient, and inexpensive carbamylation reaction. We demonstrate the utility of NeuCode carbamylation by accurately measuring quantitative ratios from tagged yeast lysates mixed in known ratios and by applying this method to quantify differential protein expression in mice fed a either control or high-fat diet.

Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage phi6.

PubMed

Makeyev, E V; Bamford, D H

2000-01-04

In nature, synthesis of both minus- and plus-sense RNA strands of all the known double-stranded RNA viruses occurs in the interior of a large protein assembly referred to as the polymerase complex. In addition to other proteins, the complex contains a putative polymerase possessing characteristic sequence motifs. However, none of the previous studies has shown template-dependent RNA synthesis directly with an isolated putative polymerase protein. In this report, recombinant protein P2 of double-stranded RNA bacteriophage phi6 was purified and demonstrated in an in vitro enzymatic assay to act as the replicase. The enzyme efficiently utilizes phage-specific, positive-sense RNA substrates to produce double-stranded RNA molecules, which are formed by newly synthesized, full-length minus-strands base paired with the plus-strand templates. P2-catalyzed replication is also shown to be very effective with a broad range of heterologous single-stranded RNA templates. The importance and implications of these results are discussed.

Site-Specific RNase A Activity Was Dramatically Reduced in Serum from Multiple Types of Cancer Patients

PubMed Central

Huang, Weiyan; Zhao, Mei; Wei, Na; Wang, Xiaoxia; Cao, Huqing; Du, Quan; Liang, Zicai

2014-01-01

Potent RNase activities were found in the serum of mammals but the physiological function of the RNases was never well illustrated, largely due to the caveats in methods of RNase activity measurement. None of the existing methods can distinguish between RNases with different target specificities. A systematic study was recently carried out in our lab to investigate the site-specificity of serum RNases on double-stranded RNA substrates, and found that serum RNases cleave double-stranded RNAs predominantly at 5′-U/A-3′ and 5′-C/A-3′ dinucleotide sites, in a manner closely resembling RNase A. Based on this finding, a FRET assay was developed in the current study to measure this site-specific serum RNase activity in human samples using a double stranded RNA substrate. We demonstrated that the method has a dynamic range of 10−5 mg/ml- 10−1 mg/ml using serial dilution of RNase A. The sera of 303 cancer patients were subjected to comparison with 128 healthy controls, and it was found that serum RNase activities visualized with this site-specific double stranded probe were found to be significantly reduced in patients with gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, ovary cancer, cervical cancer, bladder cancer, kidney cancer and lung cancer, while only minor changes were found in breast and colon cancer patients. This is the first report using double stranded RNA as probe to quantify site-specific activities of RNase A in a serum. The results illustrated that RNase A might be further evaluated to determine if it can serve as a new class of biomarkers for certain cancer types. PMID:24805924

Measuring oxidative damage to DNA and its repair with the comet assay.

PubMed

Collins, Andrew R

2014-02-01

Single cell gel electrophoresis, or the comet assay, was devised as a sensitive method for detecting DNA strand breaks, at the level of individual cells. A simple modification, incorporating a digestion of DNA with a lesion-specific endonuclease, makes it possible to measure oxidised bases. With the inclusion of formamidopyrimidine DNA glycosylase to recognise oxidised purines, or Nth (endonuclease III) to detect oxidised pyrimidines, the comet assay has been used extensively in human biomonitoring to monitor oxidative stress, usually in peripheral blood mononuclear cells. There is evidence to suggest that the enzymic approach is more accurate than chromatographic methods, when applied to low background levels of base oxidation. However, there are potential problems of over-estimation (because the enzymes are not completely specific) or under-estimation (failure to detect lesions that are close together). Attempts have been made to improve the inter-laboratory reproducibility of the comet assay. In addition to measuring DNA damage, the assay can be used to monitor the cellular or in vitro repair of strand breaks or oxidised bases. It also has applications in assessing the antioxidant status of cells. In its various forms, the comet assay is now an invaluable tool in human biomonitoring and genotoxicity testing. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. Copyright © 2013 Elsevier B.V. All rights reserved.

Analysis of Strand-Specific RNA-Seq Data Using Machine Learning Reveals the Structures of Transcription Units in Clostridium thermocellum

DOE PAGES

Chou, Wen-Chi; Ma, Qin; Yang, Shihui; ...

2015-03-12

The identification of transcription units (TUs) encoded in a bacterial genome is essential to elucidation of transcriptional regulation of the organism. To gain a detailed understanding of the dynamically composed TU structures, we have used four strand-specific RNA-seq (ssRNA-seq) datasets collected under two experimental conditions to derive the genomic TU organization of Clostridium thermocellum using a machine-learning approach. Our method accurately predicted the genomic boundaries of individual TUs based on two sets of parameters measuring the RNA-seq expression patterns across the genome: expression-level continuity and variance. A total of 2590 distinct TUs are predicted based on the four RNA-seq datasets.more » Moreover, among the predicted TUs, 44% have multiple genes. We assessed our prediction method on an independent set of RNA-seq data with longer reads. The evaluation confirmed the high quality of the predicted TUs. Functional enrichment analyses on a selected subset of the predicted TUs revealed interesting biology. To demonstrate the generality of the prediction method, we have also applied the method to RNA-seq data collected on Escherichia coli and achieved high prediction accuracies. The TU prediction program named SeqTU is publicly available athttps://code.google.com/p/seqtu/. We expect that the predicted TUs can serve as the baseline information for studying transcriptional and post-transcriptional regulation in C. thermocellum and other bacteria.« less

Magnetic Particle Spectroscopy Reveals Dynamic Changes in the Magnetic Behavior of Very Small Superparamagnetic Iron Oxide Nanoparticles During Cellular Uptake and Enables Determination of Cell-Labeling Efficacy.

PubMed

Poller, Wolfram C; Löwa, Norbert; Wiekhorst, Frank; Taupitz, Matthias; Wagner, Susanne; Möller, Konstantin; Baumann, Gert; Stangl, Verena; Trahms, Lutz; Ludwig, Antje

2016-02-01

In vivo tracking of nanoparticle-labeled cells by magnetic resonance imaging (MRI) crucially depends on accurate determination of cell-labeling efficacy prior to transplantation. Here, we analyzed the feasibility and accuracy of magnetic particle spectroscopy (MPS) for estimation of cell-labeling efficacy in living THP-1 cells incubated with very small superparamagnetic iron oxide nanoparticles (VSOP). Cell viability and proliferation capacity were not affected by the MPS measurement procedure. In VSOP samples without cell contact, MPS enabled highly accurate quantification. In contrast, MPS constantly overestimated the amount of cell associated and internalized VSOP. Analyses of the MPS spectrum shape expressed as harmonic ratio A₅/A₃ revealed distinct changes in the magnetic behavior of VSOP in response to cellular uptake. These changes were proportional to the deviation between MPS and actual iron amount, therefore allowing for adjusted iron quantification. Transmission electron microscopy provided visual evidence that changes in the magnetic properties correlated with cell surface interaction of VSOP as well as with alterations of particle structure and arrangement during the phagocytic process. Altogether, A₅/A₃-adjusted MPS enables highly accurate, cell-preserving VSOP quantification and furthermore provides information on the magnetic characteristics of internalized VSOP.

DNA double strand break repair in human bladder cancer is error prone and involves microhomology-associated end-joining

PubMed Central

Bentley, Johanne; Diggle, Christine P.; Harnden, Patricia; Knowles, Margaret A.; Kiltie, Anne E.

2004-01-01

In human cells DNA double strand breaks (DSBs) can be repaired by the non-homologous end-joining (NHEJ) pathway. In a background of NHEJ deficiency, DSBs with mismatched ends can be joined by an error-prone mechanism involving joining between regions of nucleotide microhomology. The majority of joins formed from a DSB with partially incompatible 3′ overhangs by cell-free extracts from human glioblastoma (MO59K) and urothelial (NHU) cell lines were accurate and produced by the overlap/fill-in of mismatched termini by NHEJ. However, repair of DSBs by extracts using tissue from four high-grade bladder carcinomas resulted in no accurate join formation. Junctions were formed by the non-random deletion of terminal nucleotides and showed a preference for annealing at a microhomology of 8 nt buried within the DNA substrate; this process was not dependent on functional Ku70, DNA-PK or XRCC4. Junctions were repaired in the same manner in MO59K extracts in which accurate NHEJ was inactivated by inhibition of Ku70 or DNA-PKcs. These data indicate that bladder tumour extracts are unable to perform accurate NHEJ such that error-prone joining predominates. Therefore, in high-grade tumours mismatched DSBs are repaired by a highly mutagenic, microhomology-mediated, alternative end-joining pathway, a process that may contribute to genomic instability observed in bladder cancer. PMID:15466592

Quantification of underivatised amino acids on dry blood spot, plasma, and urine by HPLC-ESI-MS/MS.

PubMed

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, accuracy, and precision. The fast run time, feasibility of high sample throughput, and small amount of sample required make this method very suitable for routine analysis in the clinical setting.

Impact of reconstruction parameters on quantitative I-131 SPECT

NASA Astrophysics Data System (ADS)

van Gils, C. A. J.; Beijst, C.; van Rooij, R.; de Jong, H. W. A. M.

2016-07-01

Radioiodine therapy using I-131 is widely used for treatment of thyroid disease or neuroendocrine tumors. Monitoring treatment by accurate dosimetry requires quantitative imaging. The high energy photons however render quantitative SPECT reconstruction challenging, potentially requiring accurate correction for scatter and collimator effects. The goal of this work is to assess the effectiveness of various correction methods on these effects using phantom studies. A SPECT/CT acquisition of the NEMA IEC body phantom was performed. Images were reconstructed using the following parameters: (1) without scatter correction, (2) with triple energy window (TEW) scatter correction and (3) with Monte Carlo-based scatter correction. For modelling the collimator-detector response (CDR), both (a) geometric Gaussian CDRs as well as (b) Monte Carlo simulated CDRs were compared. Quantitative accuracy, contrast to noise ratios and recovery coefficients were calculated, as well as the background variability and the residual count error in the lung insert. The Monte Carlo scatter corrected reconstruction method was shown to be intrinsically quantitative, requiring no experimentally acquired calibration factor. It resulted in a more accurate quantification of the background compartment activity density compared with TEW or no scatter correction. The quantification error relative to a dose calibrator derived measurement was found to be  <1%,-26% and 33%, respectively. The adverse effects of partial volume were significantly smaller with the Monte Carlo simulated CDR correction compared with geometric Gaussian or no CDR modelling. Scatter correction showed a small effect on quantification of small volumes. When using a weighting factor, TEW correction was comparable to Monte Carlo reconstruction in all measured parameters, although this approach is clinically impractical since this factor may be patient dependent. Monte Carlo based scatter correction including accurately simulated CDR modelling is the most robust and reliable method to reconstruct accurate quantitative iodine-131 SPECT images.

Indication for Computed Tomography Scan in Shoulder Instability: Sensitivity and Specificity of Standard Radiographs to Predict Bone Defects After Traumatic Anterior Glenohumeral Instability.

PubMed

Delage Royle, Audrey; Balg, Frédéric; Bouliane, Martin J; Canet-Silvestri, Fanny; Garant-Saine, Laurianne; Sheps, David M; Lapner, Peter; Rouleau, Dominique M

2017-10-01

Quantifying glenohumeral bone loss is key in preoperative surgical planning for a successful Bankart repair. Simple radiographs can accurately measure bone defects in cases of recurrent shoulder instability. Cohort study (diagnosis); Level of evidence, 2. A true anteroposterior (AP) view, alone and in combination with an axillary view, was used to evaluate the diagnostic properties of radiographs compared with computed tomography (CT) scan, the current gold standard, to predict significant bone defects in 70 patients. Sensitivity, specificity, and positive and negative predictive values were evaluated and compared. Detection of glenoid bone loss on plain film radiographs, with and without axillary view, had a sensitivity of 86% for both views and a specificity of 73% and 64% with and without the axillary view, respectively. For detection of humeral bone loss, the sensitivity was 8% and 17% and the specificity was 98% and 91% with and without the axillary view, respectively. Regular radiographs would have missed 1 instance of significant bone loss on the glenoid side and 20 on the humeral side. Interobserver reliabilities were moderate for glenoid detection (κ = 0.473-0.503) and poor for the humeral side (κ = 0.278-0.336). Regular radiographs showed suboptimal sensitivity, specificity, and reliability. Therefore, CT scan should be considered in the treatment algorithm for accurate quantification of bone loss to prevent high rates of recurrent instability.

Mismatch cleavage by single-strand specific nucleases

PubMed Central

Till, Bradley J.; Burtner, Chris; Comai, Luca; Henikoff, Steven

2004-01-01

We have investigated the ability of single-strand specific (sss) nucleases from different sources to cleave single base pair mismatches in heteroduplex DNA templates used for mutation and single-nucleotide polymorphism analysis. The TILLING (Targeting Induced Local Lesions IN Genomes) mismatch cleavage protocol was used with the LI-COR gel detection system to assay cleavage of amplified heteroduplexes derived from a variety of induced mutations and naturally occurring polymorphisms. We found that purified nucleases derived from celery (CEL I), mung bean sprouts and Aspergillus (S1) were able to specifically cleave nearly all single base pair mismatches tested. Optimal nicking of heteroduplexes for mismatch detection was achieved using higher pH, temperature and divalent cation conditions than are routinely used for digestion of single-stranded DNA. Surprisingly, crude plant extracts performed as well as the highly purified preparations for this application. These observations suggest that diverse members of the S1 family of sss nucleases act similarly in cleaving non-specifically at bulges in heteroduplexes, and single-base mismatches are the least accessible because they present the smallest single-stranded region for enzyme binding. We conclude that a variety of sss nucleases and extracts can be effectively used for high-throughput mutation and polymorphism discovery. PMID:15141034

Detection of single-nucleotide polymorphisms using gold nanoparticles and single-strand-specific nucleases.

PubMed

Chen, Yen-Ting; Hsu, Chiao-Ling; Hou, Shao-Yi

2008-04-15

The current study reports an assay approach that can detect single-nucleotide polymorphisms (SNPs) and identify the position of the point mutation through a single-strand-specific nuclease reaction and a gold nanoparticle assembly. The assay can be implemented via three steps: a single-strand-specific nuclease reaction that allows the enzyme to truncate the mutant DNA; a purification step that uses capture probe-gold nanoparticles and centrifugation; and a hybridization reaction that induces detector probe-gold nanoparticles, capture probe-gold nanoparticles, and the target DNA to form large DNA-linked three-dimensional aggregates of gold nanoparticles. At high temperature (63 degrees C in the current case), the purple color of the perfect match solution would not change to red, whereas a mismatched solution becomes red as the assembled gold nanoparticles separate. Using melting analysis, the position of the point mutation could be identified. This assay provides a convenient colorimetric detection that enables point mutation identification without the need for expensive mass spectrometry. To our knowledge, this is the first report concerning SNP detection based on a single-strand-specific nuclease reaction and a gold nanoparticle assembly.

Evaluation of a mass-balance approach to determine consumptive water use in northeastern Illinois

USGS Publications Warehouse

Mills, Patrick C.; Duncker, James J.; Over, Thomas M.; Marian Domanski,; ,; Engel, Frank

2014-01-01

Under ideal conditions, accurate quantification of consumptive use at the sewershed scale by the described mass-balance approach might be possible. Under most prevailing conditions, quantification likely would be more costly and time consuming than that of the present study, given the freely contributed technical support of the host community and relatively appropriate conditions of the study area. Essentials to quantification of consumptive use are a fully cooperative community, storm and sanitary sewers that are separate, and newer sewer infrastructure and (or) a robust program for limiting infiltration, exfiltration, and inflow.

Accurate proteome-wide protein quantification from high-resolution 15N mass spectra

PubMed Central

2011-01-01

In quantitative mass spectrometry-based proteomics, the metabolic incorporation of a single source of 15N-labeled nitrogen has many advantages over using stable isotope-labeled amino acids. However, the lack of a robust computational framework for analyzing the resulting spectra has impeded wide use of this approach. We have addressed this challenge by introducing a new computational methodology for analyzing 15N spectra in which quantification is integrated with identification. Application of this method to an Escherichia coli growth transition reveals significant improvement in quantification accuracy over previous methods. PMID:22182234

The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

NASA Astrophysics Data System (ADS)

Hari, Yvonne; Dugovič, Branislav; Istrate, Alena; Fignolé, Annabel; Leumann, Christian J.; Schürch, Stefan

2016-07-01

Tricyclo-DNA (tcDNA) is a sugar-modified analogue of DNA currently tested for the treatment of Duchenne muscular dystrophy in an antisense approach. Tandem mass spectrometry plays a key role in modern medical diagnostics and has become a widespread technique for the structure elucidation and quantification of antisense oligonucleotides. Herein, mechanistic aspects of the fragmentation of tcDNA are discussed, which lay the basis for reliable sequencing and quantification of the antisense oligonucleotide. Excellent selectivity of tcDNA for complementary RNA is demonstrated in direct competition experiments. Moreover, the kinetic stability and fragmentation pattern of matched and mismatched tcDNA heteroduplexes were investigated and compared with non-modified DNA and RNA duplexes. Although the separation of the constituting strands is the entropy-favored fragmentation pathway of all nucleic acid duplexes, it was found to be only a minor pathway of tcDNA duplexes. The modified hybrid duplexes preferentially undergo neutral base loss and backbone cleavage. This difference is due to the low activation entropy for the strand dissociation of modified duplexes that arises from the conformational constraint of the tc-sugar-moiety. The low activation entropy results in a relatively high free activation enthalpy for the dissociation comparable to the free activation enthalpy of the alternative reaction pathway, the release of a nucleobase. The gas-phase behavior of tcDNA duplexes illustrates the impact of the activation entropy on the fragmentation kinetics and suggests that tandem mass spectrometric experiments are not suited to determine the relative stability of different types of nucleic acid duplexes.

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

PubMed Central

Li, Xin; Wang, Xun; Song, Tao; Lu, Wei; Chen, Zhihua; Shi, Xiaolong

2015-01-01

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. PMID:26491602

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

PubMed

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. © 2012 American Association of Physicists in Medicine.

Flow cytomeric measurement of DNA and incorporated nucleoside analogs

DOEpatents

Dolbeare, Frank A.; Gray, Joe W.

1989-01-01

A method is provided for simultaneously measuring total cellular DNA and incorporated nucleoside analog. The method entails altering the cellular DNA of cells grown in the presence of a nucleoside analog so that single stranded and double stranded portions are present. Separate stains are used against the two portions. An immunochemical stain is used against the single stranded portion to provide a measure of incorporated nucleoside analog, and a double strand DNA-specific stain is used against the double stranded portion to simultaneously provide a measure of total cellular DNA. The method permits rapid flow cytometric analysis of cell populations, rapid identification of cycling and noncycling subpopulations, and determination of the efficacy of S phase cytotoxic anticancer agents.

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

Miller, Brian W.; Frost, Sophia; Frayo, Shani

Abstract Alpha emitting radionuclides exhibit a potential advantage for cancer treatments because they release large amounts of ionizing energy over a few cell diameters (50–80 μm) causing localized, irreparable double-strand DNA breaks that lead to cell death. Radioimmunotherapy (RIT) approaches using monoclonal antibodies labeled with alpha emitters may inactivate targeted cells with minimal radiation damage to surrounding tissues. For accurate dosimetry in alpha-RIT, tools are needed to visualize and quantify the radioactivity distribution and absorbed dose to targeted and non-targeted cells, especially for organs and tumors with heterogeneous radionuclide distributions. The aim of this study was to evaluate and characterizemore » a novel single-particle digital autoradiography imager, iQID (ionizing-radiation Quantum Imaging Detector), for use in alpha-RIT experiments. Methods: The iQID camera is a scintillator-based radiation detection technology that images and identifies charged-particle and gamma-ray/X-ray emissions spatially and temporally on an event-by-event basis. It employs recent advances in CCD/CMOS cameras and computing hardware for real-time imaging and activity quantification of tissue sections, approaching cellular resolutions. In this work, we evaluated this system’s characteristics for alpha particle imaging including measurements of spatial resolution and background count rates at various detector configurations and quantification of activity distributions. The technique was assessed for quantitative imaging of astatine-211 (211At) activity distributions in cryosections of murine and canine tissue samples. Results: The highest spatial resolution was measured at ~20 μm full width at half maximum (FWHM) and the alpha particle background was measured at a rate of (2.6 ± 0.5) × 10–4 cpm/cm2 (40 mm diameter detector area). Simultaneous imaging of multiple tissue sections was performed using a large-area iQID configuration (ø 11.5 cm). Estimation of the 211At activity distribution was demonstrated at mBq/μg levels. Conclusion: Single-particle digital autoradiography of alpha emitters has advantages over traditional autoradiographic techniques in terms of spatial resolution, sensitivity, and activity quantification capability. The system features and characterization results presented in this study show that iQID is a promising technology for microdosimetry, because it provides necessary information for interpreting alpha-RIT outcomes and for predicting the therapeutic efficacy of cell-targeted approaches using alpha emitters.« less

Development and validation of an UPLC-MS/MS method for the quantification of tamoxifen and its main metabolites in human scalp hair.

PubMed

Drooger, Jan C; Jager, Agnes; Lam, Mei-Ho; den Boer, Mathilda D; Sleijfer, Stefan; Mathijssen, Ron H J; de Bruijn, Peter

2015-10-10

The aim of this study was to validate an earlier developed high-performance highly sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method for quantification of tamoxifen and its three main metabolites (N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen) in scalp hair. This non-invasive method might, by segmental analysis of hair, be useful in the determination of the concentration of drugs and its metabolites over time, which can be used to study a wide variety of clinical relevant questions. Hair samples (150-300 hair strands, cut as close to the scalp as possible from the posterior vertex region of the head) were collected from female patients taking tamoxifen 20mg daily (n=19). The analytes were extracted using a liquid-liquid extraction procedure with carbonate buffer at pH 8.8 and a mixture of n-hexane/isopropranol method, followed by UPLC-MS/MS chromatography, based on an earlier validated method. The calibration curves were linear in the range of 1.00-200 pmol for tamoxifen and N-desmethyl-tamoxifen, with lower limit of quantitation of 1.00 pmol and 0.100-20.0 pmol with lower limit of quantitation of 0.100 pmol for endoxifen and 4-hydroxy-tamoxifen. Assay performance was fair with a within-run and between-run variability less than 9.24 at the three quality control samples and less than 15.7 for the lower limit of quantitation. Importantly, a steep linear decline was observed from distal to proximal hair segments. Probably, this is due to UV exposure as we showed degradation of tamoxifen and its metabolites after exposure to UV-light. Furthermore, higher concentrations of tamoxifen were found in black hair samples compared to blond and brown hair samples. We conclude that measurement of the concentration of tamoxifen and its main metabolites in hair is possible, with the selective, sensitive, accurate and precise UPLC-MS/MS method. However, for tamoxifen, it seems not possible to determine exposure over time with segmental analysis of hair, probably largely due to the effect of UV irradiation. Further research should therefore focus on quantification of other anticancer drugs, in segmented scalp hair, that are less sensitive to UV irradiation. Copyright © 2015 Elsevier B.V. All rights reserved.

Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos

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

Li, Zhou; Adams, Rachel M; Chourey, Karuna

2012-01-01

A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification. Isobaricmore » chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. Based on the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.« less

Systematic development of small molecules to inhibit specific microscopic steps of Aβ42 aggregation in Alzheimer's disease.

PubMed

Habchi, Johnny; Chia, Sean; Limbocker, Ryan; Mannini, Benedetta; Ahn, Minkoo; Perni, Michele; Hansson, Oskar; Arosio, Paolo; Kumita, Janet R; Challa, Pavan Kumar; Cohen, Samuel I A; Linse, Sara; Dobson, Christopher M; Knowles, Tuomas P J; Vendruscolo, Michele

2017-01-10

The aggregation of the 42-residue form of the amyloid-β peptide (Aβ42) is a pivotal event in Alzheimer's disease (AD). The use of chemical kinetics has recently enabled highly accurate quantifications of the effects of small molecules on specific microscopic steps in Aβ42 aggregation. Here, we exploit this approach to develop a rational drug discovery strategy against Aβ42 aggregation that uses as a read-out the changes in the nucleation and elongation rate constants caused by candidate small molecules. We thus identify a pool of compounds that target specific microscopic steps in Aβ42 aggregation. We then test further these small molecules in human cerebrospinal fluid and in a Caenorhabditis elegans model of AD. Our results show that this strategy represents a powerful approach to identify systematically small molecule lead compounds, thus offering an appealing opportunity to reduce the attrition problem in drug discovery.

Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination.

PubMed

Surtees, Jennifer A; Alani, Eric

2006-07-14

Genetic studies in Saccharomyces cerevisiae predict that the mismatch repair (MMR) factor MSH2-MSH3 binds and stabilizes branched recombination intermediates that form during single strand annealing and gene conversion. To test this model, we constructed a series of DNA substrates that are predicted to form during these recombination events. We show in an electrophoretic mobility shift assay that S. cerevisiae MSH2-MSH3 specifically binds branched DNA substrates containing 3' single-stranded DNA and that ATP stimulates its release from these substrates. Chemical footprinting analyses indicate that MSH2-MSH3 specifically binds at the double-strand/single-strand junction of branched substrates, alters its conformation and opens up the junction. Therefore, MSH2-MSH3 binding to its substrates creates a unique nucleoprotein structure that may signal downstream steps in repair that include interactions with MMR and nucleotide excision repair factors.

Reliable method for generating double-stranded DNA vectors containing site-specific base modifications.

PubMed

Brégeon, Damien; Doetsch, Paul W

2004-11-01

Cells of all living organisms are continuously exposed to physical and chemical agents that damage DNA and alter the integrity of their genomes. Despite the relatively high efficiency of the different repair pathways, some lesions remain in DNA when it is replicated or transcribed. Lesion bypass by DNA and RNA polymerases has been the subject of numerous investigations. However, knowledge of the in vivo mechanism of transcription lesion bypass is very limited because no robust methodology is available. Here we describe a protocol based on the synthesis of a complementary strand of a circular, single-stranded DNA molecule, which allows for the production of large amounts of double-stranded DNA containing a lesion at a specific position in a transcribed sequence. Such constructs can subsequently be used for lesion bypass studies in vivo by RNA polymerase and to ascertain how these events can be affected by the genetic background of the cells.

Highly sensitive quantification for human plasma-targeted metabolomics using an amine derivatization reagent.

PubMed

Arashida, Naoko; Nishimoto, Rumi; Harada, Masashi; Shimbo, Kazutaka; Yamada, Naoyuki

2017-02-15

Amino acids and their related metabolites play important roles in various physiological processes and have consequently become biomarkers for diseases. However, accurate quantification methods have only been established for major compounds, such as amino acids and a limited number of target metabolites. We previously reported a highly sensitive high-throughput method for the simultaneous quantification of amines using 3-aminopyridyl-N-succinimidyl carbamate as a derivatization reagent combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Herein, we report the successful development of a practical and accurate LC-MS/MS method to analyze low concentrations of 40 physiological amines in 19 min. Thirty-five of these amines showed good linearity, limits of quantification, accuracy, precision, and recovery characteristics in plasma, with scheduled selected reaction monitoring acquisitions. Plasma samples from 10 healthy volunteers were evaluated using our newly developed method. The results revealed that 27 amines were detected in one of the samples, and that 24 of these compounds could be quantified. Notably, this new method successfully quantified metabolites with high accuracy across three orders of magnitude, with lowest and highest averaged concentrations of 31.7 nM (for spermine) and 18.3 μM (for α-aminobutyric acid), respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Correlation between energy deposition and molecular damage from Auger electrons: A case study of ultra-low energy (5–18 eV) electron interactions with DNA

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

Rezaee, Mohammad, E-mail: Mohammad.Rezaee@USherbrooke.ca; Hunting, Darel J.; Sanche, Léon

2014-07-15

Purpose: The present study introduces a new method to establish a direct correlation between biologically related physical parameters (i.e., stopping and damaging cross sections, respectively) for an Auger-electron emitting radionuclide decaying within a target molecule (e.g., DNA), so as to evaluate the efficacy of the radionuclide at the molecular level. These parameters can be applied to the dosimetry of Auger electrons and the quantification of their biological effects, which are the main criteria to assess the therapeutic efficacy of Auger-electron emitting radionuclides. Methods: Absorbed dose and stopping cross section for the Auger electrons of 5–18 eV emitted by{sup 125}I withinmore » DNA were determined by developing a nanodosimetric model. The molecular damages induced by these Auger electrons were investigated by measuring damaging cross section, including that for the formation of DNA single- and double-strand breaks. Nanoscale films of pure plasmid DNA were prepared via the freeze-drying technique and subsequently irradiated with low-energy electrons at various fluences. The damaging cross sections were determined by employing a molecular survival model to the measured exposure–response curves for induction of DNA strand breaks. Results: For a single decay of{sup 125}I within DNA, the Auger electrons of 5–18 eV deposit the energies of 12.1 and 9.1 eV within a 4.2-nm{sup 3} volume of a hydrated or dry DNA, which results in the absorbed doses of 270 and 210 kGy, respectively. DNA bases have a major contribution to the deposited energies. Ten-electronvolt and high linear energy transfer 100-eV electrons have a similar cross section for the formation of DNA double-strand break, while 100-eV electrons are twice as efficient as 10 eV in the induction of single-strand break. Conclusions: Ultra-low-energy electrons (<18 eV) substantially contribute to the absorbed dose and to the molecular damage from Auger-electron emitting radionuclides; hence, they should be considered in the dosimetry calculation of such radionuclides. Moreover, absorbed dose is not an appropriate physical parameter for nanodosimetry. Instead, stopping cross section, which describes the probability of energy deposition in a target molecule can be an appropriate nanodosimetric parameter. The stopping cross section is correlated with a damaging cross section (e.g., cross section for the double-strand break formation) to quantify the number of each specific lesion in a target molecule for each nuclear decay of a single Auger-electron emitting radionuclide.« less

Correlation between energy deposition and molecular damage from Auger electrons: A case study of ultra-low energy (5–18 eV) electron interactions with DNA

PubMed Central

Rezaee, Mohammad; Hunting, Darel J.; Sanche, Léon

2015-01-01

Purpose The present study introduces a new method to establish a direct correlation between biologically related physical parameters (i.e., stopping and damaging cross sections, respectively) for an Auger-electron emitting radionuclide decaying within a target molecule (e.g., DNA), so as to evaluate the efficacy of the radionuclide at the molecular level. These parameters can be applied to the dosimetry of Auger electrons and the quantification of their biological effects, which are the main criteria to assess the therapeutic efficacy of Auger-electron emitting radionuclides. Methods Absorbed dose and stopping cross section for the Auger electrons of 5–18 eV emitted by 125I within DNA were determined by developing a nanodosimetric model. The molecular damages induced by these Auger electrons were investigated by measuring damaging cross section, including that for the formation of DNA single- and double-strand breaks. Nanoscale films of pure plasmid DNA were prepared via the freeze-drying technique and subsequently irradiated with low-energy electrons at various fluences. The damaging cross sections were determined by employing a molecular survival model to the measured exposure–response curves for induction of DNA strand breaks. Results For a single decay of 125I within DNA, the Auger electrons of 5–18 eV deposit the energies of 12.1 and 9.1 eV within a 4.2-nm3 volume of a hydrated or dry DNA, which results in the absorbed doses of 270 and 210 kGy, respectively. DNA bases have a major contribution to the deposited energies. Ten-electronvolt and high linear energy transfer 100-eV electrons have a similar cross section for the formation of DNA double-strand break, while 100-eV electrons are twice as efficient as 10 eV in the induction of single-strand break. Conclusions Ultra-low-energy electrons (<18 eV) substantially contribute to the absorbed dose and to the molecular damage from Auger-electron emitting radionuclides; hence, they should be considered in the dosimetry calculation of such radionuclides. Moreover, absorbed dose is not an appropriate physical parameter for nanodosimetry. Instead, stopping cross section, which describes the probability of energy deposition in a target molecule can be an appropriate nanodosimetric parameter. The stopping cross section is correlated with a damaging cross section (e.g., cross section for the double-strand break formation) to quantify the number of each specific lesion in a target molecule for each nuclear decay of a single Auger-electron emitting radionuclide. PMID:24989405

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

NASA Astrophysics Data System (ADS)

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.

