Amplification of telomeric arrays via rolling-circle mechanism.

PubMed

Nosek, Jozef; Rycovska, Adriana; Makhov, Alexander M; Griffith, Jack D; Tomaska, Lubomir

2005-03-18

Alternative (telomerase-independent) lengthening of telomeres mediated through homologous recombination is often accompanied by a generation of extrachromosomal telomeric circles (t-circles), whose role in direct promotion of recombinational telomere elongation has been recently demonstrated. Here we present evidence that t-circles in a natural telomerase-deficient system of mitochondria of the yeast Candida parapsilosis replicate independently of the linear chromosome via a rolling-circle mechanism. This is supported by an observation of (i) single-stranded DNA consisting of concatameric arrays of telomeric sequence, (ii) lasso-shaped molecules representing rolling-circle intermediates, and (iii) preferential incorporation of deoxyribonucleotides into telomeric fragments and t-circles. Analysis of naturally occurring variant t-circles revealed conserved motifs with potential function in driving the rolling-circle replication. These data indicate that extrachromosomal t-circles observed in a wide variety of organisms, including yeasts, plants, Xenopus laevis, and certain human cell lines, may represent independent replicons generating telomeric sequences and, thus, actively participating in telomere dynamics. Moreover, because of the promiscuous occurrence of t-circles across phyla, the results from yeast mitochondria have implications related to the primordial system of telomere maintenance, providing a paradigm for evolution of telomeres in nuclei of early eukaryotes.

Nanogrid rolling circle DNA sequencing

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

Church, George M.; Porreca, Gregory J.; Shendure, Jay

The present invention relates to methods for sequencing a polynucleotide immobilized on an array having a plurality of specific regions each having a defined diameter size, including synthesizing a concatemer of a polynucleotide by rolling circle amplification, wherein the concatemer has a cross-sectional diameter greater than the diameter of a specific region, immobilizing the concatemer to the specific region to make an immobilized concatemer, and sequencing the immobilized concatemer.

Periodic Assembly of Nanospecies on Repetitive DNA Sequences Generated on Gold Nanoparticles by Rolling Circle Amplification

NASA Astrophysics Data System (ADS)

Zhao, Weian; Brook, Michael A.; Li, Yingfu

Periodical assembly of nanospecies is desirable for the construction of nanodevices. We provide a protocol for the preparation of a gold nanoparticle (AuNP)/DNA scaffold on which nanospecies can be assembled in a periodical manner. AuNP/DNA scaffold is prepared by growing long single-stranded DNA (ssDNA) molecules (typically hundreds of nanometers to a few microns in length) on AuNPs via rolling circle amplification (RCA). Since these long ssDNA molecules contain many repetitive sequence units, complementary DNA-attached nanospecies can be assembled through specific hybridization in a controllable and periodical manner.

Rapid screening for human-pathogenic Mucorales using rolling circle amplification.

PubMed

Dolatabadi, S; Najafzadeh, M J; de Hoog, G S

2014-12-01

Mucormycosis has emerged as a relatively common severe mycosis in patients with haematological and allogeneic stem cell transplantation. Source of transmission is from unidentified sources in the environment. Early diagnosis of infection and its source of contamination are paramount for rapid and appropriate therapy. In this study, rolling circle amplification (RCA) is introduced as a sensitive, specific and reproducible isothermal DNA amplification technique for rapid molecular identification of six of the most virulent species (Rhizopus microsporus, R. arrhizus var. arrhizus, R. arrhizus var. delemar, Mucor irregularis, Mucor circinelloides, Lichtheimia ramosa, Lichtheimia corymbifera). DNAs of target species were successfully amplified, with no cross reactivity between species. RCA can be considered as a rapid detection method with high specificity and sensitivity, suitable for large screening. © 2014 Blackwell Verlag GmbH.

A microRNA detection system based on padlock probes and rolling circle amplification

PubMed Central

Jonstrup, Søren Peter; Koch, Jørn; Kjems, Jørgen

2006-01-01

The differential expression and the regulatory roles of microRNAs (miRNAs) are being studied intensively these years. Their minute size of only 19–24 nucleotides and strong sequence similarity among related species call for enhanced methods for reliable detection and quantification. Moreover, miRNA expression is generally restricted to a limited number of specific cells within an organism and therefore requires highly sensitive detection methods. Here we present a simple and reliable miRNA detection protocol based on padlock probes and rolling circle amplification. It can be performed without specialized equipment and is capable of measuring the content of specific miRNAs in a few nanograms of total RNA. PMID:16888321

A microRNA detection system based on padlock probes and rolling circle amplification.

PubMed

Jonstrup, Søren Peter; Koch, Jørn; Kjems, Jørgen

2006-09-01

The differential expression and the regulatory roles of microRNAs (miRNAs) are being studied intensively these years. Their minute size of only 19-24 nucleotides and strong sequence similarity among related species call for enhanced methods for reliable detection and quantification. Moreover, miRNA expression is generally restricted to a limited number of specific cells within an organism and therefore requires highly sensitive detection methods. Here we present a simple and reliable miRNA detection protocol based on padlock probes and rolling circle amplification. It can be performed without specialized equipment and is capable of measuring the content of specific miRNAs in a few nanograms of total RNA.

Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification.

PubMed

Ebai, Tonge; Souza de Oliveira, Felipe Marques; Löf, Liza; Wik, Lotta; Schweiger, Caroline; Larsson, Anders; Keilholtz, Ulrich; Haybaeck, Johannes; Landegren, Ulf; Kamali-Moghaddam, Masood

2017-09-01

Detecting proteins at low concentrations in plasma is crucial for early diagnosis. Current techniques in clinical routine, such as sandwich ELISA, provide sensitive protein detection because of a dependence on target recognition by pairs of antibodies, but detection of still lower protein concentrations is often called for. Proximity ligation assay with rolling circle amplification (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via binding of target proteins by 3 antibodies, and signal amplification via rolling circle amplification (RCA) in microtiter wells, easily adapted to instrumentation in use in hospitals. Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Upon target recognition these PLA probes guided oligonucleotide ligation, followed by amplification via RCA of circular DNA strands that formed in the reaction. The RCA products were detected by horseradish peroxidase-labeled oligonucleotides to generate colorimetric reaction products with readout in an absorbance microplate reader. We compared detection of interleukin (IL)-4, IL-6, IL-8, p53, and growth differentiation factor 15 (GDF-15) by PLARCA and conventional sandwich ELISA or immuno-RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared to ELISA and iRCA using the same antibodies. IL-4 and IL-6 were detected in clinical samples at femtomolar concentrations, considerably lower than for ELISA. PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories. © 2017 American Association for Clinical Chemistry.

A novel electrochemical biosensor for ultrasensitive and specific detection of DNA based on molecular beacon mediated circular strand displacement and rolling circle amplification.

PubMed

Cheng, Wei; Zhang, Wei; Yan, Yurong; Shen, Bo; Zhu, Dan; Lei, Pinhua; Ding, Shijia

2014-12-15

A novel electrochemical biosensing strategy was developed for ultrasensitive and specific detection of target DNA using a cascade signal amplification based on molecular beacon (MB) mediated circular strand displacement (CSD), rolling circle amplification (RCA), biotin-strepavidin system, and enzymatic amplification. The target DNA hybridized with the loop portion of MB probe immobilized on the gold electrode and triggered the CSD, leading to multiple biotin-tagged DNA duplex. Furthermore, via biotin-streptavidin interaction, the RCA was implemented, producing long massive tandem-repeat DNA sequences for binding numerous biotinylated detection probes. This enabled an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor showed very high sensitivity and selectivity with a dynamic response range from 1 fM to 100 pM. The proposed strategy could have the potential for applying in clinical molecular diagnostics and environmental monitoring. Copyright © 2014 Elsevier B.V. All rights reserved.

Ultrasensitive Detection of Low-Abundance Surface-Marker Protein using Isothermal Rolling Circle Amplification in Microfluidic Nano-Liter Platform

PubMed Central

Konry, Tania; Yarmush, Joel M.; Irimia, Daniel

2011-01-01

With advances in immunology and cancer biology, there is an unmet need for increasingly sensitive systems to monitor the expression of specific cell markers for the development of new diagnostic and therapeutic tools. To address this challenge, we have applied a highly sensitive labeling method that translates antigen-antibody recognition processes into DNA detection event that can be greatly amplified via isothermal Rolling Circle Amplification (RCA). By merging the single-molecule detection power of RCA reaction with microfluidic technology we were able to demonstrate that identification of specific protein markers can be achieved on tumor cell surface in miniaturized nano-liter reaction droplets. Furthermore, this combined approach of signal amplification in a microfluidic format could extend the utility of existing methods by reducing sample and reagent consumption and enhancing the sensitivities and specificities for various applications, including early diagnosis of cancer. PMID:21294269

Characterization of a novel close-to-root papillomavirus from a Florida manatee by using multiply primed rolling-circle amplification: Trichechus manatus latirostris papillomavirus type 1.

PubMed

Rector, Annabel; Bossart, Gregory D; Ghim, Shin-Je; Sundberg, John P; Jenson, A Bennett; Van Ranst, Marc

2004-11-01

By using an isothermal multiply primed rolling-circle amplification protocol, the complete genomic DNA of a novel papillomavirus was amplified from a skin lesion biopsy of a Florida manatee (Trichechus manatus latirostris), one of the most endangered marine mammals in United States coastal waters. The nucleotide sequence, genome organization, and phylogenetic position of the Trichechus manatus latirostris papillomavirus type 1 (TmPV-1) were determined. TmPV-1 is the first virus isolated from the order of Sirenia. A phylogenetic analysis shows that TmPV-1 is only distantly related to other papillomavirus sequences, and it appears in our phylogenetic tree as a novel close-to-root papillomavirus genus.

Identification of Prostate Cancer-Specific microDNAs

DTIC Science & Technology

2014-12-01

displacement amplification (MDA). 2 adopted multiple displacement amplification (MDA) with random primers for enriched circular DNA by rolling circle ... amplification (RCA) (Fig. 1) and then amplified DNA fragments were subject to deep sequencing. Sequence NO of Reads seq 1 184 seq 2 133 seq 3 2407 seq...prostate cancer cells through multiple displacement amplification .  Clone #7 is the top candidate which has been cloned in an expression vector and it

Characterization of a Novel Close-to-Root Papillomavirus from a Florida Manatee by Using Multiply Primed Rolling-Circle Amplification: Trichechus manatus latirostris Papillomavirus Type 1†

PubMed Central

Rector, Annabel; Bossart, Gregory D.; Ghim, Shin-Je; Sundberg, John P.; Jenson, A. Bennett; Van Ranst, Marc

2004-01-01

By using an isothermal multiply primed rolling-circle amplification protocol, the complete genomic DNA of a novel papillomavirus was amplified from a skin lesion biopsy of a Florida manatee (Trichechus manatus latirostris), one of the most endangered marine mammals in United States coastal waters. The nucleotide sequence, genome organization, and phylogenetic position of the Trichechus manatus latirostris papillomavirus type 1 (TmPV-1) were determined. TmPV-1 is the first virus isolated from the order of Sirenia. A phylogenetic analysis shows that TmPV-1 is only distantly related to other papillomavirus sequences, and it appears in our phylogenetic tree as a novel close-to-root papillomavirus genus. PMID:15507660

Rolling Circle Amplification For Spatially Directed Synthesis Of A Solid Phase Anchored Single-Stranded DNA Molecule

NASA Astrophysics Data System (ADS)

Reiß, Edda; Hölzel, Ralph; von Nickisch-Rosenegk, Markus; Bier, Frank F.

2006-09-01

In this article the usefulness of the enzyme phi29 DNA polymerase and the principle of rolling circle amplification (RCA) for creating single-stranded DNA (ssDNA) nanostructures is described. Currently we are working on the spatial orientation of a growing ssDNA molecule during its RCA-based synthesis by the application of a hydrodynamic force. Starting at an immobilized primer at single molecule level, the aim is to construct a nanostructure of known location and orientation, providing multiple repeating binding sites that can be addressed via complementary base-pairing. Proof-of-principle experiments demonstrate the potential of the enzymatic reaction. ssDNA molecules of more than 20 μm length were created at an immobilized primer and detected by means of fluorescence microscopy.

A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

PubMed Central

Rector, Annabel; Tachezy, Ruth; Van Ranst, Marc

2004-01-01

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with φ29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 × 104-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information. PMID:15113879

Identification of Prostate Cancer-Specific microDNAs

DTIC Science & Technology

2016-02-01

circular DNA by rolling circle amplification (RCA) and then amplified DNA fragments were subject to deep sequencing. Deep sequencing of the...demonstrate the existence of microDNAs in prostate cancer. We adopted multiple displacement amplification (MDA) with random 2 primers for enriched...prostate cancer cells through multiple displacement amplification and next generation sequencing. R e la ti v e c e ll g ro w th ( % ) 0 20

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.

Designing a Micromixer for Rolling Circle Amplification in Cancer Biomarker Detection

NASA Astrophysics Data System (ADS)

Altural, Hayriye

2015-03-01

Rolling circle amplification (RCA) is an alternative method to the Polymerase Chain Reaction based amplification for point-of-care (POC) diagnosis. In future personalized cancer diagnostic for POC applications, smaller, faster and cheaper methods are needed instead of costly and time-consuming laboratory tests. Microfluidic chips can perform the detection of cancer biomarkers within less analysis time, and provide for improvement in the sensitivity and specificity required for biochemical analysis as well. Rapid mixing is essential in the chips used in cancer diagnostic. The goal of this study is to design a micromixer for rapid RCA-based analysis and develop the assay time in cancer biomarker detection. By combining assays with micromixers, multi-step bioreactions in microfluidic chips may be achieved with minimal external control. Here, simulation results related to the micromixer are obtained by COMSOL software. The Scientific and Technological Research Council of Turkey (TUBITAK) is acknowledged for granting of H. Altural postdoctoral study in the framework of TUBITAK-BIDEB 2219-International Postdoctoral Research Scholarship Program.

DNA Hydrogel with Tunable pH-Responsive Properties Produced by Rolling Circle Amplification.

PubMed

Xu, Wanlin; Huang, Yishun; Zhao, Haoran; Li, Pan; Liu, Guoyuan; Li, Jing; Zhu, Chengshen; Tian, Leilei

2017-12-22

Recently, smart DNA hydrogels, which are generally formed by the self-assembly of oligonucleotides or through the cross-linking of oligonucleotide-polymer hybrids, have attracted tremendous attention. However, the difficulties of fabricating DNA hydrogels limit their practical applications. We report herein a novel method for producing pH-responsive hydrogels by rolling circle amplification (RCA). In this method, pH-sensitive cross-linking sites were introduced into the polymeric DNA chains during DNA synthesis. As the DNA sequence can be precisely defined by its template, the properties of such hydrogels can be finely tuned in a very facile way through template design. We have investigated the process of hydrogel formation and pH-responsiveness to provide rationales for functional hydrogel design based on the RCA reaction. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Voltammetric determination of attomolar levels of a sequence derived from the genom of hepatitis B virus by using molecular beacon mediated circular strand displacement and rolling circle amplification.

PubMed

Huang, Shan; Feng, Mengmeng; Li, Jiawen; Liu, Yi; Xiao, Qi

2018-03-03

The authors describe an electrochemical method for the determination of the single-stranded DNA (ssDNA) oligonucleotide with a sequence derived from the genom of hepatitis B virus (HBV). It is making use of circular strand displacement (CSD) and rolling circle amplification (RCA) strategies mediated by a molecular beacon (MB). This ssDNA hybridizes with the loop portion of the MB immobilized on the surface of a gold electrode, while primer DNA also hybridizes with the rest of partial DNA sequences of MB. This triggers the MB-mediated CSD. The RCA is then initiated to produce a long DNA strand with multiple tandem-repeat sequences, and this results in a significant increase of the differential pulse voltammetric response of the electrochemical probe Methylene Blue at a rather low working potential of -0.24 V (vs. Ag/AgCl). Under optimal experimental conditions, the assay displays an ultrahigh sensitivity (with a 2.6 aM detection limit) and excellent selectivity. Response is linear in the 10 to 700 aM DNA concentration range. Graphical abstract Schematic of a voltammetric method for the determination of attomolar levels of target DNA. It is based on molecular beacon mediated circular strand displacement and rolling circle amplification strategies. Under optimal experimental conditions, the assay displays an ultrahigh sensitivity with a 2.6 aM detection limit and excellent selectivity.

L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs)

PubMed Central

Qi, Xiaoquan; Bakht, Saleha; Devos, Katrien M.; Gale, Mike D.; Osbourn, Anne

2001-01-01

A flexible, non-gel-based single nucleotide polymorphism (SNP) detection method is described. The method adopts thermostable ligation for allele discrimination and rolling circle amplification (RCA) for signal enhancement. Clear allelic discrimination was achieved after staining of the final reaction mixtures with Cybr-Gold and visualisation by UV illumination. The use of a compatible buffer system for all enzymes allows the reaction to be initiated and detected in the same tube or microplate well, so that the experiment can be scaled up easily for high-throughput detection. Only a small amount of DNA (i.e. 50 ng) is required per assay, and use of carefully designed short padlock probes coupled with generic primers and probes make the SNP detection cost effective. Biallelic assay by hybridisation of the RCA products with fluorescence dye-labelled probes is demonstrated, indicating that ligation-RCA (L-RCA) has potential for multiplexed assays. PMID:11713336

Controlled Microwave Heating Accelerates Rolling Circle Amplification.

PubMed

Yoshimura, Takeo; Suzuki, Takamasa; Mineki, Shigeru; Ohuchi, Shokichi

2015-01-01

Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same.

An aptasensor for staphylococcus aureus based on nicking enzyme amplification reaction and rolling circle amplification.

PubMed

Xu, Jingguo; Guo, Jia; Maina, Sarah Wanjiku; Yang, Yumeng; Hu, Yimin; Li, Xuanxuan; Qiu, Jiarong; Xin, Zhihong

2018-05-15

An ultra-sensitive aptamer-based biosensor for the detection of staphylococcus aureus was established by adopting the nicking enzyme amplification reaction (NEAR) and the rolling circle amplification (RCA) technologies. Aptamer-probe (AP), containing an aptamer and a probe sequence, was developed to act as the recognition unit of the biosensor, which was specifically bound to S. aureus. The probe was released from AP and initiated into the subsequent DNA amplification reactions where S. aureus was present, converting the detection of S. aureus to the investigation of probe oligonucleotide. The RCA amplification products contained a G-quadruplex motif and formed a three dimensional structure in presence of hemin. The G4/hemin complex showed horseradish peroxidase (HRP)-mimic activity and catalyzed the chemiluminescence reaction of luminol mediated by H 2 O 2 . The results showed that the established biosensor could detect S. aureus specifically with a good linear correlation at 5-10 4  CFU/mL. The signal values based on NEAR-RCA two-step cycle were boosted acutely, much higher than that relied on one-cycle magnification. The limit of detection (LoD) was determined to be as low as 5 CFU/mL. The established aptasensor exhibited a good discrimination of living against dead S. aureus, and can be applied to detect S. aureus in the food industry. Copyright © 2018 Elsevier Inc. All rights reserved.

Cascade Signal Amplification Based on Copper Nanoparticle-Reported Rolling Circle Amplification for Ultrasensitive Electrochemical Detection of the Prostate Cancer Biomarker.

PubMed

Zhu, Ye; Wang, Huijuan; Wang, Lin; Zhu, Jing; Jiang, Wei

2016-02-03

An ultrasensitive and highly selective electrochemical assay was first attempted by combining the rolling circle amplification (RCA) reaction with poly(thymine)-templated copper nanoparticles (CuNPs) for cascade signal amplification. As proof of concept, prostate specific antigen (PSA) was selected as a model target. Using a gold nanoparticle (AuNP) as a carrier, we synthesized the primer-AuNP-aptamer bioconjugate for signal amplification by increasing the primer/aptamer ratio. The specific construction of primer-AuNP-aptamer/PSA/anti-PSA sandwich structure triggered the effective RCA reaction, in which thousands of tandem poly(thymine) repeats were generated and directly served as the specific templates for the subsequent CuNP formation. The signal readout was easily achieved by dissolving the RCA product-templated CuNPs and detecting the released copper ions with differential pulse stripping voltammetry. Because of the designed cascade signal amplification strategy, the newly developed method achieved a linear range of 0.05-500 fg/mL, with a remarkable detection limit of 0.020 ± 0.001 fg/mL PSA. Finally, the feasibility of the developed method for practical application was investigated by analyzing PSA in the real clinical human serum samples. The ultrasensitivity, specificity, convenience, and capability for analyzing the clinical samples demonstrate that this method has great potential for practical disease diagnosis applications.

Ultrasensitive detection of nucleic acids and proteins using quartz crystal microbalance and surface plasmon resonance sensors based on target-triggering multiple signal amplification strategy.

PubMed

Sun, Wenbo; Song, Weiling; Guo, Xiaoyan; Wang, Zonghua

2017-07-25

In this study, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) sensors were combined with template enhanced hybridization processes (TEHP), rolling circle amplification (RCA) and biocatalytic precipitation (BCP) for ultrasensitive detection of DNA and protein. The DNA complementary to the aptamer was released by the specific binding of the aptamer to the target protein and then hybridized with the capture probe and the assistant DNA to form a ternary "Y" junction structure. The initiation chain was generated by the template-enhanced hybridization process which leaded to the rolling circle amplification reaction, and a large number of repeating unit sequences were formed. Hybridized with the enzyme-labeled probes, the biocatalytic precipitation reaction was further carried out, resulting in a large amount of insoluble precipitates and amplifying the detection signal. Under the optimum conditions, detection limits as low as 43 aM for target DNA and 53 aM for lysozyme were achieved. In addition, this method also showed good selectivity and sensitivity in human serum. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular identification of Neofabraea species associated with bull's-eye rot on apple using rolling-circle amplification of partial EF-1α sequence.

PubMed

Lin, Huijiao; Jiang, Xiang; Yi, Jianping; Wang, Xinguo; Zuo, Ranling; Jiang, Zide; Wang, Weifang; Zhou, Erxun

2018-01-01

A rolling-circle amplification (RCA) method with padlock probes targeted on EF-1α regions was developed for rapid detection of apple bull's-eye rot pathogens, including Neofabraea malicorticis, N. perennans, N. kienholzii, and N. vagabunda (synonym: N. alba). Four padlock probes (PLP-Nm, PLP-Np, PLP-Nk, and PLP-Nv) were designed and tested against 28 samples, including 22 BER pathogen cultures, 4 closely related species, and 2 unrelated species that may cause serious apple decays. The assay successfully identified all the bull's-eye rot pathogenic fungi at the level of species, while no cross-reaction was observed in all target species and no false-positive reaction was observed with all strains used for reference. This study showed that the use of padlock probes and the combination of probe signal amplification by RCA provided an effective and sensitive method for the rapid identification of Neofabraea spp. The method could therefore be a useful tool for monitoring bull's-eye rot pathogens in port quarantine and orchard epidemiological studies.

NanoCluster Beacons as reporter probes in rolling circle enhanced enzyme activity detection

NASA Astrophysics Data System (ADS)

Juul, Sissel; Obliosca, Judy M.; Liu, Cong; Liu, Yen-Liang; Chen, Yu-An; Imphean, Darren M.; Knudsen, Birgitta R.; Ho, Yi-Ping; Leong, Kam W.; Yeh, Hsin-Chih

2015-04-01

As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level, Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites, Plasmodium sp. Current REEAD assays rely on organic dye-tagged linear DNA probes to report the rolling circle amplification products (RCPs), the cost of which may hinder the widespread use of REEAD. Here we show that a new class of activatable probes, NanoCluster Beacons (NCBs), can simplify the REEAD assays. Easily prepared without any need for purification and capable of large fluorescence enhancement upon hybridization, NCBs are cost-effective and sensitive. Compared to conventional fluorescent probes, NCBs are also more photostable. As demonstrated in reporting the human topoisomerases I (hTopI) cleavage-ligation reaction, the proposed NCBs suggest a read-out format attractive for future REEAD-based diagnostics.As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level, Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites, Plasmodium sp. Current REEAD assays rely on organic dye-tagged linear DNA probes to report the rolling circle amplification products (RCPs), the cost of which may hinder the widespread use of REEAD. Here we show that a new class of activatable probes, NanoCluster Beacons (NCBs), can simplify the REEAD assays. Easily prepared without any need for purification and capable of large fluorescence enhancement upon hybridization, NCBs are cost-effective and sensitive. Compared to conventional fluorescent probes, NCBs are also more photostable. As demonstrated in reporting the human topoisomerases I (hTopI) cleavage-ligation reaction, the proposed NCBs suggest a read-out format attractive for future REEAD-based diagnostics. Electronic supplementary information (ESI) available: The detailed steps of NCB preparation, REEAD assay and STEM imaging. The sequences of the sNCB and the REEAD substrate. See DOI: 10.1039/c5nr01705j

A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification.