Identification of Intermediate in Hepatitis B Virus CCC DNA Formation and Sensitive and Selective CCC DNA Detection.

PubMed

Luo, Jun; Cui, Xiuji; Gao, Lu; Hu, Jianming

2017-06-21

The hepatitis B virus (HBV) covalently closed circular (CCC) DNA functions as the only viral template capable of coding for all the viral RNA species and is thus essential to initiate and sustain viral replication. CCC DNA is converted, in a multi-step and ill-understood process, from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. To detect putative intermediates during RC to CCC DNA conversion, two 3' exonucleases Exo I and Exo III, in combination were used to degrade all DNA strands with a free 3' end, which would nevertheless preserve closed circular DNA, either single-stranded (SS) or double-stranded (DS). Indeed, a RC DNA species with a covalently closed minus strand but an open plus strand (closed minus-strand RC DNA or cM-RC DNA) was detected by this approach. Further analyses indicated that at least some of the plus strands in such a putative intermediate likely still retained the RNA primer that is attached to the 5' end of the plus strand in RC DNA, suggesting that minus strand closing can occur before plus strand processing. Furthermore, the same nuclease treatment proved to be useful for sensitive and specific detection of CCC DNA by removing all DNA species other than closed circular DNA. Application of these and similar approaches may allow the identification of additional intermediates during CCC DNA formation and facilitate specific and sensitive detection of CCC DNA, which should help elucidate the pathways of CCC DNA formation and factors involved. IMPORTANCE The hepatitis B virus (HBV) covalently closed circular (CCC) DNA is the molecular basis of viral persistence, by serving as the viral transcriptional template. CCC DNA is converted, in a multi-step and ill-understood process, from a relaxed circular (RC) DNA. Little is currently understood about the pathways or factors involved in CCC DNA formation. We have now detected a likely intermediate during the conversion of RC to CCC DNA, thus providing important clues to the pathways of CCC DNA formation. Furthermore, the same experimental approach that led to the detection of the intermediate could also facilitate specific and sensitive detection of CCC DNA, which has remained challenging. This and similar approaches will help identify additional intermediates during CCC DNA formation and elucidate the pathways and factors involved. Copyright © 2017 American Society for Microbiology.

Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

PubMed Central

Luo, Jun; Cui, Xiuji; Gao, Lu

2017-01-01

ABSTRACT Hepatitis B virus (HBV) covalently closed circular (CCC) DNA functions as the only viral template capable of coding for all the viral RNA species and is thus essential to initiate and sustain viral replication. CCC DNA is converted, in a multistep and ill-understood process, from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. To detect putative intermediates during RC DNA to CCC DNA conversion, two 3′ exonucleases, exonuclease I (Exo I) and Exo III, were used in combination to degrade all DNA strands with a free 3′ end, which would nevertheless preserve closed circular DNA in either single-stranded (SS) or double-stranded (DS) form. Indeed, an RC DNA species with a covalently closed minus strand but an open plus strand (closed minus-strand RC DNA [cM-RC DNA]) was detected by this approach. Further analyses indicated that at least some of the plus strands in such a putative intermediate likely still retained the RNA primer that is attached to the 5′ end of the plus strand in RC DNA, suggesting that minus-strand closing can occur before plus-strand processing. Furthermore, the same nuclease treatment proved to be useful for sensitive and specific detection of CCC DNA by removing all DNA species other than closed circular DNA. Application of these and similar approaches may allow the identification of additional intermediates during CCC DNA formation and facilitate specific and sensitive detection of CCC DNA, which should help elucidate the pathways of CCC DNA formation and the factors involved. IMPORTANCE The hepatitis B virus (HBV) covalently closed circular (CCC) DNA, by serving as the viral transcriptional template, is the molecular basis of viral persistence. CCC DNA is converted, in a multistep and ill-understood process, from relaxed circular (RC) DNA. Little is currently understood about the pathways or factors involved in CCC DNA formation. We have now detected a likely intermediate during the conversion of RC DNA to CCC DNA, thus providing important clues to the pathways of CCC DNA formation. Furthermore, the same experimental approach that led to the detection of the intermediate could also facilitate specific and sensitive detection of CCC DNA, which has remained challenging. This and similar approaches will help identify additional intermediates during CCC DNA formation and elucidate the pathways and factors involved. PMID:28637752

Methods and techniques for measuring gas emissions from agricultural and animal feeding operations.

PubMed

Hu, Enzhu; Babcock, Esther L; Bialkowski, Stephen E; Jones, Scott B; Tuller, Markus

2014-01-01

Emissions of gases from agricultural and animal feeding operations contribute to climate change, produce odors, degrade sensitive ecosystems, and pose a threat to public health. The complexity of processes and environmental variables affecting these emissions complicate accurate and reliable quantification of gas fluxes and production rates. Although a plethora of measurement technologies exist, each method has its limitations that exacerbate accurate quantification of gas fluxes. Despite a growing interest in gas emission measurements, only a few available technologies include real-time, continuous monitoring capabilities. Commonly applied state-of-the-art measurement frameworks and technologies were critically examined and discussed, and recommendations for future research to address real-time monitoring requirements for forthcoming regulation and management needs are provided.

An international collaboration to standardize HIV-2 viral load assays: results from the 2009 ACHI(E)V(2E) quality control study.

PubMed

Damond, F; Benard, A; Balotta, Claudia; Böni, Jürg; Cotten, Matthew; Duque, Vitor; Ferns, Bridget; Garson, Jeremy; Gomes, Perpetua; Gonçalves, Fátima; Gottlieb, Geoffrey; Kupfer, Bernd; Ruelle, Jean; Rodes, Berta; Soriano, Vicente; Wainberg, Mark; Taieb, Audrey; Matheron, Sophie; Chene, Genevieve; Brun-Vezinet, Francoise

2011-10-01

Accurate HIV-2 plasma viral load quantification is crucial for adequate HIV-2 patient management and for the proper conduct of clinical trials and international cohort collaborations. This study compared the homogeneity of HIV-2 RNA quantification when using HIV-2 assays from ACHI(E)V(2E) study sites and either in-house PCR calibration standards or common viral load standards supplied to all collaborators. Each of the 12 participating laboratories quantified blinded HIV-2 samples, using its own HIV-2 viral load assay and standard as well as centrally validated and distributed common HIV-2 group A and B standards (http://www.hiv.lanl.gov/content/sequence/HelpDocs/subtypes-more.html). Aliquots of HIV-2 group A and B strains, each at 2 theoretical concentrations (2.7 and 3.7 log(10) copies/ml), were tested. Intralaboratory, interlaboratory, and overall variances of quantification results obtained with both standards were compared using F tests. For HIV-2 group A quantifications, overall and interlaboratory and/or intralaboratory variances were significantly lower when using the common standard than when using in-house standards at the concentration levels of 2.7 log(10) copies/ml and 3.7 log(10) copies/ml, respectively. For HIV-2 group B, a high heterogeneity was observed and the variances did not differ according to the type of standard used. In this international collaboration, the use of a common standard improved the homogeneity of HIV-2 group A RNA quantification only. The diversity of HIV-2 group B, particularly in PCR primer-binding regions, may explain the heterogeneity in quantification of this strain. Development of a validated HIV-2 viral load assay that accurately quantifies distinct circulating strains is needed.

An International Collaboration To Standardize HIV-2 Viral Load Assays: Results from the 2009 ACHIEV2E Quality Control Study▿

PubMed Central

Damond, F.; Benard, A.; Balotta, Claudia; Böni, Jürg; Cotten, Matthew; Duque, Vitor; Ferns, Bridget; Garson, Jeremy; Gomes, Perpetua; Gonçalves, Fátima; Gottlieb, Geoffrey; Kupfer, Bernd; Ruelle, Jean; Rodes, Berta; Soriano, Vicente; Wainberg, Mark; Taieb, Audrey; Matheron, Sophie; Chene, Genevieve; Brun-Vezinet, Francoise

2011-01-01

Accurate HIV-2 plasma viral load quantification is crucial for adequate HIV-2 patient management and for the proper conduct of clinical trials and international cohort collaborations. This study compared the homogeneity of HIV-2 RNA quantification when using HIV-2 assays from ACHIEV2E study sites and either in-house PCR calibration standards or common viral load standards supplied to all collaborators. Each of the 12 participating laboratories quantified blinded HIV-2 samples, using its own HIV-2 viral load assay and standard as well as centrally validated and distributed common HIV-2 group A and B standards (http://www.hiv.lanl.gov/content/sequence/HelpDocs/subtypes-more.html). Aliquots of HIV-2 group A and B strains, each at 2 theoretical concentrations (2.7 and 3.7 log10 copies/ml), were tested. Intralaboratory, interlaboratory, and overall variances of quantification results obtained with both standards were compared using F tests. For HIV-2 group A quantifications, overall and interlaboratory and/or intralaboratory variances were significantly lower when using the common standard than when using in-house standards at the concentration levels of 2.7 log10 copies/ml and 3.7 log10 copies/ml, respectively. For HIV-2 group B, a high heterogeneity was observed and the variances did not differ according to the type of standard used. In this international collaboration, the use of a common standard improved the homogeneity of HIV-2 group A RNA quantification only. The diversity of HIV-2 group B, particularly in PCR primer-binding regions, may explain the heterogeneity in quantification of this strain. Development of a validated HIV-2 viral load assay that accurately quantifies distinct circulating strains is needed. PMID:21813718

A comparison of liver fat content as determined by magnetic resonance imaging-proton density fat fraction and MRS versus liver histology in non-alcoholic fatty liver disease.

PubMed

Idilman, Ilkay S; Keskin, Onur; Celik, Azim; Savas, Berna; Elhan, Atilla Halil; Idilman, Ramazan; Karcaaltincaba, Musturay

2016-03-01

Many imaging methods have been defined for quantification of hepatic steatosis in non-alcoholic fatty liver disease (NAFLD). However, studies comparing the efficiency of magnetic resonance imaging-proton density fat fraction (MRI-PDFF), magnetic resonance spectroscopy (MRS), and liver histology for quantification of liver fat content are limited. To compare the efficiency of MRI-PDFF and MRS in the quantification of liver fat content in individuals with NAFLD. A total of 19 NAFLD patients underwent MRI-PDFF, MRS, and liver biopsy for quantification of liver fat content. The MR examinations were performed on a 1.5 HDx MRI system. The MRI protocol included T1-independent volumetric multi-echo gradient-echo imaging with T2* correction and spectral fat modeling and MRS with STEAM technique. A close correlation was observed between liver MRI-PDFF- and histology- determined steatosis (r = 0.743, P < 0.001) and between liver MRS- and histology-determined steatosis (r = 0.712, P < 0.001), with no superiority between them (ƶ = 0.19, P = 0.849). For quantification of hepatic steatosis, a high correlation was observed between the two MRI methods (r = 0.986, P < 0.001). MRI-PDFF and MRS accurately differentiated moderate/severe steatosis from mild/no hepatic steatosis (P = 0.007 and 0.013, respectively), with no superiority between them (AUCMRI-PDFF = 0.881 ± 0.0856 versus AUCMRS = 0.857 ± 0.0924, P = 0.461). Both MRI-PDFF and MRS can be used for accurate quantification of hepatic steatosis. © The Foundation Acta Radiologica 2015.

K-ras gene sequence effects on the formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-DNA adducts.

PubMed

Ziegel, Rebecca; Shallop, Anthony; Jones, Roger; Tretyakova, Natalia

2003-04-01

The tobacco specific pulmonary carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolically activated to electrophilic species that form methyl and pyridyloxobutyl adducts with genomic DNA, including O(6)-methylguanine, N7-methylguanine, and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine. If not repaired, these lesions could lead to mutations and the initiation of cancer. Previous studies used ligation-mediated polymerase chain reaction (LMPCR) in combination with PAGE to examine the distribution of NNK-induced strand breaks and alkali labile lesions (e.g., N7-methylguanine) within gene sequences. However, LMPCR cannot be used to establish the distribution patterns of highly promutagenic O(6)-methylguanine and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine adducts of NNK. We have developed methods based on stable isotope labeling HPLC-electrospray ionization tandem mass spectrometry (HPLC-ESI MS/MS) that enable us to accurately quantify NNK-induced adducts at defined sites within DNA sequences. In the present study, the formation of N7-methylguanine, O(6)-methylguanine, and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine adducts at specific positions within a K-ras gene-derived double-stranded DNA sequence (5'-G(1)G(2)AG(3)CTG(4)G(5)TG(6)G(7)CG(8)TA G(9)G(10)C-3') was investigated following treatment with activated NNK metabolites. All three lesions preferentially formed at the second position of codon 12 (GGT), the major mutational hotspot for G-->A and G-->T base substitutions observed in smoking-induced lung tumors. Therefore, our data support the involvement of NNK and other tobacco specific nitrosamines in mutagenesis and carcinogenesis.

Autonomous parvovirus LuIII encapsidates equal amounts of plus and minus DNA strands

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

Bates, R.C.; Snyder, C.E.; Banerjee, P.T.

1984-02-01

Autonomous parvoviruses are thought to uniquely encapsidate single-stranded DNA of minus polarity. In contrast, the defective adeno-associated viruses separately encapsidate equal amounts of plus and minus DNA strands. The uniqueness of minus strand encapsidation is reexamined for the autonomous parvoviruses. Although it was found that Kilham rat virus and H-1 virus encapsidate varying but small amounts of complementary-strand DNA, it was unexpected to find that LuIII virus encapsidated equal amounts of plus and minus DNA. The extracted LuIII DNA possessed properties of double-stranded replicative-form DNA, including insensitivity to S1 endonuclease, cleavage by restriction enzymes, and conversion to unit-length, single-stranded DNAmore » when electrophoresed under denaturing conditions. However, the inability of this DNA to form single-stranded DNA circles when denatured and then renatured in the presence of formamide and the lack of double-stranded DNA circle formation after treatment with exonuclease III and reannealing shows a lack of sequence homology of the 3' and 5' termini of LuIII DNA, in contrast to adeno-associated virus DNA. Digestion of LuIII double-stranded DNA with EcoRI and HincII and separation of plus and minus DNA strands on composite agarose-acrylamide gels identified a heterogeneity present only in the plus DNA strand. These results suggest that strand specificity of viral DNA encapsidation is not a useful property for differentiation between the autonomous and defective parvoviruses. Furthermore, encapsidation by LuIII of equal amounts of complementary DNA strands in contrast to encapsidation of minus strands by H-1 virus, when propagated in the same host cell type, suggests that selection of strands for encapsidation is a virus-coded rather than host-controlled event.« less

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

NASA Astrophysics Data System (ADS)

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.

Effects of Homology Length in the Repeat Region on Minus-Strand DNA Transfer and Retroviral Replication

PubMed Central

Dang, Que; Hu, Wei-Shau

2001-01-01

Homology between the two repeat (R) regions in the retroviral genome mediates minus-strand DNA transfer during reverse transcription. We sought to define the effects of R homology lengths on minus-strand DNA transfer. We generated five murine leukemia virus (MLV)-based vectors that contained identical sequences but different lengths of the 3′ R (3, 6, 12, 24 and 69 nucleotides [nt]); 69 nt is the full-length MLV R. After one round of replication, viral titers from the vector with a full-length downstream R were compared with viral titers generated from the other four vectors with reduced R lengths. Viral titers generated from vectors with R lengths reduced to one-third (24 nt) or one-sixth (12 nt) that of the wild type were not significantly affected; however, viral titers generated from vectors with only 3- or 6-nt homology in the R region were significantly lower. Because expression and packaging of the RNA were similar among all the vectors, the differences in the viral titers most likely reflected the impact of the homology lengths on the efficiency of minus-strand DNA transfer. The molecular nature of minus-strand DNA transfer was characterized in 63 proviruses. Precise R-to-R transfer was observed in most proviruses generated from vectors with 12-, 24-, or 69-nt homology in R, whereas aberrant transfers were predominantly used to generate proviruses from vectors with 3- or 6-nt homology. Reverse transcription using RNA transcribed from an upstream promoter, termed read-in RNA transcripts, resulted in most of the aberrant transfers. These data demonstrate that minus-strand DNA transfer is homology driven and a minimum homology length is required for accurate and efficient minus-strand DNA transfer. PMID:11134294

Experimental Review of DNA-Based Methods for Wine Traceability and Development of a Single-Nucleotide Polymorphism (SNP) Genotyping Assay for Quantitative Varietal Authentication.

PubMed

Catalano, Valentina; Moreno-Sanz, Paula; Lorenzi, Silvia; Grando, Maria Stella

2016-09-21

The genetic varietal authentication of wine was investigated according to DNA isolation procedures reported for enological matrices and also by testing 11 commercial extraction kits and various protocol modifications. Samples were collected at different stages of the winemaking process of renowned Italian wines Brunello di Montalcino, Lambruschi Modenesi, and Trento DOC. Results demonstrated not only that grape DNA loss is produced by the fermentation process but also that clarification and stabilization operations contribute to the reduction of double-stranded DNA content on wine. Despite the presence of inhibitors, downstream PCR genotyping yielded reliable nuclear and chloroplast SSR markers for must samples, whereas no amplification or inconsistent results were obtained at later stages of the vinification. In addition, a TaqMan genotyping assay based on cultivar-specific single-nucleotide polymorphisms (SNPs) was designed, which allowed assessment of grapevine DNA mixtures. Once the wine matrix limitations are overcome, this sensitive tool may be implemented for the relative quantification of cultivars used for blend wines or frauds.

miRNA*: a passenger stranded in RNA-induced silencing complex?

PubMed

Mah, S M; Buske, C; Humphries, R K; Kuchenbauer, F

2010-01-01

Processing of the pre-microRNA (pre-miRNA) through Dicer1 generates a miRNA duplex, consisting of a miRNA and miRNA* strand (also termed guide strand and passenger strand, respectively). Despite the general consensus that miRNA*s have no regulatory activity, recent publications have provided evidence that the abundance, possible function, and physiological relevance of miRNA*s have been underestimated. This review provides an account of our current understanding of miRNA* origination and activity, mounting evidence for their unique functions and regulatory mechanisms, and examples of specific miRNA*s from the literature.

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

PubMed Central

2011-01-01

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

The fission yeast meiosis-specific Dmc1 recombinase mediates formation and branch migration of Holliday junctions by preferentially promoting strand exchange in a direction opposite to that of Rad51

PubMed Central

Murayama, Yasuto; Tsutsui, Yasuhiro; Iwasaki, Hiroshi

2011-01-01

Homologous recombination proceeds via the formation of several intermediates including Holliday junctions (HJs), which are important for creating crossover products. DNA strand exchange is a core reaction that produces these intermediates that is directly catalyzed by RecA family recombinases, of which there are two types in eukaryotes: universal Rad51 and meiosis-specific Dmc1. We demonstrated previously that Rad51 promotes four-strand exchange, mimicking the formation and branch migration of HJs. Here we show that Dmc1 from fission yeast has a similar activity, which requires ATP hydrolysis and is independent of an absolute requirement for the Swi5–Sfr1 complex. These features are critically different from three-strand exchange mediated by Dmc1, but similar to those of four-strand exchange mediated by Rad51, suggesting that strand exchange reactions between duplex–duplex and single-duplex DNAs are mechanistically different. Interestingly, despite similarities in protein structure and in reaction features, the preferential polarities of Dmc1 and Rad51 strand exchange are different (Dmc1 promotes exchange in the 5′-to-3′ direction and Rad51 promotes exchange in the 3′-to-5′ direction relative to the ssDNA region of the DNA substrate). The significance of the Dmc1 polarity is discussed within the context of the necessity for crossover production. PMID:21363965

A single-stranded DNA binding protein from mouse tumor cells specifically recognizes the C-rich strand of the (AGG:CCT)n repeats that can alter DNA conformation.

PubMed Central

Muraiso, T; Nomoto, S; Yamazaki, H; Mishima, Y; Kominami, R

1992-01-01

A protein that binds to a synthetic oligonucleotide of (CCT)12 has been purified from Ehrlich ascites tumor cells by a (CCT)12 affinity chromatography. The protein (p70) has an apparent molecular mass of 70 kDa, as assayed by Southwestern analysis. A competition experiment revealed that p70 binds to (CCT)12, (CCCT)8 and (CCTCCCT)6, but not to (CTT)12, (CT)16 and (CCTGCCT)6, suggesting that p70 has a sequence-specificity. The complementary (AGG)12 and the double stranded DNA did not show the binding. It is also confirmed by S1 nuclease analysis that the (AGG:CCT)12 duplex takes a single-stranded conformation in the absence of the protein. This raises a possibility that the duplex forms two single-stranded loops in chromosomes, the C-rich strand being bound to p70. Structural analysis of the resulting (AGG)12 strand by non-denaturing polyacrylamide gel electrophoresis demonstrated the presence of slower and faster migrated conformers in a neutral pH buffer containing 50 mM NaCl at 5 degrees C. The ratio was dependent on the DNA concentration. Both conformers disappeared in the absence of NaCl. This suggests that (AGG)12 can form intra- and inter-molecular complexes by non-Watson-Crick, guanine:guanine base-pairing. The possible biological function of the (AGG:CCT)n duplex and the p70 is discussed. Images PMID:1480484

How to perform RT-qPCR accurately in plant species? A case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population.

PubMed

De Keyser, Ellen; Desmet, Laurence; Van Bockstaele, Erik; De Riek, Jan

2013-06-24

Flower colour variation is one of the most crucial selection criteria in the breeding of a flowering pot plant, as is also the case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively in several species. In azalea, flower colour can be described by means of a 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely for studying flower colour. However, the methods used were often only semi-quantitative or quantification was not done according to the MIQE-guidelines. We aimed to develop an accurate protocol for RT-qPCR and to validate the protocol to study flower colour in an azalea mapping population. An accurate RT-qPCR protocol had to be established. RNA quality was evaluated in a combined approach by means of different techniques e.g. SPUD-assay and Experion-analysis. We demonstrated the importance of testing noRT-samples for all genes under study to detect contaminating DNA. In spite of the limited sequence information available, we prepared a set of 11 reference genes which was validated in flower petals; a combination of three reference genes was most optimal. Finally we also used plasmids for the construction of standard curves. This allowed us to calculate gene-specific PCR efficiencies for every gene to assure an accurate quantification. The validity of the protocol was demonstrated by means of the study of six genes of the flavonoid biosynthesis pathway. No correlations were found between flower colour and the individual expression profiles. However, the combination of early pathway genes (CHS, F3H, F3'H and FLS) is clearly related to co-pigmentation with flavonols. The late pathway genes DFR and ANS are to a minor extent involved in differentiating between coloured and white flowers. Concerning pink coloration, we could demonstrate that the lower intensity in this type of flowers is correlated to the expression of F3'H. Currently in plant research, validated and qualitative RT-qPCR protocols are still rare. The protocol in this study can be implemented on all plant species to assure accurate quantification of gene expression. We have been able to correlate flower colour to the combined regulation of structural genes, both in the early and late branch of the pathway. This allowed us to differentiate between flower colours in a broader genetic background as was done so far in flower colour studies. These data will now be used for eQTL mapping to comprehend even more the regulation of this pathway.

How to perform RT-qPCR accurately in plant species? A case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population

PubMed Central

2013-01-01

Background Flower colour variation is one of the most crucial selection criteria in the breeding of a flowering pot plant, as is also the case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively in several species. In azalea, flower colour can be described by means of a 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely for studying flower colour. However, the methods used were often only semi-quantitative or quantification was not done according to the MIQE-guidelines. We aimed to develop an accurate protocol for RT-qPCR and to validate the protocol to study flower colour in an azalea mapping population. Results An accurate RT-qPCR protocol had to be established. RNA quality was evaluated in a combined approach by means of different techniques e.g. SPUD-assay and Experion-analysis. We demonstrated the importance of testing noRT-samples for all genes under study to detect contaminating DNA. In spite of the limited sequence information available, we prepared a set of 11 reference genes which was validated in flower petals; a combination of three reference genes was most optimal. Finally we also used plasmids for the construction of standard curves. This allowed us to calculate gene-specific PCR efficiencies for every gene to assure an accurate quantification. The validity of the protocol was demonstrated by means of the study of six genes of the flavonoid biosynthesis pathway. No correlations were found between flower colour and the individual expression profiles. However, the combination of early pathway genes (CHS, F3H, F3'H and FLS) is clearly related to co-pigmentation with flavonols. The late pathway genes DFR and ANS are to a minor extent involved in differentiating between coloured and white flowers. Concerning pink coloration, we could demonstrate that the lower intensity in this type of flowers is correlated to the expression of F3'H. Conclusions Currently in plant research, validated and qualitative RT-qPCR protocols are still rare. The protocol in this study can be implemented on all plant species to assure accurate quantification of gene expression. We have been able to correlate flower colour to the combined regulation of structural genes, both in the early and late branch of the pathway. This allowed us to differentiate between flower colours in a broader genetic background as was done so far in flower colour studies. These data will now be used for eQTL mapping to comprehend even more the regulation of this pathway. PMID:23800303

Process of infection with bacteriophage phi chi 174. XL. Viral DNA replication of phi chi 174 mutants blocked in progeny single-stranded DNA synthesis.

PubMed Central

Fukuda, A; Sinsheimer, R L

1976-01-01

Mutation in several different cistrons of bacteriophage phi chi 174 blocks net progeny single-stranded DNA synthesis at the late period of infection (15). For the study of the functions of these cistrons in single-stranded DNA synthesis, asymmetric replication of replicative form DNA was examined at the late period of infection with amber mutants of these cistrons. While the normal, rapid process of asymmetric single-stranded viral DNA synthesis is blocked at the late period of these mutant infections, an asymmetric synthesis of the viral strand of replicative-form DNA is observed in this period, though at a reduced level, together with degradation of prelabeled viral strand. Some intermediate replicative-form molecules were also detected. Asymmetric synthesis of the viral strand of replicative-form DNA at the late period of phi chi infection is completely inhibited in the presence of a low concentration (35mug/ml) of chloramphenicol (which also blocks net single-stranded viral DNA synthesis). These results are discussed in terms of the possible role of the specific viral proteins for normal single-stranded DNA synthesis. PMID:1255871

Improving strand pairing prediction through exploring folding cooperativity

PubMed Central

Jeong, Jieun; Berman, Piotr; Przytycka, Teresa M.

2008-01-01

The topology of β-sheets is defined by the pattern of hydrogen-bonded strand pairing. Therefore, predicting hydrogen bonded strand partners is a fundamental step towards predicting β-sheet topology. At the same time, finding the correct partners is very difficult due to long range interactions involved in strand pairing. Additionally, patterns of aminoacids observed in β-sheet formations are very general and therefore difficult to use for computational recognition of specific contacts between strands. In this work, we report a new strand pairing algorithm. To address above mentioned difficulties, our algorithm attempts to mimic elements of the folding process. Namely, in addition to ensuring that the predicted hydrogen bonded strand pairs satisfy basic global consistency constraints, it takes into account hypothetical folding pathways. Consistently with this view, introducing hydrogen bonds between a pair of strands changes the probabilities of forming hydrogen bonds between other pairs of strand. We demonstrate that this approach provides an improvement over previously proposed algorithms. We also compare the performance of this method to that of a global optimization algorithm that poses the problem as integer linear programming optimization problem and solves it using ILOG CPLEX™ package. PMID:18989036

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

NASA Astrophysics Data System (ADS)

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.

Microsecond kinetics in model single- and double-stranded amylose polymers.