PubMed

Li, Xia; Song, Juan; Xue, Qing-Wang; You, Fu-Heng; Lu, Xia; Kong, Yan-Cong; Ma, Shu-Yi; Jiang, Wei; Li, Chen-Zhong

2016-10-21

Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of "G-quadruplex" in lantern-like structures. Finally, the continuously enriched "G-quadruplex lanterns" were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10 -17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

PubMed Central

Li, Xia; Song, Juan; Xue, Qing-Wang; You, Fu-Heng; Lu, Xia; Kong, Yan-Cong; Ma, Shu-Yi; Jiang, Wei; Li, Chen-Zhong

2016-01-01

Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of “G-quadruplex” in lantern-like structures. Finally, the continuously enriched “G-quadruplex lanterns” were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10−17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring. PMID:28335318

A cascade signal amplification strategy for surface enhanced Raman spectroscopy detection of thrombin based on DNAzyme assistant DNA recycling and rolling circle amplification.

PubMed

Gao, Fenglei; Du, Lili; Tang, Daoquan; Lu, Yao; Zhang, Yanzhuo; Zhang, Lixian

2015-04-15

A sensitive protocol for surface enhanced Raman spectroscopy (SERS) detection of thrombin is designed with R6G-Ag NPs as a signal tag by combining DNAzyme assistant DNA recycling and rolling circle amplification (RCA). Molecular beacon (MB) as recognition probe immobilizes on the glass slides and performs the amplification procedure. After thrombin-induced structure-switching DNA hairpins of probe 1, the DNAzyme is liberated from the caged structure, which hybridizes with the MB for cleavage of the MB in the presence of cofactor Zn(2+) and initiates the DNA recycling process, leading to the cleavage of a large number of MB and the generation of numerous primers for triggering RCA reaction. The long amplified RCA product which contained hundreds of tandem-repeat sequences, which can bind with oligonucleotide functionalized Ag NPs reporters. The attached signal tags can be easily read out by SERS. Because of the cascade signal amplification, these newly designed protocols provides a sensitive SERS detection of thrombin down to the femolar level (2.3fM) with a linear range of 5 orders of magnitude (from 10(-14) to 10(-9)M) and have high selectivity toward its target protein. The proposed method is expected to be a good clinical tool for the diagnosis of a thrombotic disease. Copyright © 2014 Elsevier B.V. All rights reserved.

Multiplexed protein profiling on microarrays by rolling-circle amplification

PubMed Central

Schweitzer, Barry; Roberts, Scott; Grimwade, Brian; Shao, Weiping; Wang, Minjuan; Fu, Qin; Shu, Quiping; Laroche, Isabelle; Zhou, Zhimin; Tchernev, Velizar T.; Christiansen, Jason; Velleca, Mark; Kingsmore, Stephen F.

2010-01-01

Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1β, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-α treatment. Because microarrays can accommodat ~1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys. PMID:11923841

Immobilized rolling circle amplification on extended-gate field-effect transistors with integrated readout circuits for early detection of platelet-derived growth factor.

PubMed

Lin, Ming-Yu; Hsu, Wen-Yang; Yang, Yuh-Shyong; Huang, Jo-Wen; Chung, Yueh-Lin; Chen, Hsin

2016-07-01

Detection of tumor-related proteins with high specificity and sensitivity is important for early diagnosis and prognosis of cancers. While protein sensors based on antibodies are not easy to keep for a long time, aptamers (single-stranded DNA) are found to be a good alternative for recognizing tumor-related protein specifically. This study investigates the feasibility of employing aptamers to recognize the platelet-derived growth factor (PDGF) specifically and subsequently triggering rolling circle amplification (RCA) of DNAs on extended-gate field-effect transistors (EGFETs) to enhance the sensitivity. The EGFETs are fabricated by the standard CMOS technology and integrated with readout circuits monolithically. The monolithic integration not only avoids the wiring complexity for a large sensor array but also enhances the sensor reliability and facilitates massive production for commercialization. With the RCA primers immobilized on the sensory surface, the protein signal is amplified as the elongation of DNA, allowing the EGFET to achieve a sensitivity of 8.8 pM, more than three orders better than that achieved by conventional EGFETs. Moreover, the responses of EGFETs are able to indicate quantitatively the reaction rates of RCA, facilitating the estimation on the protein concentration. Our experimental results demonstrate that immobilized RCA on EGFETs is a useful, label-free method for early diagnosis of diseases related to low-concentrated tumor makers (e.g., PDGF) for serum sample, as well as for monitoring the synthesis of various DNA nanostructures in real time. Graphical Abstract The tumor-related protein, PDGF, is detected by immobilizing rolling circle amplification on an EGFET with integrated readout circuit.

Repressor logic modules assembled by rolling circle amplification platform to construct a set of logic gates

PubMed Central

Wei, Hua; Hu, Bo; Tang, Suming; Zhao, Guojie; Guan, Yifu

2016-01-01

Small molecule metabolites and their allosterically regulated repressors play an important role in many gene expression and metabolic disorder processes. These natural sensors, though valuable as good logic switches, have rarely been employed without transcription machinery in cells. Here, two pairs of repressors, which function in opposite ways, were cloned, purified and used to control DNA replication in rolling circle amplification (RCA) in vitro. By using metabolites and repressors as inputs, RCA signals as outputs, four basic logic modules were constructed successfully. To achieve various logic computations based on these basic modules, we designed series and parallel strategies of circular templates, which can further assemble these repressor modules in an RCA platform to realize twelve two-input Boolean logic gates and a three-input logic gate. The RCA-output and RCA-assembled platform was proved to be easy and flexible for complex logic processes and might have application potential in molecular computing and synthetic biology. PMID:27869177

Controlled Microwave Heating Accelerates Rolling Circle Amplification

PubMed Central

Yoshimura, Takeo; Suzuki, Takamasa; Mineki, Shigeru; Ohuchi, Shokichi

2015-01-01

Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same. PMID:26348227

Homogeneous and label-free detection of microRNAs using bifunctional strand displacement amplification-mediated hyperbranched rolling circle amplification.

PubMed

Zhang, Li-rong; Zhu, Guichi; Zhang, Chun-yang

2014-07-01

MicroRNAs (miRNAs) are an emerging class of biomarkers and therapeutic targets for various diseases including cancers. Here, we develop a homogeneous and label-free method for sensitive detection of let-7a miRNA based on bifunctional strand displacement amplification (SDA)-mediated hyperbranched rolling circle amplification (HRCA). The binding of target miRNA with the linear template initiates the bifunctional SDA reaction, generating two different kinds of triggers which can hybridize with the linear template to initiate new rounds of SDA reaction for the production of more and more triggers. In the meantime, the released two different kinds of triggers can function as the first and the second primers, respectively, to initiate the HRCA reaction whose products can be simply monitored by a standard fluorometer with SYBR Green I as the fluorescent indicator. The proposed method exhibits high sensitivity with a detection limit of as low as 1.8 × 10(-13) M and a large dynamic range of 5 orders of magnitude from 0.1 pM to 10 nM, and it can even discriminate the single-base difference among the miRNA family members. Moreover, this method can be used to analyze the total RNA samples from the human lung tissues and might be further applied for sensitive detection of various proteins, small molecules, and metal ions in combination with specific aptamers.

Novel One-Tube-One-Step Real-Time Methodology for Rapid Transcriptomic Biomarker Detection: Signal Amplification by Ternary Initiation Complexes.

PubMed

Fujita, Hiroto; Kataoka, Yuka; Tobita, Seiji; Kuwahara, Masayasu; Sugimoto, Naoki

2016-07-19

We have developed a novel RNA detection method, termed signal amplification by ternary initiation complexes (SATIC), in which an analyte sample is simply mixed with the relevant reagents and allowed to stand for a short time under isothermal conditions (37 °C). The advantage of the technique is that there is no requirement for (i) heat annealing, (ii) thermal cycling during the reaction, (iii) a reverse transcription step, or (iv) enzymatic or mechanical fragmentation of the target RNA. SATIC involves the formation of a ternary initiation complex between the target RNA, a circular DNA template, and a DNA primer, followed by rolling circle amplification (RCA) to generate multiple copies of G-quadruplex (G4) on a long DNA strand like beads on a string. The G4s can be specifically fluorescence-stained with N(3)-hydroxyethyl thioflavin T (ThT-HE), which emits weakly with single- and double-stranded RNA/DNA but strongly with parallel G4s. An improved dual SATIC system, which involves the formation of two different ternary initiation complexes in the RCA process, exhibited a wide quantitative detection range of 1-5000 pM. Furthermore, this enabled visual observation-based RNA detection, which is more rapid and convenient than conventional isothermal methods, such as reverse transcription-loop-mediated isothermal amplification, signal mediated amplification of RNA technology, and RNA-primed rolling circle amplification. Thus, SATIC methodology may serve as an on-site and real-time measurement technique for transcriptomic biomarkers for various diseases.

RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection.

PubMed

Takahashi, Hirokazu; Ohkawachi, Masahiko; Horio, Kyohei; Kobori, Toshiro; Aki, Tsunehiro; Matsumura, Yukihiko; Nakashimada, Yutaka; Okamura, Yoshiko

2018-05-17

RNA-primed rolling circle amplification (RPRCA) is a useful laboratory method for RNA detection; however, the detection of RNA is limited by the lack of information on 3'-terminal sequences. We uncovered that conventional RPRCA using pre-circularized probes could potentially detect the internal sequence of target RNA molecules in combination with RNase H. However, the specificity for mRNA detection was low, presumably due to non-specific hybridization of non-target RNA with the circular probe. To overcome this technical problem, we developed a method for detecting a sequence of interest in target RNA molecules via RNase H-assisted RPRCA using padlocked probes. When padlock probes are hybridized to the target RNA molecule, they are converted to the circular form by SplintR ligase. Subsequently, RNase H creates nick sites only in the hybridized RNA sequence, and single-stranded DNA is finally synthesized from the nick site by phi29 DNA polymerase. This method could specifically detect at least 10 fmol of the target RNA molecule without reverse transcription. Moreover, this method detected GFP mRNA present in 10 ng of total RNA isolated from Escherichia coli without background DNA amplification. Therefore, this method can potentially detect almost all types of RNA molecules without reverse transcription and reveal full-length sequence information.

Ultrasensitive electrochemical detection of nucleic acids by template enhanced hybridization followed with rolling circle amplification.

PubMed

Ji, Hanxu; Yan, Feng; Lei, Jianping; Ju, Huangxian

2012-08-21

An ultrasensitive protocol for electrochemical detection of DNA is designed with quantum dots (QDs) as a signal tag by combining the template enhanced hybridization process (TEHP) and rolling circle amplification (RCA). Upon the recognition of the molecular beacon (MB) to target DNA, the MB hybridizes with assistants and target DNA to form a ternary ''Y-junction''. The target DNA can be dissociated from the structure under the reaction of nicking endonuclease to initiate the next hybridization process. The template enhanced MB fragments further act as the primers of the RCA reaction to produce thousands of repeated oligonucleotide sequences, which can bind with oligonucleotide functionalized QDs. The attached signal tags can be easily read out by square-wave voltammetry after dissolving with acid. Because of the cascade signal amplification and the specific TEHP and RCA reaction, this newly designed protocol provides an ultrasensitive electrochemical detection of DNA down to the attomolar level (11 aM) with a linear range of 6 orders of magnitude (from 1 × 10(-17) to 1 × 10(-11) M) and can discriminate mismatched DNA from perfect matched target DNA with high selectivity. The high sensitivity and specificity make this method a great potential for early diagnosis in gene-related diseases.

Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

PubMed Central

Huovinen, Tuomas; Brockmann, Eeva-Christine; Akter, Sultana; Perez-Gamarra, Susan; Ylä-Pelto, Jani; Liu, Yuan; Lamminmäki, Urpo

2012-01-01

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material. PMID:22355397

In situ rolling circle amplification detection of Crimean Congo hemorrhagic fever virus (CCHFV) complementary and viral RNA.

PubMed

Andersson, Cecilia; Henriksson, Sara; Magnusson, Karl-Eric; Nilsson, Mats; Mirazimi, Ali

2012-05-10

Crimean Congo hemorrhagic fever virus (CCHFV) is a human pathogen that causes a severe disease with high fatality rate for which there is currently no specific treatment. Knowledge regarding its replication cycle is also highly limited. In this study we developed an in situ technique for studying the different stages during the replication of CCHFV. By integrating reverse transcription, padlock probes, and rolling circle amplification, we were able to detect and differentiate between viral RNA (vRNA) and complementary RNA (cRNA) molecules, and to detect viral protein within the same cell. These data demonstrate that CCHFV nucleocapsid protein (NP) is detectable already at 6 hours post infection in vRNA- and cRNA-positive cells. Confocal microscopy showed that cRNA is enriched and co-localized to a large extent with NP in the perinuclear area, while vRNA has a more random distribution in the cytoplasm with only some co-localize with NP. However, vRNA and cRNA did not appear to co-localize directly. Copyright © 2012 Elsevier Inc. All rights reserved.

Combining isothermal rolling circle amplification and electrochemiluminescence for highly sensitive point mutation detection

NASA Astrophysics Data System (ADS)

Su, Qiang; Zhou, Xiaoming

2008-12-01

Many pathogenic and genetic diseases are associated with changes in the sequence of particular genes. We describe here a rapid and highly efficient assay for the detection of point mutation. This method is a combination of isothermal rolling circle amplification (RCA) and high sensitive electrochemluminescence (ECL) detection. In the design, a circular template generated by ligation upon the recognition of a point mutation on DNA targets was amplified isothermally by the Phi29 polymerase using a biotinylated primer. The elongation products were hybridized with tris (bipyridine) ruthenium (TBR)-tagged probes and detected in a magnetic bead based ECL platform, indicating the mutation occurrence. P53 was chosen as a model for the identification of this method. The method allowed sensitive determination of the P53 mutation from wild-type and mutant samples. The main advantage of RCA-ECL is that it can be performed under isothermal conditions and avoids the generation of false-positive results. Furthermore, ECL provides a faster, more sensitive, and economical option to currently available electrophoresis-based methods.

A valveless rotary microfluidic device for multiplex point mutation identification based on ligation-rolling circle amplification.

PubMed

Heo, Hyun Young; Chung, Soyi; Kim, Yong Tae; Kim, Do Hyun; Seo, Tae Seok

2016-04-15

Genetic variations such as single nucleotide polymorphism (SNP) and point mutations are important biomarkers to monitor disease prognosis and diagnosis. In this study, we developed a novel rotary microfluidic device which can perform multiplex SNP typing on the mutation sites of TP53 genes. The microdevice consists of three glass layers: a channel wafer, a Ti/Pt electrode-patterned resistance temperature detector (RTD) wafer, and a rotary plate in which twelve reaction chambers were fabricated. A series of sample injection, ligation-rolling circle amplification (L-RCA) reaction, and fluorescence detection of the resultant amplicons could be executed by rotating the top rotary plate, identifying five mutation points related with cancer prognosis. The use of the rotary plate eliminates the necessity of microvalves and micropumps to control the microfluidic flow in the channel, simplifying the chip design and chip operation for multiplex SNP detection. The proposed microdevice provides an advanced genetic analysis platform in terms of multiplexity, simplicity, and portability in the fields of biomedical diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

In Situ Detection of Anaplasma spp. by DNA Target-Primed Rolling-Circle Amplification of a Padlock Probe and Intracellular Colocalization with Immunofluorescently Labeled Host Cell von Willebrand Factor ▿

PubMed Central

Wamsley, Heather L.; Barbet, Anthony F.

2008-01-01

Endothelial cell culture and preliminary immunofluorescent staining of Anaplasma-infected tissues suggest that endothelial cells may be an in vivo nidus of mammalian infection. To investigate endothelial cells and other potentially cryptic sites of Anaplasma sp. infection in mammalian tissues, a sensitive and specific isothermal in situ technique to detect localized Anaplasma gene sequences by using rolling-circle amplification of circularizable, linear, oligonucleotide probes (padlock probes) was developed. Cytospin preparations of uninfected or Anaplasma-infected cell cultures were examined using this technique. Via fluorescence microscopy, the technique described here, and a combination of differential interference contrast microscopy and von Willebrand factor immunofluorescence, Anaplasma phagocytophilum and Anaplasma marginale were successfully localized in situ within intact cultured mammalian cells. This work represents the first application of this in situ method for the detection of a microorganism and forms the foundation for future applications of this technique to detect, localize, and analyze Anaplasma nucleotide sequences in the tissues of infected mammalian and arthropod hosts and in cell cultures. PMID:18495855

Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules

PubMed Central

Kühnemund, Malte; Hernández-Neuta, Iván; Sharif, Mohd Istiaq; Cornaglia, Matteo; Gijs, Martin A.M.

2017-01-01

Abstract Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope. PMID:28077562

Exponential isothermal amplification of nucleic acids and amplified assays for proteins, cells, and enzyme activities.

PubMed

Reid, Michael S; Le, X Chris; Zhang, Hongquan

2018-04-27

Isothermal exponential amplification techniques, such as strand-displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA), have great potential for on-site, point-of-care, and in-situ assay applications. These amplification techniques eliminate the need for temperature cycling required for polymerase chain reaction (PCR) while achieving comparable amplification yield. We highlight here recent advances in exponential amplification reaction (EXPAR) for the detection of nucleic acids, proteins, enzyme activities, cells, and metal ions. We discuss design strategies, enzyme reactions, detection techniques, and key features. Incorporation of fluorescence, colorimetric, chemiluminescence, Raman, and electrochemical approaches enables highly sensitive detection of a variety of targets. Remaining issues, such as undesirable background amplification resulting from non-specific template interactions, must be addressed to further improve isothermal and exponential amplification techniques. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules.

PubMed

Kühnemund, Malte; Hernández-Neuta, Iván; Sharif, Mohd Istiaq; Cornaglia, Matteo; Gijs, Martin A M; Nilsson, Mats

2017-05-05

Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Diffractometric Detection of Proteins using Microbead-based Rolling Circle Amplification

PubMed Central

Lee, Joonhyung; Icoz, Kutay; Roberts, Ana; Ellington, Andrew D.; Savran, Cagri A.

2010-01-01

We present a robust, sensitive, fluorescent or radio label-free self-assembled optical diffraction biosensor that utilizes rolling circle amplification (RCA) and magnetic microbeads as a signal enhancement method. An aptamer-based sandwich assay was performed on microcontact-printed streptavidin arranged in 15-μm-wide alternating lines, and could specifically capture and detect platelet-derived growth factor B-chain (PDGF-BB). An aptamer served as a template for the ligation of a padlock probe and the circularized probe could in turn be used as a template for RCA. The concatameric RCA product hybridized to biotinylated oligonuclotides which then captured streptavidin-labeled magnetic beads. In consequence, the signal from the captured PDGF-BB was amplified via the concatameric RCA product, and the diffraction gratings on the printed areas produced varying intensities of diffraction modes. The detected diffraction intensity and the density of the microbeads on the surface varied as a function of PDGF-BB concentration. Our results demonstrate a robust biosensing platform that is easy to construct and use, and devoid of fluorescence microscopy. The self-assembled bead patterns allow both a visual analysis of the molecular binding events under an ordinary bright-field microscope and serve as a diffraction grating biosensor. PMID:19947589

A cascade amplification strategy based on rolling circle amplification and hydroxylamine amplified gold nanoparticles enables chemiluminescence detection of adenosine triphosphate.

PubMed

Wang, Ping; Zhang, Tonghuan; Yang, Taoyi; Jin, Nan; Zhao, Yanjun; Fan, Aiping

2014-08-07

A highly sensitive and selective chemiluminescent (CL) biosensor for adenosine triphosphate (ATP) was developed by taking advantage of the ATP-dependent enzymatic reaction (ATP-DER), the powerful signal amplification capability of rolling circle amplification (RCA), and hydroxylamine-amplified gold nanoparticles (Au NPs). The strategy relies on the ability of ATP, a cofactor of T4 DNA ligase, to trigger the ligation-RCA reaction. In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction between the two ends of the padlock probe, producing a closed circular DNA template that initiates the RCA reaction with phi29 DNA polymerase and dNTP. Therein, many complementary copies of the circular template can be generated. The ATP-DER is eventually converted into a detectable CL signal after a series of processes, including gold probe hybridization, hydroxylamine amplification, and oxidative gold metal dissolution coupled with a simple and sensitive luminol CL reaction. The CL signal is directly proportional to the ATP level. The results showed that the detection limit of the assay is 100 pM of ATP, which compares favorably with those of other ATP detection techniques. In addition, by taking advantage of ATP-DER, the proposed CL sensing system exhibits extraordinary specificity towards ATP and could distinguish the target molecule ATP from its analogues. The proposed method provides a new and versatile platform for the design of novel DNA ligation reaction-based CL sensing systems for other cofactors. This novel ATP-DER based CL sensing system may find wide applications in clinical diagnosis as well as in environmental and biomedical fields.

Comparison of the rolling circle amplification and ligase-dependent reaction methods for the identification of opportunistic Exophiala species.

PubMed

Kaplan, Engin; Ilkit, Macit; de Hoog, G Sybren

2017-10-26

We developed two ligase-dependent probe amplification assays based on rolling circle amplification (RCA) and the ligase-dependent reaction (LDR) to differentiate species of Exophiala targeting the rDNA internal transcribed spacer region. We focused on Exophiala dermatitidis and E. phaeomuriformis, two opportunistic inhabitants of indoor wet cells, and further detected E. heteromorpha, E. xenobiotica, and E. crusticola; 57 reference isolates representing the five species were tested. Depending on the RCA probes used, the sensitivity was 100%, and the specificity ranged from 3.7% to 88.6% (median: 46.1%). In contrast, the sensitivity and specificity of the LDR probes targeting the same isolates were 88.6-100% (median: 95.8%) and 95.4-100% (median: 97.7%), respectively. We analyzed 198 additional environmental isolates representing the same Exophiala species. Overall, the sensitivity and specificity of LDR ranged from 89.7% to 100% (median: 94.1%) and from 93.9% to 100% (median: 96.9%), respectively. The assessment of performance and validation of LDR probes using SYBR Green quantitative polymerase chain reaction revealed high reproducibility and an acceptable range limit, in line with the guidelines of the European Network of GMO Laboratories. In conclusion, the LDR assay was more reliable and less expensive than RCA for species-level identification of Exophiala isolates. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Nucleic acid arrays and methods of synthesis

DOEpatents

Sabanayagam, Chandran R.; Sano, Takeshi; Misasi, John; Hatch, Anson; Cantor, Charles

2001-01-01

The present invention generally relates to high density nucleic acid arrays and methods of synthesizing nucleic acid sequences on a solid surface. Specifically, the present invention contemplates the use of stabilized nucleic acid primer sequences immobilized on solid surfaces, and circular nucleic acid sequence templates combined with the use of isothermal rolling circle amplification to thereby increase nucleic acid sequence concentrations in a sample or on an array of nucleic acid sequences.

Fluorescence biosensor for inorganic pyrophosphatase activity.

PubMed

Zhang, Ying; Guo, Yajuan; Zhao, Mengmeng; Lin, Cuiying; Lin, Zhenyu; Luo, Fang; Chen, Guonan

2017-02-01

A highly sensitive and selective fluorescence biosensor for inorganic pyrophosphatase (PPase) activity has been developed based on special click ligation trigger hyperbranched rolling circle amplification (CLT-HRCA). Pyrophosphate ion (PPi) can coordinate with Cu 2+ to form stable PPi/Cu 2+ complex and Cu 2+ in the complex cannot be reduced to Cu + . The addition of PPase causes the hydrolysis of PPi into orthophosphate (Pi) and therefore induces the releasing of Cu 2+ from the stable PPi/Cu 2+ complex, and the free Cu 2+ is easily reduced to Cu + by sodium ascorbate. Then Cu + catalyzes the cyclization reaction between the specially designed 5'-azide and 3'-alkyne tagged padlock probes through Cu + catalyzed azide-alkyne cycloaddition (CuAAC), which in turn initiates the hyperbranched rolling circle amplification (HRCA). Given that the CLT-HRCA products contain large amounts of double-stranded DNAs (dsDNAs), the addition of SYBR Green I resulted in the enhanced fluorescence signal. There was a linear relationship between the enhanced fluorescence intensity and the logarithm PPase activity ranging from 0.05 to 25 mU with a detection limit of 0.02 mU. Such proposed biosensor has been successfully applied to screen the potential PPase inhibitors and has accessed the related inhibit ability with high efficiency.