PubMed

Sattelle, Benedict M; Almond, Andrew

2014-05-07

Amylose, a component of starch with increasing biotechnological significance, is a linear glucose polysaccharide that self-organizes into single- and double-helical assemblies. Starch granule packing, gelation and inclusion-complex formation result from finely balanced macromolecular kinetics that have eluded precise experimental quantification. Here, graphics processing unit (GPU) accelerated multi-microsecond aqueous simulations are employed to explore conformational kinetics in model single- and double-stranded amylose. The all-atom dynamics concur with prior X-ray and NMR data while surprising and previously overlooked microsecond helix-coil, glycosidic linkage and pyranose ring exchange are hypothesized. In a dodecasaccharide, single-helical collapse was correlated with linkages and rings transitioning from their expected syn and (4)C1 chair conformers. The associated microsecond exchange rates were dependent on proximity to the termini and chain length (comparing hexa- and trisaccharides), while kinetic features of dodecasaccharide linkage and ring flexing are proposed to be a good model for polymers. Similar length double-helices were stable on microsecond timescales but the parallel configuration was sturdier than the antiparallel equivalent. In both, tertiary organization restricted local chain dynamics, implying that simulations of single amylose strands cannot be extrapolated to dimers. Unbiased multi-microsecond simulations of amylose are proposed as a valuable route to probing macromolecular kinetics in water, assessing the impact of chemical modifications on helical stability and accelerating the development of new biotechnologies.

A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma

PubMed Central

Richter, Anna; Grieu, Fabienne; Carrello, Amerigo; Amanuel, Benhur; Namdarian, Kateh; Rynska, Aleksandra; Lucas, Amanda; Michael, Victoria; Bell, Anthony; Fox, Stephen B.; Hewitt, Chelsee A.; Do, Hongdo; McArthur, Grant A.; Wong, Stephen Q.; Dobrovic, Alexander; Iacopetta, Barry

2013-01-01

Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status. We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting. Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR). Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques. CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3–4% of melanomas. HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting. CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics. PMID:23584600

COSMOS: accurate detection of somatic structural variations through asymmetric comparison between tumor and normal samples.

PubMed

Yamagata, Koichi; Yamanishi, Ayako; Kokubu, Chikara; Takeda, Junji; Sese, Jun

2016-05-05

An important challenge in cancer genomics is precise detection of structural variations (SVs) by high-throughput short-read sequencing, which is hampered by the high false discovery rates of existing analysis tools. Here, we propose an accurate SV detection method named COSMOS, which compares the statistics of the mapped read pairs in tumor samples with isogenic normal control samples in a distinct asymmetric manner. COSMOS also prioritizes the candidate SVs using strand-specific read-depth information. Performance tests on modeled tumor genomes revealed that COSMOS outperformed existing methods in terms of F-measure. We also applied COSMOS to an experimental mouse cell-based model, in which SVs were induced by genome engineering and gamma-ray irradiation, followed by polymerase chain reaction-based confirmation. The precision of COSMOS was 84.5%, while the next best existing method was 70.4%. Moreover, the sensitivity of COSMOS was the highest, indicating that COSMOS has great potential for cancer genome analysis. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Single-Stranded DNA Aptamers against Pathogens and Toxins: Identification and Biosensing Applications

PubMed Central

Hong, Ka Lok

2015-01-01

Molecular recognition elements (MREs) can be short sequences of single-stranded DNA, RNA, small peptides, or antibody fragments. They can bind to user-defined targets with high affinity and specificity. There has been an increasing interest in the identification and application of nucleic acid molecular recognition elements, commonly known as aptamers, since they were first described in 1990 by the Gold and Szostak laboratories. A large number of target specific nucleic acids MREs and their applications are currently in the literature. This review first describes the general methodologies used in identifying single-stranded DNA (ssDNA) aptamers. It then summarizes advancements in the identification and biosensing application of ssDNA aptamers specific for bacteria, viruses, their associated molecules, and selected chemical toxins. Lastly, an overview of the basic principles of ssDNA aptamer-based biosensors is discussed. PMID:26199940

Ultrasensitive Direct Quantification of Nucleobase Modifications in DNA by Surface-Enhanced Raman Scattering: The Case of Cytosine.

PubMed

Morla-Folch, Judit; Xie, Hai-nan; Gisbert-Quilis, Patricia; Gómez-de Pedro, Sara; Pazos-Perez, Nicolas; Alvarez-Puebla, Ramon A; Guerrini, Luca

2015-11-09

Recognition of chemical modifications in canonical nucleobases of nucleic acids is of key importance since such modified variants act as different genetic encoders, introducing variability in the biological information contained in DNA. Herein, we demonstrate the feasibility of direct SERS in combination with chemometrics and microfluidics for the identification and relative quantification of 4 different cytosine modifications in both single- and double-stranded DNA. The minute amount of DNA required per measurement, in the sub-nanogram regime, removes the necessity of pre-amplification or enrichment steps (which are also potential sources of artificial DNA damages). These findings show great potentials for the development of fast, low-cost and high-throughput screening analytical devices capable of detecting known and unknown modifications in nucleic acids (DNA and RNA) opening new windows of activity in several fields such as biology, medicine and forensic sciences. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Experimental Null Method to Guide the Development of Technical Procedures and to Control False-Positive Discovery in Quantitative Proteomics.

PubMed

Shen, Xiaomeng; Hu, Qiang; Li, Jun; Wang, Jianmin; Qu, Jun

2015-10-02

Comprehensive and accurate evaluation of data quality and false-positive biomarker discovery is critical to direct the method development/optimization for quantitative proteomics, which nonetheless remains challenging largely due to the high complexity and unique features of proteomic data. Here we describe an experimental null (EN) method to address this need. Because the method experimentally measures the null distribution (either technical or biological replicates) using the same proteomic samples, the same procedures and the same batch as the case-vs-contol experiment, it correctly reflects the collective effects of technical variability (e.g., variation/bias in sample preparation, LC-MS analysis, and data processing) and project-specific features (e.g., characteristics of the proteome and biological variation) on the performances of quantitative analysis. To show a proof of concept, we employed the EN method to assess the quantitative accuracy and precision and the ability to quantify subtle ratio changes between groups using different experimental and data-processing approaches and in various cellular and tissue proteomes. It was found that choices of quantitative features, sample size, experimental design, data-processing strategies, and quality of chromatographic separation can profoundly affect quantitative precision and accuracy of label-free quantification. The EN method was also demonstrated as a practical tool to determine the optimal experimental parameters and rational ratio cutoff for reliable protein quantification in specific proteomic experiments, for example, to identify the necessary number of technical/biological replicates per group that affords sufficient power for discovery. Furthermore, we assessed the ability of EN method to estimate levels of false-positives in the discovery of altered proteins, using two concocted sample sets mimicking proteomic profiling using technical and biological replicates, respectively, where the true-positives/negatives are known and span a wide concentration range. It was observed that the EN method correctly reflects the null distribution in a proteomic system and accurately measures false altered proteins discovery rate (FADR). In summary, the EN method provides a straightforward, practical, and accurate alternative to statistics-based approaches for the development and evaluation of proteomic experiments and can be universally adapted to various types of quantitative techniques.

Comparison of specific binding sites for Escherichia coli RNA polymerase with naturally occurring hairpin regions in single-stranded DNA of coliphage M13. [Aspergillus oryzae

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

Niyogi, S.K.; Mitra, S.

Escherichia coli RNA polymerase binds specifically to the single-stranded circular DNA of coliphage M13 in the presence of a saturating concentration of the bacterial DNA binding protein presumably as an essential step in the synthesis of the RNA primer required for synthesizing the complementary DNA strand in parental replicative-form DNA. The RNA polymerase-protected DNA regions were isolated after extensive digestion with pancreatic DNase, S1 endonuclease of Aspergillus oryzae, and exonuclease I of E. coli. The physicochemical properties of the RNA polymerase-protected segments (called PI and PII) were compared with those of the naturally occurring hairpin regions.

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

PubMed

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.

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

PubMed

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.

Practical quantification of necrosis in histological whole-slide images.

PubMed

Homeyer, André; Schenk, Andrea; Arlt, Janine; Dahmen, Uta; Dirsch, Olaf; Hahn, Horst K

2013-06-01

Since the histological quantification of necrosis is a common task in medical research and practice, we evaluate different image analysis methods for quantifying necrosis in whole-slide images. In a practical usage scenario, we assess the impact of different classification algorithms and feature sets on both accuracy and computation time. We show how a well-chosen combination of multiresolution features and an efficient postprocessing step enables the accurate quantification necrosis in gigapixel images in less than a minute. The results are general enough to be applied to other areas of histological image analysis as well. Copyright © 2013 Elsevier Ltd. All rights reserved.

Dynamic DNA nanotechnology using strand-displacement reactions

NASA Astrophysics Data System (ADS)

Zhang, David Yu; Seelig, Georg

2011-02-01

The specificity and predictability of Watson-Crick base pairing make DNA a powerful and versatile material for engineering at the nanoscale. This has enabled the construction of a diverse and rapidly growing set of DNA nanostructures and nanodevices through the programmed hybridization of complementary strands. Although it had initially focused on the self-assembly of static structures, DNA nanotechnology is now also becoming increasingly attractive for engineering systems with interesting dynamic properties. Various devices, including circuits, catalytic amplifiers, autonomous molecular motors and reconfigurable nanostructures, have recently been rationally designed to use DNA strand-displacement reactions, in which two strands with partial or full complementarity hybridize, displacing in the process one or more pre-hybridized strands. This mechanism allows for the kinetic control of reaction pathways. Here, we review DNA strand-displacement-based devices, and look at how this relatively simple mechanism can lead to a surprising diversity of dynamic behaviour.

Methods for the quantification of coarse woody debris and an examination of its spatial patterning: A study from the Tenderfoot Creek Experimental Forest, MT

Treesearch

Paul B. Alaback; Duncan C. Lutes

1997-01-01

Methods for the quantification of coarse woody debris volume and the description of spatial patterning were studied in the Tenderfoot Creek Experimental Forest, Montana. The line transect method was found to be an accurate, unbiased estimator of down debris volume (> 10cm diameter) on 1/4 hectare fixed-area plots, when perpendicular lines were used. The Fischer...

Whole-transcriptome, high-throughput RNA sequence analysis of the bovine macrophage response to Mycobacterium bovis infection in vitro.

PubMed

Nalpas, Nicolas C; Park, Stephen D E; Magee, David A; Taraktsoglou, Maria; Browne, John A; Conlon, Kevin M; Rue-Albrecht, Kévin; Killick, Kate E; Hokamp, Karsten; Lohan, Amanda J; Loftus, Brendan J; Gormley, Eamonn; Gordon, Stephen V; MacHugh, David E

2013-04-08

Mycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular pathogen that can persist inside host macrophages during infection via a diverse range of mechanisms that subvert the host immune response. In the current study, we have analysed and compared the transcriptomes of M. bovis-infected monocyte-derived macrophages (MDM) purified from six Holstein-Friesian females with the transcriptomes of non-infected control MDM from the same animals over a 24 h period using strand-specific RNA sequencing (RNA-seq). In addition, we compare gene expression profiles generated using RNA-seq with those previously generated by us using the high-density Affymetrix® GeneChip® Bovine Genome Array platform from the same MDM-extracted RNA. A mean of 7.2 million reads from each MDM sample mapped uniquely and unambiguously to single Bos taurus reference genome locations. Analysis of these mapped reads showed 2,584 genes (1,392 upregulated; 1,192 downregulated) and 757 putative natural antisense transcripts (558 upregulated; 119 downregulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). Of the differentially expressed genes, 694 were common to both the sense and antisense data sets, with the direction of expression (i.e. up- or downregulation) positively correlated for 693 genes and negatively correlated for the remaining gene. Gene ontology analysis of the differentially expressed genes revealed an enrichment of immune, apoptotic and cell signalling genes. Notably, the number of differentially expressed genes identified from RNA-seq sense strand analysis was greater than the number of differentially expressed genes detected from microarray analysis (2,584 genes versus 2,015 genes). Furthermore, our data reveal a greater dynamic range in the detection and quantification of gene transcripts for RNA-seq compared to microarray technology. This study highlights the value of RNA-seq in identifying novel immunomodulatory mechanisms that underlie host-mycobacterial pathogen interactions during infection, including possible complex post-transcriptional regulation of host gene expression involving antisense RNA.

Method for detecting point mutations in DNA utilizing fluorescence energy transfer

DOEpatents

Parkhurst, Lawrence J.; Parkhurst, Kay M.; Middendorf, Lyle

2001-01-01

A method for detecting point mutations in DNA using a fluorescently labeled oligomeric probe and Forster resonance energy transfer (FRET) is disclosed. The selected probe is initially labeled at each end with a fluorescence dye, which act together as a donor/acceptor pair for FRET. The fluorescence emission from the dyes changes dramatically from the duplex stage, wherein the probe is hybridized to the complementary strand of DNA, to the single strand stage, when the probe is melted to become detached from the DNA. The change in fluorescence is caused by the dyes coming into closer proximity after melting occurs and the probe becomes detached from the DNA strand. The change in fluorescence emission as a function of temperature is used to calculate the melting temperature of the complex or T.sub.m. In the case where there is a base mismatch between the probe and the DNA strand, indicating a point mutation, the T.sub.m has been found to be significantly lower than the T.sub.m for a perfectly match probelstand duplex. The present invention allows for the detection of the existence and magnitude of T.sub.m, which allows for the quick and accurate detection of a point mutation in the DNA strand and, in some applications, the determination of the approximate location of the mutation within the sequence.

Disease quantification in dermatology: in vivo near-infrared spectroscopy measures correlate strongly with the clinical assessment of psoriasis severity

NASA Astrophysics Data System (ADS)

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

2013-03-01

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 useful in quantifying disease severity, they require an extensive clinical experience and carry a risk of subjectivity. We explore the opportunity to use in vivo near-infrared (NIR) spectra as an objective and noninvasive method for local disease severity assessment in 31 psoriasis patients in whom selected plaques were scored clinically. A partial least squares (PLS) regression model was used to analyze and predict the severity scores on the NIR spectra of psoriatic and uninvolved skin. The correlation between predicted and clinically assigned scores was R=0.94 (RMSE=0.96), suggesting that in vivo NIR provides accurate clinical quantification of psoriatic plaques. Hence, NIR may be a practical solution to clinical severity assessment of psoriasis, providing a continuous, linear, numerical value of severity.

Evaluation of tamoxifen and metabolites by LC-MS/MS and HPLC methods.

PubMed

Heath, D D; Flat, S W; Wu, A H B; Pruitt, M A; Rock, C L

2014-01-01

Epidemiological and laboratory evidence suggests that quantification of serum or plasma levels of tamoxifen and its metabolites, 4-hydroxy-N-desmethyl-tamoxifen (endoxifen), Z-4-hydroxytamoxifen (4HT), N-desmethyl-tamoxifen (ND-tam), is a clinically useful tool in the assessment and monitoring of breast cancer status in patients taking adjuvant tamoxifen. A liquid chromatographic mass spectrometric method (LC-MS/MS) was used to measure the blood levels of tamoxifen and its metabolites. This fully automated analytical method is specific, accurate and sensitive. The LC-MS/MS automated technique has now become a widely accepted reference method. This study analysed a randomly selected batch of blood samples from participants enrolled in a breast cancer study to compare results from this reference method in 40 samples with those obtained from a recently developed high-performance liquid chromatography (HPLC) method with fluorescence detection. The mean (SD) concentrations for the LC-MS/MS method (endoxifen 12.6 [7.5] ng/mL, tamoxifen 105 [44] ng/mL, 4-HT 1.9 [1.0] ng/mL, ND-tam 181 [69] ng/mL) and the HPLC method (endoxifen 13.1 [7.8] ng/mL, tamoxifen 108 [55] ng/mL, 4-HT 1.8 [0.8] ng/mL, ND-tam 184 [81] ng/mL) did not show any significant differences. The results confirm that the HPLC method offers an accurate and comparable alternative for the quantification of tamoxifen and tamoxifen metabolites.

Evaluation of Tamoxifen and metabolites by LC-MS/MS and HPLC Methods

PubMed Central

Heath, D.D.; Flatt, S.W.; Wu, A.H.B.; Pruitt, M.A.; Rock, C.L.

2015-01-01

Epidemiological and laboratory evidence suggests that quantification of serum or plasma levels of tamoxifen and the metabolites of tamoxifen, 4-hydroxy-N-desmethyl-tamoxifen (endoxifen), Z-4-hydroxy-tamoxifen (4HT), N-desmethyl-tamoxifen (ND-tam) is a clinically useful tool in the assessment and monitoring of breast cancer status in patients taking adjuvant tamoxifen. A liquid chromatographic mass spectrometric method (LC-MS/MS) was used to measure the blood levels of tamoxifen and the metabolites of tamoxifen. This fully automated analytical method is specific, accurate and sensitive. The LC-MS/MS automated technique has now become a widely accepted reference method. We analyzed a randomly selected batch of blood samples from participants enrolled in a breast cancer study to compare results from this reference method in 40 samples with those obtained from a recently developed high performance liquid chromatography (HPLC) method with fluorescence detection. The mean (SD) concentration for the LC-MS/MS (endoxifen 12.6 [7.5] ng/mL, tamoxifen 105 [44] ng/mL, 4-HT 1.9 [1.0] ng/mL, ND-tam 181 [69] ng/mL) and the HPLC (endoxifen 13.1 [7.8] ng/mL, tamoxifen 108[55]ng/mL, 4-HT 1.8 [0.8] ng/mL, ND-tam 184 [81] ng/mL), the methods did not show any significant differences. Our results confirm that the HPLC method offers an accurate and comparable alternative for the quantification of tamoxifen and tamoxifen metabolites. PMID:24693573

The Zugspitze radiative closure experiment for quantifying water vapor absorption over the terrestrial and solar infrared - Part 2: Accurate calibration of high spectral-resolution infrared measurements of surface solar radiation

NASA Astrophysics Data System (ADS)

Reichert, Andreas; Rettinger, Markus; Sussmann, Ralf

2016-09-01

Quantitative knowledge of water vapor absorption is crucial for accurate climate simulations. An open science question in this context concerns the strength of the water vapor continuum in the near infrared (NIR) at atmospheric temperatures, which is still to be quantified by measurements. This issue can be addressed with radiative closure experiments using solar absorption spectra. However, the spectra used for water vapor continuum quantification have to be radiometrically calibrated. We present for the first time a method that yields sufficient calibration accuracy for NIR water vapor continuum quantification in an atmospheric closure experiment. Our method combines the Langley method with spectral radiance measurements of a high-temperature blackbody calibration source (< 2000 K). The calibration scheme is demonstrated in the spectral range 2500 to 7800 cm-1, but minor modifications to the method enable calibration also throughout the remainder of the NIR spectral range. The resulting uncertainty (2σ) excluding the contribution due to inaccuracies in the extra-atmospheric solar spectrum (ESS) is below 1 % in window regions and up to 1.7 % within absorption bands. The overall radiometric accuracy of the calibration depends on the ESS uncertainty, on which at present no firm consensus has been reached in the NIR. However, as is shown in the companion publication Reichert and Sussmann (2016), ESS uncertainty is only of minor importance for the specific aim of this study, i.e., the quantification of the water vapor continuum in a closure experiment. The calibration uncertainty estimate is substantiated by the investigation of calibration self-consistency, which yields compatible results within the estimated errors for 91.1 % of the 2500 to 7800 cm-1 range. Additionally, a comparison of a set of calibrated spectra to radiative transfer model calculations yields consistent results within the estimated errors for 97.7 % of the spectral range.

Programmable and Multiparameter DNA-Based Logic Platform For Cancer Recognition and Targeted Therapy

PubMed Central

2014-01-01

The specific inventory of molecules on diseased cell surfaces (e.g., cancer cells) provides clinicians an opportunity for accurate diagnosis and intervention. With the discovery of panels of cancer markers, carrying out analyses of multiple cell-surface markers is conceivable. As a trial to accomplish this, we have recently designed a DNA-based device that is capable of performing autonomous logic-based analysis of two or three cancer cell-surface markers. Combining the specific target-recognition properties of DNA aptamers with toehold-mediated strand displacement reactions, multicellular marker-based cancer analysis can be realized based on modular AND, OR, and NOT Boolean logic gates. Specifically, we report here a general approach for assembling these modular logic gates to execute programmable and higher-order profiling of multiple coexisting cell-surface markers, including several found on cancer cells, with the capacity to report a diagnostic signal and/or deliver targeted photodynamic therapy. The success of this strategy demonstrates the potential of DNA nanotechnology in facilitating targeted disease diagnosis and effective therapy. PMID:25361164

A Smart Detection System Based on Specific Magnetic and Rolling Cycle Amplification Signal-Amplified Dual-Aptamers to Accurately Monitor Minimal Residual Diseases in Patients with T-ALL.

PubMed

Li, Xa; Zhou, Bo; Zhao, Zilong; Hu, Zixi; Zhou, Sufang; Yang, Nuo; Huang, Yong; Zhang, Zhenghua; Su, Jing; Lan, Dan; Qin, Xue; Meng, Jinyu; Zheng, Duo; He, Jian; Huang, Xianing; Zhao, Jing; Zhang, Zhiyong; Tan, Weihong; Lu, Xiaoling; Zhao, Yongxiang

2016-12-01

It is a major clinical challenge for clinicians how to early find out minimal residual diseases (MRD) of leukemia. Here, we developed a smart detection system for MRD involving magnetic aptamer sgc8 probe (M-sgc8 probe) to capture CEM cells and rolling cycle amplification probe (RCA-sgc8 probe) to initiate RCA, producing a single-stranded tandem repeated copy of the circular template. The DNA products were hybridized with molecular beacon to generate the amplified fluorescence signal. An in vitro model to mimic MRD was established to evaluate the sensitivity of the smart detection system. The smart detection system was used to detect MRD in patients with T-ALL peri-chemotherapy, which could not only specifically captured T-ALL cells, but also significantly amplified fluorescence signals on them. The sensitivity was 1/20,000. These results indicate that the smart detection system with high specificity and sensitivity could more efficiently monitor the progress of T-ALL peri-chemotherapy.

Quantitative Detection of Streptococcus pneumoniae in Nasopharyngeal Secretions by Real-Time PCR

PubMed Central

Greiner, Oliver; Day, Philip J. R.; Bosshard, Philipp P.; Imeri, Fatime; Altwegg, Martin; Nadal, David

2001-01-01

Streptococcus pneumoniae is an important cause of community-acquired pneumonia. However, in this setting the diagnostic sensitivity of blood cultures is below 30%. Since during such infections changes in the amounts of S. pneumoniae may also occur in the upper respiratory tract, quantification of these bacteria in nasopharnygeal secretions (NPSs) may offer a suitable diagnostic approach. Real-time PCR offers a sensitive, efficient, and routinely reproducible approach to quantification. Using primers and a fluorescent probe specific for the pneumolysin gene, we were able to detect DNA from serial dilutions of S. pneumoniae cells in which the quantities of DNA ranged from the amounts extracted from 1 to 106 cells. No difference was noted when the same DNA was mixed with DNA extracted from NPSs shown to be deficient of S. pneumoniae following culture, suggesting that this bacterium can be detected and accurately quantitated in clinical samples. DNAs from Haemophilus influenzae, Moraxella catarrhalis, or alpha-hemolytic streptococci other than S. pneumoniae were not amplified or were only weakly amplified when there were ≥106 cells per reaction mixture. When the assay was applied to NPSs from patients with respiratory tract infections, the assay performed with a sensitivity of 100% and a specificity of up to 96% compared to the culture results. The numbers of S. pneumoniae organisms detected by real-time PCR correlated with the numbers detected by semiquantitative cultures. A real-time PCR that targeted the pneumolysin gene provided a sensitive and reliable means for routine rapid detection and quantification of S. pneumoniae present in NPSs. This assay may serve as a tool to study changes in the amounts of S. pneumoniae during lower respiratory tract infections. PMID:11526140

Highly sensitive and simple liquid chromatography assay with ion-pairing extraction and visible detection for quantification of gold from nanoparticles.

PubMed

Pallotta, Arnaud; Philippe, Valentin; Boudier, Ariane; Leroy, Pierre; Clarot, Igor

2018-03-01

A simple isocratic HPLC method using visible detection was developed and validated for the quantification of gold in nanoparticles (AuNP). After a first step of oxidation of nanoparticles, an ion-pair between tetrachloroaurate anion and the cationic dye Rhodamine B was formed and extracted from the aqueous media with the help of an organic solvent. The corresponding Rhodamine B was finally quantified by reversed phase liquid chromatography using a Nucleosil C18 (150mm × 4.6mm, 3µm) column and with a mobile phase containing acetonitrile and 0.1% trifluoroacetic acid aqueous solution (25/75, V/V) at 1.0mLmin -1. and at a wavelength of 555nm. The method was validated using methodology described by the International Conference on Harmonization and was shown to be specific, precise (RSD < 11%), accurate and linear in the range of 0.1 - 30.0µM with a lower limit of quantification (LLOQ) of 0.1µM. This method was in a first time applied to AuNP quality control after their synthesis. In a second time, the absence of gold leakage (either as AuNP or gold salt form) from nanostructured multilayered polyelectrolyte films under shear stress was assessed. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparison of matrix effects in HPLC-MS/MS and UPLC-MS/MS analysis of nine basic pharmaceuticals in surface waters.

PubMed

Van De Steene, Jet C; Lambert, Willy E

2008-05-01

When developing an LC-MS/MS-method matrix effects are a major issue. The effect of co-eluting compounds arising from the matrix can result in signal enhancement or suppression. During method development much attention should be paid to diminishing matrix effects as much as possible. The present work evaluates matrix effects from aqueous environmental samples in the simultaneous analysis of a group of 9 specific pharmaceuticals with HPLC-ESI/MS/MS and UPLC-ESI/MS/MS: flubendazole, propiconazole, pipamperone, cinnarizine, ketoconazole, miconazole, rabeprazole, itraconazole and domperidone. When HPLC-MS/MS is used, matrix effects are substantial and can not be compensated for with analogue internal standards. For different surface water samples different matrix effects are found. For accurate quantification the standard addition approach is necessary. Due to the better resolution and more narrow peaks in UPLC, analytes will co-elute less with interferences during ionisation, so matrix effects could be lower, or even eliminated. If matrix effects are eliminated with this technique, the standard addition method for quantification can be omitted and the overall method will be simplified. Results show that matrix effects are almost eliminated if internal standards (structural analogues) are used. Instead of the time-consuming and labour-intensive standard addition method, with UPLC the internal standardization can be used for quantification and the overall method is substantially simplified.

Development of a real-time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues.

PubMed

Carraro, R; Dalla Rovere, G; Ferraresso, S; Carraro, L; Franch, R; Toffan, A; Pascoli, F; Patarnello, T; Bargelloni, L

2018-02-01

The availability of a rapid and accurate method for the diagnosis of Photobacterium damselae subsp. piscicida (Phdp), able to discriminate its strictly correlated subsp. damselae (Phdd), formally known as Vibrio damsela, is essential for managing fish pasteurellosis outbreaks in farmed fish. A single-step, high-sensitivity real-time PCR assay for simultaneous detection and quantification of P. damselae was designed targeting partial of the sequence of the bamB gene and tested for specificity and sensitivity on laboratory-generated samples as well as on experimentally infected seabream tissue samples. With a limit of detection (LOD) of one copy in pure bacterial DNA, the sensitivity was higher than all methods previously reported. Validation in target and non-target bacterial species proved the assay was able to discriminate Phdd-Phdp subspecies from diverse hosts/geographical origins and between non-target species. In addition, two SNPs in the target amplicon region determine two distinctive qPCR dissociation curves distinguishing between Phdp-Phdd. This is the first time that a molecular method for P. damselae diagnosis combines detection, quantification and subspecies identification in one step. The assay holds the potential to improve the knowledge of infection dynamics and the development of better strategies to control an important fish disease. © 2017 John Wiley & Sons Ltd.

Antidepressants detection and quantification in whole blood samples by GC-MS/MS, for forensic purposes.

PubMed

Truta, Liliana; Castro, André L; Tarelho, Sónia; Costa, Pedro; Sales, M Goreti F; Teixeira, Helena M

2016-09-05

Depression is among the most prevalent psychiatric disorders of our society, leading to an increase in antidepressant drug consumption that needs to be accurately determined in whole blood samples in Forensic Toxicology Laboratories. For this purpose, this work presents a new gas chromatography tandem mass spectrometry (GC-MS/MS) method targeting the simultaneous and rapid determination of 14 common Antidepressants in whole blood: 13 Antidepressants (amitriptyline, citalopram, clomipramine, dothiepin, fluoxetine, imipramine, mianserin, mirtazapine, nortryptiline, paroxetine, sertraline, trimipramine and venlafaxine) and 1 Metabolite (N-desmethylclomipramine). Solid-phase extraction was used prior to chromatographic separation. Chromatographic and MS/MS parameters were selected to improve sensitivity, peak resolution and unequivocal identification of the eluted analyte. The detection was performed on a triple quadrupole tandem MS in selected ion monitoring (SIM) mode in tandem, using electronic impact ionization. Clomipramine-D3 and trimipramine-D3 were used as deutered internal standards. The validation parameters included linearity, limits of detection, lower limit of quantification, selectivity/specificity, extraction efficiency, carry-over, precision and robustness, and followed internationally accepted guidelines. Limits of quantification and detection were lower than therapeutic and sub-therapeutic concentration ranges. Overall, the method offered good selectivity, robustness and quick response (<16min) for typical concentration ranges, both for therapeutic and lethal levels. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantification issues of trace metal contaminants on silicon wafers by means of TOF-SIMS, ICP-MS, and TXRF

NASA Astrophysics Data System (ADS)

Rostam-Khani, P.; Hopstaken, M. J. P.; Vullings, P.; Noij, G.; O'Halloran, O.; Claassen, W.

2004-06-01

Measurement of surface metal contamination on silicon wafers is essential for yield enhancement in IC manufacturing. Vapor phase decomposition coupled with either inductively coupled plasma mass spectrometry (VPD-ICP-MS), or total reflection X-ray fluorescence (VPD-TXRF), TXRF and more recently time of flight secondary ion mass spectrometry (TOF-SIMS) are used to monitor surface metal contamination. These techniques complement each other in their respective strengths and weaknesses. For reliable and accurate quantification, so-called relative sensitivity factors (RSF) are required for TOF-SIMS analysis. For quantification purposes in VPD, the collection efficiency (CE) is important to ensure complete collection of contamination. A standard procedure has been developed that combines the determination of these RSFs as well as the collection efficiency using all the analytical techniques mentioned above. Therefore, sample wafers were intentionally contaminated and analyzed (by TOF-SIMS) directly after preparation. After VPD-ICP-MS, several scanned surfaces were analyzed again by TOF-SIMS. Comparing the intensities of the specific metals before and after the VPD-DC procedure on the scanned surface allows the determination of so-called removing efficiency (RE). In general, very good agreement was obtained comparing the four analytical techniques after updating the RSFs for TOF-SIMS. Progress has been achieved concerning the CE evaluation as well as determining the RSFs more precisely for TOF-SIMS.

Detection and Quantification of Graphene-Family Nanomaterials in the Environment.

PubMed

Goodwin, David G; Adeleye, Adeyemi S; Sung, Lipiin; Ho, Kay T; Burgess, Robert M; Petersen, Elijah J

2018-04-17

An increase in production of commercial products containing graphene-family nanomaterials (GFNs) has led to concern over their release into the environment. The fate and potential ecotoxicological effects of GFNs in the environment are currently unclear, partially due to the limited analytical methods for GFN measurements. In this review, the unique properties of GFNs that are useful for their detection and quantification are discussed. The capacity of several classes of techniques to identify and/or quantify GFNs in different environmental matrices (water, soil, sediment, and organisms), after environmental transformations, and after release from a polymer matrix of a product is evaluated. Extraction and strategies to combine methods for more accurate discrimination of GFNs from environmental interferences as well as from other carbonaceous nanomaterials are recommended. Overall, a comprehensive review of the techniques available to detect and quantify GFNs are systematically presented to inform the state of the science, guide researchers in their selection of the best technique for the system under investigation, and enable further development of GFN metrology in environmental matrices. Two case studies are described to provide practical examples of choosing which techniques to utilize for detection or quantification of GFNs in specific scenarios. Because the available quantitative techniques are somewhat limited, more research is required to distinguish GFNs from other carbonaceous materials and improve the accuracy and detection limits of GFNs at more environmentally relevant concentrations.

In vivo molecular photoacoustic tomography of melanomas targeted by bioconjugated gold nanocages.

PubMed

Kim, Chulhong; Cho, Eun Chul; Chen, Jingyi; Song, Kwang Hyun; Au, Leslie; Favazza, Christopher; Zhang, Qiang; Cobley, Claire M; Gao, Feng; Xia, Younan; Wang, Lihong V

2010-08-24

Early diagnosis, accurate staging, and image-guided resection of melanomas remain crucial clinical objectives for improving patient survival and treatment outcomes. Conventional techniques cannot meet this demand because of the low sensitivity, low specificity, poor spatial resolution, shallow penetration, and/or ionizing radiation. Here we overcome such limitations by combining high-resolution photoacoustic tomography (PAT) with extraordinarily optical absorbing gold nanocages (AuNCs). When bioconjugated with [Nle(4),D-Phe(7)]-alpha-melanocyte-stimulating hormone, the AuNCs can serve as a novel contrast agent for in vivo molecular PAT of melanomas with both exquisite sensitivity and high specificity. The bioconjugated AuNCs enhanced contrast approximately 300% more than the control, PEGylated AuNCs. The in vivo PAT quantification of the amount of AuNCs accumulated in melanomas was further validated with inductively coupled plasma mass spectrometry (ICP-MS).

Rapid and accurate identification by real-time PCR of biotoxin-producing dinoflagellates from the family gymnodiniaceae.

PubMed

Smith, Kirsty F; de Salas, Miguel; Adamson, Janet; Rhodes, Lesley L

2014-03-07

The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR) assays targeting the large subunit ribosomal RNA (LSU rRNA) gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.

Simultaneous determination of multi-residue and multi-class antibiotics in aquaculture shrimps by UPLC-MS/MS.

PubMed

Saxena, Sushil Kumar; Rangasamy, Rajesh; Krishnan, Anoop A; Singh, Dhirendra P; Uke, Sumedh P; Malekadi, Praveen Kumar; Sengar, Anoop S; Mohamed, D Peer; Gupta, Ananda

2018-09-15

An accurate, reliable and fast multi-residue, multi-class method using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and validated for simultaneous determination and quantification of 24 pharmacologically active substances of three different classes (Quinolones including fluoroquinolones, sulphonamides and tetracyclines) in aquaculture shrimps. Sample preparation involves extraction with acetonitrile containing 0.1% formic acid and followed by clean up with n-hexane and 0.1% methanol in water by UPLC-MS/MS within 8 min. The method was validated according to European Commission Decision 2002/657. Acceptable values were obtained for linearity (5-200 μg kg -1 ), specificity, Limit of Quantification (5-10 μg kg -1 ), recovery (between 83 and 100%), repeatability (RSD < 9%), within lab reproducibility (RSD < 15%), reproducibility (RSD ≤ 22%), decision limit (105-116 μg kg -1 ) and detection capability (110-132 μg kg -1 ). The validated method was applied to aquaculture shrimp samples from India. Copyright © 2018 Elsevier Ltd. All rights reserved.

Confocal quantification of cis-regulatory reporter gene expression in living sea urchin.

PubMed

Damle, Sagar; Hanser, Bridget; Davidson, Eric H; Fraser, Scott E

2006-11-15

Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.

Characterization of epitope specificities of reference antibodies used for the quantification of the birch pollen allergen Bet v 1.

PubMed

Brier, S; Le Mignon, M; Jain, K; Lebrun, C; Peurois, F; Kellenberger, C; Bordas-Le Floch, V; Mascarell, L; Nony, E; Moingeon, P

2018-05-01

Accurate allergen quantification is needed to document the consistency of allergen extracts used for immunotherapy. Herein, we characterize the epitope specificities of two monoclonal antibodies used in an ELISA for the quantification of the major birch pollen allergen Bet v 1, established as a reference by the BSP090 European project. The ability of mAbs 5B4 and 6H4 to recognize Bet v 1 isoforms was addressed by immunochromatography. The capacity of each mAb to compete with patients' IgE for binding to Bet v 1 was measured by ELISA inhibition. Epitope mapping was performed by pepscan analysis, site-directed mutagenesis, and hydrogen/deuterium exchange-mass spectrometry. The 5B4 epitope corresponds to a peptide sequence (I56-K68) overlapping with the binding sites of patients' serum IgEs. Mutation of residues P59, E60, and K65 abolishes 5B4 binding to Bet v 1 and reduces the level of IgE recognition. In contrast, 6H4 recognizes a conformational epitope lying opposite to the 5B4 binding site, involving residues located in segments I44-K55 and R70-F79. Substitution of E45 reduces the binding capacity of 6H4, confirming that it is critical for the interaction. Both mAbs interact with >90% of Bet v 1 content present in the birch pollen extract, while displaying a weak cross-reactivity with other allergens of the PR-10 family. MAbs 5B4 and 6H4 recognize structurally distinct epitopes present in the vast majority of Bet v 1 isoforms. These results support the relevance as a reference method of the Bet v 1-specific quantitative ELISA adopted by the European Pharmacopoeia. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

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

PubMed

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.

DNA origami metallized site specifically to form electrically conductive nanowires.

PubMed

Pearson, Anthony C; Liu, Jianfei; Pound, Elisabeth; Uprety, Bibek; Woolley, Adam T; Davis, Robert C; Harb, John N

2012-09-06

DNA origami is a promising tool for use as a template in the design and fabrication of nanoscale structures. The ability to engineer selected staple strands on a DNA origami structure provides a high density of addressable locations across the structure. Here we report a method using site-specific attachment of gold nanoparticles to modified staple strands and subsequent metallization to fabricate conductive wires from DNA origami templates. We have modified DNA origami structures by lengthening each staple strand in select regions with a 10-base nucleotide sequence and have attached DNA-modified gold nanoparticles to the lengthened staple strands via complementary base-pairing. The high density of extended staple strands allowed the gold nanoparticles to pack tightly in the modified regions of the DNA origami, where the measured median gap size between neighboring particles was 4.1 nm. Gold metallization processes were optimized so that the attached gold nanoparticles grew until gaps between particles were filled and uniform continuous nanowires were formed. Finally, electron beam lithography was used to pattern electrodes in order to measure the electrical conductivity of metallized DNA origami, which showed an average resistance of 2.4 kΩ per metallized structure.

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

PubMed

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.

Peregrine: A rapid and unbiased method to produce strand-specific RNA-Seq libraries from small quantities of starting material.

PubMed

Langevin, Stanley A; Bent, Zachary W; Solberg, Owen D; Curtis, Deanna J; Lane, Pamela D; Williams, Kelly P; Schoeniger, Joseph S; Sinha, Anupama; Lane, Todd W; Branda, Steven S

2013-04-01

Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows.

[DNA structure from A to Z--biological implications of structural diversity of DNA].

PubMed

Bukowiecka-Matusiak, Małgorzata; Woźniak, Lucyna A

2006-01-01

Deoxyribonucleic acid (DNA) is a biopolymer of nucleotides, usually adopting a double-stranded helical form in cells, with complementary base pairing holding the two strands together. The most stable is B-DNA conformation, although numerous other double helical structures can occur under specific conditions (A-DNA, Z-DNA, P-DNA). The existence of multiple-stranded (triplex, tetraplex) forms in vivo and their biological function in cells are subject of intensive studies.

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

PubMed

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.

The core microprocessor component DiGeorge syndrome critical region 8 (DGCR8) is a nonspecific RNA-binding protein.

PubMed

Roth, Braden M; Ishimaru, Daniella; Hennig, Mirko

2013-09-13

MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins.

The Core Microprocessor Component DiGeorge Syndrome Critical Region 8 (DGCR8) Is a Nonspecific RNA-binding Protein*

PubMed Central

Roth, Braden M.; Ishimaru, Daniella; Hennig, Mirko

2013-01-01

MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins. PMID:23893406

Sequence dependence of electron-induced DNA strand breakage revealed by DNA nanoarrays

PubMed Central

Keller, Adrian; Rackwitz, Jenny; Cauët, Emilie; Liévin, Jacques; Körzdörfer, Thomas; Rotaru, Alexandru; Gothelf, Kurt V.; Besenbacher, Flemming; Bald, Ilko

2014-01-01

The electronic structure of DNA is determined by its nucleotide sequence, which is for instance exploited in molecular electronics. Here we demonstrate that also the DNA strand breakage induced by low-energy electrons (18 eV) depends on the nucleotide sequence. To determine the absolute cross sections for electron induced single strand breaks in specific 13 mer oligonucleotides we used atomic force microscopy analysis of DNA origami based DNA nanoarrays. We investigated the DNA sequences 5′-TT(XYX)3TT with X = A, G, C and Y = T, BrU 5-bromouracil and found absolute strand break cross sections between 2.66 · 10−14 cm2 and 7.06 · 10−14 cm2. The highest cross section was found for 5′-TT(ATA)3TT and 5′-TT(ABrUA)3TT, respectively. BrU is a radiosensitizer, which was discussed to be used in cancer radiation therapy. The replacement of T by BrU into the investigated DNA sequences leads to a slight increase of the absolute strand break cross sections resulting in sequence-dependent enhancement factors between 1.14 and 1.66. Nevertheless, the variation of strand break cross sections due to the specific nucleotide sequence is considerably higher. Thus, the present results suggest the development of targeted radiosensitizers for cancer radiation therapy. PMID:25487346

Initial investigations into the damping characteristics of wire rope vibration isolators

NASA Technical Reports Server (NTRS)

Cutchins, M. A.; Cochran, J. E., Jr.; Kumar, K.; Fitz-Coy, N. G.; Tinker, M. L.

1987-01-01

Passive dampers composed of coils of multi-strand wire rope are investigated. Analytical results range from those produced by complex NASTRAN models to those of a Coulomb damping model with variable friction force. The latter agrees well with experiment. The Coulomb model is also utilized to generate hysteresis loops. Various other models related to early experimental investigations are described. Significant closed-form static solutions for physical properties of single-and multi-strand wire ropes are developed for certain specific geometries and loading conditions. NASTRAN models concentrate on model generation and mode shapes of 2-strand and 7-strand straight wire ropes with interfacial forces.

Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine.

PubMed

Kurashige, Tomomi; Shimamura, Mika; Nagayama, Yuji

2016-06-01

The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NAC's effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NAC's biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds. © The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

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

PubMed

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.

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.

PubMed

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 CV<9.4%. The methods require only 200μL of sample and allow a high throughput due to short analytical run (2min). These methods were successfully applied to a pig intoxicated with colchicine and treated with colchicine specific Fab fragments. Copyright © 2017 Elsevier B.V. All rights reserved.

Method for promoting specific alignment of short oligonucleotides on nucleic acids

DOEpatents

Studier, F. William; Kieleczawa, Jan; Dunn, John J.

1996-01-01

Disclosed is a method for promoting specific alignment of short oligonucleotides on a nucleic acid polymer. The nucleic acid polymer is incubated in a solution containing a single-stranded DNA-binding protein and a plurality of oligonucleotides which are perfectly complementary to distinct but adjacent regions of a predetermined contiguous nucleotide sequence in the nucleic acid polymer. The plurality of oligonucleotides anneal to the nucleic acid polymer to form a contiguous region of double stranded nucleic acid. Specific application of the methods disclosed include priming DNA synthesis and template-directed ligation.

Double strand break induction and kinetics indicate preserved hypersensitivity in keratinocytes to subtherapeutic doses for 7weeks of radiotherapy.

PubMed

Qvarnström, Fredrik; Simonsson, Martin; Nyman, Jan; Hermansson, Ingegerd; Book, Majlis; Johansson, Karl-Axel; Turesson, Ingela

2017-01-01

Previously we reported that hyper-radiosensitivity (HRS) was evidenced by quantifying DNA double strand break (DSB) foci in epidermis biopsies collected after delivering radiotherapeutic one and five dose fractions. The aim of this study was to determine whether HRS was preserved throughout a 7-week radiotherapy treatment, and also to examine the rate of foci decline and foci persistence between dose fractions. 42 patients with prostate cancer received 7-week fractionated radiotherapy treatment (RT) with daily dose fractions of 0.05-1.10Gy to the skin. Before RT, and at several times throughout treatment, skin biopsies (n=452) were collected at 30min, and 2, 3, 24, and 72h after dose fractions. DSB-foci markers, γH2AX and 53BP1, were labelled in epidermal keratinocytes with immunofluorescence and immunohistochemical staining. Foci were counted both with digital image analysis and manually. HRS in keratinocytes was evidenced by the dose-response relationships of DSB foci, observed throughout the treatment course, independent of sampling time and quantification method. Foci observed at 24h after dose fractions indicated considerable DSB persistence. Accordingly, foci significantly accumulated after 5 consecutive dose fractions. For doses below 0.3Gy, persistent foci could be observed even at 72h after damage induction. A comparison of γH2AX and 53BP1 quantifications in double-stained biopsies showed similar HRS dose-response relationships. These results represented the first evidence of preserved HRS, assessed by γH2AX- and 53BP1-labelled DSB foci, throughout a 7-week treatment course with daily repeated subtherapeutic dose fractions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A new method for determination of cocamidopropyl betaine synthesized from coconut oil through spectral shift of Eriochrome Black T

NASA Astrophysics Data System (ADS)

Gholami, Ali; Golestaneh, Mahshid; Andalib, Zeinab

2018-03-01

Cocamidopropyl betaine (CAPB) is a zwitterionic surfactant that is synthesized using coconut oil and usually supplied in form of an aqueous solution with 25-37% w/w. In this study, a novel method based on UV-visible spectroscopy is developed for an accurate determination of CAPB synthesized from coconut oil. Eriochrome Black T (EBT) as a specific color indicator was added to CAPB and a red shift and color change were observed. This shift leads in increasing wavelength selectivity of the method. The change in the color intensity depends on the concentration of CAPB. By measuring the absorbance of a solution containing CAPB, its concentration was measured. After optimizing all the effective parameters, CAPB was detected in commercial real samples. Using the proposed approach, limit of quantification (LOQ) and relative standard deviation (RSD) were obtained about 4.30 × 10- 5 M and 4.8% respectively. None of unreacted materials or by-products, which were produced in the synthesis of CAPB, showed any interference in the determination of CAPB. This shows that the proposed method is specific and accurate, and can potentially be used for quantitative determination of CAPB in commercial samples with satisfactory results.

Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems

PubMed Central

Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B.

2015-01-01

Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing. PMID:26053390

Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems.

PubMed

Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B

2015-01-01

Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing.

The Human DNA glycosylases NEIL1 and NEIL3 Excise Psoralen-Induced DNA-DNA Cross-Links in a Four-Stranded DNA Structure.

PubMed

Martin, Peter R; Couvé, Sophie; Zutterling, Caroline; Albelazi, Mustafa S; Groisman, Regina; Matkarimov, Bakhyt T; Parsons, Jason L; Elder, Rhoderick H; Saparbaev, Murat K

2017-12-12

Interstrand cross-links (ICLs) are highly cytotoxic DNA lesions that block DNA replication and transcription by preventing strand separation. Previously, we demonstrated that the bacterial and human DNA glycosylases Nei and NEIL1 excise unhooked psoralen-derived ICLs in three-stranded DNA via hydrolysis of the glycosidic bond between the crosslinked base and deoxyribose sugar. Furthermore, NEIL3 from Xenopus laevis has been shown to cleave psoralen- and abasic site-induced ICLs in Xenopus egg extracts. Here we report that human NEIL3 cleaves psoralen-induced DNA-DNA cross-links in three-stranded and four-stranded DNA substrates to generate unhooked DNA fragments containing either an abasic site or a psoralen-thymine monoadduct. Furthermore, while Nei and NEIL1 also cleave a psoralen-induced four-stranded DNA substrate to generate two unhooked DNA duplexes with a nick, NEIL3 targets both DNA strands in the ICL without generating single-strand breaks. The DNA substrate specificities of these Nei-like enzymes imply the occurrence of long uninterrupted three- and four-stranded crosslinked DNA-DNA structures that may originate in vivo from DNA replication fork bypass of an ICL. In conclusion, the Nei-like DNA glycosylases unhook psoralen-derived ICLs in various DNA structures via a genuine repair mechanism in which complex DNA lesions can be removed without generation of highly toxic double-strand breaks.

A liquid chromatography-tandem mass spectrometry-based targeted proteomics assay for monitoring P-glycoprotein levels in human breast tissue.

PubMed

Yang, Ting; Chen, Fei; Xu, Feifei; Wang, Fengliang; Xu, Qingqing; Chen, Yun

2014-09-25

P-glycoprotein (P-gp) can efflux drugs from cancer cells, and its overexpression is commonly associated with multi-drug resistance (MDR). Thus, the accurate quantification of P-gp would help predict the response to chemotherapy and for prognosis of breast cancer patients. An advanced liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based targeted proteomics assay was developed and validated for monitoring P-gp levels in breast tissue. Tryptic peptide 368IIDNKPSIDSYSK380 was selected as a surrogate analyte for quantification, and immuno-depleted tissue extract was used as a surrogate matrix. Matched pairs of breast tissue samples from 60 patients who were suspected to have drug resistance were subject to analysis. The levels of P-gp were quantified. Using data from normal tissue, we suggested a P-gp reference interval. The experimental values of tumor tissue samples were compared with those obtained from Western blotting and immunohistochemistry (IHC). The result indicated that the targeted proteomics approach was comparable to IHC but provided a lower limit of quantification (LOQ) and could afford more reliable results at low concentrations than the other two methods. LC/MS/MS-based targeted proteomics may allow the quantification of P-gp in breast tissue in a more accurate manner. Copyright © 2014 Elsevier B.V. All rights reserved.

miR-MaGiC improves quantification accuracy for small RNA-seq.

PubMed

Russell, Pamela H; Vestal, Brian; Shi, Wen; Rudra, Pratyaydipta D; Dowell, Robin; Radcliffe, Richard; Saba, Laura; Kechris, Katerina

2018-05-15

Many tools have been developed to profile microRNA (miRNA) expression from small RNA-seq data. These tools must contend with several issues: the small size of miRNAs, the small number of unique miRNAs, the fact that similar miRNAs can be transcribed from multiple loci, and the presence of miRNA isoforms known as isomiRs. Methods failing to address these issues can return misleading information. We propose a novel quantification method designed to address these concerns. We present miR-MaGiC, a novel miRNA quantification method, implemented as a cross-platform tool in Java. miR-MaGiC performs stringent mapping to a core region of each miRNA and defines a meaningful set of target miRNA sequences by collapsing the miRNA space to "functional groups". We hypothesize that these two features, mapping stringency and collapsing, provide more optimal quantification to a more meaningful unit (i.e., miRNA family). We test miR-MaGiC and several published methods on 210 small RNA-seq libraries, evaluating each method's ability to accurately reflect global miRNA expression profiles. We define accuracy as total counts close to the total number of input reads originating from miRNAs. We find that miR-MaGiC, which incorporates both stringency and collapsing, provides the most accurate counts.

Molecular diagnostic toolkit for Rhizophagus irregularis isolate DAOM-197198 using quantitative PCR assay targeting the mitochondrial genome.

PubMed

Badri, Amine; Stefani, Franck O P; Lachance, Geneviève; Roy-Arcand, Line; Beaudet, Denis; Vialle, Agathe; Hijri, Mohamed

2016-10-01

Rhizophagus irregularis (previously named Glomus irregulare) is one of the most widespread and common arbuscular mycorrhizal fungal (AMF) species. It has been recovered worldwide in agricultural and natural soils, and the isolate DAOM-197198 has been utilized as a commercial inoculant for two decades. Despite the ecological and economical importance of this taxon, specific markers for quantification of propagules by quantitative real-time PCR (qPCR) are extremely limited and none have been rigorously validated for quality control of manufactured products such as biofertilizers. From the sequencing of 14 complete AMF mitochondrial (mt) genomes, a qPCR assay using a hydrolysis probe designed in the single copy cox3-rnl intergenic region was tested and validated to specifically and accurately quantify the spores of R. irregularis isolate DAOM-197198. Specificity tests were performed using standard PCR and qPCR, and results clearly showed that the primers specifically amplified the isolate DAOM-197198, yielding a PCR product of 106 bp. According to the qPCR analyses on spores produced in vitro, the average copy number of mt genomes per spore was 3172 ± 304 SE (n = 6). Quantification assays were successfully undertaken on known and unknown samples in liquid suspensions and commercial dry formulations to show the accuracy, precision, robustness, and reproducibility of the qPCR assay. This study provides a powerful molecular toolkit specifically designed to quantify spores of the model AMF isolate DAOM-197198. The approach of molecular toolkit used in our study could be applied to other AMF taxa and will be useful to research institutions and governmental and industrial laboratories running routine quality control of AMF-based products.

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

PubMed

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.

Structural properties of oligonucleotide monolayers on gold surfaces probed by fluorescence investigations.

PubMed

Rant, Ulrich; Arinaga, Kenji; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard; Tornow, Marc

2004-11-09

We present optical investigations on the conformation of oligonucleotide layers on Au surfaces. Our studies concentrate on the effect of varying surface coverage densities on the structural properties of layers of 12- and 24mer single-stranded DNA, tethered to the Au surface at one end while being labeled with a fluorescent marker at the opposing end. The distance-dependent energy transfer from the marker dye to the metal surface, which causes quenching of the observed fluorescence, is used to provide information on the orientation of the DNA strands relative to the surface. Variations in the oligonucleotide coverage density, as determined from electrochemical quantification, over 2 orders of magnitude are achieved by employing different preparation conditions. The observed enhancement in fluorescence intensity with increasing DNA coverage can be related to a model involving mutual steric interactions of oligonucleotides on the surface, as well as fluorescence quenching theory. Finally, the applicability of the presented concepts for investigations of heterogeneous monolayers is demonstrated by means of studying the coadsorption of mercaptohexanol onto DNA-modified Au surfaces.

In-vitro radiosensitivity in patients with systemic lupus erythematosus.

PubMed

Carrillo-Alascio, P L; Sabio, J M; Núñez-Torres, M I; López, E; Muñoz-Gámez, J A; Hidalgo-Tenorio, C; Jáimez, L; Martín, J; Jiménez-Alonso, J

2009-06-01

To determine the "in-vitro" intrinsic cell radiosensitivity (RS) as a risk indicator of radiation-related side-effects in patients with systemic lupus erythematosus (SLE) compared with healthy subjects (control group). Moreover, we elucidated if clinical, therapeutic and biological parameters could affect the "in-vitro" intrinsic RS in patients with SLE. Intrinsic RS was determined by the quantification of the initial radiation-induced DNA double-strand breaks in peripheral lymphocytes, measured by pulsed-field gel electrophoresis from 52 patients with SLE and a control group consisting of 48 sex- and age-matched healthy subjects. No difference in intrinsic RS was found among both groups. However, SLE patients with anaemia, increased erythrocyte sedimentation rate and those with positive result for anti-La/SSB and anti-RNP antibodies showed significantly higher DNA double-strand breaks than those without them. In our study, patients with SLE did not have a higher intrinsic RS than healthy subjects. According to these results, and with the caution of being a limited laboratory study, the use of radiotherapy should not be avoided in patients with SLE when it is clinically needed.

Double hull grounding experiments

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

Rodd, J.L.; Sikora, J.P.

1995-12-31

In the last few years the public and governments of many nations have become increasingly aware of the need for improving oil tanker safety. The requirements for double hull tankers are an attempt to address this need through legislation. Even though a number of investigations on the mechanics of collisions have been done in the past, until recently very little research supported the development of structural improvements to reduce oil tanker damage during grounding and stranding accidents. An aggressive evaluation of double hull tanker crashworthiness in stranding and grounding accidents is underway at CD/NSWC (formerly the David Taylor Research Center).more » The ability to predict damage from grounding accidents accurately is not currently available. The objective of this paper is to present qualitatively the structural failure mechanisms associated with stranding and grounding events for candidate double hull tanker structures and to present some simple methods for comparing damage scenarios. A comparison of the structural performance of key features in several very different designs will provide useful information toward this understanding.« less

A quantification model for the structure of clay materials.

PubMed

Tang, Liansheng; Sang, Haitao; Chen, Haokun; Sun, Yinlei; Zhang, Longjian

2016-07-04

In this paper, the quantification for clay structure is explicitly explained, and the approach and goals of quantification are also discussed. The authors consider that the purpose of the quantification for clay structure is to determine some parameters that can be used to quantitatively characterize the impact of clay structure on the macro-mechanical behaviour. According to the system theory and the law of energy conservation, a quantification model for the structure characteristics of clay materials is established and three quantitative parameters (i.e., deformation structure potential, strength structure potential and comprehensive structure potential) are proposed. And the corresponding tests are conducted. The experimental results show that these quantitative parameters can accurately reflect the influence of clay structure on the deformation behaviour, strength behaviour and the relative magnitude of structural influence on the above two quantitative parameters, respectively. These quantitative parameters have explicit mechanical meanings, and can be used to characterize the structural influences of clay on its mechanical behaviour.

Misleading measures in Vitamin D analysis: a novel LC-MS/MS assay to account for epimers and isobars.

PubMed

Shah, Iltaf; James, Ricky; Barker, James; Petroczi, Andrea; Naughton, Declan P

2011-05-14

Recently, the accuracies of many commercially available immunoassays for Vitamin D have been questioned. Liquid chromatography tandem mass spectrometry (LC- MS/MS) has been shown to facilitate accurate separation and quantification of the major circulating metabolite 25-hydroxyvitamin-D3 (25OHD3) and 25-hydroxyvitamin-D2 (25OHD2) collectively termed as 25OHD. However, among other interferents, this method may be compromised by overlapping peaks and identical masses of epimers and isobars, resulting in inaccuracies in circulating 25OHD measurements. The aim of this study was to develop a novel LC-MS/MS method that can accurately identify and quantitate 25OHD3 and 25OHD2 through chromatographic separation of 25OHD from its epimers and isobars. A positive ion electrospray ionisation (ESI) LC-MS/MS method was used in the Multiple Reaction Monitoring (MRM) mode for quantification. It involved i) liquid-liquid extraction, ii) tandem columns (a high resolution ZORBAX C18 coupled to an ULTRON chiral, with guard column and inlet filter), iii) Stanozolol-D3 as internal standard, and iv) identification via ESI and monitoring of three fragmentation transitions. To demonstrate the practical usefulness of our method, blood samples were collected from 5 healthy male Caucasian volunteers; age range 22 to 37 years and 25OHD2, 25OHD3 along with co-eluting epimers and analogues were quantified. The new method allowed chromatographic separation and quantification of 25OHD2, 25OHD3, along with 25OHD3 epimer 3-epi-25OHD3 and isobars 1-α-hydroxyvitamin-D3 (1αOHD3), and 7-α-hydroxy-4-cholesten-3-one (7αC4). The new assay was capable of detecting 0.25 ng/mL of all analytes in serum. To our knowledge, this is the first specific, reliable, reproducible and robust LC-MS/MS method developed for the accurate detection of 25OHD (Vitamin D). The method is capable of detecting low levels of 25OHD3 and 25OHD2 together with chromatographic separation from the co-eluting epimers and isobars and circumvents other instrumental/analytical interferences. This analytical method does not require time-consuming derivatisation and complex extraction techniques and could prove very useful in clinical studies.