Characterization of Two Novel Polyomaviruses of Birds by Using Multiply Primed Rolling-Circle Amplification of Their Genomes

PubMed Central

Johne, Reimar; Wittig, Walter; Fernández-de-Luco, Daniel; Höfle, Ursula; Müller, Hermann

2006-01-01

Polyomaviruses are small nonenveloped particles with a circular double-stranded genome, approximately 5 kbp in size. The mammalian polyomaviruses mainly cause persistent subclinical infections in their natural nonimmunocompromised hosts. In contrast, the polyomaviruses of birds—avian polyomavirus (APV) and goose hemorrhagic polyomavirus (GHPV)—are the primary agents of acute and chronic disease with high mortality rates in young birds. Screening of field samples of diseased birds by consensus PCR revealed the presence of two novel polyomaviruses in the liver of an Eurasian bullfinch (Pyrrhula pyrrhula griseiventris) and in the spleen of a Eurasian jackdaw (Corvus monedula), tentatively designated as finch polyomavirus (FPyV) and crow polyomavirus (CPyV), respectively. The genomes of the viruses were amplified by using multiply primed rolling-circle amplification and cloned. Analysis of the FPyV and CPyV genome sequences revealed a close relationship to APV and GHPV, indicating the existence of a distinct avian group among the polyomaviruses. The main characteristics of this group are (i) involvement in fatal disease, (ii) the existence of an additional open reading frame in the 5′ region of the late mRNAs, and (iii) a different manner of DNA binding of the large tumor antigen compared to that of the mammalian polyomaviruses. PMID:16537620

Double-probe signal enhancing strategy for toxin aptasensing based on rolling circle amplification.

PubMed

Tong, Ping; Zhao, Wei-Wei; Zhang, Lan; Xu, Jing-Juan; Chen, Hong-Yuan

2012-03-15

On the basis of aptamer-based rolling circle amplification (RCA) and magnetic beads (MBs), a highly sensitive electrochemical method was developed for the determination of Ochratoxin A (OTA). Initially, an amino-modified capture DNA was immobilized onto MBs for the following hybridization with an OTA aptamer and a phosphate labeled padlock DNA. In the presence of OTA, the aptamer would dissociate from the bioconjugate, and the padlock DNA would subsequently hybridize with the capture DNA to form a circular template with the aid of the T4 ligase. Next, capture DNA would act as primer to initiate a linear RCA reaction and hence generate a long tandem repeated sequences by phi29 DNA polymerase and dNTPs. Then, two quantum dots (QDs) labeled DNA probes were tagged on the resulted RCA product to indicate the OTA recognition event by electrochemical readout. This strategy, based on the novel design of OTA-mediated DNA circularization, the combination of RCA and double signal probes introduction, could detect OTA down to the level of 0.2 pg mL(-1) with a dynamic range spanning more than 4 orders of magnitude. The proposed approach is tested to determine OTA in red wines and shows good application potential in real samples. Copyright © 2011 Elsevier B.V. All rights reserved.

Ferrocenemonocarboxylic-HRP@Pt nanoparticles labeled RCA for multiple amplification of electro-immunosensing.

PubMed

Su, Huilan; Yuan, Ruo; Chai, Yaqin; Mao, Li; Zhuo, Ying

2011-07-15

A multiple amplification immunoassay was proposed to detect alpha-fetoprotein (AFP), which was based on ferrocenemonocarboxylic-HRP conjugated on Pt nanoparticles as labels for rolling circle amplification (RCA). Firstly, the capture antibody (anti-AFP) was immobilized on glass carbon electrode (GCE) deposited nano-sized gold particles. After a typical immuno-sandwich protocol, primary DNA was immobilized by labeling secondary antibody, which acted as a precursor to initiate RCA. The products of RCA provide large amount of sites to link detection DNAs, which were labeled by signal probes (ferrocenemonocarboxylic) and horseradish peroxidase (HRP). Moreover, the enzymatic amplification signals could be produced by the catalysis of HRP and Pt nanoparticles with the addition of H₂O₂. These lead to multiple amplification signals monitoring by electrochemical instrument and further resulted in high sensitivity of the immunoassay with the detection limit of 1.7 pg/mL. Copyright © 2011 Elsevier B.V. All rights reserved.

Rolling circle amplification-based analysis of Sri Lankan cassava mosaic virus isolates from Tamil Nadu, India, suggests a low level of genetic variability.

PubMed

Kushawaha, Akhilesh Kumar; Rabindran, Ramalingam; Dasgupta, Indranil

2018-03-01

Cassava mosaic disease is a widespread disease of cassava in south Asia and the African continent. In India, CMD is known to be caused by two single-stranded DNA viruses (geminiviruses), Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosdaic virus (SLCMV). Previously, the diversity of ICMV and SLCMV in India has been studied using PCR, a sequence-dependent method. To have a more in-depth study of the variability of the above viruses and to detect any novel geminiviruses associated with CMD, sequence-independent amplification using rolling circle amplification (RCA)-based methods were used. CMD affected cassava plants were sampled across eighty locations in nine districts of the southern Indian state of Tamil Nadu. Twelve complete sequence of coat protein genes of the resident geminiviruses, comprising 256 amino acid residues were generated from the above samples, which indicated changes at only six positions. RCA followed by RFLP of the 80 samples indicated that most samples (47) contained only SLCMV, followed by 8, which were infected jointly with ICMV and SLCMV. In 11 samples, the pattern did not match the expected patterns from either of the two viruses and hence, were variants. Sequence analysis of an average of 700 nucleotides from 31 RCA-generated fragments of the variants indicated identities of 97-99% with the sequence of a previously reported infectious clone of SLCMV. The evidence suggests low levels of genetic variability in the begomoviruses infecting cassava, mainly in the form of scattered single nucleotide changes.

Rolling Circle Translation of Circular RNA in Living Human Cells.

PubMed

Abe, Naoko; Matsumoto, Ken; Nishihara, Mizuki; Nakano, Yukiko; Shibata, Aya; Maruyama, Hideto; Shuto, Satoshi; Matsuda, Akira; Yoshida, Minoru; Ito, Yoshihiro; Abe, Hiroshi

2015-11-10

We recently reported that circular RNA is efficiently translated by a rolling circle amplification (RCA) mechanism in a cell-free Escherichia coli translation system. Recent studies have shown that circular RNAs composed of exonic sequences are abundant in human cells. However, whether these circular RNAs can be translated into proteins within cells remains unclear. In this study, we prepared circular RNAs with an infinite open reading frame and tested their translation in eukaryotic systems. Circular RNAs were translated into long proteins in rabbit reticulocyte lysate in the absence of any particular element for internal ribosome entry, a poly-A tail, or a cap structure. The translation systems in eukaryote can accept much simpler RNA as a template for protein synthesis by cyclisation. Here, we demonstrated that the circular RNA is efficiently translated in living human cells to produce abundant protein product by RCA mechanism. These findings suggest that translation of exonic circular RNAs present in human cells is more probable than previously thought.

Rolling Circle Translation of Circular RNA in Living Human Cells

PubMed Central

Abe, Naoko; Matsumoto, Ken; Nishihara, Mizuki; Nakano, Yukiko; Shibata, Aya; Maruyama, Hideto; Shuto, Satoshi; Matsuda, Akira; Yoshida, Minoru; Ito, Yoshihiro; Abe, Hiroshi

2015-01-01

We recently reported that circular RNA is efficiently translated by a rolling circle amplification (RCA) mechanism in a cell-free Escherichia coli translation system. Recent studies have shown that circular RNAs composed of exonic sequences are abundant in human cells. However, whether these circular RNAs can be translated into proteins within cells remains unclear. In this study, we prepared circular RNAs with an infinite open reading frame and tested their translation in eukaryotic systems. Circular RNAs were translated into long proteins in rabbit reticulocyte lysate in the absence of any particular element for internal ribosome entry, a poly-A tail, or a cap structure. The translation systems in eukaryote can accept much simpler RNA as a template for protein synthesis by cyclisation. Here, we demonstrated that the circular RNA is efficiently translated in living human cells to produce abundant protein product by RCA mechanism. These findings suggest that translation of exonic circular RNAs present in human cells is more probable than previously thought. PMID:26553571

Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries

PubMed Central

Schmidt, Thorsten L.; Beliveau, Brian J.; Uca, Yavuz O.; Theilmann, Mark; Da Cruz, Felipe; Wu, Chao-Ting; Shih, William M.

2015-01-01

Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology, genetics and synthetic biology, which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nanomole total oligonucleotides produced) and is dominated by the use of commercial enzymes. PMID:26567534

Terahertz spectroscopy for the isothermal detection of bacterial DNA by magnetic bead-based rolling circle amplification.

PubMed

Yang, Xiang; Yang, Ke; Zhao, Xiang; Lin, Zhongquan; Liu, Zhiyong; Luo, Sha; Zhang, Yang; Wang, Yunxia; Fu, Weiling

2017-12-04

The demand for rapid and sensitive bacterial detection is continuously increasing due to the significant requirements of various applications. In this study, a terahertz (THz) biosensor based on rolling circle amplification (RCA) was developed for the isothermal detection of bacterial DNA. The synthetic bacterium-specific sequence of 16S rDNA hybridized with a padlock probe (PLP) that contains a sequence fully complementary to the target sequence at the 5' and 3' ends. The linear PLP was circularized by ligation to form a circular PLP upon recognition of the target sequence; then the capture probe (CP) immobilized on magnetic beads (MBs) acted as a primer to initialize RCA. As DNA molecules are much less absorptive than water molecules in the THz range, the RCA products on the surface of the MBs cause a significant decrease in THz absorption, which can be sensitively probed by THz spectroscopy. Our results showed that 0.12 fmol of synthetic bacterial DNA and 0.05 ng μL -1 of genomic DNA could be effectively detected using this assay. In addition, the specificity of this strategy was demonstrated by its low signal response to interfering bacteria. The proposed strategy not only represents a new method for the isothermal detection of the target bacterial DNA but also provides a general methodology for sensitive and specific DNA biosensing using THz spectroscopy.

Quantitative Single-Cell mRNA Analysis in Hydrogel Beads.

PubMed

Rakszewska, Agata; Stolper, Rosa J; Kolasa, Anna B; Piruska, Aigars; Huck, Wilhelm T S

2016-06-01

In recent years, technologies capable of analyzing single cells have emerged that are transforming many fields of biological research. Herein we report how DNA-functionalized hydrogel beads can serve as a matrix to capture mRNA from lysed single cells. mRNA quantification free of pre-amplification bias is ensured by using padlock probes and rolling circle amplification followed by hybridization with fluorescent probes. The number of transcripts in individual cells is assessed by simply counting fluorescent dots inside gel beads. The method extends the potential of existing techniques and provides a general platform for capturing molecules of interest from single cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification

PubMed Central

Maruyama, Fumito; Kenzaka, Takehiko; Yamaguchi, Nobuyasu; Tani, Katsuji; Nasu, Masao

2005-01-01

Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 106-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria. PMID:16332770

Ligation-rolling circle amplification combined with γ-cyclodextrin mediated stemless molecular beacon for sensitive and specific genotyping of single-nucleotide polymorphism.

PubMed

Zou, Zhen; Qing, Zhihe; He, Xiaoxiao; Wang, Kemin; He, Dinggeng; Shi, Hui; Yang, Xue; Qing, Taiping; Yang, Xiaoxiao

2014-07-01

A novel approach for highly sensitive and selective genotyping of single-nucleotide polymorphism (SNP) has been developed based on ligation-rolling circle amplification (L-RCA) and stemless molecular beacon. In this approach, two tailored DNA probes were involved. The stemless molecular beacon, formed through the inclusion interactions of γ-cyclodextrin (γ-CD) and bis-pyrene labeled DNA fragment, was served as signal probe. In the absence of mutant target, the two pyrene molecules were bound in the γ-CD cavity to form an excimer and showed a strong fluorescence at 475 nm. It was here named γ-CD-P-MB. The padlock DNA probe was designed as recognition probe. Upon the recognition of a point mutation DNA targets, the padlock probe was ligated to generate a circular template. An RCA amplification was then initiated using the circular template in the presence of Phi29 polymerase and dNTPs. The L-RCA products, containing repetitive sequence units, subsequently hybridized with the γ-CD-P-MB. This made pyrene molecules away from γ-CD cavity and caused a decrease of excimer fluorescence. As a proof-of-concept, SNP typing of β-thalassemia gene at position -28 was investigated using this approach. The detection limit of mutated target was determined to be 40 fM. In addition, DNA ligase offered high fidelity in distinguishing the mismatched bases at the ligation site, resulting in positive detection of mutant target even when the ratio of the wildtype to the mutant is 999:1. Given these attractive characteristics, the developed approach might provide a great genotyping platform for pathogenic diagnosis and genetic analysis. Copyright © 2014 Elsevier B.V. All rights reserved.

An "off-on" electrochemiluminescent biosensor based on DNAzyme-assisted target recycling and rolling circle amplifications for ultrasensitive detection of microRNA.

PubMed

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

2015-03-17

In this study, an off-on switching of a dual amplified electrochemiluminescence (ECL) biosensor based on Pb(2+)-induced DNAzyme-assisted target recycling and rolling circle amplification (RCA) was constructed for microRNA (miRNA) detection. First, the primer probe with assistant probe and miRNA formed Y junction which was cleaved with the addition of Pb(2+) to release miRNA. Subsequently, the released miRNA could initiate the next recycling process, leading to the generation of numerous intermediate DNA sequences (S2). Afterward, bare glassy carbon electrode (GCE) was immersed into HAuCl4 solution to electrodeposit a Au nanoparticle layer (depAu), followed by the assembly of a hairpin probe (HP). Then, dopamine (DA)-modified DNA sequence (S1) was employed to hybridize with HP, which switching off the sensing system. This is the first work that employs DA to quench luminol ECL signal, possessing the biosensor ultralow background signal. Afterward, S2 produced by the target recycling process was loaded onto the prepared electrode to displace S1 and served as an initiator for RCA. With rational design, numerous repeated DNA sequences coupling with hemin to form hemin/G-quadruplex were generated, which could exhibit strongly catalytic toward H2O2, thus amplified the ECL signal and switched the ON state of the sensing system. The liner range for miRNA detection was from 1.0 fM to 100 pM with a low detection limit down to 0.3 fM. Moreover, with the high sensitivity and specificity induced by the dual signal amplification, the proposed miRNA biosensor holds great potential for analysis of other interesting tumor markers.

Magnetic nanobeads present during enzymatic amplification and labeling for a simplified DNA detection protocol based on AC susceptometry

NASA Astrophysics Data System (ADS)

Bejhed, Rebecca S.; Strømme, Maria; Svedlindh, Peter; Ahlford, Annika; Strömberg, Mattias

2015-12-01

Magnetic biosensors are promising candidates for low-cost point-of-care biodiagnostic devices. For optimal efficiency it is crucial to minimize the time and complexity of the assay protocol including target recognition, amplification, labeling and read-out. In this work, possibilities for protocol simplifications for a DNA biodetection principle relying on hybridization of magnetic nanobeads to rolling circle amplification (RCA) products are investigated. The target DNA is recognized through a padlock ligation assay resulting in DNA circles serving as templates for the RCA process. It is found that beads can be present during amplification without noticeably interfering with the enzyme used for RCA (phi29 polymerase). As a result, the bead-coil hybridization can be performed immediately after amplification in a one-step manner at elevated temperature within a few minutes prior to read-out in an AC susceptometer setup, i.e. a combined protocol approach. Moreover, by recording the phase angle ξ = arctan(χ″/χ'), where χ and χ″ are the in-phase and out-of-phase components of the AC susceptibility, respectively, at one single frequency the total assay time for the optimized combined protocol would be no more than 1.5 hours, often a relevant time frame for diagnosis of cancer and infectious disease. Also, applying the phase angle method normalization of AC susceptibility data is not needed. These findings are useful for the development of point-of-care biodiagnostic devices relying on bead-coil binding and magnetic AC susceptometry.

Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection.

PubMed

Lau, Han Yih; Botella, Jose R

2017-01-01

Diagnostic technologies for the detection of plant pathogens with point-of-care capability and high multiplexing ability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and high-throughput multiplex detection capability. This article describes and discusses various DNA-based point-of care diagnostic methods for applications in plant disease detection. Polymerase chain reaction (PCR) is the most common DNA amplification technology used for detecting various plant and animal pathogens. However, subsequent to PCR based assays, several types of nucleic acid amplification technologies have been developed to achieve higher sensitivity, rapid detection as well as suitable for field applications such as loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, recombinase polymerase amplification, and molecular inversion probe. The principle behind these technologies has been thoroughly discussed in several review papers; herein we emphasize the application of these technologies to detect plant pathogens by outlining the advantages and disadvantages of each technology in detail.

Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection

PubMed Central

Lau, Han Yih; Botella, Jose R.

2017-01-01

Diagnostic technologies for the detection of plant pathogens with point-of-care capability and high multiplexing ability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and high-throughput multiplex detection capability. This article describes and discusses various DNA-based point-of care diagnostic methods for applications in plant disease detection. Polymerase chain reaction (PCR) is the most common DNA amplification technology used for detecting various plant and animal pathogens. However, subsequent to PCR based assays, several types of nucleic acid amplification technologies have been developed to achieve higher sensitivity, rapid detection as well as suitable for field applications such as loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, recombinase polymerase amplification, and molecular inversion probe. The principle behind these technologies has been thoroughly discussed in several review papers; herein we emphasize the application of these technologies to detect plant pathogens by outlining the advantages and disadvantages of each technology in detail. PMID:29375588

Recent advances in signal amplification strategy based on oligonucleotide and nanomaterials for microRNA detection-a review.

PubMed

Chen, Ying-Xu; Huang, Ke-Jing; Niu, Ke-Xin

2018-01-15

MicroRNAs (MiRNAs) play multiple crucial regulating roles in cell which can regulate one third of protein-coding genes. MiRNAs participate in the developmental and physiological processes of human body, while their aberrant adjustment will be more likely to trigger diseases such as cancers, kidney disease, central nervous system diseases, cardiovascular diseases, diabetes, viral infections and so on. What's worse, for the detection of miRNAs, their small size, high sequence similarity, low abundance and difficult extraction from cells impose great challenges in the analysis. Hence, it's necessary to fabricate accurate and sensitive biosensing platform for miRNAs detection. Up to now, researchers have developed many signal-amplification strategies for miRNAs detection, including hybridization chain reaction, nuclease amplification, rolling circle amplification, catalyzed hairpin assembly amplification and nanomaterials based amplification. These methods are typical, feasible and frequently used. In this review, we retrospect recent advances in signal amplification strategies for detecting miRNAs and point out the pros and cons of them. Furthermore, further prospects and promising developments of the signal-amplification strategies for detecting miRNAs are proposed. Copyright © 2017 Elsevier B.V. All rights reserved.

Novel readout method for molecular diagnostic assays based on optical measurements of magnetic nanobead dynamics.

PubMed

Donolato, Marco; Antunes, Paula; Bejhed, Rebecca S; Zardán Gómez de la Torre, Teresa; Østerberg, Frederik W; Strömberg, Mattias; Nilsson, Mats; Strømme, Maria; Svedlindh, Peter; Hansen, Mikkel F; Vavassori, Paolo

2015-02-03

We demonstrate detection of DNA coils formed from a Vibrio cholerae DNA target at picomolar concentrations using a novel optomagnetic approach exploiting the dynamic behavior and optical anisotropy of magnetic nanobead (MNB) assemblies. We establish that the complex second harmonic optical transmission spectra of MNB suspensions measured upon application of a weak uniaxial AC magnetic field correlate well with the rotation dynamics of the individual MNBs. Adding a target analyte to the solution leads to the formation of permanent MNB clusters, namely, to the suppression of the dynamic MNB behavior. We prove that the optical transmission spectra are highly sensitive to the formation of permanent MNB clusters and, thereby to the target analyte concentration. As a specific clinically relevant diagnostic case, we detect DNA coils formed via padlock probe recognition and isothermal rolling circle amplification and benchmark against a commercial equipment. The results demonstrate the fast optomagnetic readout of rolling circle products from bacterial DNA utilizing the dynamic properties of MNBs in a miniaturized and low-cost platform requiring only a transparent window in the chip.

Rapid Identification of Pseudallescheria and Scedosporium Strains by Using Rolling Circle Amplification

PubMed Central

Lackner, Michaela; Najafzadeh, Mohammad Javad; Sun, Jiufeng; Lu, Qiaoyun

2012-01-01

The Pseudallescheria boydii complex, comprising environmental pathogens with Scedosporium anamorphs, has recently been subdivided into five main species: Scedosporium dehoogii, S. aurantiacum, Pseudallescheria minutispora, P. apiosperma, and P. boydii, while the validity of some other taxa is being debated. Several Pseudallescheria and Scedosporium species are indicator organisms of pollution in soil and water. Scedosporium dehoogii in particular is enriched in soils contaminated by aliphatic hydrocarbons. In addition, the fungi may cause life-threatening infections involving the central nervous system in severely impaired patients. For screening purposes, rapid and economic tools for species recognition are needed. Our aim is to establish rolling circle amplification (RCA) as a screening tool for species-specific identification of Pseudallescheria and Scedosporium. With this aim, a set of padlock probes was designed on the basis of the internal transcribed spacer (ITS) region, differing by up to 13 fixed mutations. Padlock probes were unique as judged from sequence comparison by BLAST search in GenBank and in dedicated research databases at CBS (Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre). RCA was applied as an in vitro tool, tested with pure DNA amplified from cultures. The species-specific padlock probes designed in this study yielded 100% specificity. The method presented here was found to be an attractive alternative to identification by restriction fragment length polymorphism (RFLP) or sequencing. The rapidity (<1 day), specificity, and low costs make RCA a promising screening tool for environmentally and clinically relevant fungi. PMID:22057865

Rapid identification of Pseudallescheria and Scedosporium strains by using rolling circle amplification.

PubMed

Lackner, Michaela; Najafzadeh, Mohammad Javad; Sun, Jiufeng; Lu, Qiaoyun; Hoog, G Sybren de

2012-01-01

The Pseudallescheria boydii complex, comprising environmental pathogens with Scedosporium anamorphs, has recently been subdivided into five main species: Scedosporium dehoogii, S. aurantiacum, Pseudallescheria minutispora, P. apiosperma, and P. boydii, while the validity of some other taxa is being debated. Several Pseudallescheria and Scedosporium species are indicator organisms of pollution in soil and water. Scedosporium dehoogii in particular is enriched in soils contaminated by aliphatic hydrocarbons. In addition, the fungi may cause life-threatening infections involving the central nervous system in severely impaired patients. For screening purposes, rapid and economic tools for species recognition are needed. Our aim is to establish rolling circle amplification (RCA) as a screening tool for species-specific identification of Pseudallescheria and Scedosporium. With this aim, a set of padlock probes was designed on the basis of the internal transcribed spacer (ITS) region, differing by up to 13 fixed mutations. Padlock probes were unique as judged from sequence comparison by BLAST search in GenBank and in dedicated research databases at CBS (Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre). RCA was applied as an in vitro tool, tested with pure DNA amplified from cultures. The species-specific padlock probes designed in this study yielded 100% specificity. The method presented here was found to be an attractive alternative to identification by restriction fragment length polymorphism (RFLP) or sequencing. The rapidity (<1 day), specificity, and low costs make RCA a promising screening tool for environmentally and clinically relevant fungi.

Exploitation of rolling circle amplification for the construction of large phage-display antibody libraries.