Misleading measures in Vitamin D analysis: A novel LC-MS/MS assay to account for epimers and isobars

PubMed Central

2011-01-01

Background Recently, the accuracies of many commercially available immunoassays for Vitamin D have been questioned. Liquid chromatography tandem mass spectrometry (LC- MS/MS) has been shown to facilitate accurate separation and quantification of the major circulating metabolite 25-hydroxyvitamin-D3 (25OHD3) and 25-hydroxyvitamin-D2 (25OHD2) collectively termed as 25OHD. However, among other interferents, this method may be compromised by overlapping peaks and identical masses of epimers and isobars, resulting in inaccuracies in circulating 25OHD measurements. The aim of this study was to develop a novel LC-MS/MS method that can accurately identify and quantitate 25OHD3 and 25OHD2 through chromatographic separation of 25OHD from its epimers and isobars. Methods A positive ion electrospray ionisation (ESI) LC-MS/MS method was used in the Multiple Reaction Monitoring (MRM) mode for quantification. It involved i) liquid-liquid extraction, ii) tandem columns (a high resolution ZORBAX C18 coupled to an ULTRON chiral, with guard column and inlet filter), iii) Stanozolol-D3 as internal standard, and iv) identification via ESI and monitoring of three fragmentation transitions. To demonstrate the practical usefulness of our method, blood samples were collected from 5 healthy male Caucasian volunteers; age range 22 to 37 years and 25OHD2, 25OHD3 along with co-eluting epimers and analogues were quantified. Results The new method allowed chromatographic separation and quantification of 25OHD2, 25OHD3, along with 25OHD3 epimer 3-epi-25OHD3 and isobars 1-α-hydroxyvitamin-D3 (1αOHD3), and 7-α-hydroxy-4-cholesten-3-one (7αC4). The new assay was capable of detecting 0.25 ng/mL of all analytes in serum. Conclusions To our knowledge, this is the first specific, reliable, reproducible and robust LC-MS/MS method developed for the accurate detection of 25OHD (Vitamin D). The method is capable of detecting low levels of 25OHD3 and 25OHD2 together with chromatographic separation from the co-eluting epimers and isobars and circumvents other instrumental/analytical interferences. This analytical method does not require time-consuming derivatisation and complex extraction techniques and could prove very useful in clinical studies. PMID:21569549

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

PubMed

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.

Reproducible surface-enhanced Raman quantification of biomarkers in multicomponent mixtures.

PubMed

De Luca, Anna Chiara; Reader-Harris, Peter; Mazilu, Michael; Mariggiò, Stefania; Corda, Daniela; Di Falco, Andrea

2014-03-25

Direct and quantitative detection of unlabeled glycerophosphoinositol (GroPIns), an abundant cytosolic phosphoinositide derivative, would allow rapid evaluation of several malignant cell transformations. Here we report label-free analysis of GroPIns via surface-enhanced Raman spectroscopy (SERS) with a sensitivity of 200 nM, well below its apparent concentration in cells. Crucially, our SERS substrates, based on lithographically defined gold nanofeatures, can be used to predict accurately the GroPIns concentration even in multicomponent mixtures, avoiding the preliminary separation of individual compounds. Our results represent a critical step toward the creation of SERS-based biosensor for rapid, label-free, and reproducible detection of specific molecules, overcoming limits of current experimental methods.

Applications of PET CT in clinical practice: Present and future

NASA Astrophysics Data System (ADS)

Costa, Durval Campos

2007-02-01

Radionuclide imaging and specially positron emission tomography (PET) has already demonstrated its benefits in three major medical subjects, i.e. neurology, cardiology and particularly clinical oncology. More recently the combination of PET and X-ray computed tomography (CT) as PET-CT led to a significant increment of the already large number of clinical applications of this imaging modality. This "anatomy-metabolic fusion" also known as Metabolic Imaging has its future assured if we can: (1) improve resolution reducing partial volume effect, (2) achieve very fast whole body imaging, (3) obtain accurate quantification of specific functions with higher contrast resolution and, if possible, (4) reduce exposure rates due to the unavoidable use of ionizing radiation.

Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection

PubMed Central

KC, R.; Srivastava, A.; Wilkowski, J. M.; Richter, C. E.; Shavit, J. A.; Burke, D. T.; Bielas, S. L.

2016-01-01

CRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies. PMID:27557703

More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny.

PubMed

Vahidi Ferdousi, Leyla; Rocheteau, Pierre; Chayot, Romain; Montagne, Benjamin; Chaker, Zayna; Flamant, Patricia; Tajbakhsh, Shahragim; Ricchetti, Miria

2014-11-01

The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite) cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs) via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs. Copyright © 2014. Published by Elsevier B.V.

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.

PubMed

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.

Rapid quantification of vesicle concentration for DOPG/DOPC and Cardiolipin/DOPC mixed lipid systems of variable composition.

PubMed

Elmer-Dixon, Margaret M; Bowler, Bruce E

2018-05-19

A novel approach to quantify mixed lipid systems is described. Traditional approaches to lipid vesicle quantification are time consuming, require large amounts of material and are destructive. We extend our recently described method for quantification of pure lipid systems to mixed lipid systems. The method only requires a UV-Vis spectrometer and does not destroy sample. Mie scattering data from absorbance measurements are used as input into a Matlab program to calculate the total vesicle concentration and the concentrations of each lipid in the mixed lipid system. The technique is fast and accurate, which is essential for analytical lipid binding experiments. Copyright © 2018. Published by Elsevier Inc.

Lesion Quantification in Dual-Modality Mammotomography

NASA Astrophysics Data System (ADS)

Li, Heng; Zheng, Yibin; More, Mitali J.; Goodale, Patricia J.; Williams, Mark B.

2007-02-01

This paper describes a novel x-ray/SPECT dual modality breast imaging system that provides 3D structural and functional information. While only a limited number of views on one side of the breast can be acquired due to mechanical and time constraints, we developed a technique to compensate for the limited angle artifact in reconstruction images and accurately estimate both the lesion size and radioactivity concentration. Various angular sampling strategies were evaluated using both simulated and experimental data. It was demonstrated that quantification of lesion size to an accuracy of 10% and quantification of radioactivity to an accuracy of 20% are feasible from limited-angle data acquired with clinically practical dosage and acquisition time

Specificity of cell–cell adhesion by classical cadherins: Critical role for low-affinity dimerization through β-strand swapping

PubMed Central

Chen, Chien Peter; Posy, Shoshana; Ben-Shaul, Avinoam; Shapiro, Lawrence; Honig, Barry H.

2005-01-01

Cadherins constitute a family of cell-surface proteins that mediate intercellular adhesion through the association of protomers presented from juxtaposed cells. Differential cadherin expression leads to highly specific intercellular interactions in vivo. This cell–cell specificity is difficult to understand at the molecular level because individual cadherins within a given subfamily are highly similar to each other both in sequence and structure, and they dimerize with remarkably low binding affinities. Here, we provide a molecular model that accounts for these apparently contradictory observations. The model is based in part on the fact that cadherins bind to one another by “swapping” the N-terminal β-strands of their adhesive domains. An inherent feature of strand swapping (or, more generally, the domain swapping phenomenon) is that “closed” monomeric conformations act as competitive inhibitors of dimer formation, thus lowering affinities even when the dimer interface has the characteristics of high-affinity complexes. The model describes quantitatively how small affinity differences between low-affinity cadherin dimers are amplified by multiple cadherin interactions to establish large specificity effects at the cellular level. It is shown that cellular specificity would not be observed if cadherins bound with high affinities, thus emphasizing the crucial role of strand swapping in cell–cell adhesion. Numerical estimates demonstrate that the strength of cellular adhesion is extremely sensitive to the concentration of cadherins expressed at the cell surface. We suggest that the domain swapping mechanism is used by a variety of cell-adhesion proteins and that related mechanisms to control affinity and specificity are exploited in other systems. PMID:15937105

Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication

PubMed Central

Shinbrot, Eve; Henninger, Erin E.; Weinhold, Nils; Covington, Kyle R.; Göksenin, A. Yasemin; Schultz, Nikolaus; Chao, Hsu; Doddapaneni, HarshaVardhan; Muzny, Donna M.; Gibbs, Richard A.; Sander, Chris; Pursell, Zachary F.

2014-01-01

Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication. PMID:25228659

Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

DOEpatents

Gray, Joe W.; Pinkel, Daniel

1991-01-01

A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. Probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations.

DNA purification by triplex-affinity capture and affinity capture electrophoresis

DOEpatents

Cantor, Charles R.; Ito, Takashi; Smith, Cassandra L.

1996-01-01

The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel.

Centrifuge: rapid and sensitive classification of metagenomic sequences.

PubMed

Kim, Daehwan; Song, Li; Breitwieser, Florian P; Salzberg, Steven L

2016-12-01

Centrifuge is a novel microbial classification engine that enables rapid, accurate, and sensitive labeling of reads and quantification of species on desktop computers. The system uses an indexing scheme based on the Burrows-Wheeler transform (BWT) and the Ferragina-Manzini (FM) index, optimized specifically for the metagenomic classification problem. Centrifuge requires a relatively small index (4.2 GB for 4078 bacterial and 200 archaeal genomes) and classifies sequences at very high speed, allowing it to process the millions of reads from a typical high-throughput DNA sequencing run within a few minutes. Together, these advances enable timely and accurate analysis of large metagenomics data sets on conventional desktop computers. Because of its space-optimized indexing schemes, Centrifuge also makes it possible to index the entire NCBI nonredundant nucleotide sequence database (a total of 109 billion bases) with an index size of 69 GB, in contrast to k-mer-based indexing schemes, which require far more extensive space. © 2016 Kim et al.; Published by Cold Spring Harbor Laboratory Press.

Determination of Carbonyl Functional Groups in Bio-oils by Potentiometric Titration: The Faix Method.

PubMed

Black, Stuart; Ferrell, Jack R

2017-02-07

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls cause an increase in viscosity (often referred to as 'aging') during storage of bio-oils. As such, carbonyl content has previously been used as a method of tracking bio-oil aging and condensation reactions with less variability than viscosity measurements. Additionally, carbonyls are also responsible for coke formation in bio-oil upgrading processes. Given the importance of carbonyls in bio-oils, accurate analytical methods for their quantification are very important for the bio-oil community. Potentiometric titration methods based on carbonyl oximation have long been used for the determination of carbonyl content in pyrolysis bio-oils. Here, we present a modification of the traditional carbonyl oximation procedures that results in less reaction time, smaller sample size, higher precision, and more accurate carbonyl determinations. While traditional carbonyl oximation methods occur at room temperature, the Faix method presented here occurs at an elevated temperature of 80 °C.

Using iRT, a normalized retention time for more targeted measurement of peptides

PubMed Central

Escher, Claudia; Reiter, Lukas; MacLean, Brendan; Ossola, Reto; Herzog, Franz; Chilton, John; MacCoss, Michael J.; Rinner, Oliver

2014-01-01

Multiple reaction monitoring (MRM) has recently become the method of choice for targeted quantitative measurement of proteins using mass spectrometry. The method, however, is limited in the number of peptides that can be measured in one run. This number can be markedly increased by scheduling the acquisition if the accurate retention time (RT) of each peptide is known. Here we present iRT, an empirically derived dimensionless peptide-specific value that allows for highly accurate RT prediction. The iRT of a peptide is a fixed number relative to a standard set of reference iRT-peptides that can be transferred across laboratories and chromatographic systems. We show that iRT facilitates the setup of multiplexed experiments with acquisition windows more than 4 times smaller compared to in silico RT predictions resulting in improved quantification accuracy. iRTs can be determined by any laboratory and shared transparently. The iRT concept has been implemented in Skyline, the most widely used software for MRM experiments. PMID:22577012

Quantification of breast density with spectral mammography based on a scanned multi-slit photon-counting detector: a feasibility study.

PubMed

Ding, Huanjun; Molloi, Sabee

2012-08-07

A simple and accurate measurement of breast density is crucial for the understanding of its impact in breast cancer risk models. The feasibility to quantify volumetric breast density with a photon-counting spectral mammography system has been investigated using both computer simulations and physical phantom studies. A computer simulation model involved polyenergetic spectra from a tungsten anode x-ray tube and a Si-based photon-counting detector has been evaluated for breast density quantification. The figure-of-merit (FOM), which was defined as the signal-to-noise ratio of the dual energy image with respect to the square root of mean glandular dose, was chosen to optimize the imaging protocols, in terms of tube voltage and splitting energy. A scanning multi-slit photon-counting spectral mammography system has been employed in the experimental study to quantitatively measure breast density using dual energy decomposition with glandular and adipose equivalent phantoms of uniform thickness. Four different phantom studies were designed to evaluate the accuracy of the technique, each of which addressed one specific variable in the phantom configurations, including thickness, density, area and shape. In addition to the standard calibration fitting function used for dual energy decomposition, a modified fitting function has been proposed, which brought the tube voltages used in the imaging tasks as the third variable in dual energy decomposition. For an average sized 4.5 cm thick breast, the FOM was maximized with a tube voltage of 46 kVp and a splitting energy of 24 keV. To be consistent with the tube voltage used in current clinical screening exam (∼32 kVp), the optimal splitting energy was proposed to be 22 keV, which offered a FOM greater than 90% of the optimal value. In the experimental investigation, the root-mean-square (RMS) error in breast density quantification for all four phantom studies was estimated to be approximately 1.54% using standard calibration function. The results from the modified fitting function, which integrated the tube voltage as a variable in the calibration, indicated a RMS error of approximately 1.35% for all four studies. The results of the current study suggest that photon-counting spectral mammography systems may potentially be implemented for an accurate quantification of volumetric breast density, with an RMS error of less than 2%, using the proposed dual energy imaging technique.

Induced nanoparticle aggregation for short nucleic acid quantification by depletion isotachophoresis.

PubMed

Marczak, Steven; Senapati, Satyajyoti; Slouka, Zdenek; Chang, Hsueh-Chia

2016-12-15

A rapid (<20min) gel-membrane biochip platform for the detection and quantification of short nucleic acids is presented based on a sandwich assay with probe-functionalized gold nanoparticles and their separation into concentrated bands by depletion-generated gel isotachophoresis. The platform sequentially exploits the enrichment and depletion phenomena of an ion-selective cation-exchange membrane created under an applied electric field. Enrichment is used to concentrate the nanoparticles and targets at a localized position at the gel-membrane interface for rapid hybridization. The depletion generates an isotachophoretic zone without the need for different conductivity buffers, and is used to separate linked nanoparticles from isolated ones in the gel medium and then by field-enhanced aggregation of only the linked particles at the depletion front. The selective field-induced aggregation of the linked nanoparticles during the subsequent depletion step produces two lateral-flow like bands within 1cm for easy visualization and quantification as the aggregates have negligible electrophoretic mobility in the gel and the isolated nanoparticles are isotachophoretically packed against the migrating depletion front. The detection limit for 69-base single-stranded DNA targets is 10 pM (about 10 million copies for our sample volume) with high selectivity against nontargets and a three decade linear range for quantification. The selectivity and signal intensity are maintained in heterogeneous mixtures where the nontargets outnumber the targets 10,000 to 1. The selective field-induced aggregation of DNA-linked nanoparticles at the ion depletion front is attributed to their trailing position at the isotachophoretic front with a large field gradient. Copyright © 2016 Elsevier B.V. All rights reserved.

Sensitive quantification of Clostridium perfringens in human feces by quantitative real-time PCR targeting alpha-toxin and enterotoxin genes.

PubMed

Nagpal, Ravinder; Ogata, Kiyohito; Tsuji, Hirokazu; Matsuda, Kazunori; Takahashi, Takuya; Nomoto, Koji; Suzuki, Yoshio; Kawashima, Kazunari; Nagata, Satoru; Yamashiro, Yuichiro

2015-10-19

Clostridium perfringens is a widespread pathogen, but the precise quantification of this subdominant gut microbe remains difficult due to its low fecal count (particularly in asymptomatic subjects) and also due to the presence of abundant polymerase-inhibitory substances in human feces. Also, information on the intestinal carriage of toxigenic C. perfringens strains in healthy subjects is sparse. Therefore, we developed a sensitive quantitative real-time PCR assays for quantification of C. perfringens in human feces by targeting its α-toxin and enterotoxin genes. To validate the assays, we finally observed the occurrence of α-toxigenic and enterotoxigenic C. perfringens in the fecal microbiota of healthy Japanese infants and young adults. The plc-specific qPCR assay was newly validated, while primers for 16S rRNA and cpe genes were retrieved from literature. The assays were validated for specificity and sensitivity in pre-inoculated fecal samples, and were finally applied to quantify C. perfringens in stool samples from apparently healthy infants (n 124) and young adults (n 221). The qPCR assays were highly specific and sensitive, with a minimum detection limit of 10(3) bacterial cells/g feces. Alpha-toxigenic C. perfringens was detected in 36% infants and 33% adults, with counts ranging widely (10(3)-10(7) bacterial cells/g). Intriguingly, the mean count of α-toxigenic C. perfringens was significantly higher in infants (6.0±1.5 log10 bacterial cells/g), as compared to that in adults (4.8±1.2). Moreover, the prevalence of enterotoxigenic C. perfringens was also found to be significantly higher in infants, as compared to that in adults. The mean enterotoxigenic C. perfringens count was 5.9±1.9 and 4.8±0.8 log10 bacterial cells/g in infants and adults, respectively. These data indicate that some healthy infants and young adults carry α-toxigenic and enterotoxigenic C. perfringens at significant levels, and may be predisposed to related diseases. Thus, high fecal carriage of toxigenic C. perfringens in healthy children warrants further investigation on its potential sources and clinical significance in these subjects. In summary, we present a novel qPCR assay for sensitive and accurate quantification of α-toxigenic and enterotoxigenic C. perfringens in human feces, which should facilitate prospective studies of the gut microbiota.

Single-molecule dilution and multiple displacement amplification for molecular haplotyping.

PubMed

Paul, Philip; Apgar, Josh

2005-04-01

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.

An Electrochemical Genosensing Assay Based on Magnetic Beads and Gold Nanoparticle-Loaded Latex Microspheres for Vibrio cholerae Detection.

PubMed

Low, Kim-Fatt; Rijiravanich, Patsamon; Singh, Kirnpal Kaur Banga; Surareungchai, Werasak; Yean, Chan Yean

2015-04-01

An ultrasensitive electrochemical genosensing assay was developed for the sequence-specific detection of Vibrio cholerae DNA using magnetic beads as the biorecognition surface and gold nanoparticle-loaded latex microspheres (latex-AuNPs) as a signal-amplified hybridization tag. This biorecognition surface was prepared by immobilizing specific biotinylated capturing probes onto the streptavidin-coupled magnetic beads. Fabricating a hybridization tag capable of amplifying the electrochemical signal involved loading multiple AuNPs onto polyelectrolyte multilayer film-coated poly(styrene-co-acrylic acid) latex microspheres as carrier particles. The detection targets, single-stranded 224-bp asymmetric PCR amplicons of the V. cholerae lolB gene, were sandwich-hybridized to magnetic bead-functionalized capturing probes and fluorescein-labeled detection probes and tagged with latex-AuNPs. The subsequent electrochemical stripping analysis of chemically dissolved AuNPs loaded onto the latex microspheres allowed for the quantification of the target amplicons. The high-loading capacity of the AuNPs on the latex microspheres for sandwich-type dual-hybridization genosensing provided eminent signal amplification. The genosensing variables were optimized, and the assay specificity was demonstrated. The clinical applicability of the assay was evaluated using spiked stool specimens. The current signal responded linearly to the different V. cholerae concentrations spiked into stool specimens with a detection limit of 2 colony-forming units (CFU)/ml. The proposed latex-AuNP-based magnetogenosensing platform is promising, exhibits an effective amplification performance, and offers new opportunities for the ultrasensitive detection of other microbial pathogens.

Quantifying circular RNA expression from RNA-seq data using model-based framework.

PubMed

Li, Musheng; Xie, Xueying; Zhou, Jing; Sheng, Mengying; Yin, Xiaofeng; Ko, Eun-A; Zhou, Tong; Gu, Wanjun

2017-07-15

Circular RNAs (circRNAs) are a class of non-coding RNAs that are widely expressed in various cell lines and tissues of many organisms. Although the exact function of many circRNAs is largely unknown, the cell type-and tissue-specific circRNA expression has implicated their crucial functions in many biological processes. Hence, the quantification of circRNA expression from high-throughput RNA-seq data is becoming important to ascertain. Although many model-based methods have been developed to quantify linear RNA expression from RNA-seq data, these methods are not applicable to circRNA quantification. Here, we proposed a novel strategy that transforms circular transcripts to pseudo-linear transcripts and estimates the expression values of both circular and linear transcripts using an existing model-based algorithm, Sailfish. The new strategy can accurately estimate transcript expression of both linear and circular transcripts from RNA-seq data. Several factors, such as gene length, amount of expression and the ratio of circular to linear transcripts, had impacts on quantification performance of circular transcripts. In comparison to count-based tools, the new computational framework had superior performance in estimating the amount of circRNA expression from both simulated and real ribosomal RNA-depleted (rRNA-depleted) RNA-seq datasets. On the other hand, the consideration of circular transcripts in expression quantification from rRNA-depleted RNA-seq data showed substantial increased accuracy of linear transcript expression. Our proposed strategy was implemented in a program named Sailfish-cir. Sailfish-cir is freely available at https://github.com/zerodel/Sailfish-cir . tongz@medicine.nevada.edu or wanjun.gu@gmail.com. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway.

PubMed

Yi, Lian; Shi, Tujin; Gritsenko, Marina A; X'avia Chan, Chi-Yuet; Fillmore, Thomas L; Hess, Becky M; Swensen, Adam C; Liu, Tao; Smith, Richard D; Wiley, H Steven; Qian, Wei-Jun

2018-04-17

Large-scale phosphoproteomics with coverage of over 10,000 sites of phosphorylation have now been routinely achieved with advanced mass spectrometry (MS)-based workflows. However, accurate targeted MS-based quantification of phosphorylation dynamics, an important direction for gaining quantitative understanding of signaling pathways or networks, has been much less investigated. Herein, we report an assessment of the targeted workflow in the context of signal transduction pathways, using the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway as our model. A total of 43 phosphopeptides from the EGFR-MAPK pathway were selected for the study. The recovery and sensitivity of two commonly used enrichment methods, immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO 2 ), combined with selected reaction monitoring (SRM)-MS were evaluated. The recovery of phosphopeptides by IMAC and TiO 2 enrichment was quantified to be 38 ± 5% and 58 ± 20%, respectively, based on internal standards. Moreover, both enrichment methods provided comparable sensitivity from 1 to 100 μg starting peptides. Robust quantification was consistently achieved for most targeted phosphopeptides when starting with 25-100 μg peptides. However, the numbers of quantified targets significantly dropped when peptide samples were in the 1-25 μg range. Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK pathway in Hs578T cells following 10 ng/mL EGF treatment. The kinetics of phosphorylation clearly revealed early and late phases of phosphorylation, even for very low abundance proteins. These results demonstrate the feasibility of robust targeted quantification of phosphorylation dynamics for specific pathways, even starting with relatively small amounts of protein.

Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway

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

Yi, Lian; Shi, Tujin; Gritsenko, Marina A.

2018-03-27

Large-scale phosphoproteomics with coverage of over 10,000 sites of phosphorylation have now been routinely achieved with advanced mass spectrometry (MS)-based workflows. However, accurate targeted MS-based quantification of phosphorylation dynamics, an important direction for gaining quantitative understanding of signaling pathways or networks, has been much less investigated. Herein, we report an assessment of the targeted workflow in the context of signal transduction pathways, using the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) pathway as our model. 43 phosphopeptides from the EGFR–MAPK pathway were selected for the study. The recovery and sensitivity of a workflow consisted of two commonly used enrichmentmore » methods, immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO2), combined with selected reaction monitoring (SRM)-MS, were evaluated. The recovery of phosphopeptides by IMAC and TiO2 enrichment was quantified to be 38 ± 5% and 58 ± 20%, respectively, based on internal standards. Moreover, both enrichment methods provided comparable sensitivity from 1-100 g starting peptides. Robust quantification was consistently achieved for most targeted phosphopeptides when starting with 25-100 g peptides. However, the numbers of quantified targets significantly dropped when peptide samples were in the 1-25g range. Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK pathway in Hs578T cells following 3 ng/mL EGF treatment. The kinetics of phosphorylation clearly revealed early and late phases of phosphorylation, even for very low abundance proteins. These results demonstrate the feasibility of robust targeted quantification of phosphorylation dynamics for specific pathways, even starting with relatively small amounts of protein.« less

Markers of anthropogenic contamination: A validated method for quantification of pharmaceuticals, illicit drug metabolites, perfluorinated compounds, and plasticisers in sewage treatment effluent and rain runoff.

PubMed

Wilkinson, John L; Swinden, Julian; Hooda, Peter S; Barker, James; Barton, Stephen

2016-09-01

An effective, specific and accurate method is presented for the quantification of 13 markers of anthropogenic contaminants in water using solid phase extraction (SPE) followed by high performance liquid chromatography (HPLC) tandem mass spectrometry (MS/MS). Validation was conducted according to the International Conference on Harmonisation (ICH) guidelines. Method recoveries ranged from 77 to 114% and limits of quantification between 0.75 and 4.91 ng/L. A study was undertaken to quantify the concentrations and loadings of the selected contaminants in 6 sewage treatment works (STW) effluent discharges as well as concentrations in 5 rain-driven street runoffs and field drainages. Detection frequencies in STW effluent ranged from 25% (ethinylestradiol) to 100% (benzoylecgonine, bisphenol-A (BPA), bisphenol-S (BPS) and diclofenac). Average concentrations of detected compounds in STW effluents ranged from 3.62 ng/L (ethinylestradiol) to 210 ng/L (BPA). Levels of perfluorinated compounds (PFCs) perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA) as well as the plasticiser BPA were found in street runoff at maximum levels of 1160 ng/L, 647 ng/L and 2405 ng/L respectively (8.52, 3.09 and 2.7 times more concentrated than maximum levels in STW effluents respectively). Rain-driven street runoff may have an effect on levels of PFCs and plasticisers in receiving rivers and should be further investigated. Together, this method with the 13 selected contaminants enables the quantification of various markers of anthropogenic pollutants: inter alia pharmaceuticals, illicit drugs and their metabolites from humans and improper disposal of drugs, while the plasticisers and perfluorinated compounds may also indicate contamination from industrial and transport activity (street runoff). Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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

Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch; Gallati, Sabina, E-mail: sabina.gallati@insel.ch; Schaller, Andre, E-mail: andre.schaller@insel.ch

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 serialmore » 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 degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.« less

PicoGreen dye as an active medium for plastic lasers

NASA Astrophysics Data System (ADS)

Pradeep, C.; Vallabhan, C. P. G.; Radhakrishnan, P.; Nampoori, V. P. N.

2015-08-01

Deoxyribonucleic acid lipid complex thin films are used as a host material for laser dyes. We tested PicoGreen dye, which is commonly used for the quantification of single and double stranded DNA, for its applicability as lasing medium. PicoGreen dye exhibits enhanced fluorescence on intercalation with DNA. This enormous fluorescence emission is amplified in a planar microcavity to achieve yellow lasing. Here the role of DNA is not only a host medium, but also as a fluorescence dequencher. With the obtained results we have ample reasons to propose PicoGreen dye as a lasing medium, which can lead to the development of DNA based bio-lasers.

Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork

PubMed Central

Schauer, Grant D.; O’Donnell, Michael E.

2017-01-01

The eukaryotic genome is primarily replicated by two DNA polymerases, Pol ε and Pol δ, that function on the leading and lagging strands, respectively. Previous studies have established recruitment mechanisms whereby Cdc45-Mcm2-7-GINS (CMG) helicase binds Pol ε and tethers it to the leading strand, and PCNA (proliferating cell nuclear antigen) binds tightly to Pol δ and recruits it to the lagging strand. The current report identifies quality control mechanisms that exclude the improper polymerase from a particular strand. We find that the replication factor C (RFC) clamp loader specifically inhibits Pol ε on the lagging strand, and CMG protects Pol ε against RFC inhibition on the leading strand. Previous studies show that Pol δ is slow and distributive with CMG on the leading strand. However, Saccharomyces cerevisiae Pol δ–PCNA is a rapid and processive enzyme, suggesting that CMG may bind and alter Pol δ activity or position it on the lagging strand. Measurements of polymerase binding to CMG demonstrate Pol ε binds CMG with a Kd value of 12 nM, but Pol δ binding CMG is undetectable. Pol δ, like bacterial replicases, undergoes collision release upon completing replication, and we propose Pol δ–PCNA collides with the slower CMG, and in the absence of a stabilizing Pol δ–CMG interaction, the collision release process is triggered, ejecting Pol δ on the leading strand. Hence, by eviction of incorrect polymerases at the fork, the clamp machinery directs quality control on the lagging strand and CMG enforces quality control on the leading strand. PMID:28069954

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

PubMed

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.

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

PubMed Central

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

2014-01-01

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. PMID:25313033

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

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

Venema, J.; van Hoffen, A.; Karcagi, V.

1991-08-01

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{prime} 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% dimersmore » 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{prime} 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.« less

Biotransformation of lignan glycoside to its aglycone by Woodfordia fruticosa flowers: quantification of compounds using a validated HPTLC method.

PubMed

Mishra, Shikha; Aeri, Vidhu

2017-12-01

Saraca asoca Linn. (Caesalpiniaceae) is an important traditional remedy for gynaecological disorders and it contains lyoniside, an aryl tetralin lignan glycoside. The aglycone of lyoniside, lyoniresinol possesses structural similarity to enterolignan precursors which are established phytoestrogens. This work illustrates biotransformation of lyoniside to lyoniresinol using Woodfordia fruticosa Kurz. (Lythraceae) flowers and simultaneous quantification of lyoniside and lyoniresinol using a validated HPTLC method. The aqueous extract prepared from S. asoca bark was fermented using W. fruticosa flowers. The substrate and fermented product both were simultaneously analyzed using solvent system:toluene:ethyl acetate:formic acid (4:3:0.4) at 254 nm. The method was validated for specificity, accuracy, precision, linearity, sensitivity and robustness as per ICH guidelines. The substrate showed the presence of lyoniside, however, it decreased as the fermentation proceeded. On 3rd day, lyoniresinol starts appearing in the medium. In 8 days duration most of the lyoniside converted to lyoniresinol. The developed method was specific for lyoniside and lyoniresinol. Lyoniside and lyoniresinol showed linearity in the range of 250-3000 and 500-2500 ng. The method was accurate as resulted in 99.84% and 99.83% recovery, respectively, for lyoniside and lyoniresinol. Aryl tetralin lignan glycoside, lyoniside was successfully transformed into lyoniresinol using W. fruticosa flowers and their contents were simultaneously analyzed using developed validated HPTLC method.

Cutaneous T cell lymphoma: Current practices in blood assessment and the utility of T-cell receptor Vβ chain restriction

PubMed Central

Gibson, Juliet F; Huang, Jing; Liu, Kristina J; Carlson, Kacie R; Foss, Francine; Choi, Jaehyuk; Edelson, Richard; Hussong, Jerry W.; Mohl, Ramsey; Hill, Sally; Girardi, Sally

2016-01-01

Background Accurate quantification of malignant cells in the peripheral blood of patients with cutaneous T cell lymphoma (CTCL) is important for early detection, prognosis, and monitoring disease burden. Objective Determine the spectrum of current clinical practices; critically evaluate elements of current ISCL B1 and B2 staging criteria; and assess the potential role of TCR-Vβ analysis by flow cytometry. Methods We assessed current clinical practices by survey, and performed a retrospective analysis of 161 patients evaluated at Yale (2011-2014) to compare the sensitivity, specificity, PPV, and NPV of parameters for ISCL B2 staging. Results There was heterogeneity in clinical practices among institutions. ISCL B1 criteria did not capture five Yale cohort patients with immunophenotypic abnormalities who later progressed. TCR-Vβ testing was more specific than PCR and aided diagnosis in detecting clonality, but was of limited benefit in quantification of tumor burden. Limitations Because of limited follow-up involving a single center, further investigation will be necessary to conclude whether our proposed diagnostic algorithm is of general clinical benefit. Conclusion We propose further study of “modified B1 criteria”: CD4/CD8 ratio ≥5, %CD4+/CD26- ≥ 20%, %CD4+/CD7- ≥ 20%, with evidence of clonality. TCR-Vβ testing should be considered in future diagnostic and staging algorithms. PMID:26874819

A Simple and Specific Stability- Indicating RP-HPLC Method for Routine Assay of Adefovir Dipivoxil in Bulk and Tablet Dosage Form.

PubMed

Darsazan, Bahar; Shafaati, Alireza; Mortazavi, Seyed Alireza; Zarghi, Afshin

2017-01-01

A simple and reliable stability-indicating RP-HPLC method was developed and validated for analysis of adefovir dipivoxil (ADV).The chromatographic separation was performed on a C 18 column using a mixture of acetonitrile-citrate buffer (10 mM at pH 5.2) 36:64 (%v/v) as mobile phase, at a flow rate of 1.5 mL/min. Detection was carried out at 260 nm and a sharp peak was obtained for ADV at a retention time of 5.8 ± 0.01 min. No interferences were observed from its stress degradation products. The method was validated according to the international guidelines. Linear regression analysis of data for the calibration plot showed a linear relationship between peak area and concentration over the range of 0.5-16 μg/mL; the regression coefficient was 0.9999and the linear regression equation was y = 24844x-2941.3. The detection (LOD) and quantification (LOQ) limits were 0.12 and 0.35 μg/mL, respectively. The results proved the method was fast (analysis time less than 7 min), precise, reproducible, and accurate for analysis of ADV over a wide range of concentration. The proposed specific method was used for routine quantification of ADV in pharmaceutical bulk and a tablet dosage form.

The unfolding mechanism of monomeric mutant SOD1 by simulated force spectroscopy.

PubMed

Habibi, Mona; Rottler, Jörg; Plotkin, Steven S

2017-11-01

Mechanical unfolding of mutated apo, disulfide-reduced, monomeric superoxide dismutase 1 protein (SOD1) has been simulated via force spectroscopy techniques, using both an all-atom (AA), explicit solvent model and a coarse-grained heavy-atom Gō (HA-Gō) model. The HA-Gō model was implemented at two different pulling speeds for comparison. The most-common sequence of unfolding in the AA model agrees well with the most-common unfolding sequence of the HA-Gō model, when the same normalized pulling rate was used. Clustering of partially-native structures as the protein unfolds shows that the AA and HA-Gō models both exhibit a dominant pathway for early unfolding, which eventually bifurcates repeatedly to multiple branches after the protein is about half-unfolded. The force-extension curve exhibits multiple force drops, which are concomitant with jumps in the local interaction potential energy between specific β-strands in the protein. These sudden jumps in the potential energy coincide with the dissociation of specific pairs of β-strands, and thus intermediate unfolding events. The most common sequence of β-strand dissociation in the unfolding pathway of the AA model is β-strands 5, 4, 8, 7, 1, 2, then finally β-strands 3 and 6. The observation that β-strand 5 is among the first to unfold here, but the last to unfold in simulations of loop-truncated SOD1, could imply the existence of an evolutionary compensation mechanism, which would stabilize β-strands flanking long loops against their entropic penalty by strengthening intramolecular interactions. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Feasibility and accuracy of dual-layer spectral detector computed tomography for quantification of gadolinium: a phantom study.

PubMed

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 < 0.001; ICCs > 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.

Phosphorylation-specific status of RNAi triggers in pharmacokinetic and biodistribution analyses

PubMed Central

Trubetskoy, Vladimir S.; Griffin, Jacob B.; Nicholas, Anthony L.; Nord, Eric M.; Xu, Zhao; Peterson, Ryan M.; Wooddell, Christine I.; Rozema, David B.; Wakefield, Darren H.; Lewis, David L.

2017-01-01

Abstract The RNA interference (RNAi)-based therapeutic ARC-520 for chronic hepatitis B virus (HBV) infection consists of a melittin-derived peptide conjugated to N-acetylgalactosamine for hepatocyte targeting and endosomal escape, and cholesterol-conjugated RNAi triggers, which together result in HBV gene silencing. To characterize the kinetics of RNAi trigger delivery and 5΄-phosphorylation of guide strands correlating with gene knockdown, we employed a peptide-nucleic acid (PNA) hybridization assay. A fluorescent sense strand PNA probe binding to RNAi duplex guide strands was coupled with anion exchange high performance liquid chromatography to quantitate guide strands and metabolites. Compared to PCR- or ELISA-based methods, this assay enables separate quantitation of non-phosphorylated full-length guide strands from 5΄-phosphorylated forms that may associate with RNA-induced silencing complexes (RISC). Biodistribution studies in mice indicated that ARC-520 guide strands predominantly accumulated in liver. 5΄-phosphorylation of guide strands was observed within 5 min after ARC-520 injection, and was detected for at least 4 weeks corresponding to the duration of HBV mRNA silencing. Guide strands detected in RISC by AGO2 immuno-isolation represented 16% of total 5΄-phosphorylated guide strands in liver, correlating with a 2.7 log10 reduction of HBsAg. The PNA method enables pharmacokinetic analysis of RNAi triggers, elucidates potential metabolic processing events and defines pharmacokinetic-pharmacodynamic relationships. PMID:28180327

Frequency domain near-infrared multiwavelength imager design using high-speed, direct analog-to-digital conversion

NASA Astrophysics Data System (ADS)

Zimmermann, Bernhard B.; Fang, Qianqian; Boas, David A.; Carp, Stefan A.

2016-01-01

Frequency domain near-infrared spectroscopy (FD-NIRS) has proven to be a reliable method for quantification of tissue absolute optical properties. We present a full-sampling direct analog-to-digital conversion FD-NIR imager. While we developed this instrument with a focus on high-speed optical breast tomographic imaging, the proposed design is suitable for a wide-range of biophotonic applications where fast, accurate quantification of absolute optical properties is needed. Simultaneous dual wavelength operation at 685 and 830 nm is achieved by concurrent 67.5 and 75 MHz frequency modulation of each laser source, respectively, followed by digitization using a high-speed (180 MS/s) 16-bit A/D converter and hybrid FPGA-assisted demodulation. The instrument supports 25 source locations and features 20 concurrently operating detectors. The noise floor of the instrument was measured at <1.4 pW/√Hz, and a dynamic range of 115+ dB, corresponding to nearly six orders of magnitude, has been demonstrated. Titration experiments consisting of 200 different absorption and scattering values were conducted to demonstrate accurate optical property quantification over the entire range of physiologically expected values.

Frequency domain near-infrared multiwavelength imager design using high-speed, direct analog-to-digital conversion

PubMed Central

Zimmermann, Bernhard B.; Fang, Qianqian; Boas, David A.; Carp, Stefan A.

2016-01-01

Abstract. Frequency domain near-infrared spectroscopy (FD-NIRS) has proven to be a reliable method for quantification of tissue absolute optical properties. We present a full-sampling direct analog-to-digital conversion FD-NIR imager. While we developed this instrument with a focus on high-speed optical breast tomographic imaging, the proposed design is suitable for a wide-range of biophotonic applications where fast, accurate quantification of absolute optical properties is needed. Simultaneous dual wavelength operation at 685 and 830 nm is achieved by concurrent 67.5 and 75 MHz frequency modulation of each laser source, respectively, followed by digitization using a high-speed (180  MS/s) 16-bit A/D converter and hybrid FPGA-assisted demodulation. The instrument supports 25 source locations and features 20 concurrently operating detectors. The noise floor of the instrument was measured at <1.4  pW/√Hz, and a dynamic range of 115+ dB, corresponding to nearly six orders of magnitude, has been demonstrated. Titration experiments consisting of 200 different absorption and scattering values were conducted to demonstrate accurate optical property quantification over the entire range of physiologically expected values. PMID:26813081

Quantification of Global DNA Methylation Levels by Mass Spectrometry.

PubMed

Fernandez, Agustin F; Valledor, Luis; Vallejo, Fernando; Cañal, Maria Jesús; Fraga, Mario F

2018-01-01

Global DNA methylation was classically considered the relative percentage of 5-methylcysine (5mC) with respect to total cytosine (C). Early approaches were based on the use of high-performance separation technologies and UV detection. However, the recent development of protocols using mass spectrometry for the detection has increased sensibility and permitted the precise identification of peak compounds based on their molecular masses. This allows work to be conducted with much less genomic DNA starting material and also to quantify 5-hydroxymethyl-cytosine (5hmC), a recently identified form of methylated cytosine that could play an important role in active DNA demethylation. Here, we describe the protocol that we currently use in our laboratory to analyze 5mC and 5hmC by mass spectrometry. The protocol, which is based on the method originally developed by Le and colleagues using Ultra Performance Liquid Chromatography (UPLC) and mass spectrometry (triple Quadrupole (QqQ)) detection, allows for the rapid and accurate quantification of relative global 5mC and 5hmC levels starting from just 1 μg of genomic DNA, which allows for the rapid and accurate quantification of relative global 5mC and 5hmC levels.

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

PubMed

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.

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

PubMed

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.

Avian-specific real-time PCR assay for authenticity control in farm animal feeds and pet foods.

PubMed

Pegels, Nicolette; González, Isabel; García, Teresa; Martín, Rosario

2014-01-01

A highly sensitive TaqMan real-time PCR assay targeting the mitochondrial 12S rRNA gene was developed for detection of an avian-specific DNA fragment (68bp) in farm animal and pet feeds. The specificity of the assay was verified against a wide representation of animal and plant species. Applicability assessment of the avian real-time PCR was conducted through representative analysis of two types of compound feeds: industrial farm animal feeds (n=60) subjected to extreme temperatures, and commercial dog and cat feeds (n=210). Results obtained demonstrated the suitability of the real-time PCR assay to detect the presence of low percentages of highly processed avian material in the feed samples analysed. Although quantification results were well reproducible under the experimental conditions tested, an accurate estimation of the target content in feeds is impossible in practice. Nevertheless, the method may be useful as an alternative tool for traceability purposes within the framework of feed control. Copyright © 2013 Elsevier Ltd. All rights reserved.

Systematic development of small molecules to inhibit specific microscopic steps of Aβ42 aggregation in Alzheimer’s disease

PubMed Central

Habchi, Johnny; Chia, Sean; Limbocker, Ryan; Mannini, Benedetta; Ahn, Minkoo; Perni, Michele; Hansson, Oskar; Arosio, Paolo; Kumita, Janet R.; Challa, Pavan Kumar; Cohen, Samuel I. A.; Dobson, Christopher M.; Knowles, Tuomas P. J.; Vendruscolo, Michele

2017-01-01

The aggregation of the 42-residue form of the amyloid-β peptide (Aβ42) is a pivotal event in Alzheimer’s disease (AD). The use of chemical kinetics has recently enabled highly accurate quantifications of the effects of small molecules on specific microscopic steps in Aβ42 aggregation. Here, we exploit this approach to develop a rational drug discovery strategy against Aβ42 aggregation that uses as a read-out the changes in the nucleation and elongation rate constants caused by candidate small molecules. We thus identify a pool of compounds that target specific microscopic steps in Aβ42 aggregation. We then test further these small molecules in human cerebrospinal fluid and in a Caenorhabditis elegans model of AD. Our results show that this strategy represents a powerful approach to identify systematically small molecule lead compounds, thus offering an appealing opportunity to reduce the attrition problem in drug discovery. PMID:28011763

Development and Validation of an HPLC Method for Karanjin in Pongamia pinnata linn. Leaves.

PubMed

Katekhaye, S; Kale, M S; Laddha, K S

2012-01-01

A rapid, simple and specific reversed-phase HPLC method has been developed for analysis of karanjin in Pongamia pinnata Linn. leaves. HPLC analysis was performed on a C(18) column using an 85:13.5:1.5 (v/v) mixtures of methanol, water and acetic acid as isocratic mobile phase at a flow rate of 1 ml/min. UV detection was at 300 nm. The method was validated for accuracy, precision, linearity, specificity. Validation revealed the method is specific, accurate, precise, reliable and reproducible. Good linear correlation coefficients (r(2)>0.997) were obtained for calibration plots in the ranges tested. Limit of detection was 4.35 μg and limit of quantification was 16.56 μg. Intra and inter-day RSD of retention times and peak areas was less than 1.24% and recovery was between 95.05 and 101.05%. The established HPLC method is appropriate enabling efficient quantitative analysis of karanjin in Pongamia pinnata leaves.

Development and Validation of an HPLC Method for Karanjin in Pongamia pinnata linn. Leaves

PubMed Central

Katekhaye, S; Kale, M. S.; Laddha, K. S.

2012-01-01

A rapid, simple and specific reversed-phase HPLC method has been developed for analysis of karanjin in Pongamia pinnata Linn. leaves. HPLC analysis was performed on a C18 column using an 85:13.5:1.5 (v/v) mixtures of methanol, water and acetic acid as isocratic mobile phase at a flow rate of 1 ml/min. UV detection was at 300 nm. The method was validated for accuracy, precision, linearity, specificity. Validation revealed the method is specific, accurate, precise, reliable and reproducible. Good linear correlation coefficients (r2>0.997) were obtained for calibration plots in the ranges tested. Limit of detection was 4.35 μg and limit of quantification was 16.56 μg. Intra and inter-day RSD of retention times and peak areas was less than 1.24% and recovery was between 95.05 and 101.05%. The established HPLC method is appropriate enabling efficient quantitative analysis of karanjin in Pongamia pinnata leaves. PMID:23204626

TU-AB-201-11: A Novel Theoretical Framework for MRI-Only Image Guided LDR Prostate and Breast Brachytherapy Implant Dosimetry

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

Soliman, A; Elzibak, A; Fatemi, A

Purpose: To propose a novel framework for accurate model-based dose calculations using only MR images for LDR prostate and breast seed implant brachytherapy. Methods: Model-based dose calculation methodologies recommended by TG-186 require further knowledge about specific tissue composition, which is challenging with MRI. However, relying on MRI-only for implant dosimetry would reduce the soft tissue delineation uncertainty, costs, and uncertainties associated with multi-modality registration and fusion processes. We propose a novel framework to address this problem using quantitative MRI acquisitions and reconstruction techniques. The framework includes three steps: (1) Identify the locations of seeds(2) Identify the presence (or absence) ofmore » calcification(s)(3) Quantify the water and fat content in the underlying tissueSteps (1) and (2) consider the sources that limit patient dosimetry, particularly the inter-seed attenuation and the calcified regions; while step (3) targets the quantification of the tissue composition to consider the heterogeneities in the medium. Our preliminary work has shown that the seeds and the calcifications can be identified with MRI using both the magnitude and the phase images. By employing susceptibility-weighted imaging with specific post-processing techniques, the phase images can be further explored to distinguish the seeds from the calcifications. Absolute quantification of tissue, water, and fat content is feasible and was previously demonstrated in phantoms and in-vivo applications, particularly for brain diseases. The approach relies on the proportionality of the MR signal to the number of protons in an image volume. By employing appropriate correction algorithms for T1 - and T2*-related biases, B1 transmit and receive field inhomogeneities, absolute water/fat content can be determined. Results: By considering calcification and interseed attenuation, and through the knowledge of water and fat mass density, accurate patient-specific implant dosimetry can be achieved with MRI-only. Conclusion: The proposed framework showed that model-based dose calculation is feasible using MRI-only state-of-the-art techniques.« less

Microstructural Quantification, Property Prediction, and Stochastic Reconstruction of Heterogeneous Materials Using Limited X-Ray Tomography Data

NASA Astrophysics Data System (ADS)

Li, Hechao

An accurate knowledge of the complex microstructure of a heterogeneous material is crucial for quantitative structure-property relations establishment and its performance prediction and optimization. X-ray tomography has provided a non-destructive means for microstructure characterization in both 3D and 4D (i.e., structural evolution over time). Traditional reconstruction algorithms like filtered-back-projection (FBP) method or algebraic reconstruction techniques (ART) require huge number of tomographic projections and segmentation process before conducting microstructural quantification. This can be quite time consuming and computationally intensive. In this thesis, a novel procedure is first presented that allows one to directly extract key structural information in forms of spatial correlation functions from limited x-ray tomography data. The key component of the procedure is the computation of a "probability map", which provides the probability of an arbitrary point in the material system belonging to specific phase. The correlation functions of interest are then readily computed from the probability map. Using effective medium theory, accurate predictions of physical properties (e.g., elastic moduli) can be obtained. Secondly, a stochastic optimization procedure that enables one to accurately reconstruct material microstructure from a small number of x-ray tomographic projections (e.g., 20 - 40) is presented. Moreover, a stochastic procedure for multi-modal data fusion is proposed, where both X-ray projections and correlation functions computed from limited 2D optical images are fused to accurately reconstruct complex heterogeneous materials in 3D. This multi-modal reconstruction algorithm is proved to be able to integrate the complementary data to perform an excellent optimization procedure, which indicates its high efficiency in using limited structural information. Finally, the accuracy of the stochastic reconstruction procedure using limited X-ray projection data is ascertained by analyzing the microstructural degeneracy and the roughness of energy landscape associated with different number of projections. Ground-state degeneracy of a microstructure is found to decrease with increasing number of projections, which indicates a higher probability that the reconstructed configurations match the actual microstructure. The roughness of energy landscape can also provide information about the complexity and convergence behavior of the reconstruction for given microstructures and projection number.

An accurate proteomic quantification method: fluorescence labeling absolute quantification (FLAQ) using multidimensional liquid chromatography and tandem mass spectrometry.

PubMed

Liu, Junyan; Liu, Yang; Gao, Mingxia; Zhang, Xiangmin

2012-08-01

A facile proteomic quantification method, fluorescent labeling absolute quantification (FLAQ), was developed. Instead of using MS for quantification, the FLAQ method is a chromatography-based quantification in combination with MS for identification. Multidimensional liquid chromatography (MDLC) with laser-induced fluorescence (LIF) detection with high accuracy and tandem MS system were employed for FLAQ. Several requirements should be met for fluorescent labeling in MS identification: Labeling completeness, minimum side-reactions, simple MS spectra, and no extra tandem MS fragmentations for structure elucidations. A fluorescence dye, 5-iodoacetamidofluorescein, was finally chosen to label proteins on all cysteine residues. The fluorescent dye was compatible with the process of the trypsin digestion and MALDI MS identification. Quantitative labeling was achieved with optimization of reacting conditions. A synthesized peptide and model proteins, BSA (35 cysteines), OVA (five cysteines), were used for verifying the completeness of labeling. Proteins were separated through MDLC and quantified based on fluorescent intensities, followed by MS identification. High accuracy (RSD% < 1.58) and wide linearity of quantification (1-10(5) ) were achieved by LIF detection. The limit of quantitation for the model protein was as low as 0.34 amol. Parts of proteins in human liver proteome were quantified and demonstrated using FLAQ. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fibrin-Targeted Magnetic Resonance Imaging Allows In Vivo Quantification of Thrombus Fibrin Content and Identifies Thrombi Amenable for Thrombolysis

PubMed Central

Jenkins, Julia; Modarai, Bijan; Wiethoff, Andrea J.; Phinikaridou, Alkystis; Grover, Steven P.; Patel, Ashish S.; Schaeffter, Tobias; Smith, Alberto; Botnar, Rene M.

2014-01-01

Objective Deep venous thrombosis is a major health problem. Thrombolytic therapies are effective in recanalizing the veins and preventing post-thrombotic complications, but there is no consensus on selection criteria. The aim of this study was to investigate a fibrin-specific MRI contrast agent (EP-2104R) for the accurate quantification of thrombus’ fibrin content in vivo and for the identification of thrombus suitable for thrombolysis. Approach and Results Venous thrombosis was induced in the inferior vena cava of 8- to 10-week-old male BALB/C mice and MRI performed 2, 4, 7, 10, 14, and 21 days later. Eighteen mice were scanned at each time point pre and 2 hours post injection of EP-2104R (8.0 μmol/kg) with 12 mice at each time point used to correlate fibrin contrast uptake with thrombus’ histological stage and fibrin content. Six mice at each time point were immediately subjected to intravascular thrombolytic therapy (10 mg/kg of tissue-type plasminogen activator). Mice were imaged to assess response to lytic therapy 24 hours after thrombolytic treatment. Two mice at each time point were scanned post injection of 0.2 mmol/kg of Gd-DTPA (gadolinium with diethylenetriaminepentacetate, Magnevist, Schering AG, Berlin, Germany) for control purpose. Contrast uptake was correlated positively with the fibrin content of the thrombus measured by Western blotting (R2=0.889; P<0.001). Thrombus relaxation rate (R1) post contrast and the change in visualized thrombus size on late gadolinium enhancement inversion recovery MRI pre–EP-2104R and post–EP-2104R injection were the best predictors for successful thrombolysis (area under the curve, 0.989 [95% confidence interval, 0.97–1.00] and 0.994 [95% confidence interval, 0.98–1.00] respectively). Conclusions MRI with a fibrin-specific contrast agent accurately estimates thrombus fibrin content in vivo and identifies thrombi that are amenable for thrombolysis. PMID:24723557

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

PubMed Central

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

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

PubMed

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.

In vivo molecular photoacoustic tomography of melanomas targeted by bio-conjugated gold nanocages

PubMed Central

Kim, Chulhong; Cho, Eun Chul; Chen, Jingyi; Song, Kwang Hyun; Au, Leslie; Favazza, Christopher; Zhang, Qiang; Cobley, Claire M.; Gao, Feng; Xia, Younan; Wang, Lihong V.

2010-01-01

Early diagnosis, accurate staging, and image-guided resection of melanomas remain crucial clinical objectives for improving patient survival and treatment outcomes. Conventional techniques cannot meet this demand because of the low sensitivity, low specificity, poor spatial resolution, shallow penetration, and/or ionizing radiation. Here we overcome such limitations by combining high-resolution photoacoustic tomography (PAT) with extraordinarily optical absorbing gold nanocages (AuNCs). When bio-conjugated with [Nle4,D-Phe7]-α-melanocyte-stimulating hormone, the AuNCs can serve as a novel contrast agent for in vivo molecular PAT of melanomas with both exquisite sensitivity and high specificity. The bio-conjugated AuNCs enhanced contrast ~300% more than the control, PEGylated AuNCs. The in vivo PAT quantification of the amount of AuNCs accumulated in melanomas was further validated with inductively coupled plasma mass spectrometry (ICP-MS). PMID:20731439

Novel rolling circle amplification and DNA origami-based DNA belt-involved signal amplification assay for highly sensitive detection of prostate-specific antigen (PSA).

PubMed

Yan, Juan; Hu, Chongya; Wang, Ping; Liu, Rui; Zuo, Xiaolei; Liu, Xunwei; Song, Shiping; Fan, Chunhai; He, Dannong; Sun, Gang

2014-11-26

Prostate-specific antigen (PSA) is one of the most important biomarkers for the early diagnosis and prognosis of prostate cancer. Although many efforts have been made to achieve significant progress for the detection of PSA, challenges including relative low sensitivity, complicated operation, sophisticated instruments, and high cost remain unsolved. Here, we have developed a strategy combining rolling circle amplification (RCA)-based DNA belts and magnetic bead-based enzyme-linked immunosorbent assay (ELISA) for the highly sensitive and specific detection of PSA. At first, a 96-base circular DNA template was designed and prepared for the following RCA. Single stranded DNA (ssDNA) products from RCA were used as scaffold strand for DNA origami, which was hybridized with three staple strands of DNA. The resulting DNA belts were conjugated with multiple enzymes for signal amplification and then employed to magnetic bead based ELISA for PSA detection. Through our strategy, as low as 50 aM of PSA can be detected with excellent specificity.

Surface shapes and surrounding environment analysis of single- and double-stranded DNA-binding proteins in protein-DNA interface.

PubMed

Wang, Wei; Liu, Juan; Sun, Lin

2016-07-01

Protein-DNA bindings are critical to many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the residues shape (peak, flat, or valley) and the surrounding environment of double-stranded DNA-binding proteins (DSBs) and single-stranded DNA-binding proteins (SSBs) in protein-DNA interfaces. In the results, we found that the interface shapes, hydrogen bonds, and the surrounding environment present significant differences between the two kinds of proteins. Built on the investigation results, we constructed a random forest (RF) classifier to distinguish DSBs and SSBs with satisfying performance. In conclusion, we present a novel methodology to characterize protein interfaces, which will deepen our understanding of the specificity of proteins binding to ssDNA (single-stranded DNA) or dsDNA (double-stranded DNA). Proteins 2016; 84:979-989. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

DOEpatents

Gray, J.W.; Pinkel, D.

1991-07-02

A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. The probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations. No Drawings

DNA purification by triplex-affinity capture and affinity capture electrophoresis

DOEpatents

Cantor, C.R.; Ito, Takashi; Smith, C.L.

1996-01-09

The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel. 6 figs.

Specific functions of the Rep and Rep׳ proteins of porcine circovirus during copy-release and rolling-circle DNA replication.

PubMed

Cheung, Andrew K

2015-07-01

The roles of two porcine circovirus replication initiator proteins, Rep and Rep׳, in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replication) and generates the single-stranded circular (ssc) genome from the displaced DNA strand. In the process, a minus-genome primer (MGP) necessary for complementary-strand synthesis, from ssc to ccc, is synthesized. Rep׳ cleaves the growing nascent-strand to regenerate the parent ccc molecule. In the process, a Rep׳-DNA hybrid containing the right palindromic sequence (at the origin of DNA replication) is generated. Analysis of the virus particle showed that it is composed of four components: ssc, MGP, capsid protein and a novel Rep-related protein (designated Protein-3). Copyright © 2015. Published by Elsevier Inc.

Single DNA imaging and length quantification through a mobile phone microscope

NASA Astrophysics Data System (ADS)

Wei, Qingshan; Luo, Wei; Chiang, Samuel; Kappel, Tara; Mejia, Crystal; Tseng, Derek; Chan, Raymond Yan L.; Yan, Eddie; Qi, Hangfei; Shabbir, Faizan; Ozkan, Haydar; Feng, Steve; Ozcan, Aydogan

2016-03-01

The development of sensitive optical microscopy methods for the detection of single DNA molecules has become an active research area which cultivates various promising applications including point-of-care (POC) genetic testing and diagnostics. Direct visualization of individual DNA molecules usually relies on sophisticated optical microscopes that are mostly available in well-equipped laboratories. For POC DNA testing/detection, there is an increasing need for the development of new single DNA imaging and sensing methods that are field-portable, cost-effective, and accessible for diagnostic applications in resource-limited or field-settings. For this aim, we developed a mobile-phone integrated fluorescence microscopy platform that allows imaging and sizing of single DNA molecules that are stretched on a chip. This handheld device contains an opto-mechanical attachment integrated onto a smartphone camera module, which creates a high signal-to-noise ratio dark-field imaging condition by using an oblique illumination/excitation configuration. Using this device, we demonstrated imaging of individual linearly stretched λ DNA molecules (48 kilobase-pair, kbp) over 2 mm2 field-of-view. We further developed a robust computational algorithm and a smartphone app that allowed the users to quickly quantify the length of each DNA fragment imaged using this mobile interface. The cellphone based device was tested by five different DNA samples (5, 10, 20, 40, and 48 kbp), and a sizing accuracy of <1 kbp was demonstrated for DNA strands longer than 10 kbp. This mobile DNA imaging and sizing platform can be very useful for various diagnostic applications including the detection of disease-specific genes and quantification of copy-number-variations at POC settings.

Asymmetric single-strand polymorphism: an accurate and cost-effective method to amplify and sequence allelic variants

USDA-ARS?s Scientific Manuscript database

We needed to obtain an alternative to conventional cloning to generate high-quality DNA sequences from a variety of nuclear orthologs for phylogenetic studies in potato, to save time and money and to avoid problems typically encountered in cloning. We tested a variety of SSCP protocols to include pu...