PubMed

Shahsavarian, Melody A; Le Minoux, Damien; Matti, Kalyankumar M; Kaveri, Srini; Lacroix-Desmazes, Sébastien; Boquet, Didier; Friboulet, Alain; Avalle, Bérangère; Padiolleau-Lefèvre, Séverine

2014-05-01

Phage display antibody libraries have proven to have a significant role in the discovery of therapeutic antibodies and polypeptides with desired biological and physicochemical properties. Obtaining a large and diverse phage display antibody library, however, is always a challenging task. Various steps of this technique can still undergo optimization in order to obtain an efficient library. In the construction of a single chain fragment variable (scFv) phage display library, the cloning of the scFv fragments into a phagemid vector is of crucial importance. An efficient restriction enzyme digestion of the scFv DNA leads to its proper ligation with the phagemid followed by its successful cloning and expression. Here, we are reporting a different approach to enhance the efficiency of the restriction enzyme digestion step. We have exploited rolling circle amplification (RCA) to produce a long strand of DNA with tandem repeats of scFv sequences, which is found to be highly susceptible to restriction digestion. With this important modification, we are able to construct a large phage display antibody library of naive SJL/J mice. The size of the library is estimated as ~10(8) clones. The number of clones containing a scFv fragment is estimated at 90%. Hence, the present results could considerably aid the utilization of the phage-display technique in order to get an efficiently large antibody library. Copyright © 2014 Elsevier B.V. All rights reserved.

Protein Detection via Direct Enzymatic Amplification of Short DNA Aptamers

PubMed Central

Fischer, Nicholas O.; Tarasow, Theodore M.; Tok, Jeffrey B.-H.

2008-01-01

Aptamers are single-stranded nucleic acids that fold into defined tertiary structures to bind target molecules with high specificities and affinities. DNA aptamers have garnered much interest as recognition elements for biodetection and diagnostic applications due to their small size, ease of discovery and synthesis, and chemical and thermal stability. Herein, we describe the design and application of a short DNA molecule capable of both protein target binding and amplifiable bioreadout processes. As both recognition and readout capabilities are incorporated into a single DNA molecule, tedious conjugation procedures required for protein-DNA hybrids can be omitted. The DNA aptamer is designed to be amplified directly by either the polymerase chain reaction (PCR) or rolling circle amplification (RCA) processes, taking advantage of real-time amplification monitoring techniques for target detection. A combination of both RCA and PCR provides a wide protein target dynamic range (1 μM to 10 pM). PMID:17980857

Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities.

PubMed

Wang, Guoping; Ding, Xiong; Hu, Jiumei; Wu, Wenshuai; Sun, Jingjing; Mu, Ying

2017-10-24

Existing isothermal nucleic acid amplification (INAA) relying on the strand displacement activity of DNA polymerase usually requires at least two primers. However, in this paper, we report an unusual isothermal multimerization and amplification (UIMA) which only needs one primer and is efficiently initiated by the strand-displacing DNA polymerases with reverse transcription activities. On electrophoresis, the products of UIMA present a cascade-shape band and they are confirmed to be multimeric DNAs with repeated target sequences. In contrast to current methods, UIMA is simple to product multimeric DNA, due to the independent of multiple primers and rolling circle structures. Through assaying the synthesized single-stranded DNA targets, UIMA performs high sensitivity and specificity, as well as the universality. In addition, a plausible mechanism of UIMA is proposed, involving short DNA bending, mismatch extension, and template slippage. UIMA is a good explanation for why nonspecific amplification easily happens in existing INAAs. As the simplest INAA till now, UIMA provides a new insight for deeply understanding INAA and opens a new avenue for thoroughly addressing nonspecific amplification.

Miniaturized isothermal nucleic acid amplification, a review.

PubMed

Asiello, Peter J; Baeumner, Antje J

2011-04-21

Micro-Total Analysis Systems (µTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented.

Rolling chain amplification based signal-enhanced electrochemical aptasensor for ultrasensitive detection of ochratoxin A.

PubMed

Huang, Lin; Wu, Jingjing; Zheng, Lei; Qian, Haisheng; Xue, Feng; Wu, Yucheng; Pan, Daodong; Adeloju, Samuel B; Chen, Wei

2013-11-19

A novel electrochemical aptasensor is described for rapid and ultrasensitive detection of ochratoxin A (OTA) based on signal enhancement with rolling circle amplification (RCA). The primer for RCA was designed to compose of a two-part sequence, one part of the aptamer sequence directed against OTA while the other part was complementary to the capture probe on the electrode surface. In the presence of target OTA, the primer, originally hybridized with the RCA padlock, is replaced to combine with OTA. This induces the inhibition of RCA and decreases the OTA sensing signal obtained with the electrochemical aptasensor. Under the optimized conditions, ultrasensitive detection of OTA was achieved with a limit of detection (LOD) of 0.065 ppt (pg/mL), which is much lower than previously reported. The electrochemical aptasensor was also successfully applied to the determination of OTA in wine samples. This ultrasensitive electrochemical aptasensor is of great practical importance in food safety and could be widely extended to the detection of other toxins by replacing the sequence of the recognition aptamer.

Investigation on papillomavirus infection in dromedary camels in Al-Ahsa, Saudi Arabia

PubMed Central

Khalafalla, Abdelmalik Ibrahim; Ramadan, Ramadan Omer; Rector, Annabel; Barakat, Seif

2017-01-01

We investigated two outbreaks of papillomatosis between 2013 and 2015 in Al Ahsa region of eastern Saudi Arabia involving fourteen dromedary camels. The disease affected both young and adult animals and occurred in coincidence with demodectic mange infestation. Diagnosis was made based on gross and histopathological characteristics of the wart lesion and was confirmed by PCR. Rolling circle amplification followed by degenerate primer PCR and sequencing of the amplicons revealed the presence of both Camelus dromedarius papillomavirus types 1 and 2, previously identified in infected dromedaries in Sudan. PMID:28717601

An enhanced chemiluminescence resonance energy transfer aptasensor based on rolling circle amplification and WS2 nanosheet for Staphylococcus aureus detection.

PubMed

Hao, Liling; Gu, Huajie; Duan, Nuo; Wu, Shijia; Ma, Xiaoyuan; Xia, Yu; Tao, Zui; Wang, Zhouping

2017-03-22

A chemiluminescence resonance energy transfer aptasensor was fabricated for the detection of Staphylococcus aureus (S. aureus) with Co 2+ enhanced N-(aminobutyl)-N-(ethylisoluminol) (ABEI) functional flowerlike gold nanoparticles (Co 2+ /ABEI-AuNFs) as donor and WS 2 nanosheet as acceptor. In the presence of S. aureus, rolling circle amplification (RCA) can be started. Partially complementary sequence of RCA product functional ABEI-AuNFs (cDNA-ABEI-AuNFs) were then annealed to multiple sites of the RCA product to form duplex complex. This complex is less adsorbed onto the WS 2 nanosheet, thus attenuating the quenching of ABEI-AuNFs chemiluminescence by WS 2 nanosheet. In the absence of target S. aureus (and hence the absence of RCA and duplex formation), the free cDNA-ABEI-AuNFs is completely adsorbed onto the WS 2 nanosheet and chemiluminescence quenching ensues. Under optimal conditions, the logarithmic correlation between the concentration of S. aureus and the CL signal was found to be linear within the range of 50 cfu/mL to 1.5 × 10 5  cfu/mL (R 2  = 0.9913). The limits of detection of the developed method were found to be 15 cfu/mL for S. aureus. The selectivity and the capability of the biosensor in meat samples were also studied. Therefore, this simple and easy operation method can be used to detect S. aureus with high sensitivity and specificity. Copyright © 2017 Elsevier B.V. All rights reserved.

A graphene-based biosensing platform based on the release of DNA probes and rolling circle amplification.

PubMed

Liu, Meng; Song, Jinping; Shuang, Shaomin; Dong, Chuan; Brennan, John D; Li, Yingfu

2014-06-24

We report a versatile biosensing platform capable of achieving ultrasensitive detection of both small-molecule and macromolecular targets. The system features three components: reduced graphene oxide for its ability to adsorb single-stranded DNA molecules nonspecifically, DNA aptamers for their ability to bind reduced graphene oxide but undergo target-induced conformational changes that facilitate their release from the reduced graphene oxide surface, and rolling circle amplification (RCA) for its ability to amplify a primer-template recognition event into repetitive sequence units that can be easily detected. The key to the design is the tagging of a short primer to an aptamer sequence, which results in a small DNA probe that allows for both effective probe adsorption onto the reduced graphene oxide surface to mask the primer domain in the absence of the target, as well as efficient probe release in the presence of the target to make the primer available for template binding and RCA. We also made an observation that the circular template, which on its own does not cause a detectable level of probe release from the reduced graphene oxide, augments target-induced probe release. The synergistic release of DNA probes is interpreted to be a contributing factor for the high detection sensitivity. The broad utility of the platform is illustrated though engineering three different sensors that are capable of achieving ultrasensitive detection of a protein target, a DNA sequence and a small-molecule analyte. We envision that the approach described herein will find useful applications in the biological, medical, and environmental fields.

Sensitive detection of T4 polynucleotide kinase activity based on multifunctional magnetic probes and polymerization nicking reactions mediated hyperbranched rolling circle amplification.

PubMed

Li, Xia; Xu, Xiaowen; Song, Juan; Xue, Qingwang; Li, Chenzhong; Jiang, Wei

2017-05-15

T4 polynucleotide kinase (PNK) plays critical roles in regulating DNA phosphorylation modes during the repair of DNA lesions. The aberrant activity of T4 PNK has been proven to be associated with a variety of human pathologies. Sensitive detection of T4 PNK activity is critical to both clinical diagnosis and therapeutics. Herein, a background-eliminated fluorescence assay for sensitive detection of T4 PNK activity has been developed by multifunctional magnetic probes and polymerization nicking reactions mediated hyperbranched rolling circle amplification (HRCA). First, the streptavidin-magnetic nanobeads (MBs) were functionalized with the biotin modified hairpin probe (HP) with 3'-phosphoryl, forming multifunctional magnetic probes (HP-MBs). Then, in the presence of T4 PNK, the 3'-phosphoryl of HP-MBs was hydrolyzed to 3'-hydroxyl, thus serving as primers to initiate the polymerization extension and nicking endonuclease cleavage reaction. Next, the primers released from above "polymerization-nicking" cycles were separated out to trigger the subsequently HRCA process, producing plenty of dsDNA. Finally, the intercalating dye SYBR Green I (SG) was inserted into the dsDNA, generating enhanced fluorescence signals. In our design, the HP-MBs here serve together as the T4 PNK, DNA polymerase, and endonuclease recognition probe, and thus avoid the demands of utilizing multiple probes design. Moreover, it performed primary "polymerization-nicking" amplification and mediate secondary HRCA. In addition to, performing the separation function, the binding of HP-MBs and SG could be avoided while a low background was acquired. This method showed excellent sensitivity with a detection limit of 0.0436 mU/mL, and accomplished exceptional characterization T4 PNK activity in cell extracts, offering a powerful tool for biomedical research and clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Dew inspired breathing-based detection of genetic point mutation visualized by naked eye

PubMed Central

Xie, Liping; Wang, Tongzhou; Huang, Tianqi; Hou, Wei; Huang, Guoliang; Du, Yanan

2014-01-01

A novel label-free method based on breathing-induced vapor condensation was developed for detection of genetic point mutation. The dew-inspired detection was realized by integration of target-induced DNA ligation with rolling circle amplification (RCA). The vapor condensation induced by breathing transduced the RCA-amplified variances in DNA contents into visible contrast. The image could be recorded by a cell phone for further or even remote analysis. This green assay offers a naked-eye-reading method potentially applied for point-of-care liver cancer diagnosis in resource-limited regions. PMID:25199907

Dew inspired breathing-based detection of genetic point mutation visualized by naked eye

NASA Astrophysics Data System (ADS)

Xie, Liping; Wang, Tongzhou; Huang, Tianqi; Hou, Wei; Huang, Guoliang; Du, Yanan

2014-09-01

A novel label-free method based on breathing-induced vapor condensation was developed for detection of genetic point mutation. The dew-inspired detection was realized by integration of target-induced DNA ligation with rolling circle amplification (RCA). The vapor condensation induced by breathing transduced the RCA-amplified variances in DNA contents into visible contrast. The image could be recorded by a cell phone for further or even remote analysis. This green assay offers a naked-eye-reading method potentially applied for point-of-care liver cancer diagnosis in resource-limited regions.

Dew inspired breathing-based detection of genetic point mutation visualized by naked eye.

PubMed

Xie, Liping; Wang, Tongzhou; Huang, Tianqi; Hou, Wei; Huang, Guoliang; Du, Yanan

2014-09-09

A novel label-free method based on breathing-induced vapor condensation was developed for detection of genetic point mutation. The dew-inspired detection was realized by integration of target-induced DNA ligation with rolling circle amplification (RCA). The vapor condensation induced by breathing transduced the RCA-amplified variances in DNA contents into visible contrast. The image could be recorded by a cell phone for further or even remote analysis. This green assay offers a naked-eye-reading method potentially applied for point-of-care liver cancer diagnosis in resource-limited regions.

Rapid detection of IHNV by molecular padlock recognition and surface-associated isothermal amplification

NASA Astrophysics Data System (ADS)

McCarthy, Erik L.; Egeler, Teressa J.; Bickerstaff, Lee E.; Pereira da Cunha, Mauricio; Millard, Paul J.

2005-11-01

RNA sequences derived from infectious hematopoeitic necrosis virus (IHNV) could be detected using a combination of surface-associated molecular padlock DNA probes (MPP) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV were recognized by MPP. Circularized MPP were then captured on the inner surface of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA gave rise to DNA concatamers, which were in turn bound by the fluorescent reporter SYBR Green II nucleic acid stain, and measured by microfluorimetry. Surface-associated molecular padlock technology, combined with isothermal RCA, exhibited high selectivity and sensitivity without thermal cycling. This technology is applicable to direct RNA and DNA detection, permitting detection of a variety of viral or bacterial pathogens.

Massive amplification of rolling-circle transposons in the lineage of the bat Myotis lucifugus

PubMed Central

Pritham, Ellen J.; Feschotte, Cédric

2007-01-01

Rolling-circle (RC) transposons, or Helitrons, are a newly recognized group of eukaryotic transposable elements abundant in the genomes of plants, invertebrates, and zebrafish. We provide evidence for the colonization of a mammalian genome by Helitrons, which has not been reported previously. We identified and characterized two families of Helitrons in the little brown bat Myotis lucifugus. The consensus sequence for the first family, HeliBat1, displays the hallmarks of an autonomous Helitron, including coding capacity for an ≈1,500-aa protein with an RC replication motif and a region related to the SF1 superfamily of DNA helicases. The HeliBatN1 family is a nonautonomous Helitron family that is only distantly related to HeliBat1. The two HeliBat families have attained high copy numbers (≈15,000 and > 100,000 copies, respectively) and make up at least ≈3% of the M. lucifugus genome. Sequence divergence and cross-species analyses indicate that both HeliBat families have amplified within the last ≈30–36 million years and are restricted to the lineage of vesper bats. We could not detect the presence of Helitrons in any other order of placental mammals, despite the broad representation of these taxa in the databases. We describe an instance of HeliBat-mediated transduction of a host gene fragment that was subsequently dispersed in ≈1,000 copies throughout the M. lucifugus genome. Given the demonstrated propensity of RC transposons to mediate the duplication and shuffling of host genes in bacteria and maize, it is tempting to speculate that the massive amplification of Helitrons in vesper bats has influenced the evolutionary trajectory of these mammals. PMID:17261799

CdTe/CdSe quantum dot-based fluorescent aptasensor with hemin/G-quadruplex DNzyme for sensitive detection of lysozyme using rolling circle amplification and strand hybridization.

PubMed

Qiu, Zhenli; Shu, Jian; He, Yu; Lin, Zhenzhen; Zhang, Kangyao; Lv, Shuzhen; Tang, Dianping

2017-01-15

Lysozyme with a small monomeric globular enzymatic protein is part of the innate immune system, and its deficiency can cause the increased incidence of disease. Herein, we devise a new signal-enhanced fluorescence aptasensing platform for quantitative screening of lysozyme by coupling with rolling circle amplification (RCA) and strand hybridization reaction, accompanying the assembly of CdTe/CdSe quantum dots (QDs) and hemin/G-quadruplex DNzyme. Initially, target-triggered release of the primer was carried out from DNA duplex via the reaction of the aptamer with the analyte, and the released primer could be then utilized as the template to produce numerous repeated oligonucleotide sequences by the RCA reaction. Following that, the formed long-stranded DNA simultaneously hybridized with the CdTe/CdSe QD-labeled probe and hemin/G-quadruplex DNzyme strand in the system, thereby resulting in the quenching of QD fluorescent signal through the proximity hemin/G-quadruplex DNzyme on the basis of transferring photoexcited conduction band electrons of quantum dots to Fe(III)/Fe(II)-protoporphyrin IX (hemin) complex. Under optimal conditions, the fluorescent signal decreased with the increasing target lysozyme within the dynamic range from 5.0 to 500nM with a detection limit (LOD) of 2.6nM at the 3s blank criterion. Intra-assay and interassay coefficients of variation (CVs) were below 8.5% and 11.5%, respectively. Finally, the system was applied to analyze spiked human serum samples, and the recoveries in all cases were 85-111.9%. Copyright © 2016 Elsevier B.V. All rights reserved.

Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides.

PubMed

Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M; Högberg, Björn

2013-07-01

Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.

Enzymatic Production of Monoclonal Stoichiometric Single-Stranded DNA Oligonucleotides

PubMed Central

Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M.; Högberg, Björn

2013-01-01

Single-stranded oligonucleotides are important as research tools as probes for diagnostics and gene therapy. Today, production of oligonucleotides is done via solid-phase synthesis. However, the capabilities of current polymer chemistry are limited in comparison to what can be produced in biological systems. The errors in synthetic DNA increases with oligonucleotide length, and sequence diversity can often be a problem. Here, we present the Monoclonal Stoichiometric (MOSIC) method for enzymatic DNA oligonucleotide production. Using this method, we amplify oligonucleotides from clonal templates followed by digestion of a cutter-hairpin, resulting in pools of monoclonal oligonucleotides with precisely controlled relative stoichiometric ratios. We present data where MOSIC oligonucleotides, 14–378 nt long, were prepared either by in vitro rolling-circle amplification, or by amplification in Escherichia coli in the form of phagemid DNA. The formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides. PMID:23727986

Novel electrochemiluminescence of perylene derivative and its application to mercury ion detection based on a dual amplification strategy.

PubMed

Zhao, Jing; Lei, Yan-Mei; Chai, Ya-Qin; Yuan, Ruo; Zhuo, Ying

2016-12-15

In this paper, a novel covalently crosslinked perylene derivative (PTC-PEI) composed of polyethylenimine (PEI) and perylenetetracarboxylic acid (PTCA) has been first investigated for cathodic electrochemiluminescence (ECL) in an aqueous system with dissolved O2 as coreactant. The promising novel ECL materials of PTC-PEI exhibited admirable physical and chemical stability and high ECL intensity, which held an alternative way to construct ECL sensor with improved sensitivity. Thus, it was applied to construct a dual amplified "signal-on" mercury ion (Hg(2+)) sensor by the employment of nicking endonuclease (NEase)-assisted target recycling and rolling circle amplification (RCA) for signal amplification. Herein, a long G-rich sequence was generated by RCA process to capture abundant hemin on the electrode surface, and then a significantly amplified ECL signal of PTC-PEI was obtained. Based on dual signal amplification strategy, the devised sensor showed a linear range from 0.1pM to 0.1μΜ with a detection limit down to 33fM (S/N=3), and was successfully used in the direct detection of real water sample with high sensitivity and selectivity. Copyright © 2016 Elsevier B.V. All rights reserved.

Graphene Nanoprobes for Real-Time Monitoring of Isothermal Nucleic Acid Amplification.

PubMed

Li, Fan; Liu, Xiaoguo; Zhao, Bin; Yan, Juan; Li, Qian; Aldalbahi, Ali; Shi, Jiye; Song, Shiping; Fan, Chunhai; Wang, Lihua

2017-05-10

Isothermal amplification is an efficient way to amplify DNA with high accuracy; however, the real-time monitoring for quantification analysis mostly relied on expensive and precisely designed probes. In the present study, a graphene oxide (GO)-based nanoprobe was used to real-time monitor the isothermal amplification process. The interaction between GO and different DNA structures was systematically investigated, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), DNA 3-helix, and long rolling circle amplification (RCA) and hybridization chain reaction (HCR) products, which existed in one-, two-, and three-dimensional structures. It was found that the high rigid structures exhibited much lower affinity with GO than soft ssDNA, and generally the rigidity was dependent on the length of targets and the hybridization position with probe DNA. On the basis of these results, we successfully monitored HCR amplification process, RCA process, and the enzyme restriction of RCA products with GO nanoprobe; other applications including the detection of the assembly/disassembly of DNA 3-helix structures were also performed. Compared to the widely used end-point detection methods, the GO-based sensing platform is simple, sensitive, cost-effective, and especially in a real-time monitoring mode. We believe such studies can provide comprehensive understandings and evocation on design of GO-based biosensors for broad application in various fields.

Rapid Identification and Differentiation of Trichophyton Species, Based on Sequence Polymorphisms of the Ribosomal Internal Transcribed Spacer Regions, by Rolling-Circle Amplification▿

PubMed Central

Kong, Fanrong; Tong, Zhongsheng; Chen, Xiaoyou; Sorrell, Tania; Wang, Bin; Wu, Qixuan; Ellis, David; Chen, Sharon

2008-01-01

DNA sequencing analyses have demonstrated relatively limited polymorphisms within the fungal internal transcribed spacer (ITS) regions among Trichophyton spp. We sequenced the ITS region (ITS1, 5.8S, and ITS2) for 42 dermatophytes belonging to seven species (Trichophyton rubrum, T. mentagrophytes, T. soudanense, T. tonsurans, Epidermophyton floccosum, Microsporum canis, and M. gypseum) and developed a novel padlock probe and rolling-circle amplification (RCA)-based method for identification of single nucleotide polymorphisms (SNPs) that could be exploited to differentiate between Trichophyton spp. Sequencing results demonstrated intraspecies genetic variation for T. tonsurans, T. mentagrophytes, and T. soudanense but not T. rubrum. Signature sets of SNPs between T. rubrum and T. soudanense (4-bp difference) and T. violaceum and T. soudanense (3-bp difference) were identified. The RCA assay correctly identified five Trichophyton species. Although the use of two “group-specific” probes targeting both the ITS1 and the ITS2 regions were required to identify T. soudanense, the other species were identified by single ITS1- or ITS2-targeted species-specific probes. There was good agreement between ITS sequencing and the RCA assay. Despite limited genetic variation between Trichophyton spp., the sensitive, specific RCA-based SNP detection assay showed potential as a simple, reproducible method for the rapid (2-h) identification of Trichophyton spp. PMID:18234865

The rolling-circle melting-pot model for porcine circovirus DNA replication

USDA-ARS?s Scientific Manuscript database

A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication (Ori) among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. Porcine circo...

Hybridization chain reaction: a versatile molecular tool for biosensing, bioimaging, and biomedicine.

PubMed

Bi, Sai; Yue, Shuzhen; Zhang, Shusheng

2017-07-17

Developing powerful, simple and low-cost DNA amplification techniques is of great significance to bioanalysis and biomedical research. Thus far, many signal amplification strategies have been developed, such as polymerase chain reaction (PCR), rolling circle amplification (RCA), and DNA strand displacement amplification (SDA). In particular, hybridization chain reaction (HCR), a type of toehold-mediated strand displacement (TMSD) reaction, has attracted great interest because of its enzyme-free nature, isothermal conditions, simple protocols, and excellent amplification efficiency. In a typical HCR, an analyte initiates the cross-opening of two DNA hairpins, yielding nicked double helices that are analogous to alternating copolymers. As an efficient amplification platform, HCR has been utilized for the sensitive detection of a wide variety of analytes, including nucleic acids, proteins, small molecules, and cells. In recent years, more complicated sets of monomers have been designed to develop nonlinear HCR, such as branched HCR and even dendritic systems, achieving quadratic and exponential growth mechanisms. In addition, HCR has attracted enormous attention in the fields of bioimaging and biomedicine, including applications in fluorescence in situ hybridization (FISH) imaging, live cell imaging, and targeted drug delivery. In this review, we introduce the fundamentals of HCR and examine the visualization and analysis techniques for HCR products in detail. The most recent HCR developments in biosensing, bioimaging, and biomedicine are subsequently discussed with selected examples. Finally, the review provides insight into the challenges and future perspectives of HCR.

Highly sensitive MicroRNA 146a detection using a gold nanoparticle-based CTG repeat probing system and isothermal amplification.