Development and validation of a fast and simple multi-analyte procedure for quantification of 40 drugs relevant to emergency toxicology using GC-MS and one-point calibration.

PubMed

Meyer, Golo M J; Weber, Armin A; Maurer, Hans H

2014-05-01

Diagnosis and prognosis of poisonings should be confirmed by comprehensive screening and reliable quantification of xenobiotics, for example by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS). The turnaround time should be short enough to have an impact on clinical decisions. In emergency toxicology, quantification using full-scan acquisition is preferable because this allows screening and quantification of expected and unexpected drugs in one run. Therefore, a multi-analyte full-scan GC-MS approach was developed and validated with liquid-liquid extraction and one-point calibration for quantification of 40 drugs relevant to emergency toxicology. Validation showed that 36 drugs could be determined quickly, accurately, and reliably in the range of upper therapeutic to toxic concentrations. Daily one-point calibration with calibrators stored for up to four weeks reduced workload and turn-around time to less than 1 h. In summary, the multi-analyte approach with simple liquid-liquid extraction, GC-MS identification, and quantification over fast one-point calibration could successfully be applied to proficiency tests and real case samples. Copyright © 2013 John Wiley & Sons, Ltd.

Replication of tobacco mosaic virus. VII. Further characterization of single- and double-stranded virus-related RNAs from TMV-infected plants.

PubMed

Palukaitis, P; García-Arenal, F; Sulzinski, M A; Zaitlin, M

1983-12-01

The single-stranded (ss) and double-stranded (ds) RNAs produced in tobacco tissue as a result of infection by tobacco mosaic virus (TMV) have been reinvestigated. 32P-labeled probes consisting of either cDNA or viral RNA, complementary to specific regions of either the viral RNA or its negative strand, respectively, were used in "Northern" hybridization experiments. Of the 10 ssRNA bands observed, all but four appeared to be artifacts of electrophoresis. These four RNAs were found on polyribosomes and are presumed to be true mRNAs; three were identified as the well-known genomic RNA, the I2-mRNA and the coat protein mRNA, or LMC. The fourth RNA species of MW approximately 1.2 x 10(6) had not previously been specifically identified as a subgenomic RNA of TMV. The viral RNA which gave rise to the six artifactual ssRNA bands was heterogeneous in size and was shown to be encapsidated in vivo. Upon electrophoresis, these heterogeneous RNA fragments comigrated approximately with plant rRNAs also present in the extracts, generating the observed artifactual bands. Four dsRNAs were also identified. From molecular weight and hybridization analyses, they appeared to be double-stranded forms of the above four polyribosome-associated ssRNAs. Attempts to translate proteins from the denatured dsRNAs in vitro were unsuccessful. A population of low-molecular-weight, TMV-specific ssRNAs, (+) and (-) in sequence, was generated during infection; however these RNAs were believed to be breakdown products.

In vivo imaging of cancer cell size and cellularity using temporal diffusion spectroscopy.

PubMed

Jiang, Xiaoyu; Li, Hua; Xie, Jingping; McKinley, Eliot T; Zhao, Ping; Gore, John C; Xu, Junzhong

2017-07-01

A temporal diffusion MRI spectroscopy based approach has been developed to quantify cancer cell size and density in vivo. A novel imaging microstructural parameters using limited spectrally edited diffusion (IMPULSED) method selects a specific limited diffusion spectral window for an accurate quantification of cell sizes ranging from 10 to 20 μm in common solid tumors. In practice, it is achieved by a combination of a single long diffusion time pulsed gradient spin echo (PGSE) and three low-frequency oscillating gradient spin echo (OGSE) acquisitions. To validate our approach, hematoxylin and eosin staining and immunostaining of cell membranes, in concert with whole slide imaging, were used to visualize nuclei and cell boundaries, and hence, enabled accurate estimates of cell size and cellularity. Based on a two compartment model (incorporating intra- and extracellular spaces), accurate estimates of cell sizes were obtained in vivo for three types of human colon cancers. The IMPULSED-derived apparent cellularities showed a stronger correlation (r = 0.81; P < 0.0001) with histology-derived cellularities than conventional ADCs (r = -0.69; P < 0.03). The IMPULSED approach samples a specific region of temporal diffusion spectra with enhanced sensitivity to length scales of 10-20 μm, and enables measurements of cell sizes and cellularities in solid tumors in vivo. Magn Reson Med 78:156-164, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

Meiotic Crossover Control by Concerted Action of Rad51-Dmc1 in Homolog Template Bias and Robust Homeostatic Regulation

PubMed Central

Huang, Chu-Chun; Grubb, Jennifer; Thacker, Drew; Lee, Chih-Ying; Dresser, Michael E.; Hunter, Neil; Bishop, Douglas K.

2013-01-01

During meiosis, repair of programmed DNA double-strand breaks (DSBs) by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1's strand exchange activity, while its own exchange activity is inhibited. However, in a dmc1 mutant, elimination of inhibitory factor, Hed1, activates Rad51's strand exchange activity and results in high levels of recombination without participation of Dmc1. Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1's chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1's dominance in promoting strand exchange between homologs involves repression of Rad51's strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation in response to defective homolog interactions. Thus, robust crossover homeostasis is conferred by integrated regulation at initiation, strand-exchange and maturation steps of meiotic recombination. PMID:24367271

A Novel Real-Time PCR for Listeria monocytogenes That Monitors Analytical Performance via an Internal Amplification Control

PubMed Central

Rodríguez-Lázaro, David; Pla, Maria; Scortti, Mariela; Monzó, Héctor J.; Vázquez-Boland, José A.

2005-01-01

We describe a novel quantitative real-time (Q)-PCR assay for Listeria monocytogenes based on the coamplification of a target hly gene fragment and an internal amplification control (IAC). The IAC is a chimeric double-stranded DNA containing a fragment of the rapeseed BnACCg8 gene flanked by the hly-specific target sequences. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the hly and IAC signals. The hly-IAC assay had a specificity and sensitivity of 100%, as assessed using 49 L. monocytogenes isolates of different serotypes and 96 strains of nontarget bacteria, including 51 Listeria isolates. The detection and quantification limits were 8 and 30 genome equivalents, and the coefficients for PCR linearity (R2) and efficiency (E) were 0.997 and 0.80, respectively. We tested the performance of the hly-IAC Q-PCR assay using various broth media and food matrices. Fraser and half-Fraser media, raw pork, and raw or cold-smoked salmon were strongly PCR-inhibitory. This Q-PCR assay for L. monocytogenes, the first incorporating an IAC to be described for quantitative detection of a food-borne pathogen, is a simple and robust tool facilitating the identification of false negatives or underestimations of contamination loads due to PCR failure. PMID:16332910

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids.

PubMed

Yang, Litao; Liang, Wanqi; Jiang, Lingxi; Li, Wenquan; Cao, Wei; Wilson, Zoe A; Zhang, Dabing

2008-06-04

Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. We describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.

Event-specific real-time detection and quantification of genetically modified Roundup Ready soybean.

PubMed

Huang, Chia-Chia; Pan, Tzu-Ming

2005-05-18

The event-specific real-time detection and quantification of Roundup Ready soybean (RRS) using an ABI PRISM 7700 sequence detection system with light upon extension (LUX) primer was developed in this study. The event-specific primers were designed, targeting the junction of the RRS 5' integration site and the endogenous gene lectin1. Then, a standard reference plasmid was constructed that carried both of the targeted sequences for quantitative analysis. The detection limit of the LUX real-time PCR system was 0.05 ng of 100% RRS genomic DNA, which was equal to 20.5 copies. The range of quantification was from 0.1 to 100%. The sensitivity and range of quantification successfully met the requirement of the labeling rules in the European Union and Taiwan.

Simultaneous multiplexed quantification of nicotine and its metabolites using surface enhanced Raman scattering.

PubMed

Alharbi, Omar; Xu, Yun; Goodacre, Royston

2014-10-07

The detection and quantification of xenobiotics and their metabolites in man is important for drug dosing, therapy and for substance abuse monitoring where longer-lived metabolic products from illicit materials can be assayed after the drug of abuse has been cleared from the system. Raman spectroscopy offers unique specificity for molecular characterization and this usually weak signal can be significantly enhanced using surface enhanced Raman scattering (SERS). We report here the novel development of SERS with chemometrics for the simultaneous analysis of the drug nicotine and its major xenometabolites cotinine and trans-3'-hydroxycotinine. Initial experiments optimized the SERS conditions and we found that when these three determinands were analysed individually that the maximum SERS signals were found at three different pH. These were pH 3 for nicotine and pH 10 and 11 for cotinine and trans-3'-hydroxycotinine, respectively. Tertiary mixtures containing nicotine, cotinine and trans-3'-hydroxycotinine were generated in the concentration range 10(-7)-10(-5) M and SERS spectra were collected at all three pH values. Chemometric analysis using kernel-partial least squares (K-PLS) and artificial neural networks (ANNs) were conducted and these models were validated using bootstrap resampling. All three analytes were accurately quantified with typical root mean squared error of prediction on the test set data being 5-9%; nicotine was most accurately predicted followed by cotinine and then trans-3'-hydroxycotinine. We believe that SERS is a powerful approach for the simultaneous analysis of multiple determinands without recourse to lengthy chromatography, as demonstrated here for the xenobiotic nicotine and its two major xenometabolites.

Proceedings from the Communication Across the Curriculum Strand. NCA 2001 Summer Conference: Engaging 21st Century Communication Students.

ERIC Educational Resources Information Center

Dannels, Deanna; Jackson, Nancy; Robertson, Terry; Sheckles, Ted; Tomlinson, Stephanie

This Proceedings from the Communication across the Curriculum (CXC) strand of the National Communication Association's 2001 Summer Conference first highlights and describes five of the most common types of programs across the nation: Speaking Intensive Programs, Combined Speaking and Writing Programs, Discipline-Specific Programs, Faculty…

Characterization of Sulfolobus islandicus rod-shaped virus 2 gp19, a single-strand specific endonuclease.

PubMed

Gardner, Andrew F; Prangishvili, David; Jack, William E

2011-09-01

The hyperthermophilic Sulfolobus islandicus rod-shaped virus 2 (SIRV2) encodes a 25-kDa protein (SIRV2gp19) annotated as a hypothetical protein with sequence homology to the RecB nuclease superfamily. Even though SIRV2gp19 homologs are conserved throughout the rudivirus family and presumably play a role in the viral life cycle, SIRV2gp19 has not been functionally characterized. To define the minimal requirements for activity, SIRV2gp19 was purified and tested under varying conditions. SIRV2gp19 is a single-strand specific endonuclease that requires Mg(2+) for activity and is inactive on double-stranded DNA. A conserved aspartic acid in RecB nuclease superfamily Motif II (D89) is also essential for SIRV2gp19 activity and mutation to alanine (D89A) abolishes activity. Therefore, the SIRV2gp19 cleavage mechanism is similar to previously described RecB nucleases. Finally, SIRV2gp19 single-stranded DNA endonuclease activity could play a role in host chromosome degradation during SIRV2 lytic infection.

Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

PubMed Central

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

Efficient synthesis of supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication

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

Kodadek, T.; Gamper, H.

The authors report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, they report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. Theymore » demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.« less

A liquid chromatography-tandem mass spectrometry assay for the detection and quantification of trehalose in biological samples.

PubMed

Kretschmer, Philip M; Bannister, Austin M; O Brien, Molly K; MacManus-Spencer, Laura A; Paulick, Margot G

2016-10-15

Trehalose is an important disaccharide that is used as a cellular protectant by many different organisms, helping these organisms better survive extreme conditions, such as dehydration, oxidative stress, and freezing temperatures. Methods to detect and accurately measure trehalose from different organisms will help us gain a better understanding of the mechanisms behind trehalose's ability to act as a cellular protectant. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay using selected reaction monitoring mode for the detection and quantification of trehalose using maltose as an internal standard has been developed. This assay uses a commercially available LC column for trehalose separation and a standard triple quadrupole mass spectrometer, thus allowing many scientists to take advantage of this simple assay. The calibration curve from 3 to 100μM trehalose was fit best by a single polynomial. This LC-MS/MS assay directly detects and accurately quantifies trehalose, with an instrument limit of detection (LOD) that is 2-1000 times more sensitive than the most commonly-used assays for trehalose detection and quantification. Furthermore, this assay was used to detect and quantify endogenous trehalose produced by Escherichia coli (E. coli) cells, which were found to have an intracellular concentration of 8.5±0.9mM trehalose. This method thus shows promise for the reliable detection and quantification of trehalose from different biological sources. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a targeted method for twenty-three metabolites related to polyphenol gut microbial metabolism in biological samples, using SPE and UHPLC-ESI-MS/MS.

PubMed

Gasperotti, Mattia; Masuero, Domenico; Guella, Graziano; Mattivi, Fulvio; Vrhovsek, Urska

2014-10-01

An increasing number of studies have concerned the profiling of polyphenol microbial metabolites, especially in urine or plasma, but only a few have regarded their accurate quantification. This study reports on a new ultra-performance liquid chromatography tandem mass spectrometry method with electrospray ionisation (UHPLC-ESI-MS/MS) using a simple clean-up step with solid phase extraction (SPE) and validation on different biological matrices. The method was tested with spiked samples of liver, heart, kidneys, brain, blood and urine. The purification procedure, after the evaluation of three different cartridges, makes it possible to obtain cleaner samples and better quantification of putative trace metabolites, especially related to dietary studies, with concentrations below ng/g in tissue and for urine and blood, starting from ng/ml. Limits of detection and linear range were also assessed using mixed polyphenol metabolite standards. Short chromatographic separation was carried out for 23 target compounds related to the polyphenol microbial metabolism, coupled with a triple quadrupole mass spectrometer for their accurate quantification. By analysing different spiked biological samples we were able to test metabolite detection in the matrix and validate the overall recovery of the method, from purification to quantification. The method developed can be successfully applied and is suitable for high-throughput targeted metabolomics analysis related to nutritional intervention, or the study of the metabolic mechanism in response to a polyphenol-rich diet. Copyright © 2014 Elsevier B.V. All rights reserved.

Critical assessment of digital PCR for the detection and quantification of genetically modified organisms.

PubMed

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.

Multiscale recurrence quantification analysis of order recurrence plots

NASA Astrophysics Data System (ADS)

Xu, Mengjia; Shang, Pengjian; Lin, Aijing

2017-03-01

In this paper, we propose a new method of multiscale recurrence quantification analysis (MSRQA) to analyze the structure of order recurrence plots. The MSRQA is based on order patterns over a range of time scales. Compared with conventional recurrence quantification analysis (RQA), the MSRQA can show richer and more recognizable information on the local characteristics of diverse systems which successfully describes their recurrence properties. Both synthetic series and stock market indexes exhibit their properties of recurrence at large time scales that quite differ from those at a single time scale. Some systems present more accurate recurrence patterns under large time scales. It demonstrates that the new approach is effective for distinguishing three similar stock market systems and showing some inherent differences.

Quantification of SAA1 and SAA2 in lung cancer plasma using the isotype-specific PRM assays.

PubMed

Kim, Yeoun Jin; Gallien, Sebastien; El-Khoury, Victoria; Goswami, Panchali; Sertamo, Katriina; Schlesser, Marc; Berchem, Guy; Domon, Bruno

2015-09-01

The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based parallel reaction monitoring (PRM) method provides an immediate benefit over the conventional SRM-based method in terms of selectivity. In this study, multiplexed PRM assays were developed to analyze isotypes of serum amyloid A (SAA) proteins in human plasma with a focus on SAA1 and SAA2. Elevated plasma levels of these proteins in patients diagnosed with lung cancer have been reported in previous studies. Since SAA1 and SAA2 are highly homologous, the available immunoassays tend to overestimate their concentrations due to cross-reactivity. On the other hand, when mass spectrometry (MS)-based assays are used, the presence of the several allelic variants may result in a problem of underestimation. In the present study, eight peptides that represent the target proteins at three different levels: isotype-specific (SAA1α,  SAA 1β,  SAA1γ,  SAA2α,  SAA2β), protein-specific (SAA1 or SAA2), and pan SAA (SAA1 and SAA2) were chosen to differentiate SAAs in lung cancer plasma samples using a panel of PRM assays. The measurement of specific isotypes, leveraging the analytical performance of PRM, allowed to quantify the allelic variants of both target proteins. The isotypes detected were corroborated with the genetic information obtained from the same samples. The combination of SAA2α and SAA2β assays representing the total SAA2 concentration demonstrated a superior analytical outcome than the previously used assay on the common peptide when applied to the detection of lung cancer. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Zero-Echo-Time and Dixon Deep Pseudo-CT (ZeDD CT): Direct Generation of Pseudo-CT Images for Pelvic PET/MRI Attenuation Correction Using Deep Convolutional Neural Networks with Multiparametric MRI.

PubMed

Leynes, Andrew P; Yang, Jaewon; Wiesinger, Florian; Kaushik, Sandeep S; Shanbhag, Dattesh D; Seo, Youngho; Hope, Thomas A; Larson, Peder E Z

2018-05-01

Accurate quantification of uptake on PET images depends on accurate attenuation correction in reconstruction. Current MR-based attenuation correction methods for body PET use a fat and water map derived from a 2-echo Dixon MRI sequence in which bone is neglected. Ultrashort-echo-time or zero-echo-time (ZTE) pulse sequences can capture bone information. We propose the use of patient-specific multiparametric MRI consisting of Dixon MRI and proton-density-weighted ZTE MRI to directly synthesize pseudo-CT images with a deep learning model: we call this method ZTE and Dixon deep pseudo-CT (ZeDD CT). Methods: Twenty-six patients were scanned using an integrated 3-T time-of-flight PET/MRI system. Helical CT images of the patients were acquired separately. A deep convolutional neural network was trained to transform ZTE and Dixon MR images into pseudo-CT images. Ten patients were used for model training, and 16 patients were used for evaluation. Bone and soft-tissue lesions were identified, and the SUV max was measured. The root-mean-squared error (RMSE) was used to compare the MR-based attenuation correction with the ground-truth CT attenuation correction. Results: In total, 30 bone lesions and 60 soft-tissue lesions were evaluated. The RMSE in PET quantification was reduced by a factor of 4 for bone lesions (10.24% for Dixon PET and 2.68% for ZeDD PET) and by a factor of 1.5 for soft-tissue lesions (6.24% for Dixon PET and 4.07% for ZeDD PET). Conclusion: ZeDD CT produces natural-looking and quantitatively accurate pseudo-CT images and reduces error in pelvic PET/MRI attenuation correction compared with standard methods. © 2018 by the Society of Nuclear Medicine and Molecular Imaging.

RNA protects a nucleoprotein complex against radiation damage.

PubMed

Bury, Charles S; McGeehan, John E; Antson, Alfred A; Carmichael, Ian; Gerstel, Markus; Shevtsov, Mikhail B; Garman, Elspeth F

2016-05-01

Radiation damage during macromolecular X-ray crystallographic data collection is still the main impediment for many macromolecular structure determinations. Even when an eventual model results from the crystallographic pipeline, the manifestations of radiation-induced structural and conformation changes, the so-called specific damage, within crystalline macromolecules can lead to false interpretations of biological mechanisms. Although this has been well characterized within protein crystals, far less is known about specific damage effects within the larger class of nucleoprotein complexes. Here, a methodology has been developed whereby per-atom density changes could be quantified with increasing dose over a wide (1.3-25.0 MGy) range and at higher resolution (1.98 Å) than the previous systematic specific damage study on a protein-DNA complex. Specific damage manifestations were determined within the large trp RNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein. Over a large dose range, the RNA was found to be far less susceptible to radiation-induced chemical changes than the protein. The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility. The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. Additionally, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density.

RNA protects a nucleoprotein complex against radiation damage

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

Bury, Charles S.; McGeehan, John E.; Antson, Alfred A.

Radiation damage during macromolecular X-ray crystallographic data collection is still the main impediment for many macromolecular structure determinations. Even when an eventual model results from the crystallographic pipeline, the manifestations of radiation-induced structural and conformation changes, the so-called specific damage, within crystalline macromolecules can lead to false interpretations of biological mechanisms. Although this has been well characterized within protein crystals, far less is known about specific damage effects within the larger class of nucleoprotein complexes. We developed a methodology whereby per-atom density changes could be quantified with increasing dose over a wide (1.3–25.0 MGy) range and at higher resolution (1.98more » Å) than the previous systematic specific damage study on a protein–DNA complex. Specific damage manifestations were determined within the largetrpRNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein. Over a large dose range, the RNA was found to be far less susceptible to radiation-induced chemical changes than the protein. The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility. The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. In addition, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density.« less

RNA protects a nucleoprotein complex against radiation damage

DOE PAGES

Bury, Charles S.; McGeehan, John E.; Antson, Alfred A.; ...

2016-04-26

Radiation damage during macromolecular X-ray crystallographic data collection is still the main impediment for many macromolecular structure determinations. Even when an eventual model results from the crystallographic pipeline, the manifestations of radiation-induced structural and conformation changes, the so-called specific damage, within crystalline macromolecules can lead to false interpretations of biological mechanisms. Although this has been well characterized within protein crystals, far less is known about specific damage effects within the larger class of nucleoprotein complexes. We developed a methodology whereby per-atom density changes could be quantified with increasing dose over a wide (1.3–25.0 MGy) range and at higher resolution (1.98more » Å) than the previous systematic specific damage study on a protein–DNA complex. Specific damage manifestations were determined within the largetrpRNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein. Over a large dose range, the RNA was found to be far less susceptible to radiation-induced chemical changes than the protein. The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility. The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. In addition, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density.« less

Quantitative single-particle digital autoradiography with α-particle emitters for targeted radionuclide therapy using the iQID camera

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

Miller, Brian W., E-mail: brian.miller@pnnl.gov; Frost, Sofia H. L.; Frayo, Shani L.

2015-07-15

Purpose: Alpha-emitting radionuclides exhibit a potential advantage for cancer treatments because they release large amounts of ionizing energy over a few cell diameters (50–80 μm), causing localized, irreparable double-strand DNA breaks that lead to cell death. Radioimmunotherapy (RIT) approaches using monoclonal antibodies labeled with α emitters may thus inactivate targeted cells with minimal radiation damage to surrounding tissues. Tools are needed to visualize and quantify the radioactivity distribution and absorbed doses to targeted and nontargeted cells for accurate dosimetry of all treatment regimens utilizing α particles, including RIT and others (e.g., Ra-223), especially for organs and tumors with heterogeneous radionuclidemore » distributions. The aim of this study was to evaluate and characterize a novel single-particle digital autoradiography imager, the ionizing-radiation quantum imaging detector (iQID) camera, for use in α-RIT experiments. Methods: The iQID camera is a scintillator-based radiation detection system that images and identifies charged-particle and gamma-ray/x-ray emissions spatially and temporally on an event-by-event basis. It employs CCD-CMOS cameras and high-performance computing hardware for real-time imaging and activity quantification of tissue sections, approaching cellular resolutions. In this work, the authors evaluated its characteristics for α-particle imaging, including measurements of intrinsic detector spatial resolutions and background count rates at various detector configurations and quantification of activity distributions. The technique was assessed for quantitative imaging of astatine-211 ({sup 211}At) activity distributions in cryosections of murine and canine tissue samples. Results: The highest spatial resolution was measured at ∼20 μm full width at half maximum and the α-particle background was measured at a rate as low as (2.6 ± 0.5) × 10{sup −4} cpm/cm{sup 2} (40 mm diameter detector area). Simultaneous imaging of multiple tissue sections was performed using a large-area iQID configuration (ø 11.5 cm). Estimation of the {sup 211}At activity distribution was demonstrated at mBq/μg-levels. Conclusions: Single-particle digital autoradiography of α emitters has advantages over traditional film-based autoradiographic techniques that use phosphor screens, in terms of spatial resolution, sensitivity, and activity quantification capability. The system features and characterization results presented in this study show that the iQID is a promising technology for microdosimetry, because it provides necessary information for interpreting alpha-RIT outcomes and for predicting the therapeutic efficacy of cell-targeted approaches using α emitters.« less

Quantitative single-particle digital autoradiography with α-particle emitters for targeted radionuclide therapy using the iQID camera.

PubMed

Miller, Brian W; Frost, Sofia H L; Frayo, Shani L; Kenoyer, Aimee L; Santos, Erlinda; Jones, Jon C; Green, Damian J; Hamlin, Donald K; Wilbur, D Scott; Fisher, Darrell R; Orozco, Johnnie J; Press, Oliver W; Pagel, John M; Sandmaier, Brenda M

2015-07-01

Alpha-emitting radionuclides exhibit a potential advantage for cancer treatments because they release large amounts of ionizing energy over a few cell diameters (50-80 μm), causing localized, irreparable double-strand DNA breaks that lead to cell death. Radioimmunotherapy (RIT) approaches using monoclonal antibodies labeled with α emitters may thus inactivate targeted cells with minimal radiation damage to surrounding tissues. Tools are needed to visualize and quantify the radioactivity distribution and absorbed doses to targeted and nontargeted cells for accurate dosimetry of all treatment regimens utilizing α particles, including RIT and others (e.g., Ra-223), especially for organs and tumors with heterogeneous radionuclide distributions. The aim of this study was to evaluate and characterize a novel single-particle digital autoradiography imager, the ionizing-radiation quantum imaging detector (iQID) camera, for use in α-RIT experiments. The iQID camera is a scintillator-based radiation detection system that images and identifies charged-particle and gamma-ray/x-ray emissions spatially and temporally on an event-by-event basis. It employs CCD-CMOS cameras and high-performance computing hardware for real-time imaging and activity quantification of tissue sections, approaching cellular resolutions. In this work, the authors evaluated its characteristics for α-particle imaging, including measurements of intrinsic detector spatial resolutions and background count rates at various detector configurations and quantification of activity distributions. The technique was assessed for quantitative imaging of astatine-211 ((211)At) activity distributions in cryosections of murine and canine tissue samples. The highest spatial resolution was measured at ∼20 μm full width at half maximum and the α-particle background was measured at a rate as low as (2.6 ± 0.5) × 10(-4) cpm/cm(2) (40 mm diameter detector area). Simultaneous imaging of multiple tissue sections was performed using a large-area iQID configuration (ø 11.5 cm). Estimation of the (211)At activity distribution was demonstrated at mBq/μg-levels. Single-particle digital autoradiography of α emitters has advantages over traditional film-based autoradiographic techniques that use phosphor screens, in terms of spatial resolution, sensitivity, and activity quantification capability. The system features and characterization results presented in this study show that the iQID is a promising technology for microdosimetry, because it provides necessary information for interpreting alpha-RIT outcomes and for predicting the therapeutic efficacy of cell-targeted approaches using α emitters.

Automated solid-phase extraction coupled online with HPLC-FLD for the quantification of zearalenone in edible oil.

PubMed

Drzymala, Sarah S; Weiz, Stefan; Heinze, Julia; Marten, Silvia; Prinz, Carsten; Zimathies, Annett; Garbe, Leif-Alexander; Koch, Matthias

2015-05-01

Established maximum levels for the mycotoxin zearalenone (ZEN) in edible oil require monitoring by reliable analytical methods. Therefore, an automated SPE-HPLC online system based on dynamic covalent hydrazine chemistry has been developed. The SPE step comprises a reversible hydrazone formation by ZEN and a hydrazine moiety covalently attached to a solid phase. Seven hydrazine materials with different properties regarding the resin backbone, pore size, particle size, specific surface area, and loading have been evaluated. As a result, a hydrazine-functionalized silica gel was chosen. The final automated online method was validated and applied to the analysis of three maize germ oil samples including a provisionally certified reference material. Important performance criteria for the recovery (70-120 %) and precision (RSDr <25 %) as set by the Commission Regulation EC 401/2006 were fulfilled: The mean recovery was 78 % and RSDr did not exceed 8 %. The results of the SPE-HPLC online method were further compared to results obtained by liquid-liquid extraction with stable isotope dilution analysis LC-MS/MS and found to be in good agreement. The developed SPE-HPLC online system with fluorescence detection allows a reliable, accurate, and sensitive quantification (limit of quantification, 30 μg/kg) of ZEN in edible oils while significantly reducing the workload. To our knowledge, this is the first report on an automated SPE-HPLC method based on a covalent SPE approach.

Interpreting the spatio-temporal patterns of sea turtle strandings: Going with the flow

USGS Publications Warehouse

Hart, K.M.; Mooreside, P.; Crowder, L.B.

2006-01-01

Knowledge of the spatial and temporal distribution of specific mortality sources is crucial for management of species that are vulnerable to human interactions. Beachcast carcasses represent an unknown fraction of at-sea mortalities. While a variety of physical (e.g., water temperature) and biological (e.g., decomposition) factors as well as the distribution of animals and their mortality sources likely affect the probability of carcass stranding, physical oceanography plays a major role in where and when carcasses strand. Here, we evaluate the influence of nearshore physical oceanographic and wind regimes on sea turtle strandings to decipher seasonal trends and make qualitative predictions about stranding patterns along oceanfront beaches. We use results from oceanic drift-bottle experiments to check our predictions and provide an upper limit on stranding proportions. We compare predicted current regimes from a 3D physical oceanographic model to spatial and temporal locations of both sea turtle carcass strandings and drift bottle landfalls. Drift bottle return rates suggest an upper limit for the proportion of sea turtle carcasses that strand (about 20%). In the South Atlantic Bight, seasonal development of along-shelf flow coincides with increased numbers of strandings of both turtles and drift bottles in late spring and early summer. The model also predicts net offshore flow of surface waters during winter - the season with the fewest relative strandings. The drift bottle data provide a reasonable upper bound on how likely carcasses are to reach land from points offshore and bound the general timeframe for stranding post-mortem (< two weeks). Our findings suggest that marine turtle strandings follow a seasonal regime predictable from physical oceanography and mimicked by drift bottle experiments. Managers can use these findings to reevaluate incidental strandings limits and fishery takes for both nearshore and offshore mortality sources. ?? 2005 Elsevier Ltd. All rights reserved.