PubMed

Le, Binh Huy; Seo, Young Jun

2018-01-25

We have developed a gold nanoparticle (AuNP)-based CTG repeat probing system displaying high quenching capability and combined it with isothermal amplification for the detection of miRNA 146a. This method of using a AuNP-based CTG repeat probing system with isothermal amplification allowed the highly sensitive (14 aM) and selective detection of miRNA 146a. A AuNP-based CTG repeat probing system having a hairpin structure and a dT F fluorophore exhibited highly efficient quenching because the CTG repeat-based stable hairpin structure imposed a close distance between the AuNP and the dT F residue. A small amount of miRNA 146a induced multiple copies of the CAG repeat sequence during rolling circle amplification; the AuNP-based CTG repeat probing system then bound to the complementary multiple-copy CAG repeat sequence, thereby inducing a structural change from a hairpin to a linear structure with amplified fluorescence. This AuNP-based CTG probing system combined with isothermal amplification could also discriminate target miRNA 146a from one- and two-base-mismatched miRNAs (ORN 1 and ORN 2, respectively). This simple AuNP-based CTG probing system, combined with isothermal amplification to induce a highly sensitive change in fluorescence, allows the detection of miRNA 146a with high sensitivity (14 aM) and selectivity. Copyright © 2017 Elsevier B.V. All rights reserved.

Specific functions of the Rep and Rep' proteins of porcine circovirus during copy-release and rolling-circle DNA replication

USDA-ARS?s Scientific Manuscript database

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 replica...

Carbon nanotube enhanced label-free detection of microRNAs based on hairpin probe triggered solid-phase rolling-circle amplification

NASA Astrophysics Data System (ADS)

Tian, Qianqian; Wang, Ying; Deng, Ruijie; Lin, Lei; Liu, Yang; Li, Jinghong

2014-12-01

The detection of microRNAs (miRNAs) is imperative for gaining a better understanding of the functions of these biomarkers and has great potential for the early diagnosis of human disease. High sensitivity and selectivity for miRNA detection brings new challenges. Herein, an ultrasensitive protocol for electrochemical detection of miRNA is designed through carbon nanotube (CNT) enhanced label-free detection based on hairpin probe triggered solid-phase rolling-circle amplification (RCA). Traditionally, RCA, widely applied for signal enhancement in the construction of a variety of biosensors, has an intrinsic limitation of ultrasensitive detection, as it is difficult to separate the enzymes, templates, and padlock DNAs from the RCA products in the homogeneous solution. We purposely designed a solid-phase RCA strategy, using CNTs as the solid substrate, integrated with a hairpin structured probe to recognize target miRNA. In the presence of miRNA the stem-loop structure will be unfolded, triggering the CNT based RCA process. Due to the efficient blocking effect originating from the polymeric RCA products, the label-free assay of miRNA exhibits an ultrasensitive detection limit of 1.2 fM. Furthermore, the protocol possesses excellent specificity for resolving lung cancer-related let-7 family members which have only one-nucleotide variations. The high sensitivity and selectivity give the method great potential for applications in online diagnostics and in situ detection in long-term development.The detection of microRNAs (miRNAs) is imperative for gaining a better understanding of the functions of these biomarkers and has great potential for the early diagnosis of human disease. High sensitivity and selectivity for miRNA detection brings new challenges. Herein, an ultrasensitive protocol for electrochemical detection of miRNA is designed through carbon nanotube (CNT) enhanced label-free detection based on hairpin probe triggered solid-phase rolling-circle amplification (RCA). Traditionally, RCA, widely applied for signal enhancement in the construction of a variety of biosensors, has an intrinsic limitation of ultrasensitive detection, as it is difficult to separate the enzymes, templates, and padlock DNAs from the RCA products in the homogeneous solution. We purposely designed a solid-phase RCA strategy, using CNTs as the solid substrate, integrated with a hairpin structured probe to recognize target miRNA. In the presence of miRNA the stem-loop structure will be unfolded, triggering the CNT based RCA process. Due to the efficient blocking effect originating from the polymeric RCA products, the label-free assay of miRNA exhibits an ultrasensitive detection limit of 1.2 fM. Furthermore, the protocol possesses excellent specificity for resolving lung cancer-related let-7 family members which have only one-nucleotide variations. The high sensitivity and selectivity give the method great potential for applications in online diagnostics and in situ detection in long-term development. Electronic supplementary information (ESI) available: Preparation of the chemically modified multi-walled carbon nanotubes (CNTs), characterization of the CNTs and modified CNTs, preparation of the circular probe, gel electrophoresis of the RCA products, and DNA probes as noted in the text. See DOI: 10.1039/c4nr05243a

Initiator-catalyzed self-assembly of duplex-looped DNA hairpin motif based on strand displacement reaction for logic operations and amplified biosensing.

PubMed

Bi, Sai; Yue, Shuzhen; Wu, Qiang; Ye, Jiayan

2016-09-15

Here we program an initiator-catalyzed self-assembly of duplex-looped DNA hairpin motif based on strand displacement reaction. Due to the recycling of initiator and performance in a cascade manner, this system is versatilely extended to logic operations, including the construction of concatenated logic circuits with a feedback function and a biocomputing keypad-lock security system. Compared with previously reported molecular security systems, the prominent feature of our keypad lock is that it can be spontaneously reset and recycled with no need of any external stimulus and human intervention. Moreover, through integrating with an isothermal amplification technique of rolling circle amplification (RCA), this programming catalytic DNA self-assembly strategy readily achieves sensitive and selective biosensing of initiator. Importantly, a magnetic graphene oxide (MGO) is introduced to remarkably reduced background, which plays an important role in enhancing the signal-to-noise ratio and improving the detection sensitivity. Therefore, the proposed sophisticated DNA strand displacement-based methodology with engineering dynamic functions may find broad applications in the construction of programming DNA nanostructures, amplification biosensing platform, and large-scale DNA circuits. Copyright © 2016 Elsevier B.V. All rights reserved.

Novel Polyomavirus associated with Brain Tumors in Free-Ranging Raccoons, Western United States

PubMed Central

Dela Cruz, Florante N.; Giannitti, Federico; Li, Linlin; Woods, Leslie W.; Del Valle, Luis; Delwart, Eric

2013-01-01

Tumors of any type are exceedingly rare in raccoons. High-grade brain tumors, consistently located in the frontal lobes and olfactory tracts, were detected in 10 raccoons during March 2010–May 2012 in California and Oregon, suggesting an emerging, infectious origin. We have identified a candidate etiologic agent, dubbed raccoon polyomavirus, that was present in the tumor tissue of all affected animals but not in tissues from 20 unaffected animals. Southern blot hybridization and rolling circle amplification showed the episomal viral genome in the tumors. The multifunctional nuclear protein large T-antigen was detectable by immunohistochemical analyses in a subset of neoplastic cells. Raccoon polyomavirus may contribute to the development of malignant brain tumors of raccoons. PMID:23260029

Novel polyomavirus associated with brain tumors in free-ranging raccoons, western United States.

PubMed

Dela Cruz, Florante N; Giannitti, Federico; Li, Linlin; Woods, Leslie W; Del Valle, Luis; Delwart, Eric; Pesavento, Patricia A

2013-01-01

Tumors of any type are exceedingly rare in raccoons. High-grade brain tumors, consistently located in the frontal lobes and olfactory tracts, were detected in 10 raccoons during March 2010-May 2012 in California and Oregon, suggesting an emerging, infectious origin. We have identified a candidate etiologic agent, dubbed raccoon polyomavirus, that was present in the tumor tissue of all affected animals but not in tissues from 20 unaffected animals. Southern blot hybridization and rolling circle amplification showed the episomal viral genome in the tumors. The multifunctional nuclear protein large T-antigen was detectable by immunohistochemical analyses in a subset of neoplastic cells. Raccoon polyomavirus may contribute to the development of malignant brain tumors of raccoons.

Error baseline rates of five sample preparation methods used to characterize RNA virus populations.

PubMed

Kugelman, Jeffrey R; Wiley, Michael R; Nagle, Elyse R; Reyes, Daniel; Pfeffer, Brad P; Kuhn, Jens H; Sanchez-Lockhart, Mariano; Palacios, Gustavo F

2017-01-01

Individual RNA viruses typically occur as populations of genomes that differ slightly from each other due to mutations introduced by the error-prone viral polymerase. Understanding the variability of RNA virus genome populations is critical for understanding virus evolution because individual mutant genomes may gain evolutionary selective advantages and give rise to dominant subpopulations, possibly even leading to the emergence of viruses resistant to medical countermeasures. Reverse transcription of virus genome populations followed by next-generation sequencing is the only available method to characterize variation for RNA viruses. However, both steps may lead to the introduction of artificial mutations, thereby skewing the data. To better understand how such errors are introduced during sample preparation, we determined and compared error baseline rates of five different sample preparation methods by analyzing in vitro transcribed Ebola virus RNA from an artificial plasmid-based system. These methods included: shotgun sequencing from plasmid DNA or in vitro transcribed RNA as a basic "no amplification" method, amplicon sequencing from the plasmid DNA or in vitro transcribed RNA as a "targeted" amplification method, sequence-independent single-primer amplification (SISPA) as a "random" amplification method, rolling circle reverse transcription sequencing (CirSeq) as an advanced "no amplification" method, and Illumina TruSeq RNA Access as a "targeted" enrichment method. The measured error frequencies indicate that RNA Access offers the best tradeoff between sensitivity and sample preparation error (1.4-5) of all compared methods.

A split recognition mode combined with cascade signal amplification strategy for highly specific, sensitive detection of microRNA.

PubMed

Wang, Rui; Wang, Lei; Zhao, Haiyan; Jiang, Wei

2016-12-15

MicroRNAs (miRNAs) are vital for many biological processes and have been regarded as cancer biomarkers. Specific and sensitive detection of miRNAs is essential for cancer diagnosis and therapy. Herein, a split recognition mode combined with cascade signal amplification strategy is developed for highly specific and sensitive detection of miRNA. The split recognition mode possesses two specific recognition processes, which are based on toehold-mediated strand displacement reaction (TSDR) and direct hybridization reaction. Two recognition probes, hairpin probe (HP) with overhanging toehold domain and assistant probe (AP), are specially designed. Firstly, the toehold domain of HP and AP recognize part of miRNA simultaneously, accompanied with TSDR to unfold the HP and form the stable DNA Y-shaped junction structure (YJS). Then, the AP in YJS can further act as primer to initiate strand displacement amplification, releasing numerous trigger sequences. Finally, the trigger sequences hybridize with padlock DNA to initiate circular rolling circle amplification and generate enhanced fluorescence responses. In this strategy, the dual recognition effect of split recognition mode guarantees the excellent selectivity to discriminate let-7b from high-homology sequences. Furthermore, the high amplification efficiency of cascade signal amplification guarantees a high sensitivity with the detection limit of 3.2 pM and the concentration of let-7b in total RNA sample extracted from Hela cells is determined. These results indicate our strategy will be a promising miRNA detection strategy in clinical diagnosis and disease treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

Direct electrical and mechanical characterization of in situ generated DNA between the tips of silicon nanotweezers (SNT).

PubMed

Karsten, Stanislav L; Kumemura, Momoko; Jalabert, Laurent; Lafitte, Nicolas; Kudo, Lili C; Collard, Dominique; Fujita, Hiroyuki

2016-05-24

Previously, we reported the application of micromachined silicon nanotweezers (SNT) integrated with a comb-drive actuator and capacitive sensors for capturing and mechanical characterization of DNA bundles. Here, we demonstrate direct DNA amplification on such a MEMS structure with subsequent electrical and mechanical characterization of a single stranded DNA (ssDNA) bundle generated between the tips of SNT via isothermal rolling circle amplification (RCA) and dielectrophoresis (DEP). An in situ generated ssDNA bundle was visualized and evaluated via electrical conductivity (I-V) and mechanical frequency response measurements. Colloidal gold nanoparticles significantly enhanced (P < 0.01) the electrical properties of thin ssDNA bundles. The proposed technology allows direct in situ synthesis of DNA with a predefined sequence on the tips of a MEMS sensor device, such as SNT, followed by direct DNA electrical and mechanical characterization. In addition, our data provides a "proof-of-principle" for the feasibility of the on-chip label free DNA detection device that can be used for a variety of biomedical applications focused on sequence specific DNA detection.

Simplified methods for the construction of RNA and DNA virus infectious clones.

PubMed

Nagata, Tatsuya; Inoue-Nagata, Alice Kazuko

2015-01-01

Infectious virus clones are one of the most powerful tools in plant pathology, molecular biology, and biotechnology. The construction of infectious clones of RNA and DNA viruses, however, usually requires laborious cloning and subcloning steps. In addition, instability of the RNA virus genome is frequently reported after its introduction into the vector and transference to Escherichia coli. These difficulties hamper the cloning procedures, making it tedious and cumbersome. This chapter describes two protocols for a simple construction of infectious viruses, an RNA virus, the tobamovirus Pepper mild mottle virus, and a DNA virus, a bipartite begomovirus. For this purpose, the strategy of overlap-extension PCR was used for the construction of infectious tobamovirus clone and of rolling circle amplification (RCA) for the construction of a dimeric form of the begomovirus clone.

Label-free electrical detection of pyrophosphate generated from DNA polymerase reactions on field-effect devices.

PubMed

Credo, Grace M; Su, Xing; Wu, Kai; Elibol, Oguz H; Liu, David J; Reddy, Bobby; Tsai, Ta-Wei; Dorvel, Brian R; Daniels, Jonathan S; Bashir, Rashid; Varma, Madoo

2012-03-21

We introduce a label-free approach for sensing polymerase reactions on deoxyribonucleic acid (DNA) using a chelator-modified silicon-on-insulator field-effect transistor (SOI-FET) that exhibits selective and reversible electrical response to pyrophosphate anions. The chemical modification of the sensor surface was designed to include rolling-circle amplification (RCA) DNA colonies for locally enhanced pyrophosphate (PPi) signal generation and sensors with immobilized chelators for capture and surface-sensitive detection of diffusible reaction by-products. While detecting arrays of enzymatic base incorporation reactions is typically accomplished using optical fluorescence or chemiluminescence techniques, our results suggest that it is possible to develop scalable and portable PPi-specific sensors and platforms for broad biomedical applications such as DNA sequencing and microbe detection using surface-sensitive electrical readout techniques.

Detection of the 35S promoter in transgenic maize via various isothermal amplification techniques: a practical approach.

PubMed

Zahradnik, Celine; Kolm, Claudia; Martzy, Roland; Mach, Robert L; Krska, Rudolf; Farnleitner, Andreas H; Brunner, Kurt

2014-11-01

In 2003 the European Commission introduced a 0.9% threshold for food and feed products containing genetically modified organism (GMO)-derived components. For commodities containing GMO contents higher than this threshold, labelling is mandatory. To provide a DNA-based rapid and simple detection method suitable for high-throughput screening of GMOs, several isothermal amplification approaches for the 35S promoter were tested: strand displacement amplification, nicking-enzyme amplification reaction, rolling circle amplification, loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA). The assays developed were tested for specificity in order to distinguish between samples containing genetically modified (GM) maize and non-GM maize. For those assays capable of this discrimination, tests were performed to determine the lower limit of detection. A false-negative rate was determined to rule out whether GMO-positive samples were incorrectly classified as GMO-negative. A robustness test was performed to show reliable detection independent from the instrument used for amplification. The analysis of three GM maize lines showed that only LAMP and HDA were able to differentiate between the GMOs MON810, NK603, and Bt11 and non-GM maize. Furthermore, with the HDA assay it was possible to realize a detection limit as low as 0.5%. A false-negative rate of only 5% for 1% GM maize for all three maize lines shows that HDA has the potential to be used as an alternative strategy for the detection of transgenic maize. All results obtained with the LAMP and HDA assays were compared with the results obtained with a previously reported real-time PCR assay for the 35S promoter in transgenic maize. This study presents two new screening assays for detection of the 35S promoter in transgenic maize by applying the isothermal amplification approaches HDA and LAMP.

Self-locked aptamer probe mediated cascade amplification strategy for highly sensitive and selective detection of protein and small molecule.

PubMed

Li, Wei; Jiang, Wei; Wang, Lei

2016-10-12

In this work, a novel self-locked aptamer probe mediated cascade amplification strategy has been constructed for highly sensitive and specific detection of protein. First, the self-locked aptamer probe was designed with three functions: one was specific molecular recognition attributed to the aptamer sequence, the second was signal transduction owing to the transduction sequence, and the third was self-locking through the hybridization of the transduction sequence and part of the aptamer sequence. Then, the aptamer sequence specific recognized the target and folded into a three-way helix junction, leading to the release of the transduction sequence. Next, the 3'-end of this three-way junction acted as primer to trigger the strand displacement amplification (SDA), yielding a large amount of primers. Finally, the primers initiated the dual-exponential rolling circle amplification (DE-RCA) and generated numerous G-quadruples sequences. By inserting the fluorescent dye N-methyl mesoporphyrin IX (NMM), enhanced fluorescence signal was achieved. In this strategy, the self-locked aptamer probe was more stable to reduce the interference signals generated by the uncontrollable folding in unbounded state. Through the cascade amplification of SDA and DE-RCA, the sensitivity was further improved with a detection limit of 3.8 × 10(-16) mol/L for protein detection. Furthermore, by changing the aptamer sequence of the probe, sensitive and selective detection of adenosine has been also achieved, suggesting that the proposed strategy has good versatility and can be widely used in sensitive and selective detection of biomolecules. Copyright © 2016 Elsevier B.V. All rights reserved.

Colocalization recognition-activated cascade signal amplification strategy for ultrasensitive detection of transcription factors.

PubMed

Zhu, Desong; Wang, Lei; Xu, Xiaowen; Jiang, Wei

2017-03-15

Transcription factors (TFs) bind to specific double-stranded DNA (dsDNA) sequences in the regulatory regions of genes to regulate the process of gene transcription. Their expression levels sensitively reflect cell developmental situation and disease state. TFs have become potential diagnostic markers and therapeutic targets of cancers and some other diseases. Hence, high sensitive detection of TFs is of vital importance for early diagnosis of diseases and drugs development. The traditional exonucleases-assisted signal amplification methods suffered from the false positives caused by incomplete digestion of excess recognition probes. Herein, based on a new recognition way-colocalization recognition (CR)-activated dual signal amplification, an ultrasensitive fluorescent detection strategy for TFs was developed. TFs-induced the colocalization of three split recognition components resulted in noticeable increases of local effective concentrations and hybridization of three split components, which activated the subsequent cascade signal amplification including strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). This strategy eliminated the false positive influence and achieved ultra-high sensitivity towards the purified NF-κB p50 with detection limit of 2.0×10 -13 M. Moreover, NF-κB p50 can be detected in as low as 0.21ngμL -1 HeLa cell nuclear extracts. In addition, this proposed strategy could be used for the screening of NF-κB p50 activity inhibitors and potential anti-NF-κB p50 drugs. Finally, our proposed strategy offered a potential method for reliable detection of TFs in medical diagnosis and treatment research of cancers and other related diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Assign Roles to Get Literature Circles Rolling

ERIC Educational Resources Information Center

Curriculum Review, 2005

2005-01-01

This article briefly describes a role-playing exercise designed to break the ice in a classroom literature circle from The Ultimate Small-Group Reading How-To Book: Building Comprehension through SmallGroup Instruction, written by Gail Saunders-Smith. The members of a literature circle participate in the group discussion according to the…

A simple and efficient method for agroinfection of Vernonia cinerea with infectious clones of Vernonia yellow vein virus.

PubMed

Packialakshmi, R M; Usha, R

2011-12-01

Vernonia yellow vein virus (VeYVV) is a distinct monopartite begomovirus associated with a satellite DNA β. After constructing dimers of both DNA A and DNA β in binary vectors, a number of infection methods were attempted. However, only a modified stem-prick method produced up to 83% infection in the natural host Vernonia cinerea, thus, fulfilling the Koch's postulate. The presence of the viral DNA in the agroinfected plants was confirmed by rolling circle amplification (RCA), followed by Southern hybridization. DNA β induces typical symptoms of Vernonia yellow vein disease (VeYVD) when co-agroinoculated with the begomovirus to Vernonia and also leads to the accumulation of DNA A systemically. VeYVV represents a new member of the emerging group of monopartite begomoviruses requiring a satellite component for symptom induction.

A sensitive colorimetric assay system for nucleic acid detection based on isothermal signal amplification technology.

PubMed

Hu, Bo; Guo, Jing; Xu, Ying; Wei, Hua; Zhao, Guojie; Guan, Yifu

2017-08-01

Rapid and accurate detection of microRNAs in biological systems is of great importance. Here, we report the development of a visual colorimetric assay which possesses the high amplification capabilities and high selectivity of the rolling circle amplification (RCA) method and the simplicity and convenience of gold nanoparticles used as a signal indicator. The designed padlock probe recognizes the target miRNA and is circularized, and then acts as the template to extend the target miRNA into a long single-stranded nucleotide chain of many tandem repeats of nucleotide sequences. Next, the RCA product is hybridized with oligonucleotides tagged onto gold nanoparticles. This interaction leads to the aggregation of gold nanoparticles, and the color of the system changes from wine red to dark blue according to the abundance of miRNA. A linear correlation between fluorescence and target oligonucleotide content was obtained in the range 0.3-300 pM, along with a detection limit of 0.13 pM (n = 7) and a RSD of 3.9% (30 pM, n = 9). The present approach provides a simple, rapid, and accurate visual colorimetric assay that allows sensitive biodetection and bioanalysis of DNA and RNA nucleotides of interest in biologically important samples. Graphical abstract The colorimetric assay system for analyzing target oligonucleotides.

Unveiling the mystery of mitochondrial DNA replication in yeasts.

PubMed

Chen, Xin Jie; Clark-Walker, George Desmond

2018-01-01

Conventional DNA replication is initiated from specific origins and requires the synthesis of RNA primers for both the leading and lagging strands. In contrast, the replication of yeast mitochondrial DNA is origin-independent. The replication of the leading strand is likely primed by recombinational structures and proceeded by a rolling circle mechanism. The coexistent linear and circular DNA conformers facilitate the recombination-based initiation. The replication of the lagging strand is poorly understood. Re-evaluation of published data suggests that the rolling circle may also provide structures for the synthesis of the lagging-strand by mechanisms such as template switching. Thus, the coupling of recombination with rolling circle replication and possibly, template switching, may have been selected as an economic replication mode to accommodate the reductive evolution of mitochondria. Such a replication mode spares the need for conventional replicative components, including those required for origin recognition/remodelling, RNA primer synthesis and lagging-strand processing. Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

DNAzymes in DNA Nanomachines and DNA Analysis

NASA Astrophysics Data System (ADS)

He, Yu; Tian, Ye; Chen, Yi; Mao, Chengde

This chapter discusses our efforts in using DNAzymes in DNA nano-machines and DNA analysis systems. 10-23 DNAzymes can cleave specific phos-phodiester bonds in RNA. We use them to construct an autonomous DNA-RNA chimera nanomotor, which constantly extracts chemical energy from RNA substrates and transduces the energy into a mechanical motion: cycles of contraction and extension. The motor's motion can be reversibly turned on and off by a DNA analogue (brake) of the RNA substrate. Addition and removal of the brake stops and restarts, respectively, the motor's motion. Furthermore, when the RNA substrates are preorganized into a one-dimensional track, a DNAzyme can continuously move along the track so long as there are substrates available ahead. Based on a similar mechanism, a novel DNA detection system has been developed. A target DNA activates a DNAzyme to cleave RNA-containing molecular beacons (MB), which generates an enhanced fluorescence signal. A following work integrates two steps of signal amplifications: a rolling-circle amplification (RCA) to synthesize multiple copies of DNAzymes, and the DNAzymes catalyze a chemical reaction to generate a colorimetric signal. This method allows detection of DNA analytes whose concentration is as low as 1 pM.

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

PubMed

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

2015-07-07

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

A method for the determination of the coefficient of rolling friction using cycloidal pendulum

NASA Astrophysics Data System (ADS)

Ciornei, M. C.; Alaci, S.; Ciornei, F. C.; Romanu, I. C.

2017-08-01

The paper presents a method for experimental finding of coefficient of rolling friction appropriate for biomedical applications based on the theory of cycloidal pendulum. When a mobile circle rolls over a fixed straight line, the points from the circle describe trajectories called normal cycloids. To materialize this model, it is sufficient that a small region from boundary surfaces of a moving rigid body is spherical. Assuming pure rolling motion, the equation of motion of the cycloidal pendulum is obtained - an ordinary nonlinear differential equation. The experimental device is composed by two interconnected balls rolling over the material to be studied. The inertial characteristics of the pendulum can be adjusted via weights placed on a rod. A laser spot oscillates together to the pendulum and provides the amplitude of oscillations. After finding the experimental parameters necessary in differential equation of motion, it can be integrated using the Runge-Kutta of fourth order method. The equation was integrated for several materials and found values of rolling friction coefficients. Two main conclusions are drawn: the coefficient of rolling friction influenced significantly the amplitude of oscillation but the effect upon the period of oscillation is practically imperceptible. A methodology is proposed for finding the rolling friction coefficient and the pure rolling condition is verified.

Circular RNAs: diversity of form and function.

PubMed

Lasda, Erika; Parker, Roy

2014-12-01

It is now clear that there is a diversity of circular RNAs in biological systems. Circular RNAs can be produced by the direct ligation of 5' and 3' ends of linear RNAs, as intermediates in RNA processing reactions, or by "backsplicing," wherein a downstream 5' splice site (splice donor) is joined to an upstream 3' splice site (splice acceptor). Circular RNAs have unique properties including the potential for rolling circle amplification of RNA, the ability to rearrange the order of genomic information, protection from exonucleases, and constraints on RNA folding. Circular RNAs can function as templates for viroid and viral replication, as intermediates in RNA processing reactions, as regulators of transcription in cis, as snoRNAs, and as miRNA sponges. Herein, we review the breadth of circular RNAs, their biogenesis and metabolism, and their known and anticipated functions. © 2014 Lasda and Parker; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Circular RNAs: diversity of form and function

PubMed Central

Lasda, Erika

2014-01-01

It is now clear that there is a diversity of circular RNAs in biological systems. Circular RNAs can be produced by the direct ligation of 5′ and 3′ ends of linear RNAs, as intermediates in RNA processing reactions, or by “backsplicing,” wherein a downstream 5′ splice site (splice donor) is joined to an upstream 3′ splice site (splice acceptor). Circular RNAs have unique properties including the potential for rolling circle amplification of RNA, the ability to rearrange the order of genomic information, protection from exonucleases, and constraints on RNA folding. Circular RNAs can function as templates for viroid and viral replication, as intermediates in RNA processing reactions, as regulators of transcription in cis, as snoRNAs, and as miRNA sponges. Herein, we review the breadth of circular RNAs, their biogenesis and metabolism, and their known and anticipated functions. PMID:25404635

Programming a topologically constrained DNA nanostructure into a sensor

NASA Astrophysics Data System (ADS)

Liu, Meng; Zhang, Qiang; Li, Zhongping; Gu, Jimmy; Brennan, John D.; Li, Yingfu

2016-06-01

Many rationally engineered DNA nanostructures use mechanically interlocked topologies to connect individual DNA components, and their physical connectivity is achieved through the formation of a strong linking duplex. The existence of such a structural element also poses a significant topological constraint on functions of component rings. Herein, we hypothesize and confirm that DNA catenanes with a strong linking duplex prevent component rings from acting as the template for rolling circle amplification (RCA). However, by using an RNA-containing DNA [2] catenane with a strong linking duplex, we show that a stimuli-responsive RNA-cleaving DNAzyme can linearize one component ring, and thus enable RCA, producing an ultra-sensitive biosensing system. As an example, a DNA catenane biosensor is engineered to detect the model bacterial pathogen Escherichia coli through binding of a secreted protein, with a detection limit of 10 cells ml-1, thus establishing a new platform for further applications of mechanically interlocked DNA nanostructures.

Rapid Detection and Identification of a Pathogen's DNA Using Phi29 DNA Polymerase

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

Xu, Y.; Dunn, J.; Gao, S.

2008-10-31

Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing asmore » little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.« less

Programming a topologically constrained DNA nanostructure into a sensor

PubMed Central

Liu, Meng; Zhang, Qiang; Li, Zhongping; Gu, Jimmy; Brennan, John D.; Li, Yingfu

2016-01-01

Many rationally engineered DNA nanostructures use mechanically interlocked topologies to connect individual DNA components, and their physical connectivity is achieved through the formation of a strong linking duplex. The existence of such a structural element also poses a significant topological constraint on functions of component rings. Herein, we hypothesize and confirm that DNA catenanes with a strong linking duplex prevent component rings from acting as the template for rolling circle amplification (RCA). However, by using an RNA-containing DNA [2] catenane with a strong linking duplex, we show that a stimuli-responsive RNA-cleaving DNAzyme can linearize one component ring, and thus enable RCA, producing an ultra-sensitive biosensing system. As an example, a DNA catenane biosensor is engineered to detect the model bacterial pathogen Escherichia coli through binding of a secreted protein, with a detection limit of 10 cells ml−1, thus establishing a new platform for further applications of mechanically interlocked DNA nanostructures. PMID:27337657

Rolling Reloaded

ERIC Educational Resources Information Center

Jones, Simon A.; Nieminen, John M.

2008-01-01

Not so long ago a new observation about rolling motion was described: for a rolling wheel, there is a set of points with instantaneous velocities directed at or away from the centre of the wheel; these points form a circle whose diameter connects the centre of the wheel to the wheel's point of contact with the ground (Sharma 1996 "Eur. J. Phys."…

To Believe the Past or to Trust the Future

ERIC Educational Resources Information Center

Jin, Fengtao; Zhou, Zhaoyan

2012-01-01

A small ball rolls down from a quarter-circle to a frictionless plane. What will be the magnitude of the normal force when the ball arrives at the tangent point of the circle and the plane? According to the centripetal force formula, the normal force will be 3 "mg" when the curvature radius of the circle is considered, but will be "mg" instead…

Error baseline rates of five sample preparation methods used to characterize RNA virus populations

PubMed Central

Kugelman, Jeffrey R.; Wiley, Michael R.; Nagle, Elyse R.; Reyes, Daniel; Pfeffer, Brad P.; Kuhn, Jens H.; Sanchez-Lockhart, Mariano; Palacios, Gustavo F.

2017-01-01

Individual RNA viruses typically occur as populations of genomes that differ slightly from each other due to mutations introduced by the error-prone viral polymerase. Understanding the variability of RNA virus genome populations is critical for understanding virus evolution because individual mutant genomes may gain evolutionary selective advantages and give rise to dominant subpopulations, possibly even leading to the emergence of viruses resistant to medical countermeasures. Reverse transcription of virus genome populations followed by next-generation sequencing is the only available method to characterize variation for RNA viruses. However, both steps may lead to the introduction of artificial mutations, thereby skewing the data. To better understand how such errors are introduced during sample preparation, we determined and compared error baseline rates of five different sample preparation methods by analyzing in vitro transcribed Ebola virus RNA from an artificial plasmid-based system. These methods included: shotgun sequencing from plasmid DNA or in vitro transcribed RNA as a basic “no amplification” method, amplicon sequencing from the plasmid DNA or in vitro transcribed RNA as a “targeted” amplification method, sequence-independent single-primer amplification (SISPA) as a “random” amplification method, rolling circle reverse transcription sequencing (CirSeq) as an advanced “no amplification” method, and Illumina TruSeq RNA Access as a “targeted” enrichment method. The measured error frequencies indicate that RNA Access offers the best tradeoff between sensitivity and sample preparation error (1.4−5) of all compared methods. PMID:28182717

Quantifying the benefits of improved rolling of chip seals : final report, June 2008.

DOT National Transportation Integrated Search

2008-06-01

This report presents an improvement in the rolling protocol for chip seals based on an evaluation of aggregate : retention performance and aggregate embedment depth. The flip-over test (FOT), Vialit test, modified sand circle : test, digital image pr...

Digital Storytelling: Reinventing Literature Circles

ERIC Educational Resources Information Center

Tobin, Maryann Tatum

2012-01-01

New literacies in reading research demand the study of comprehension skills using multiple modalities through a more complex, multi-platform view of reading. Taking into account the robust roll of technology in our daily lives, this article presents an update to the traditional literature circle lesson to include digital storytelling and…

Investigation of the Influence of Shapes-Texture on Surface Deformation of UHMWPE as a Bearing Material in Static Normal Load and Rolling Contact

NASA Astrophysics Data System (ADS)

Lestari, W. D.; Ismail, R.; Jamari, J.; Bayuseno, A. P.

2017-05-01

Surface texture is a common method for improving wear properties of a tribo-pair of soft and hard bearing material. The reduction of wear rates on the contacting surface material is becoming important issues. In the present study, analysis of the contact pressure on the flat surface of UHMWPE (Ultra High Molecular Weight Polyethylene) under the static- and rolling motion with the surface of steel ball used the 3D finite element method (FEM) (the ABAQUS software version 6.12). Five shaped-texture models (square, circle, ellipse, triangle, and chevron) were presented on the flat surface for analysis. The normal load of 17, 30 and 50 N was deliberately set-up for static and rolling contact analysis. The contact pressure was determined to predict the wear behavior of the shaped-texture on the flat surface of UHMWPE. The results have shown that the static normal load yielded the lowest von-Mises stress distribution on the shaped-texture of the ellipse for all values applied a load, while the square shape experienced the highest stress distribution. Under rolling contact, however, the increasing load yielded the increasing von Mises stress distribution for the texture with a triangle shape. Moreover, the texture shapes for circle, ellipse, and chevron respectively, may undergo the lowest stress distribution for all load. The wear calculation provided that the circle and square shape may undergo the highest wear rates. Obviously, the surface texture of circle, ellipse, and chevron may experience the lowest wear rates and is potential for use in the surface engineering of bearing materials.

Synthesis of a multi-functional DNA nanosphere barcode system for direct cell detection.

PubMed

Han, Sangwoo; Lee, Jae Sung; Lee, Jong Bum

2017-09-28

Nucleic acid-based technologies have been applied to numerous biomedical applications. As a novel material for target detection, DNA has been used to construct a barcode system with a range of structures. This paper reports multi-functionalized DNA nanospheres (DNANSs) by rolling circle amplification (RCA) with several functionalized nucleotides. DNANSs with a barcode system were designed to exhibit fluorescence for coding enhanced signals and contain biotin for more functionalities, including targeting through the biotin-streptavidin (biotin-STA) interaction. Functionalized deoxynucleotide triphosphates (dNTPs) were mixed in the RCA process and functional moieties can be expressed on the DNANSs. The anti-epidermal growth factor receptor antibodies (anti-EGFR Abs) can be conjugated on DNANSs for targeting cancer cells specifically. As a proof of concept, the potential of the multi-functional DNANS barcode was demonstrated by direct cell detection as a simple detection method. The DNANS barcode provides a new route for the simple and rapid selective recognition of cancer cells.

Light-dependent segregation of begomoviruses in Asystasia gangetica leaves.

PubMed

Wyant, Patricia; Strohmeier, Stephan; Fischer, Alexander; Schäfer, Benjamin; Briddon, Rob W; Krenz, Björn; Jeske, Holger

2015-01-02

Asystasia gangetica (Acanthaceae) from tropical Africa and Asia is used as source of food and for medical applications. Plants collected in West Africa in the 1980s with typical geminivirus symptoms showed an unusual symptom segregation that included vein yellowing, curling and mosaic, which were present simultaneously or separately on different leaves of the same plant or on different plants propagated as cuttings from a single plant. Rolling-circle amplification in combination with restriction fragment length polymorphism analysis followed by deep sequencing of the RCA products identified two geminiviruses in these plants. One with a bipartite genome, Asystasia begomovirus 1, and the other with a monopartite genome together with its defective DNA, Asystasia begomovirus 2. The relationship between leaf symptoms and virus distribution under different light regimes was investigated, and showed for the first time an unusual segregation of symptoms and viruses, either within a single plant, or even within a leaf. Copyright © 2014 Elsevier B.V. All rights reserved.

How many papillomavirus species can go undetected in papilloma lesions?

PubMed

Daudt, Cíntia; da Silva, Flavio R C; Streck, André F; Weber, Matheus N; Mayer, Fabiana Q; Cibulski, Samuel P; Canal, Cláudio W

2016-11-03

A co-infection comprising to at least seven papillomavirus (PV) types was detected by next generation sequencing (NGS) of randomly primed rolling circle amplification (RCA) products of a bovine (Bos taurus) papilloma lesion from the Brazilian Amazon region. Six putative new PV types that could not be detected by commonly used PCR protocols were identified. Their overall L1 nucleotide identities were less than 90% compared to described PV species and types. L1 nucleotide BLAST sequence hits showed that each new type was related to Beta, Gamma, Dyokappa, Dyoeta, and Xipapillomavirus, as well as two likely new unclassified genera. Our results show that the employment of NGS is relevant to the detection and characterization of distantly related PV and is of major importance in co-infection studies. This knowledge will help us understand the biology and pathogenesis of PV, as well as contribute to disease control. Moreover, we can also conclude that there are many unknown circulating PVs.

A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities.

PubMed

Wu, Yushu; Yan, Ping; Xu, Xiaowen; Jiang, Wei

2016-03-07

Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL(-1) for UDG and 0.11 U mL(-1) for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses.

Singularity of the time-energy uncertainty in adiabatic perturbation and cycloids on a Bloch sphere

PubMed Central

Oh, Sangchul; Hu, Xuedong; Nori, Franco; Kais, Sabre

2016-01-01

Adiabatic perturbation is shown to be singular from the exact solution of a spin-1/2 particle in a uniformly rotating magnetic field. Due to a non-adiabatic effect, its quantum trajectory on a Bloch sphere is a cycloid traced by a circle rolling along an adiabatic path. As the magnetic field rotates more and more slowly, the time-energy uncertainty, proportional to the length of the quantum trajectory, calculated by the exact solution is entirely different from the one obtained by the adiabatic path traced by the instantaneous eigenstate. However, the non-adiabatic Aharonov- Anandan geometric phase, measured by the area enclosed by the exact path, approaches smoothly the adiabatic Berry phase, proportional to the area enclosed by the adiabatic path. The singular limit of the time-energy uncertainty and the regular limit of the geometric phase are associated with the arc length and arc area of the cycloid on a Bloch sphere, respectively. Prolate and curtate cycloids are also traced by different initial states outside and inside of the rolling circle, respectively. The axis trajectory of the rolling circle, parallel to the adiabatic path, is shown to be an example of transitionless driving. The non-adiabatic resonance is visualized by the number of cycloid arcs. PMID:26916031

Genome-Based Genetic Tool Development for Bacillus methanolicus: Theta- and Rolling Circle-Replicating Plasmids for Inducible Gene Expression and Application to Methanol-Based Cadaverine Production.

PubMed

Irla, Marta; Heggeset, Tonje M B; Nærdal, Ingemar; Paul, Lidia; Haugen, Tone; Le, Simone B; Brautaset, Trygve; Wendisch, Volker F

2016-01-01

Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.

Genome-Based Genetic Tool Development for Bacillus methanolicus: Theta- and Rolling Circle-Replicating Plasmids for Inducible Gene Expression and Application to Methanol-Based Cadaverine Production

PubMed Central

Irla, Marta; Heggeset, Tonje M. B.; Nærdal, Ingemar; Paul, Lidia; Haugen, Tone; Le, Simone B.; Brautaset, Trygve; Wendisch, Volker F.

2016-01-01

Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium. PMID:27713731

Wide diversity of PAX5 alterations in B-ALL: a Groupe Francophone de Cytogenetique Hematologique study.

PubMed

Coyaud, Etienne; Struski, Stephanie; Prade, Nais; Familiades, Julien; Eichner, Ruth; Quelen, Cathy; Bousquet, Marina; Mugneret, Francine; Talmant, Pascaline; Pages, Marie-Pierre; Lefebvre, Christine; Penther, Dominique; Lippert, Eric; Nadal, Nathalie; Taviaux, Sylvie; Poppe, Bruce; Luquet, Isabelle; Baranger, Laurence; Eclache, Virginie; Radford, Isabelle; Barin, Carole; Mozziconacci, Marie-Joëlle; Lafage-Pochitaloff, Marina; Antoine-Poirel, Hélène; Charrin, Christiane; Perot, Christine; Terre, Christine; Brousset, Pierre; Dastugue, Nicole; Broccardo, Cyril

2010-04-15

PAX5 is the main target of somatic mutations in acute B lymphoblastic leukemia (B-ALL). We analyzed 153 adult and child B-ALL harboring karyotypic abnormalities at chromosome 9p, to determine the frequency and the nature of PAX5 alterations. We found PAX5 internal rearrangements in 21% of the cases. To isolate fusion partners, we used classic and innovative techniques (rolling circle amplification-rapid amplification of cDNA ends) and single nucleotide polymorphism-comparative genomic hybridization arrays. Recurrent and novel fusion partners were identified, including NCoR1, DACH2, GOLGA6, and TAOK1 genes showing the high variability of the partners. We noted that half the fusion genes can give rise to truncated PAX5 proteins. Furthermore, malignant cells carrying PAX5 fusion genes displayed a simple karyotype. These data strongly suggest that PAX5 fusion genes are early players in leukemogenesis. In addition, PAX5 deletion was observed in 60% of B-ALL with 9p alterations. Contrary to cases with PAX5 fusions, deletions were associated with complex karyotypes and common recurrent translocations. This supports the hypothesis of the secondary nature of the deletion. Our data shed more light on the high variability of PAX5 alterations in B-ALL. Therefore, it is probable that gene fusions occur early, whereas deletions should be regarded as a late/secondary event.

Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection.

PubMed

Wang, Xingyu; Li, Can; Gao, Xiaomeng; Wang, Jing; Liang, Xingguo

2015-01-13

A facile and robust RNA preparation protocol was developed by combining rolling circle transcription (RCT) with RNA cleavage by RNase H. Circular DNA with a complementary sequence was used as the template for promoter-free transcription. With the aid of a 2'-O-methylated DNA, the RCT-generated tandem repeats of the desired RNA sequence were disconnected at the exact end-to-end position to harvest the desired RNA oligomers. Compared with the template DNA, more than 4 × 10(3) times the amount of small RNA products were obtained when modest cleavage was carried out during transcription. Large amounts of RNA oligomers could easily be obtained by simply increasing the reaction volume.

Direct and precise measurement of displacement and velocity of flexible web in roll-to-roll manufacturing systems

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

Kang, Dongwoo; Lee, Eonseok; Choi, Young-Man

Interest in the production of printed electronics using a roll-to-roll system has gradually increased due to its low mass-production costs and compatibility with flexible substrate. To improve the accuracy of roll-to-roll manufacturing systems, the movement of the web needs to be measured precisely in advance. In this paper, a novel measurement method is developed to measure the displacement and velocity of the web precisely and directly. The proposed algorithm is based on the traditional single field encoder principle, and the scale grating has been replaced with a printed grating on the web. Because a printed grating cannot be as accuratemore » as a scale grating in a traditional encoder, there will inevitably be variations in pitch and line-width, and the motion of the web should be measured even though there are variations in pitch and line-width in the printed grating patterns. For this reason, the developed algorithm includes a precise method of estimating the variations in pitch. In addtion, a method of correcting the Lissajous curve is presented for precision phase interpolation to improve measurement accuracy by correcting Lissajous circle to unit circle. The performance of the developed method is evaluated by simulation and experiment. In the experiment, the displacement error was less than 2.5 μm and the velocity error of 1σ was about 0.25%, while the grating scale moved 30 mm.« less

Direct and precise measurement of displacement and velocity of flexible web in roll-to-roll manufacturing systems

NASA Astrophysics Data System (ADS)

Kang, Dongwoo; duk Kim, Young; Lee, Eonseok; Choi, Young-Man; Lee, Taik-Min; Kim, Dongmin

2013-12-01

Interest in the production of printed electronics using a roll-to-roll system has gradually increased due to its low mass-production costs and compatibility with flexible substrate. To improve the accuracy of roll-to-roll manufacturing systems, the movement of the web needs to be measured precisely in advance. In this paper, a novel measurement method is developed to measure the displacement and velocity of the web precisely and directly. The proposed algorithm is based on the traditional single field encoder principle, and the scale grating has been replaced with a printed grating on the web. Because a printed grating cannot be as accurate as a scale grating in a traditional encoder, there will inevitably be variations in pitch and line-width, and the motion of the web should be measured even though there are variations in pitch and line-width in the printed grating patterns. For this reason, the developed algorithm includes a precise method of estimating the variations in pitch. In addtion, a method of correcting the Lissajous curve is presented for precision phase interpolation to improve measurement accuracy by correcting Lissajous circle to unit circle. The performance of the developed method is evaluated by simulation and experiment. In the experiment, the displacement error was less than 2.5 μm and the velocity error of 1σ was about 0.25%, while the grating scale moved 30 mm.

Application of round grating angle measurement composite error amendment in the online measurement accuracy improvement of large diameter

NASA Astrophysics Data System (ADS)

Wang, Biao; Yu, Xiaofen; Li, Qinzhao; Zheng, Yu

2008-10-01

The paper aiming at the influence factor of round grating dividing error, rolling-wheel produce eccentricity and surface shape errors provides an amendment method based on rolling-wheel to get the composite error model which includes all influence factors above, and then corrects the non-circle measurement angle error of the rolling-wheel. We make soft simulation verification and have experiment; the result indicates that the composite error amendment method can improve the diameter measurement accuracy with rolling-wheel theory. It has wide application prospect for the measurement accuracy higher than 5 μm/m.

Theoretical damping in roll and rolling moment due to differential wing incidence for slender cruciform wings and wing-body combinations

NASA Technical Reports Server (NTRS)

Adams, Gaynor J; DUGAN DUANE W

1952-01-01

A method of analysis based on slender-wing theory is developed to investigate the characteristics in roll of slender cruciform wings and wing-body combinations. The method makes use of the conformal mapping processes of classical hydrodynamics which transform the region outside a circle and the region outside an arbitrary arrangement of line segments intersecting at the origin. The method of analysis may be utilized to solve other slender cruciform wing-body problems involving arbitrarily assigned boundary conditions. (author)

Evidence of recombination and positive selection in cetacean papillomaviruses.

PubMed

Robles-Sikisaka, Refugio; Rivera, Rebecca; Nollens, Hendrik H; St Leger, Judy; Durden, Wendy N; Stolen, Megan; Burchell, Jennifer; Wellehan, James F X

2012-06-05

Papillomaviruses (PVs) are small DNA viruses that have been associated with increased epithelial proliferation. Over one hundred PV types have been identified in humans; however, only three have been identified in bottlenose dolphins (Tursiops truncatus) to date. Using rolling circle amplification and degenerate PCR, we identified four novel PV genomes of bottlenose dolphins. TtPV4, TtPV5 and TtPV6 were identified in genital lesions while TtPV7 was identified in normal genital mucosa. Bayesian analysis of the full-length L1 genes found that TtPV4 and TtPV7 group within the Upsilonpapillomavirus genus while TtPV5 and TtPV6 group with Omikronpapillomavirus. However, analysis of the E1 gene did not distinguish these genera, implying that these genes may not share a common history, consistent with recombination. Recombination analyses identified several probable events. Signals of positive selection were found mostly in the E1 and E2 genes. Recombination and diversifying selection pressures constitute important driving forces of cetacean PV evolution. Copyright © 2012 Elsevier Inc. All rights reserved.

Molecular Characterization of Watermelon Chlorotic Stunt Virus (WmCSV) from Palestine

PubMed Central

Ali-Shtayeh, Mohammed S.; Jamous, Rana M.; Mallah, Omar B.; Abu-Zeitoun, Salam Y.

2014-01-01

The incidence of watermelon chlorotic stunt disease and molecular characterization of the Palestinian isolate of Watermelon chlorotic stunt virus (WmCSV-[PAL]) are described in this study. Symptomatic leaf samples obtained from watermelon Citrullus lanatus (Thunb.), and cucumber (Cucumis sativus L.) plants were tested for WmCSV-[PAL] infection by polymerase chain reaction (PCR) and Rolling Circle Amplification (RCA). Disease incidence ranged between 25%–98% in watermelon fields in the studied area, 77% of leaf samples collected from Jenin were found to be mixed infected with WmCSV-[PAL] and SLCV. The full-length DNA-A and DNA-B genomes of WmCSV-[PAL] were amplified and sequenced, and the sequences were deposited in the GenBank. Sequence analysis of virus genomes showed that DNA-A and DNA-B had 97.6%–99.42% and 93.16%–98.26% nucleotide identity with other virus isolates in the region, respectively. Sequence analysis also revealed that the Palestinian isolate of WmCSV shared the highest nucleotide identity with an isolate from Israel suggesting that the virus was introduced to Palestine from Israel. PMID:24956181

Evidence of recombination and positive selection in cetacean papillomaviruses

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

Robles-Sikisaka, Refugio, E-mail: refugio.robles1@gmail.com; Rivera, Rebecca, E-mail: RRivera@hswri.org; Nollens, Hendrik H., E-mail: Hendrik.Nollens@SeaWorld.com

2012-06-05

Papillomaviruses (PVs) are small DNA viruses that have been associated with increased epithelial proliferation. Over one hundred PV types have been identified in humans; however, only three have been identified in bottlenose dolphins (Tursiops truncatus) to date. Using rolling circle amplification and degenerate PCR, we identified four novel PV genomes of bottlenose dolphins. TtPV4, TtPV5 and TtPV6 were identified in genital lesions while TtPV7 was identified in normal genital mucosa. Bayesian analysis of the full-length L1 genes found that TtPV4 and TtPV7 group within the Upsilonpapillomavirus genus while TtPV5 and TtPV6 group with Omikronpapillomavirus. However, analysis of the E1 genemore » did not distinguish these genera, implying that these genes may not share a common history, consistent with recombination. Recombination analyses identified several probable events. Signals of positive selection were found mostly in the E1 and E2 genes. Recombination and diversifying selection pressures constitute important driving forces of cetacean PV evolution.« less

A Rolling Circle Replication Mechanism Produces Multimeric Lariats of Mitochondrial DNA in Caenorhabditis elegans

PubMed Central

Lewis, Samantha C.; Joers, Priit; Willcox, Smaranda; Griffith, Jack D.; Jacobs, Howard T.; Hyman, Bradley C.