The New Aptima HBV Quant Real-Time TMA Assay Accurately Quantifies Hepatitis B Virus DNA from Genotypes A to F.

PubMed

Chevaliez, Stéphane; Dauvillier, Claude; Dubernet, Fabienne; Poveda, Jean-Dominique; Laperche, Syria; Hézode, Christophe; Pawlotsky, Jean-Michel

2017-04-01

Sensitive and accurate hepatitis B virus (HBV) DNA detection and quantification are essential to diagnose HBV infection, establish the prognosis of HBV-related liver disease, and guide the decision to treat and monitor the virological response to antiviral treatment and the emergence of resistance. Currently available HBV DNA platforms and assays are generally designed for batching multiple specimens within an individual run and require at least one full day of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed, fully automated, one-step Aptima HBV Quant assay to accurately detect and quantify HBV DNA in a large series of patients infected with different HBV genotypes. The limit of detection of the assay was estimated to be 4.5 IU/ml. The specificity of the assay was 100%. Intra-assay and interassay coefficients of variation ranged from 0.29% to 5.07% and 4.90% to 6.85%, respectively. HBV DNA levels from patients infected with HBV genotypes A to F measured with the Aptima HBV Quant assay strongly correlated with those measured by two commercial real-time PCR comparators (Cobas AmpliPrep/Cobas TaqMan HBV test, version 2.0, and Abbott RealTi m e HBV test). In conclusion, the Aptima HBV Quant assay is sensitive, specific, and reproducible and accurately quantifies HBV DNA in plasma samples from patients with chronic HBV infections of all genotypes, including patients on antiviral treatment with nucleoside or nucleotide analogues. The Aptima HBV Quant assay can thus confidently be used to detect and quantify HBV DNA in both clinical trials with new anti-HBV drugs and clinical practice. Copyright © 2017 American Society for Microbiology.

The New Aptima HCV Quant Dx Real-time TMA Assay Accurately Quantifies Hepatitis C Virus Genotype 1-6 RNA.

PubMed

Chevaliez, Stéphane; Dubernet, Fabienne; Dauvillier, Claude; Hézode, Christophe; Pawlotsky, Jean-Michel

2017-06-01

Sensitive and accurate hepatitis C virus (HCV) RNA detection and quantification is essential for the management of chronic hepatitis C therapy. Currently available platforms and assays are usually batched and require at least 5hours of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed Aptima HCV Quant Dx assay that eliminates the need for batch processing and automates all aspects of nucleic acid testing in a single step, to accurately detect and quantify HCV RNA in a large series of patients infected with different HCV genotypes. The limit of detection was estimated to be 2.3 IU/mL. The specificity of the assay was 98.6% (95% confidence interval: 96.1%-99.5%). Intra-assay and inter-assay coefficients of variation ranged from 0.09% to 5.61%, and 1.05% to 3.65%, respectively. The study of serum specimens from patients infected with HCV genotypes 1 to 6 showed a satisfactory relationship between HCV RNA levels measured by the Aptima HCV Quant Dx assay, and both real-time PCR comparators (Abbott RealTime HCV and Cobas AmpliPrep/Cobas TaqMan HCV Test, version 2.0, assays). the new Aptima HCV Quant Dx assay is rapid, sensitive, reasonably specific and reproducible and accurately quantifies HCV RNA in serum samples from patients with chronic HCV infection, including patients on antiviral treatment. The Aptima HCV Quant Dx assay can thus be confidently used to detect and quantify HCV RNA in both clinical trials with new anti-HCV drugs and clinical practice in Europe and the US. Copyright © 2017 Elsevier B.V. All rights reserved.

The New Aptima HBV Quant Real-Time TMA Assay Accurately Quantifies Hepatitis B Virus DNA from Genotypes A to F

PubMed Central

Dauvillier, Claude; Dubernet, Fabienne; Poveda, Jean-Dominique; Laperche, Syria; Hézode, Christophe; Pawlotsky, Jean-Michel

2017-01-01

ABSTRACT Sensitive and accurate hepatitis B virus (HBV) DNA detection and quantification are essential to diagnose HBV infection, establish the prognosis of HBV-related liver disease, and guide the decision to treat and monitor the virological response to antiviral treatment and the emergence of resistance. Currently available HBV DNA platforms and assays are generally designed for batching multiple specimens within an individual run and require at least one full day of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed, fully automated, one-step Aptima HBV Quant assay to accurately detect and quantify HBV DNA in a large series of patients infected with different HBV genotypes. The limit of detection of the assay was estimated to be 4.5 IU/ml. The specificity of the assay was 100%. Intra-assay and interassay coefficients of variation ranged from 0.29% to 5.07% and 4.90% to 6.85%, respectively. HBV DNA levels from patients infected with HBV genotypes A to F measured with the Aptima HBV Quant assay strongly correlated with those measured by two commercial real-time PCR comparators (Cobas AmpliPrep/Cobas TaqMan HBV test, version 2.0, and Abbott RealTime HBV test). In conclusion, the Aptima HBV Quant assay is sensitive, specific, and reproducible and accurately quantifies HBV DNA in plasma samples from patients with chronic HBV infections of all genotypes, including patients on antiviral treatment with nucleoside or nucleotide analogues. The Aptima HBV Quant assay can thus confidently be used to detect and quantify HBV DNA in both clinical trials with new anti-HBV drugs and clinical practice. PMID:28202793

Identification of diagnostic peptide regions that distinguish Zika virus from related mosquito-borne Flaviviruses

PubMed Central

Lee, Alexandra J.; Bhattacharya, Roshni; Scheuermann, Richard H.

2017-01-01

Zika virus (ZIKV) is a member of the Flavivirus genus of positive-sense single-stranded RNA viruses, which includes Dengue, West Nile, Yellow Fever, and other mosquito-borne arboviruses. Infection by ZIKV can be difficult to distinguish from infection by other mosquito-borne Flaviviruses due to high sequence similarity, serum antibody cross-reactivity, and virus co-circulation in endemic areas. Indeed, existing serological methods are not able to consistently differentiate ZIKV from other Flaviviruses, which makes it extremely difficult to accurately calculate the incidence rate of Zika-associated Guillain-Barre in adults, microcephaly in newborns, or asymptomatic infections within a geographical area. In order to identify Zika-specific peptide regions that could be used as serology reagents, we have applied comparative genomics and protein structure analyses to identify amino acid residues that distinguish each of 10 Flavivirus species and subtypes from each other by calculating the specificity, sensitivity, and surface exposure of each residue in relevant target proteins. For ZIKV we identified 104 and 116 15-mer peptides in the E glycoprotein and NS1 non-structural protein, respectively, that contain multiple diagnostic sites and are located in surface-exposed regions in the tertiary protein structure. These sensitive, specific, and surface-exposed peptide regions should serve as useful reagents for seroprevalence studies to better distinguish between prior infections with any of these mosquito-borne Flaviviruses. The development of better detection methods and diagnostic tools will enable clinicians and public health workers to more accurately estimate the true incidence rate of asymptomatic infections, neurological syndromes, and birth defects associated with ZIKV infection. PMID:28562637

Speech graphs provide a quantitative measure of thought disorder in psychosis.

PubMed

Mota, Natalia B; Vasconcelos, Nivaldo A P; Lemos, Nathalia; Pieretti, Ana C; Kinouchi, Osame; Cecchi, Guillermo A; Copelli, Mauro; Ribeiro, Sidarta

2012-01-01

Psychosis has various causes, including mania and schizophrenia. Since the differential diagnosis of psychosis is exclusively based on subjective assessments of oral interviews with patients, an objective quantification of the speech disturbances that characterize mania and schizophrenia is in order. In principle, such quantification could be achieved by the analysis of speech graphs. A graph represents a network with nodes connected by edges; in speech graphs, nodes correspond to words and edges correspond to semantic and grammatical relationships. To quantify speech differences related to psychosis, interviews with schizophrenics, manics and normal subjects were recorded and represented as graphs. Manics scored significantly higher than schizophrenics in ten graph measures. Psychopathological symptoms such as logorrhea, poor speech, and flight of thoughts were grasped by the analysis even when verbosity differences were discounted. Binary classifiers based on speech graph measures sorted schizophrenics from manics with up to 93.8% of sensitivity and 93.7% of specificity. In contrast, sorting based on the scores of two standard psychiatric scales (BPRS and PANSS) reached only 62.5% of sensitivity and specificity. The results demonstrate that alterations of the thought process manifested in the speech of psychotic patients can be objectively measured using graph-theoretical tools, developed to capture specific features of the normal and dysfunctional flow of thought, such as divergence and recurrence. The quantitative analysis of speech graphs is not redundant with standard psychometric scales but rather complementary, as it yields a very accurate sorting of schizophrenics and manics. Overall, the results point to automated psychiatric diagnosis based not on what is said, but on how it is said.

Improved uncertainty quantification in nondestructive assay for nonproliferation

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

Burr, Tom; Croft, Stephen; Jarman, Ken

2016-12-01

This paper illustrates methods to improve uncertainty quantification (UQ) for non-destructive assay (NDA) measurements used in nuclear nonproliferation. First, it is shown that current bottom-up UQ applied to calibration data is not always adequate, for three main reasons: (1) Because there are errors in both the predictors and the response, calibration involves a ratio of random quantities, and calibration data sets in NDA usually consist of only a modest number of samples (3–10); therefore, asymptotic approximations involving quantities needed for UQ such as means and variances are often not sufficiently accurate; (2) Common practice overlooks that calibration implies a partitioningmore » of total error into random and systematic error, and (3) In many NDA applications, test items exhibit non-negligible departures in physical properties from calibration items, so model-based adjustments are used, but item-specific bias remains in some data. Therefore, improved bottom-up UQ using calibration data should predict the typical magnitude of item-specific bias, and the suggestion is to do so by including sources of item-specific bias in synthetic calibration data that is generated using a combination of modeling and real calibration data. Second, for measurements of the same nuclear material item by both the facility operator and international inspectors, current empirical (top-down) UQ is described for estimating operator and inspector systematic and random error variance components. A Bayesian alternative is introduced that easily accommodates constraints on variance components, and is more robust than current top-down methods to the underlying measurement error distributions.« less

Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay.

PubMed

Hendricks, D A; Stowe, B J; Hoo, B S; Kolberg, J; Irvine, B D; Neuwald, P D; Urdea, M S; Perrillo, R P

1995-11-01

The aim of this study was to establish the performance characteristics of a nonradioisotopic branched DNA (bDNA) signal amplification assay for quantitation of hepatitis B virus (HBV) DNA in human serum. Quantitation was determined from a standard curve and expressed as HBV DNA equivalents/mL (Eq/mL; 285,000 Eq = 1 pg of double stranded HBV DNA). The bDNA assay exhibited a nearly four log dynamic range of quantitation and an analytical detection limit of approximately 100,000 Eq/mL. To ensure a specificity of 99.7%, the quantitation limit was set at 700,000 Eq/mL. The interassay percent coefficient of variance for quantification values ranged from 10% to 15% when performed by novice users with different sets of reagents. Using the bDNA assay, HBV DNA was detected in 94% to 100% of hepatitis B e antigen-positive specimens and 27% to 31% of hepatitis B e antigen-negative specimens from chronic HBV-infected patients. The bDNA assay may be useful as a prognostic and therapy monitoring tool for the management of HBV-infected patients undergoing antiviral treatment.

Temperature modulation and quadrature detection for selective titration of two-state exchanging reactants.

PubMed

Zrelli, K; Barilero, T; Cavatore, E; Berthoumieux, H; Le Saux, T; Croquette, V; Lemarchand, A; Gosse, C; Jullien, L

2011-04-01

Biological samples exhibit huge molecular diversity over large concentration ranges. Titrating a given compound in such mixtures is often difficult, and innovative strategies emphasizing selectivity are thus demanded. To overcome limitations inherent to thermodynamics, we here present a generic technique where discrimination relies on the dynamics of interaction between the target of interest and a probe introduced in excess. Considering an ensemble of two-state exchanging reactants submitted to temperature modulation, we first demonstrate that the amplitude of the out-of-phase concentration oscillations is maximum for every compound involved in a reaction whose equilibrium constant is equal to unity and whose relaxation time is equal to the inverse of the excitation angular frequency. Taking advantage of this feature, we next devise a highly specific detection protocol and validate it using a microfabricated resistive heater and an epifluorescence microscope, as well as labeled oligonucleotides to model species displaying various dynamic properties. As expected, quantification of a sought for strand is obtained even if interfering reagents are present in similar amounts. Moreover, our approach does not require any separation and is compatible with imaging. It could then benefit some of the numerous binding assays performed every day in life sciences.

Replication of avocado sunblotch viroid: evidence for a symmetric pathway with two rolling circles and hammerhead ribozyme processing.

PubMed Central

Daròs, J A; Marcos, J F; Hernández, C; Flores, R

1994-01-01

The structure of a series of RNAs extracted from avocado infected by the 247-nt avocado sunblotch viroid (ASBVd) was investigated. The identification of multistranded complexes containing circular ASBVd RNAs of (+) and (-) polarity suggests that replication of ASBVd proceeds through a symmetric pathway with two rolling circles where these two circular RNAs are the templates. This is in contrast to the replication of potato spindle tuber viroid and probably of most of its related viroids, which proceeds via an asymmetric pathway where circular (+)-strand and linear multimeric (-)-strand RNAs are the two templates. Linear (+) and (-) ASBVd RNAs of subgenomic length (137 nt and about 148 nt, respectively) and one linear (+)-strand ASBVd RNA of supragenomic length (383-384 nt) were also found in viroid-infected tissue. The two linear (+)-strand RNAs have the same 5'- and 3'-terminal sequences, with the supragenomic species being a fusion product of the monomeric and subgenomic (+)-strand ASBVd RNAs. The 3' termini of these two (+)-strand molecules, which at least in the subgenomic RNA has an extra nontemplate cytidylate residue, could represent sites of either premature termination of the (+)-strands or specific initiation of the (-)-strands. The 5' termini of sub- and supragenomic (+)-strand and the 5' terminus of the subgenomic (-)-strand ASBVd RNA are identical to those produced in the in vitro self-cleavage reactions of (+) and (-) dimeric ASBVd RNAs, respectively. These observations strongly suggest that the hammerhead structures which mediate the in vitro self-cleavage reactions are also operative in vivo. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:7809126

Statistical analysis of the Nb3Sn strand production for the ITER toroidal field coils

NASA Astrophysics Data System (ADS)

Vostner, A.; Jewell, M.; Pong, I.; Sullivan, N.; Devred, A.; Bessette, D.; Bevillard, G.; Mitchell, N.; Romano, G.; Zhou, C.

2017-04-01

The ITER toroidal field (TF) strand procurement initiated the largest Nb3Sn superconducting strand production hitherto. The industrial-scale production started in Japan in 2008 and finished in summer 2015. Six ITER partners (so-called Domestic Agencies, or DAs) are in charge of the procurement and involved eight different strand suppliers all over the world, of which four are using the bronze route (BR) process and four the internal-tin (IT) process. In total more than 500 tons have been produced including excess material covering losses during the conductor manufacturing process, in particular the cabling. The procurement is based on a functional specification where the main strand requirements like critical current, hysteresis losses, Cu ratio and residual resistance ratio are specified but not the strand production process or layout. This paper presents the analysis on the data acquired during the quality control (QC) process that was carried out to ensure the same conductor performance requirements are met by the different strand suppliers regardless of strand design. The strand QC is based on 100% billet testing and on applying statistical process control (SPC) limits. Throughout the production, samples adjacent to the strand pieces tested by the suppliers are cross-checked (‘verified’) by their respective DAs reference labs. The level of verification was lowered from 100% at the beginning of the procurement progressively to approximately 25% during the final phase of production. Based on the complete dataset of the TF strand production, an analysis of the SPC limits of the critical strand parameters is made and the related process capability indices are calculated. In view of the large-scale production and costs, key manufacturing parameters such as billet yield, number of breakages and piece-length distribution are also discussed. The results are compared among all the strand suppliers, focusing on the difference between BR and IT processes. Following the completion of the largest Nb3Sn strand production, our experience gained from monitoring the execution of the QC activities and from auditing the results from the measurements is summarised for future superconducting strand material procurement activities.

A sun-tracking environmental chamber for the outdoor quantification of CPV modules

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

Faiman, David, E-mail: faiman@bgu.ac.il; Melnichak, Vladimir, E-mail: faiman@bgu.ac.il; Bokobza, Dov, E-mail: faiman@bgu.ac.il

2014-09-26

The paper describes a sun-tracking environmental chamber and its associated fast electronics, devised for the accurate outdoor characterization of CPV cells, receivers, mono-modules, and modules. Some typical measurement results are presented.

DNA gyrase with a single catalytic tyrosine can catalyze DNA supercoiling by a nicking-closing mechanism

PubMed Central

Gubaev, Airat; Weidlich, Daniela; Klostermeier, Dagmar

2016-01-01

The topological state of DNA is important for replication, recombination and transcription, and is regulated in vivo by DNA topoisomerases. Gyrase introduces negative supercoils into DNA at the expense of ATP hydrolysis. It is the accepted view that gyrase achieves supercoiling by a strand passage mechanism, in which double-stranded DNA is cleaved, and a second double-stranded segment is passed through the gap, converting a positive DNA node into a negative node. We show here that gyrase with only one catalytic tyrosine that cleaves a single strand of its DNA substrate can catalyze DNA supercoiling without strand passage. We propose an alternative mechanism for DNA supercoiling via nicking and closing of DNA that involves trapping, segregation and relaxation of two positive supercoils. In contrast to DNA supercoiling, ATP-dependent relaxation and decatenation of DNA by gyrase lacking the C-terminal domains require both tyrosines and strand passage. Our results point towards mechanistic plasticity of gyrase and might pave the way for finding novel and specific mechanism-based gyrase inhibitors. PMID:27557712

Resonant Formation of Strand Breaks in Sensitized Oligonucleotides Induced by Low-Energy Electrons (0.5-9 eV).

PubMed

Schürmann, Robin; Tsering, Thupten; Tanzer, Katrin; Denifl, Stephan; Kumar, S V K; Bald, Ilko

2017-08-28

Halogenated nucleobases are used as radiosensitizers in cancer radiation therapy, enhancing the reactivity of DNA to secondary low-energy electrons (LEEs). LEEs induce DNA strand breaks at specific energies (resonances) by dissociative electron attachment (DEA). Although halogenated nucleobases show intense DEA resonances at various electron energies in the gas phase, it is inherently difficult to investigate the influence of halogenated nucleobases on the actual DNA strand breakage over the broad range of electron energies at which DEA can take place (<12 eV). By using DNA origami nanostructures, we determined the energy dependence of the strand break cross-section for oligonucleotides modified with 8-bromoadenine ( 8Br A). These results were evaluated against DEA measurements with isolated 8Br A in the gas phase. Contrary to expectations, the major contribution to strand breaks is from resonances at around 7 eV while resonances at very low energy (<2 eV) have little influence on strand breaks. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A major role of DNA polymerase δ in replication of both the leading and lagging DNA strands

PubMed Central

Prakash, Louise; Prakash, Satya

2015-01-01

SUMMARY 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. PMID:26145172

Quantification of meat proportions by measuring DNA contents in raw and boiled sausages using matrix-adapted calibrators and multiplex real-time PCR.

PubMed

Köppel, René; Eugster, Albert; Ruf, Jürg; Rentsch, Jürg

2012-01-01

The quantification of meat proportions in raw and boiled sausage according to the recipe was evaluated using three different calibrators. To measure the DNA contents from beef, pork, sheep (mutton), and horse, a tetraplex real-time PCR method was applied. Nineteen laboratories analyzed four meat products each made of different proportions of beef, pork, sheep, and horse meat. Three kinds of calibrators were used: raw and boiled sausages of known proportions ranging from 1 to 55% of meat, and a dilution series of DNA from muscle tissue. In general, results generated using calibration sausages were more accurate than those resulting from the use of DNA from muscle tissue, and exhibited smaller measurement uncertainties. Although differences between uses of raw and boiled calibration sausages were small, the most precise and accurate results were obtained by calibration with fine-textured boiled reference sausages.

Analyses of the radiation of birnaviruses from diverse host phyla and of their evolutionary affinities with other double-stranded RNA and positive strand RNA viruses using robust structure-based multiple sequence alignments and advanced phylogenetic methods

PubMed Central

2013-01-01

Background Birnaviruses form a distinct family of double-stranded RNA viruses infecting animals as different as vertebrates, mollusks, insects and rotifers. With such a wide host range, they constitute a good model for studying the adaptation to the host. Additionally, several lines of evidence link birnaviruses to positive strand RNA viruses and suggest that phylogenetic analyses may provide clues about transition. Results We characterized the genome of a birnavirus from the rotifer Branchionus plicalitis. We used X-ray structures of RNA-dependent RNA polymerases and capsid proteins to obtain multiple structure alignments that allowed us to obtain reliable multiple sequence alignments and we employed “advanced” phylogenetic methods to study the evolutionary relationships between some positive strand and double-stranded RNA viruses. We showed that the rotifer birnavirus genome exhibited an organization remarkably similar to other birnaviruses. As this host was phylogenetically very distant from the other known species targeted by birnaviruses, we revisited the evolutionary pathways within the Birnaviridae family using phylogenetic reconstruction methods. We also applied a number of phylogenetic approaches based on structurally conserved domains/regions of the capsid and RNA-dependent RNA polymerase proteins to study the evolutionary relationships between birnaviruses, other double-stranded RNA viruses and positive strand RNA viruses. Conclusions We show that there is a good correlation between the phylogeny of the birnaviruses and that of their hosts at the phylum level using the RNA-dependent RNA polymerase (genomic segment B) on the one hand and a concatenation of the capsid protein, protease and ribonucleoprotein (genomic segment A) on the other hand. This correlation tends to vanish within phyla. The use of advanced phylogenetic methods and robust structure-based multiple sequence alignments allowed us to obtain a more accurate picture (in terms of probability of the tree topologies) of the evolutionary affinities between double-stranded RNA and positive strand RNA viruses. In particular, we were able to show that there exists a good statistical support for the claims that dsRNA viruses are not monophyletic and that viruses with permuted RdRps belong to a common evolution lineage as previously proposed by other groups. We also propose a tree topology with a good statistical support describing the evolutionary relationships between the Picornaviridae, Caliciviridae, Flaviviridae families and a group including the Alphatetraviridae, Nodaviridae, Permutotretraviridae, Birnaviridae, and Cystoviridae families. PMID:23865988

Electrochemical Branched-DNA Assay for Polymerase Chain Reaction-Free Detection and Quantification of Oncogenes in Messenger RNA

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

Lee, Ai Cheng; Dai, Ziyu; Chen, Baowei

2008-12-01

We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcript in the population of messenger RNA (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify targets signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-napthyl-phosphate. The specificity and sensitivity of assay enabled direct detection ofmore » target transcript in as little as 4.6 ng mRNA without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcript in total mRNA population. The approach thus provides a simple, sensitive, accurate and quantitative tool alternate to the RQ-PCR for early disease diagnosis.« less

Development of validated high-performance thin layer chromatography for quantification of aristolochic acid in different species of the Aristolochiaceae family.

PubMed

Agrawal, Poonam; Laddha, Kirti

2017-04-01

This study was undertaken to isolate and quantify aristolochic acid in Aristolochia indica stem and Apama siliquosa root. Aristolochic acid is an important biomarker component present in the Aristolochiaceae family. The isolation method involved simple solvent extraction, precipitation and further purification, using recrystallization. The structure of the compound was confirmed using infrared spectroscopy, mass spectrometry and nuclear magnetic resonance. A specific and rapid high-performance thin layer chromatography (HPTLC) method was developed for analysis of aristolochic acid. The method involved separation on the silica gel 60 F 254 plates using the single solvent system of n-hexane: chloroform: methanol. The method showed good linear relationship in the range 0.4-2.0 μg/spot with r 2  = 0.998. The limit of detection and limit of quantification were 62.841 ng/spot and 209.47 ng/spot, respectively. The proposed validated HPTLC method was found to be an easy to use, accurate and convenient method that could be successfully used for standardization and quality assessment of herbal material as well as formulations containing different species of the Aristolochiaceae family. Copyright © 2016. Published by Elsevier B.V.

Residual urinary extracellular vesicles in ultracentrifugation supernatants after hydrostatic filtration dialysis enrichment.

PubMed

Musante, Luca; Tataruch-Weinert, Dorota; Kerjaschki, Dontscho; Henry, Michael; Meleady, Paula; Holthofer, Harry

2017-01-01

Urinary extracellular vesicles (UEVs) appear an ideal source of biomarkers for kidney and urogenital diseases. The majority of protocols designed for their isolation are based on differential centrifugation steps. However, little is still known of the type and amount of vesicles left in the supernatant. Here we used an isolation protocol for UEVs which uses hydrostatic filtration dialysis as first pre-enrichment step, followed by differential centrifugation. Transmission electron microscopy (TEM), mass spectrometry (MS), western blot, ELISA assays and tuneable resistive pulse sensing (TRPS) were used to characterise and quantify UEVs in the ultracentrifugation supernatant. TEM showed the presence of a variety of small size vesicles in the supernatant while protein identification by MS matched accurately with the protein list available in Vesiclepedia. Screening and relative quantification for specific vesicle markers showed that the supernatant was preferentially positive for CD9 and TSG101. ELISA tests for quantification of exosome revealed that 14%, was left in the supernatant with a particle diameter of 110 nm and concentration of 1.54 × 10 10 /ml. Here we show a comprehensive characterisation of exosomes and other small size urinary vesicles which the conventional differential centrifugation protocol may lose.

Residual urinary extracellular vesicles in ultracentrifugation supernatants after hydrostatic filtration dialysis enrichment

PubMed Central

Musante, Luca; Tataruch-Weinert, Dorota; Kerjaschki, Dontscho; Henry, Michael; Meleady, Paula; Holthofer, Harry

2017-01-01

ABSTRACT Urinary extracellular vesicles (UEVs) appear an ideal source of biomarkers for kidney and urogenital diseases. The majority of protocols designed for their isolation are based on differential centrifugation steps. However, little is still known of the type and amount of vesicles left in the supernatant. Here we used an isolation protocol for UEVs which uses hydrostatic filtration dialysis as first pre-enrichment step, followed by differential centrifugation. Transmission electron microscopy (TEM), mass spectrometry (MS), western blot, ELISA assays and tuneable resistive pulse sensing (TRPS) were used to characterise and quantify UEVs in the ultracentrifugation supernatant. TEM showed the presence of a variety of small size vesicles in the supernatant while protein identification by MS matched accurately with the protein list available in Vesiclepedia. Screening and relative quantification for specific vesicle markers showed that the supernatant was preferentially positive for CD9 and TSG101. ELISA tests for quantification of exosome revealed that 14%, was left in the supernatant with a particle diameter of 110 nm and concentration of 1.54 × 1010/ml. Here we show a comprehensive characterisation of exosomes and other small size urinary vesicles which the conventional differential centrifugation protocol may lose. PMID:28326167

A fast and validated method for the determination of malondialdehyde in fish liver using high-performance liquid chromatography with a photodiode array detector.

PubMed

Faizan, Mohammad; Esatbeyoglu, Tuba; Bayram, Banu; Rimbach, Gerald

2014-04-01

Malondialdehyde (MDA) is a biomarker of lipid peroxidation and is present in foods and biological samples such as plasma. A high-performance liquid chromatography (HPLC) method was applied to determine MDA in fish liver samples after derivatization with 2,4-dinitrophenylhydrazine (DNPH) using a ODS2 column (10 cm × 4.6 mm, 3 μm) and a photodiode array detector. The mobile phase consisted of 0.2% acetic acid (v/v) in distilled water and acetonitrile (42:58, v/v). The present method was validated in terms of linearity, lower limit of quantification, lower limit of detection, precision, accuracy, recovery, and stability of MDA according to U.S. Food and Drug Administration (FDA) guidelines. The limit of quantification of MDA was 0.39 μmol/L, which is comparable to other methods. The recovery of the spiked MDA liver samples was in the range of 92.4% to 104.2%. This newly modified HPLC method is specific, sensitive, and accurate and allows the analysis of MDA within 4 min in fish liver but also in other tissues and plasma. © 2014 Institute of Food Technologists®

Ultrasensitive NIR-SERRS Probes with Multiplexed Ratiometric Quantification for In Vivo Antibody Leads Validation.

PubMed

Kang, Homan; Jeong, Sinyoung; Jo, Ahla; Chang, Hyejin; Yang, Jin-Kyoung; Jeong, Cheolhwan; Kyeong, San; Lee, Youn Woo; Samanta, Animesh; Maiti, Kaustabh Kumar; Cha, Myeong Geun; Kim, Taek-Keun; Lee, Sukmook; Jun, Bong-Hyun; Chang, Young-Tae; Chung, Junho; Lee, Ho-Young; Jeong, Dae Hong; Lee, Yoon-Sik

2018-02-01

Immunotargeting ability of antibodies may show significant difference between in vitro and in vivo. To select antibody leads with high affinity and specificity, it is necessary to perform in vivo validation of antibody candidates following in vitro antibody screening. Herein, a robust in vivo validation of anti-tetraspanin-8 antibody candidates against human colon cancer using ratiometric quantification method is reported. The validation is performed on a single mouse and analyzed by multiplexed surface-enhanced Raman scattering using ultrasensitive and near infrared (NIR)-active surface-enhanced resonance Raman scattering nanoprobes (NIR-SERRS dots). The NIR-SERRS dots are composed of NIR-active labels and Au/Ag hollow-shell assembled silica nanospheres. A 93% of NIR-SERRS dots is detectable at a single-particle level and signal intensity is 100-fold stronger than that from nonresonant molecule-labeled spherical Au NPs (80 nm). The result of SERRS-based antibody validation is comparable to that of the conventional method using single-photon-emission computed tomography. The NIR-SERRS-based strategy is an alternate validation method which provides cost-effective and accurate multiplexing measurements for antibody-based drug development. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.