2015-01-01

Mitochondrial DNA (mtDNA) encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s) of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s) of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans. PMID:25693201

Break-induced replication and recombinational telomere elongation in yeast.

PubMed

McEachern, Michael J; Haber, James E

2006-01-01

When a telomere becomes unprotected or if only one end of a chromosomal double-strand break succeeds in recombining with a template sequence, DNA can be repaired by a recombination-dependent DNA replication process termed break-induced replication (BIR). In budding yeasts, there are two BIR pathways, one dependent on the Rad51 recombinase protein and one Rad51 independent; these two repair processes lead to different types of survivors in cells lacking the telomerase enzyme that is required for normal telomere maintenance. Recombination at telomeres is triggered by either excessive telomere shortening or disruptions in the function of telomere-binding proteins. Telomere elongation by BIR appears to often occur through a "roll and spread" mechanism. In this process, a telomeric circle produced by recombination at a dysfunctional telomere acts as a template for a rolling circle BIR event to form an elongated telomere. Additional BIR events can then copy the elongated sequence to all other telomeres.

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.

Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode.

PubMed

Hernández-Neuta, Iván; Pereiro, Iago; Ahlford, Annika; Ferraro, Davide; Zhang, Qiongdi; Viovy, Jean-Louis; Descroix, Stéphanie; Nilsson, Mats

2018-04-15

Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA analysis in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification. This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions with high throughput processing up to 120μL of DNA dilution at flow rates ranging from 1 to 5μL/min without compromising performance. The fluidized bed was 20-50% more efficient than a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based microarray and tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

Ubiquiter circovirus sequences raise challenges in laboratory diagnosis: the case of honey bee and bee mite, reptiles, and free living amoebae.

PubMed

Marton, Szilvia; Ihász, Katalin; Lengyel, György; Farkas, Szilvia L; Dán, Ádám; Paulus, Petra; Bányai, Krisztián; Fehér, Enikő

2015-03-01

Circoviruses of pigs and birds are established pathogens, however, the exact role of other, recently described circoviruses and circovirus-like viruses remains to be elucidated. The aim of this study was the detection of circoviruses in neglected host species, including honey bees, exotic reptiles and free-living amoebae by widely used broad-spectrum polymerase chain reaction (PCR) assays specific for the replication initiation protein coding gene of these viruses. The majority of sequences obtained from honey bees were highly similar to canine and porcine circoviruses, or, were distantly related to dragonfly cycloviruses. Other rep sequences detected in some honey bees, reptiles and amoebae showed similarities to various rep sequences deposited in the GenBank. Back-to-back PCR primers designed for the amplification of whole viral genomes failed to work that suggested the existence of integrated rep-like elements in many samples. Rolling circle amplification and exonuclease treatment confirmed the absence of small circular DNA genomes in the specimens analysed. In case of honey bees Varroa mite DNA contamination might be a source of the identified endogenous rep-like elements. The reptile and amoebae rep-like sequences were nearly identical with each other and with sequences detected in chimpanzee feces raising the possibility that detection of novel or unusual rep-like elements in some host species might originate from the microbial community of the host. Our results indicate that attention is needed when broad-spectrum rep gene specific polymerase chain reaction is chosen for laboratory diagnosis of circovirus infections.

To believe the past or to trust the future

NASA Astrophysics Data System (ADS)

Jin, Fengtao; Zhou, Zhaoyan

2012-01-01

A small ball rolls down from a quarter-circle to a frictionless plane. What will be the magnitude of the normal force when the ball arrives at the tangent point of the circle and the plane? According to the centripetal force formula, the normal force will be 3mg when the curvature radius of the circle is considered, but will be mg instead when the curvature radius of the plane is considered. Which one is the correct answer? The difficulty is that Newton's second law requires the second time derivative of displacement to be continuous; however, this condition is not fulfilled at the tangent point. In this paper we will discuss several possible solutions to this problem in detail.

The genomes of four novel begomoviruses and a new Sida micrantha mosaic virus strain from Bolivian weeds.

PubMed

Wyant, Patrícia Soares; Gotthardt, Diether; Schäfer, Benjamin; Krenz, Björn; Jeske, Holger

2011-02-01

Begomovirus is the largest genus within the family Geminiviridae and includes economically important plant DNA viruses infecting a broad range of plant species and causing devastating crop diseases, mainly in subtropical and tropical countries. Besides cultivated plants, many weeds act as virus reservoirs. Eight begomovirus isolates from Bolivian weeds were examined using rolling-circle amplification (RCA) and restriction fragment length polymorphism (RFLP). An efficient, novel cloning strategy using limited Sau3A digestion to obtain tandem-repeat inserts allowed the sequencing of the complete genomes. The viruses were classified by phylogenetic analysis as typical bipartite New World begomoviruses. Four of them represented distinct new virus species, for which the names Solanum mosaic Bolivia virus, Sida mosaic Bolivia virus 1, Sida mosaic Bolivia virus 2, and Abutilon mosaic Bolivia virus are proposed. Three were variants of a new strain of Sida micrantha mosaic virus (SimMV), SimMV-rho[BoVi07], SimMV-rho[Bo:CF1:07] and SimMV-rho[Bo:CF2:07], and one was a new variant of a previously described SimMV, SimMV-MGS2:07-Bo.

Characterization of a novel papillomavirus species (ZcPV1) from two California sea lions (Zalophus californianus).

PubMed

Rivera, Rebecca; Robles-Sikisaka, Refugio; Hoffman, Elizabeth M; Stacy, Brian A; Jensen, Eric D; Nollens, Hendrik H; Wellehan, James F X

2012-03-23

A seven-year old California sea lion (Zalophus californianus) presented with focally extensive, bilaterally symmetric, proliferative axillary skin lesions and preputial lesions. A second California sea lion in the same population presented with similar proliferative lesions on the underside of the tail. Histopathology revealed epidermal hyperplasia with severe hyperkeratosis, with proliferating keratinocytes forming broad, branching pegs that extended into the dermis. Pan-papillomaviral consensus PCR was used to obtain initial E1 sequence template and the complete genome was determined using a combination of rolling circle amplification and specific-primer PCR. Analysis revealed a novel papillomavirus, Zalophus californianus papillomavirus 1 (ZcPV1), with seven open reading frames encoding five early proteins (E6, E7, E1, E2 and E4) and two late proteins (L1 and L2). Phylogenetic analysis revealed that (ZcPV1) is most closely related to Equine papillomavirus 1 (EcPV1) in the genus Zetapapillomavirus, and Canine papillomaviruses 3 and 4 (CPV3, CPV4) in the genus Chipapillomavirus. The lesions regressed without intervention over a period of several months. Copyright © 2011 Elsevier B.V. All rights reserved.

Characterization of Human Papillomavirus Type 154 and Tissue Tropism of Gammapapillomaviruses

PubMed Central

Ure, Agustín Enrique; Forslund, Ola

2014-01-01

The novel human papillomavirus type 154 (HPV154) was characterized from a wart on the crena ani of a three-year-old boy. It was previously designated as the putative HPV type FADI3 by sequencing of a subgenomic FAP amplicon. We obtained the complete genome by combined methods including rolling circle amplification (RCA), genome walking through an adapted method for detection of integrated papillomavirus sequences by ligation-mediated PCR (DIPS-PCR), long-range PCR, and finally by cloning of four overlapping amplicons. Phylogenetically, the HPV154 genome clustered together with members of the proposed species Gammapapillomavirus 11, and demonstrated the highest identity in L1 to HPV136 (68.6%). The HPV154 was detected in 3% (2/62) of forehead skin swabs from healthy children. In addition, the different detection sites of 62 gammapapillomaviruses were summarized in order to analyze their tissue tropism. Several of these HPV types have been detected from multiple sources such as skin, oral, nasal, and genital sites, suggesting that the gammapapillomaviruses are generalists with a broader tissue tropism than previously appreciated. The study expands current knowledge concerning genetic diversity and tropism among HPV types in the rapidly growing gammapapillomavirus genus. PMID:24551244

Organization and evolution of highly repeated satellite DNA sequences in plant chromosomes.

PubMed

Sharma, S; Raina, S N

2005-01-01

A major component of the plant nuclear genome is constituted by different classes of repetitive DNA sequences. The structural, functional and evolutionary aspects of the satellite repetitive DNA families, and their organization in the chromosomes is reviewed. The tandem satellite DNA sequences exhibit characteristic chromosomal locations, usually at subtelomeric and centromeric regions. The repetitive DNA family(ies) may be widely distributed in a taxonomic family or a genus, or may be specific for a species, genome or even a chromosome. They may acquire large-scale variations in their sequence and copy number over an evolutionary time-scale. These features have formed the basis of extensive utilization of repetitive sequences for taxonomic and phylogenetic studies. Hybrid polyploids have especially proven to be excellent models for studying the evolution of repetitive DNA sequences. Recent studies explicitly show that some repetitive DNA families localized at the telomeres and centromeres have acquired important structural and functional significance. The repetitive elements are under different evolutionary constraints as compared to the genes. Satellite DNA families are thought to arise de novo as a consequence of molecular mechanisms such as unequal crossing over, rolling circle amplification, replication slippage and mutation that constitute "molecular drive". Copyright 2005 S. Karger AG, Basel.

Immobilized-free miniaturized electrochemical sensing system for Pb2+ detection based on dual Pb2+-DNAzyme assistant feedback amplification strategy.

PubMed

Cai, Wei; Xie, Shunbi; Zhang, Jin; Tang, Dianyong; Tang, Ying

2018-06-08

We presented a novel dual-DNAzyme feedback amplification (DDFA) strategy for Pb 2+ detection based on a micropipette tip-based miniaturized homogeneous electrochemical device. The DDFA system involves two rolling circle amplification (RCA) processes in which two circular DNA templates (C1 and C2) have been designed with a Pb 2+ -DNAzyme sequence (8-17 DNAzyme, anti-GR-5 DNAzyme) and an antisense sequence of G-quadruplex. And a linear DNA (L-DNA), which consists of a primer sequence and a Pb 2+ -DNAzyme substrate sequence, could hybridize with C1 and C2 to form two DNA complexes. In presence of Pb 2+ , the Pb 2+ -DNAzyme exhibited excellent cleavage specificity toward the substrate sequence in L-DNA, leaving primer sequence to trigger two paths of RCA process and finally resulting in massive long nanosolo DNA strands with reduplicated G-quadruplex sequences. And then, methylene blue (MB) could selectively intercalate into G-quadruplex to reduce the free MB concentration in the solution. Thereafter, a carbon fiber microelectrode-based miniaturized electrochemical device was constructed to record the decrease of electrochemical signal due to the much lower diffusion rate of MB/G-quadruplex complex than that of free MB. Therefore, the concentration of Pb 2+ could be correctively and sensitively determined in a homogeneous solution by combining DDFA with miniaturized electrochemical device. This protocol not only exhibited high selectivity and sensitivity toward Pb 2+ with a detection limit of 0.048 pM, but also reduced sample volume to 10 µL. In addition, this sensing system has been successfully applied to Pb 2+ detection in Yangtze River with desirable quantitative manners, which matched well with the atomic absorption spectrometry (AAS). Copyright © 2018 Elsevier B.V. All rights reserved.

A Century of Shope Papillomavirus in Museum Rabbit Specimens

PubMed Central

Escudero Duch, Clara; Williams, Richard A. J.; Timm, Robert M.; Perez-Tris, Javier; Benitez, Laura

2015-01-01

Sylvilagus floridanus Papillomavirus (SfPV) causes growth of large horn-like tumors on rabbits. SfPV was described in cottontail rabbits (probably Sylvilagus floridanus) from Kansas and Iowa by Richard Shope in 1933, and detected in S. audubonii in 2011. It is known almost exclusively from the US Midwest. We explored the University of Kansas Natural History Museum for historical museum specimens infected with SfPV, using molecular techniques, to assess if additional wild species host SfPV, and whether SfPV occurs throughout the host range, or just in the Midwest. Secondary aims were to detect distinct strains, and evidence for strain spatio-temporal specificity. We found 20 of 1395 rabbits in the KU collection SfPV symptomatic. Three of 17 lagomorph species (S. nuttallii, and the two known hosts) were symptomatic, while Brachylagus, Lepus and eight additional Sylvilagus species were not. 13 symptomatic individuals were positive by molecular testing, including the first S. nuttallii detection. Prevalence of symptomatic individuals was significantly higher in Sylvilagus (1.8%) than Lepus. Half of these specimens came from Kansas, though new molecular detections were obtained from Jalisco—Mexico’s first—and Nebraska, Nevada, New Mexico, and Texas, USA. We document the oldest lab-confirmed case (Kansas, 1915), pre-dating Shope’s first case. SfPV amplification was possible from 63.2% of symptomatic museum specimens. Using multiple methodologies, rolling circle amplification and, multiple isothermal displacement amplification in addition to PCR, greatly improved detection rates. Short sequences were obtained from six individuals for two genes. L1 gene sequences were identical to all previously detected sequences; E7 gene sequences, were more variable, yielding five distinct SfPV1 strains that differing by less than 2% from strains circulating in the Midwest and Mexico, between 1915 and 2005. Our results do not clarify whether strains are host species specific, though they are consistent with SfPV specificity to genus Sylvilagus. PMID:26147570

Porcine circovirus: transcription and rolling-circle DNA replication

USDA-ARS?s Scientific Manuscript database

This review summarizes the molecular studies pertaining to porcine circovirus (PCV) transcription and DNA replication. The genome of PCV is circular, single-stranded DNA and contains 1759-1768 nucleotides. Both the genome-strand (packaged in the virus particle) and the complementary-strand (synthesi...

Strengthening Resilience in Families

ERIC Educational Resources Information Center

Guild, Diane; Espiner, Deborah

2014-01-01

Rolling with Resilience (RwR) provides a springboard for developing strategies that build strengths and supports to foster developmental assets in children and youth (Benson, Scales, & Roehlkepartain, 2011). In Circle of Courage terms, resilience is strengthened by opportunities for Belonging, Mastery, Independence, and Generosity (Brendtro,…

Design of a platform technology for systemic delivery of siRNA to tumours using rolling circle transcription

NASA Astrophysics Data System (ADS)

Jang, Mihue; Kim, Jong Hwan; Nam, Hae Yun; Kwon, Ick Chan; Ahn, Hyung Jun

2015-08-01

For therapeutic applications of siRNA, there are technical challenges with respect to targeted and systemic delivery. We here report a new siRNA carrier, RNAtr NPs, in a way that multiple tandem copies of RNA hairpins as a result of rolling circle transcription (RCT) can be readily adapted in tumour-targeted and systemic siRNA delivery. RNAtr NPs provide a means of condensing large amounts of multimeric RNA transcripts into the compact nanoparticles, especially without the aid of polycationic agents, and thus reduce the risk of immunogenicity and cytotoxicity by avoiding the use of synthetic polycationic reagents. This strategy allows the design of a platform technology for systemic delivery of siRNA to tumour sites, because RCT reaction, which enzymatically generates RNA polymers in multiple copy numbers at low cost, can lead to directly accessible routes to targeted and systemic delivery. Therefore, RNAtr NPs suggest great potentials as the siRNA therapeutics for cancer treatment.

Electron microscopic studies of bacteriophage M13 DNA replication. [Escherichia coli

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

Allison, D.P.; Ganesan, A.T.; Olson, A.C.

Intracellular forms of M13 phage DNA isolated after infection of Escherichia coli with wild-type phage have been studied by electron microscopy and ultracentrifugation. The data indicate the involvement of rolling-circle intermediates in single-stranded DNA synthesis. In addition to single-stranded, circular DNA, we observed covalently closed and nicked replicative-form (RF) DNAs, dimer RF DNAs, concatenated RF DNAs, RF DNAs with single-stranded tails (sigma, rolling circles), and, occasionally, RF DNAs with theta structures. The tails in sigma molecules are always single stranded and are never longer than the DNA from mature phage; the proportion of sigma to other RF molecules does notmore » change significantly with time after infection. The origin of single-stranded DNA synthesis has been mapped by electron microscopy at a unique location on RF DNA by use of partial denaturation mapping and restriction endonuclease digestion. This location is between gene IV and gene II, and synthesis proceeds in a counterclockwise direction on the conventional genetic map.« less

Specific RNA self-cleavage in coconut cadang cadang viroid: potential for a role in rolling circle replication.

PubMed Central

Liu, Y H; Symons, R H

1998-01-01

The rolling circle replication of the small, single-stranded viroid RNAs requires a specific processing reaction to produce monomeric RNAs that are ligated into the final circular form. For avocado sunblotch viroid, peach latent mosaic viroid, and chrysanthemum chlorotic mottle viroid, the hammerhead self-cleavage reaction is considered to provide this processing reaction. We have searched for a similar type of reaction in the 246-nt coconut cadang cadang viroid, the smallest viroid of the 24-member potato spindle tuber viroid (PSTV) group. RNA transcripts prepared from the cloned central or C domain of this viroid self-cleaved specifically after denaturation with methylmercuric hydroxide followed by incubation in the presence of spermidine but in the absence of added magnesium ions. The unique cleavage site was located in the bottom strand of the C domain within a potential hairpin structure that is conserved within members of all three subgroups of the PSTV group of viroids. PMID:9630248

The partitioning and copy number control systems of the selfish yeast plasmid: an optimized molecular design for stable persistence in host cells

PubMed Central

Yen-Ting-Liu; Sau, Saumitra; Ma, Chien-Hui; Kachroo, Aashiq H; Rowley, Paul A; Chang, Keng-Ming; Fan, Hsiu-Fang; Jayaram, Makkuni

2014-01-01

Summary The multi-copy 2 micron plasmid of Saccharomyces cerevisiae, a resident of the nucleus, is remarkable for its high chromosome-like stability. The plasmid does not appear to contribute to the fitness of the host, nor does it impose a significant metabolic burden on the host at its steady state copy number. The plasmid may be viewed as a highly optimized selfish DNA element whose genome design is devoted entirely towards efficient replication, equal segregation and copy number maintenance. A partitioning system comprised of two plasmid coded proteins, Rep1 and Rep2, and a partitioning locus STB is responsible for equal or nearly equal segregation of plasmid molecules to mother and daughter cells. Current evidence supports a model in which the Rep-STB system promotes the physical association of the plasmid with chromosomes and thus plasmid segregation by a hitchhiking mechanism. The Flp site-specific recombination system housed by the plasmid plays a critical role in maintaining steady state plasmid copy number. A decrease in plasmid population due to rare missegregation events is rectified by plasmid amplification via a recombination induced rolling circle replication mechanism. Appropriate plasmid amplification, without runaway increase in copy number, is ensured by positive and negative regulation of FLP gene expression by plasmid coded proteins and by the control of Flp level/activity through host mediated post-translational modification(s) of Flp. The Flp system has been successfully utilized to understand mechanisms of site-specific recombination, to bring about directed genetic alterations for addressing fundamental problems in biology, and as a tool in biotechnological applications. PMID:25541598

The partitioning and copy number control systems of the selfish yeast plasmid: an optimized molecular design for stable persistence in host cells.

PubMed

Yen-Ting-Liu; Sau, Saumitra; Ma, Chien-Hui; Kachroo, Aashiq H; Rowley, Paul A; Chang, Keng-Ming; Fan, Hsiu-Fang; Jayaram, Makkuni

2014-10-01

The multi-copy 2 micron plasmid of Saccharomyces cerevisiae, a resident of the nucleus, is remarkable for its high chromosome-like stability. The plasmid does not appear to contribute to the fitness of the host, nor does it impose a significant metabolic burden on the host at its steady state copy number. The plasmid may be viewed as a highly optimized selfish DNA element whose genome design is devoted entirely towards efficient replication, equal segregation and copy number maintenance. A partitioning system comprised of two plasmid coded proteins, Rep1 and Rep2, and a partitioning locus STB is responsible for equal or nearly equal segregation of plasmid molecules to mother and daughter cells. Current evidence supports a model in which the Rep-STB system promotes the physical association of the plasmid with chromosomes and thus plasmid segregation by a hitchhiking mechanism. The Flp site-specific recombination system housed by the plasmid plays a critical role in maintaining steady state plasmid copy number. A decrease in plasmid population due to rare missegregation events is rectified by plasmid amplification via a recombination induced rolling circle replication mechanism. Appropriate plasmid amplification, without runaway increase in copy number, is ensured by positive and negative regulation of FLP gene expression by plasmid coded proteins and by the control of Flp level/activity through host mediated post-translational modification(s) of Flp. The Flp system has been successfully utilized to understand mechanisms of site-specific recombination, to bring about directed genetic alterations for addressing fundamental problems in biology, and as a tool in biotechnological applications.

Evaluation of two molecular techniques for rapid detection of the main dermatophytic agents of tinea capitis.

PubMed

Deng, S; Zhou, Z; de Hoog, G S; Wang, X; Abliz, P; Sun, J; Najafzadeh, M J; Pan, W; Lei, W; Zhu, S; Hasimu, H; Zhang, P; Guo, Y; Deng, D; Liao, W

2015-12-01

Tinea capitis is very common in Western China, with the most widespread aetiological agent being Trichophyton violaceum, while Microsporum canis is prevalent in the remainder of China. Conventional diagnostics and internal transcribed spacer (ITS) sequencing analyses have proven relatively limited due to the close phylogenetic relationship of anthropophilic dermatophytes. Therefore, alternative molecular tools with sufficient specificity, reproducibility and sensitivity are necessary. To evaluate two molecular techniques [multiplex ligation-dependent probe amplification (MLPA) and rolling circle amplification (RCA)] for rapid detection of the aetiological agents of tinea capitis, T. violaceum and M. canis. Probes of RCA and MLPA were designed with target sequences in the rDNA ITS gene region. Strains tested consist of 31 T. violaceum, 22 M. canis and 24 reference strains of species that are taxonomically close to the target species. The specificity and reproducibility of RCA and MLPA in detection of T. violaceum and M. canis were both 100% in both species. Sensitivity testing showed that RCA was positive at concentrations down to 1·68 × 10(6) copies of DNA in the TvioRCA probe, and 2·7 × 10(8) copies of DNA in McRCA. MLPA yielded positive results at concentrations of DNA down to 1·68 × 10(1) copies in the TvioMLPA probe and 2·7 × 10(2) in McMLPA. The two techniques were sufficiently specific and sensitive for discriminating the target DNA of T. violaceum and M. canis from that of closely related dermatophytes. RCA and MLPA are advantageous in their reliability and ease of operation compared with standard polymerase chain reaction and conventional methods. © 2015 British Association of Dermatologists.

A theoretical study for the propagation of rolling noise over a porous road pavement

NASA Astrophysics Data System (ADS)

Keung Lui, Wai; Ming Li, Kai

2004-07-01

A simplified model based on the study of sound diffracted by a sphere is proposed for investigating the propagation of noise in a hornlike geometry between porous road surfaces and rolling tires. The simplified model is verified by comparing its predictions with the published numerical and experimental results of studies on the horn amplification of sound over a road pavement. In a parametric study, a point monopole source is assumed to be localized on the surface of a tire. In the frequency range of interest, a porous road pavement can effectively reduce the level of amplified sound due to the horn effect. It has been shown that an increase in the thickness and porosity of a porous layer, or the use of a double layer of porous road pavement, attenuates the horn amplification of sound. However, a decrease in the flow resistivity of a porous road pavement does little to reduce the horn amplification of sound. It has also been demonstrated that the horn effect over a porous road pavement is less dependent on the angular position of the source on the surface of tires.

Hamburger polyomaviruses

PubMed Central

Peretti, Alberto; FitzGerald, Peter C.; Bliskovsky, Valery

2015-01-01

Epidemiological studies have suggested that consumption of beef may correlate with an increased risk of colorectal cancer. One hypothesis to explain this proposed link might be the presence of a carcinogenic infectious agent capable of withstanding cooking. Polyomaviruses are a ubiquitous family of thermostable non-enveloped DNA viruses that are known to be carcinogenic. Using virion enrichment, rolling circle amplification (RCA) and next-generation sequencing, we searched for polyomaviruses in meat samples purchased from several supermarkets. Ground beef samples were found to contain three polyomavirus species. One species, bovine polyomavirus 1 (BoPyV1), was originally discovered as a contaminant in laboratory FCS. A previously unknown species, BoPyV2, occupies the same clade as human Merkel cell polyomavirus and raccoon polyomavirus, both of which are carcinogenic in their native hosts. A third species, BoPyV3, is related to human polyomaviruses 6 and 7. Examples of additional DNA virus families, including herpesviruses, adenoviruses, circoviruses and gyroviruses were also detected either in ground beef samples or in comparison samples of ground pork and ground chicken. The results suggest that the virion enrichment/RCA approach is suitable for random detection of essentially any DNA virus with a detergent-stable capsid. It will be important for future studies to address the possibility that animal viruses commonly found in food might be associated with disease. PMID:25568187

Spatial sexual dimorphism of X and Y homolog gene expression in the human central nervous system during early male development.

PubMed

Johansson, Martin M; Lundin, Elin; Qian, Xiaoyan; Mirzazadeh, Mohammadreza; Halvardson, Jonatan; Darj, Elisabeth; Feuk, Lars; Nilsson, Mats; Jazin, Elena

2016-01-01

Renewed attention has been directed to the functions of the Y chromosome in the central nervous system during early human male development, due to the recent proposed involvement in neurodevelopmental diseases. PCDH11Y and NLGN4Y are of special interest because they belong to gene families involved in cell fate determination and formation of dendrites and axon. We used RNA sequencing, immunocytochemistry and a padlock probing and rolling circle amplification strategy, to distinguish the expression of X and Y homologs in situ in the human brain for the first time. To minimize influence of androgens on the sex differences in the brain, we focused our investigation to human embryos at 8-11 weeks post-gestation. We found that the X- and Y-encoded genes are expressed in specific and heterogeneous cellular sub-populations of both glial and neuronal origins. More importantly, we found differential distribution patterns of X and Y homologs in the male developing central nervous system. This study has visualized the spatial distribution of PCDH11X/Y and NLGN4X/Y in human developing nervous tissue. The observed spatial distribution patterns suggest the existence of an additional layer of complexity in the development of the male CNS.

Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ

PubMed Central

Mignardi, Marco; Mezger, Anja; Qian, Xiaoyan; La Fleur, Linnea; Botling, Johan; Larsson, Chatarina; Nilsson, Mats

2015-01-01

In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ. PMID:26240388

Molecular characterization and pathogenicity of a carrot (Daucus carota) infecting begomovirus and associated betasatellite from India.

PubMed

Kumar, Jitendra; Gunapati, Samatha; Singh, Sudhir P; Gadre, Rekha; Sharma, Naresh C; Tuli, Rakesh

2013-10-24

The yellow mosaic pattern and shortening of leaf petiole are common disease symptoms associated with begomovirus infection in carrot. DNA from field infected carrot leaves was analyzed by rolling circle amplification and sequencing. The results established the presence of ageratum enation virus (AEV), which is referred to here as ageratum enation virus-carrot (AEV-Car). Symptomatic ageratum (Ageratum conyzoides) plants, growing adjacent to the carrot fields, also showed the presence of AEV (AEV-Age). Ageratum yellow leaf curl betasatellite (AYLCB) was also detected in the AEV infected carrot and ageratum samples. AEV-Car and AEV-Age are 95-97% identical in their DNA sequences, represents groups of isolates from the respective plant hosts (carrot and ageratum). Agroinoculation using infectious clones of AEV-Car plus AYLCB or AEV-Age plus AYLCB in carrot, ageratum, tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum) produced yellow mosaic and curling symptoms in leaves of inoculated plants. Agroinoculation of the two isolates together, along with the betasatellite (AEV-Car plus AEV-Age plus AYLCB) resulted in the enhancement of symptoms in comparison to the plants inoculated with single isolate. Plants with more severe symptoms showed a higher level of viral DNA accumulation, suggesting synergistic interactions between the two isolates of AEV. Copyright © 2013. Published by Elsevier B.V.

Detection of a novel circovirus in mute swans (Cygnus olor) by using nested broad-spectrum PCR.

PubMed

Halami, M Y; Nieper, H; Müller, H; Johne, R

2008-03-01

Circoviruses are the causative agents of acute and chronic diseases in several animal species. Clinical symptoms of circovirus infections range from depression and diarrhoea to immunosuppression and feather disorders in birds. Eleven different members of the genus Circovirus are known so far, which infect pigs and birds in a species-specific manner. Here, a nested PCR was developed for the detection of a broad range of different circoviruses in clinical samples. Using this assay, a novel circovirus was detected in mute swans (Cygnus olor) found dead in Germany in 2006. Sequence analysis of the swan circovirus (SwCV) genome, amplified by multiply primed rolling-circle amplification and PCR, indicates that SwCV is a distinct virus most closely related to the goose circovirus (73.2% genome sequence similarity). Sequence variations between SwCV genomes derived from two different individuals were high (15.5% divergence) and mainly confined to the capsid protein-encoding region. By PCR testing of 32 samples derived from swans found dead in two different regions of Germany, detection rates of 20.0 and 77.3% were determined, thus indicating a high incidence of SwCV infection. The clinical significance of SwCV infection, however, needs to be investigated further.

Unstable spiral modes in disk-shaped galaxies

PubMed Central

Lau, Y. Y.; Lin, C. C.; Mark, James W.-K.

1976-01-01

The mechanisms for the maintenance and the excitation of trailing spiral modes of density waves in diskshaped galaxies, as proposed by Lin in 1969 and by Mark recently, are substantiated by an analysis of the gas-dynamical model of the galaxy. The self-excitation of the unstable mode in caused by waves propagating outwards from the corotation circle, which carry away angular momentum of a sign opposite to that contained in the wave system inside that circle. Specifically, a simple dispersion relationship is given as a definite integral, which allows the immediate determination of the pattern frequency and the amplification rate, once the basic galactic model is known. PMID:16592313

Going Around On Circles: Mathematics and Computer Art. Part 2.

ERIC Educational Resources Information Center

Gordon, Sheldon P.; Gordon, Florence S.

1984-01-01

Discusses properties of epicycloids. (The easiest way to picture them is to think of a piece of radioactive bubble gum attached to a wheel which is rolling around the outside of a larger wheel.) Includes a computer program (TRS-80 color computer) that will graph any epicycloid with integer values for the radii. (JN)

Replicative intermediates of porcine circovirus in animal tissue cultured cells or in bacteria undergoing copy-release replication

USDA-ARS?s Scientific Manuscript database

Porcine circovirus (PCV) has been assumed to replicate its genome via the rolling-circle replication (RCR) mechanism because it encodes a Rep protein that contains several amino acid motifs commonly found in other RCR biological systems. Two proteins, Rep and Rep', are essential for PCV DNA replicat...

Biopower and Pedagogy: Local Spaces and Institutional Technologies

ERIC Educational Resources Information Center

Wittman, John

2006-01-01

While sitting on a bench outside the university's museum grappling with his own emotional numbness and inability to comprehend the traumatic events of 9-11, John Wittman watched a group of 4 students circling one of the main buildings on campus with an American flag on the back of the roll bar of a truck. Having thought about the flag-waving…

Transition to chaos of natural convection between two infinite differentially heated vertical plates

NASA Astrophysics Data System (ADS)

Gao, Zhenlan; Sergent, Anne; Podvin, Berengere; Xin, Shihe; Le Quéré, Patrick; Tuckerman, Laurette S.

2013-08-01

Natural convection of air between two infinite vertical differentially heated plates is studied analytically in two dimensions (2D) and numerically in two and three dimensions (3D) for Rayleigh numbers Ra up to 3 times the critical value Rac=5708. The first instability is a supercritical circle pitchfork bifurcation leading to steady 2D corotating rolls. A Ginzburg-Landau equation is derived analytically for the flow around this first bifurcation and compared with results from direct numerical simulation (DNS). In two dimensions, DNS shows that the rolls become unstable via a Hopf bifurcation. As Ra is further increased, the flow becomes quasiperiodic, and then temporally chaotic for a limited range of Rayleigh numbers, beyond which the flow returns to a steady state through a spatial modulation instability. In three dimensions, the rolls instead undergo another pitchfork bifurcation to 3D structures, which consist of transverse rolls connected by counter-rotating vorticity braids. The flow then becomes time dependent through a Hopf bifurcation, as exchanges of energy occur between the rolls and the braids. Chaotic behavior subsequently occurs through two competing mechanisms: a sequence of period-doubling bifurcations leading to intermittency or a spatial pattern modulation reminiscent of the Eckhaus instability.

Self-amplification of nigral degeneration in Parkinson's disease: a hypothesis.

PubMed

Ionov, Ilya D

2008-12-01

This review analyzes current evidence regarding possible mechanisms of nigral damage in idiopathic Parkinson's disease (iPD). In normal brain, a specific interplay among the blood-brain barrier (BBB), substantia nigra (SN), and locus coeruleus (LC) creates the condition for a self-accelerating damage to the SN. Three vicious circles involving SN-BBB, LC-SN-BBB, and histamine-BBB-SN interactions are described. In iPD, a self-accelerating loss of nigral cells can be triggered by brain hypoperfusion and by an increased blood histamine level. iPD-associated factors such as decreased CSF levels of substance P, somatostatin, and glutamate can aggravate the vicious-circle-induced damage to the SN.

A variant of Rubus yellow net virus with altered genomic organization.

PubMed

Diaz-Lara, Alfredo; Mosier, Nola J; Keller, Karen E; Martin, Robert R

2015-02-01

Rubus yellow net virus (RYNV) is a member of the genus Badnavirus (family: Caulimoviridae). RYNV infects Rubus species causing chlorosis of the tissue along the leaf veins, giving an unevenly distributed netted symptom in some cultivars of red and black raspberry. Recently, a strain of RYNV was sequenced from a Rubus idaeus plant in Alberta, Canada, exhibiting such symptoms. The viral genome contained seven open reading frames (ORFs) with five of them in the sense-strand, including a large polyprotein. Here we describe a graft-transmissible strain of RYNV from Europe infecting cultivar 'Baumforth's Seedling A' (named RYNV-BS), which was sequenced using rolling circle amplification, enzymatic digestion, cloning and primer walking, and it was resequenced at a 5X coverage. This sequence was then compared with the RYNV-Ca genome and significant differences were observed. Genomic analysis identified differences in the arrangement of coding regions, promoter elements, and presence of motifs. The genomic organization of RYNV-BS consisted of five ORFs (four ORFs in the sense-strand and one ORF in the antisense-strand). ORFs 1, 2, and 3 showed a high degree of homology to RYNV-Ca, while ORFs 4 and 6 of RYNV-BS were quite distinct. Also, the predicted ORFs 5 and 7 in the RYNV-Ca were absent in the RYNV-BS sequence. These differences may account for the lack of aphid transmissibility of RYNV-BS.

ELISA-PLA: A novel hybrid platform for the rapid, highly sensitive and specific quantification of proteins and post-translational modifications.

PubMed

Tong, Qing-He; Tao, Tao; Xie, Li-Qi; Lu, Hao-Jie

2016-06-15

Detection of low-abundance proteins and their post-translational modifications (PTMs) remains a great challenge. A conventional enzyme-linked immunosorbent assay (ELISA) is not sensitive enough to detect low-abundance PTMs and suffers from nonspecific detection. Herein, a rapid, highly sensitive and specific platform integrating ELISA with a proximity ligation assay (PLA), termed ELISA-PLA, was developed. Using ELISA-PLA, the specificity was improved by the simultaneous and proximate recognition of targets through multiple probes, and the sensitivity was significantly improved by rolling circle amplification (RCA). For GFP, the limit of detection (LOD) was decreased by two orders of magnitude compared to that of ELISA. Using site-specific phospho-antibody and pan-specific phospho-antibody, ELISA-PLA was successfully applied to quantify the phosphorylation dynamics of ERK1/2 and the overall tyrosine phosphorylation level of ERK1/2, respectively. ELISA-PLA was also used to quantify the O-GlcNAcylation of AKT, c-Fos, CREB and STAT3, which is faster and more sensitive than the conventional immunoprecipitation and western blotting (IP-WB) method. As a result, the sample consumption of ELISA-PLA was reduced 40-fold compared to IP-WB. Therefore, ELISA-PLA could be a promising platform for the rapid, sensitive and specific detection of proteins and PTMs. Copyright © 2016 Elsevier B.V. All rights reserved.

23. VIEW OF SECTION DRAWINGS. THE SECTION LINES CUT THE ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

23. VIEW OF SECTION DRAWINGS. THE SECTION LINES CUT THE BUILDING EAST-WEST. THE ORIGINAL DRAWING HAS BEEN ARCHIVED ON MICROFILM. THE DRAWING WAS REPRODUCED AT THE BEST QUALITY POSSIBLE. LETTERS AND NUMBERS IN THE CIRCLES INDICATE FOOTER AND/OR COLUMN LOCATIONS. - Rocky Flats Plant, Uranium Rolling & Forming Operations, Southeast section of plant, southeast quadrant of intersection of Central Avenue & Eighth Street, Golden, Jefferson County, CO

Vestibular Responses and Motion Sickness during Pitch, Roll, and Yaw Sinusoidal Whole-Body Oscillation.

DTIC Science & Technology

1990-03-01

vestibulo-ocular reflex) pitch, ’- 11’, 1 3c\\ " ’ I...[ 16. PRC C nystagmus 1 ’>"( k 1.,’ roll V-) , __r_ ,_t_ _ _ 17. SECURITY CLASSIFICATION 1S SECURITY...of amplification of EOG was 3.0 s. Because of the position of subjEcts’ heads relative to the axis of rotation, vertical nystagmus comprised the VOR...response in Groups I and III. To achieve measurement of the VOR in Groups IV and V, subjects were instructed to gaze downward, 30 deg in Group IV, and

21. VIEW OF THE FIRST FLOOR PLAN. THE FIRST FLOOR ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

21. VIEW OF THE FIRST FLOOR PLAN. THE FIRST FLOOR WAS USED FOR DEPLETED AND ENRICHED URANIUM FABRICATION. THE ORIGINAL DRAWING HAS BEEN ARCHIVED ON MICROFILM. THE DRAWING WAS REPRODUCED AT THE BEST QUALITY POSSIBLE. LETTERS AND NUMBERS IN THE CIRCLES INDICATE FOOTER AND/OR COLUMN LOCATIONS. - Rocky Flats Plant, Uranium Rolling & Forming Operations, Southeast section of plant, southeast quadrant of intersection of Central Avenue & Eighth Street, Golden, Jefferson County, CO

DNA Replication During Conjugal Transfer of R1162

DTIC Science & Technology

2002-01-01

transport out of the cell. This idea is stimulated by the similarity between the intermediates of single-stranded phage DNA replication and conjugative...the tra gene clusters ( Miele et al., 1991). The protein is not required for replication of the plasmid, and it seems to be dispensable for transfer...donor, in order to reattach to the transport mechanism prior to the start of a new round of transfer. Rolling circle replication itself might also be

22. VIEW OF THE SECOND FLOOR PLAN. THE SECOND FLOOR ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

22. VIEW OF THE SECOND FLOOR PLAN. THE SECOND FLOOR CONTAINS THE AIR PLENUM ND SOME OFFICE SPACE. THE ORIGINAL DRAWING HAS BEEN ARCHIVED ON MICROFILM. THE DRAWING WAS REPRODUCED AT THE BEST QUALITY POSSIBLE. LETTERS AND NUMBERS IN THE CIRCLES INDICATE FOOTER AND/OR COLUMN LOCATIONS. - Rocky Flats Plant, Uranium Rolling & Forming Operations, Southeast section of plant, southeast quadrant of intersection of Central Avenue & Eighth Street, Golden, Jefferson County, CO

Begomovirus diversity in tomato crops and weeds in Ecuador and the detection of a recombinant isolate of rhynchosia golden mosaic Yucatan virus infecting tomato.

PubMed

Paz-Carrasco, Lenin C; Castillo-Urquiza, Gloria P; Lima, Alison T M; Xavier, Cesar A D; Vivas-Vivas, Leticia M; Mizubuti, Eduardo S G; Zerbini, F Murilo

2014-08-01

Viral diseases caused by begomoviruses are of economic importance due to their adverse effects on the production of tropical and subtropical crops. In Ecuador, despite reports of significant infestations of Bemisia tabaci in the late 1990s, only very recently has a begomovirus, tomato leaf deformation virus (ToLDeV, also present in Peru), been reported in tomato. ToLDeV is the first monopartite begomovirus discovered that originated in the Americas, and its presence in Ecuador highlights the need for a wider survey of tomato-infecting begomoviruses in this country. Tomato and weed samples were collected in 2010 and 2011 in six provinces of Ecuador, and begomovirus genomes were cloned and sequenced using a rolling-circle-amplification-based approach. Most tomato samples from the provinces of Guayas, Loja, Manabi and Santa Elena were infected with tomato leaf deformation virus (ToLDeV). One sample from Manabi had a triple infection with ToLDeV, rhynchosia golden mosaic Yucatan virus (RhGMYuV) and an isolate that was a recombinant between the two. A new begomovirus was detected in another tomato sample from Manabi. Samples of Rhynchosia sp. from the provinces of Guayas and Manabi were infected by RhGMYuV. These results indicate not only the prevalence of ToLDeV in tomato in Ecuador but also the presence of other viruses, albeit at a much lower frequency.

Causative Role of Grapevine Red Blotch Virus in Red Blotch Disease.

PubMed

Yepes, Luz Marcela; Cieniewicz, Elizabeth; Krenz, Björn; McLane, Heather; Thompson, Jeremy R; Perry, Keith Lloyd; Fuchs, Marc

2018-05-17

Grapevine red blotch virus (GRBV) has a monopartite single-stranded DNA genome and is the type species of the genus Grablovirus in the family Geminiviridae. To address the etiological role of GRBV in the recently recognized red blotch disease of grapevine, infectious GRBV clones were engineered from the genome of each of the two previously identified phylogenetic clades for Agrobacterium tumefaciens-mediated inoculations of tissue culture-grown Vitis spp. plants. Following agroinoculation and one or two dormancy cycles, systemic GRBV infection was detected by multiplex polymerase chain reaction (PCR) in Vitis vinifera exhibiting foliar disease symptoms but not in asymptomatic vines. Infected rootstock genotype SO4 (V. berlandieri × V. riparia) exhibited leaf chlorosis and cupping, while infection was asymptomatic in agroinoculated 110R (V. berlandieri × V. rupestris), 3309C (V. riparia × V. rupestris), and V. rupestris. Spliced GRBV transcripts of the replicase-associated protein coding region accumulated in leaves of agroinfected vines, as shown by reverse-transcription PCR; this was consistent with systemic infection resulting from virus replication. Additionally, a virus progeny identical in nucleotide sequence to the infectious GRBV clones was recovered from agroinfected vines by rolling circle amplification, cloning, and sequencing. Concomitantly, subjecting naturally infected grapevines to microshoot tip culture resulted in an asymptomatic plant progeny that tested negative for GRBV in multiplex PCR. Altogether, our agroinoculation and therapeutic experiments fulfilled Koch's postulates and revealed the causative role of GRBV in red blotch disease.

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.

DNA double-strand break in vivo at the 3' extremity of exons located upstream of group II introns. Senescence and circular DNA introns in Podospora mitochondria.

PubMed

Sainsard-Chanet, A; Begel, O; Belcour, L

1994-10-07

In the filamentous fungus Podospora anserina, the unavoidable phenomenon of senescence is associated with the amplification of the first intron of the mitochondrial cox1 that accumulates as circular DNA molecules consisting of tandem repeats. This group II intron (cox1-i1 or alpha) is able to transpose and contains an open reading frame with significant amino acid similarity with reverse transcriptases. The generation of these intronic circular DNA molecules, their amplification and their involvement in the senescence process are unresolved questions. We demonstrate here that: (1) another group II intron, the fourth intron of gene cox1, cox1-i4, is also able to give precise DNA end to end junctions; (2) this intronic sequence can be found amplified during senescence, although to a lesser extent than cox1-i1; (3) the amplification of the DNA multimeric cox1-i1 molecules likely does not proceed by autonomous replication; (4) the generation of the DNA intronic circles does not require efficient intron splicing; (5) a DNA double-strand break occurs in vivo at the 3' extremity of the cox1-e1 and cox1-e4 exons preceding the group II introns that form circular DNAs. On the whole, these results show that the ability to form DNA circular molecules is a property of some group II introns and they demonstrate the occurrence of a specific DNA cleavage at or near the integration site of these group II introns. The results strongly suggest that this cleavage is involved in the formation of the group II intronic DNA circles and could also be involved in the phenomenon of group II intron homing.

Rolling Circle Transcription of Ribozymes Targeted to ras and mdr-1

DTIC Science & Technology

2001-09-01

ssDNA) to direct transcription of an tion-PCR, and recyclization were carried out to optimize active hammerhead ribozyme in E. coli cells. transcription...transcription I hammerhead ribozyme I in vitro selection and 12.5 units/ml RNase inhibitor (Promega), in a total reaction volume of 15 tk1. After a...sequence encoding a ssDNA, and splint ssDNA were ethanol-precipitated and used as hammerhead ribozyme . templates to begin the next round of in vitro

20. VIEW OF THE BASEMENT FLOOR PLAN. THE BASEMENT AREA ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

20. VIEW OF THE BASEMENT FLOOR PLAN. THE BASEMENT AREA INCLUDES A UTILITY ROOM, PROCESS WASTE STORAGE AND MAINTENANCE AREAS, AND THE ENTRANCE TO AN UNDERGROUND TUNNEL LEADING TO BUILDING 881. THE ORIGINAL DRAWING HAS BEEN ARCHIVED ON MICROFILM. THE DRAWING WAS REPRODUCED AT THE BEST QUALITY POSSIBLE. LETTERS AND NUMBERS IN THE CIRCLES INDICATE FOOTER AND/OR COLUMN LOCATIONS. - Rocky Flats Plant, Uranium Rolling & Forming Operations, Southeast section of plant, southeast quadrant of intersection of Central Avenue & Eighth Street, Golden, Jefferson County, CO

Programmable oligonucleotide probes design and applications for in situ and in vivo RNA imaging in cells

NASA Astrophysics Data System (ADS)

Cheglakov, Zoya

Unequal spreading of mRNA is a frequent experience observed in varied cell lines. The study of cellular processes dynamics and precise localization of mRNAs offers a vital toolbox to target specific proteins in precise cytoplasmic areas and provides a convenient instrument to uncover their mechanisms and functions. Latest methodological innovations have allowed imaging of a single mRNA molecule in situ and in vivo. Today, Fluorescent In Situ Hybridization (FISH) methods allow the studying of mRNA expression and offer a vital toolbox for accurate biological models. Studies enable analysis of the dynamics of an individual mRNA, have uncovered the multiplex RNA transport systems. With all current approaches, a single mRNA tracking in the mammalian cells is still challenging. This thesis describes mRNA detection methods based on programmable fluorophore-labeled DNA structures complimentary to native targets providing an accurate mRNA imaging in mammalian cells. First method represents beta-actin (ACTB) transcripts in situ detection in human cells, the technique strategy is based on programmable DNA probes, amplified by rolling circle amplification (RCA). The method reports precise localization of molecule of interest with an accuracy of a single-cell. Visualization and localization of specific endogenous mRNA molecules in real-time in vivo has the promising to innovate cellular biology studies, medical analysis and to provide a vital toolbox in drugs invention area. Second method described in this thesis represents miR-21 miRNA detection within a single live-cell resolution. The method using fluorophore-labeled short synthetic DNAs probes forming a stem-loop shape and generating Fluorescent Resonance Energy Transfer (FRET) as a result of target-probes hybridization. Catalytic nucleic acid (DNAzymes) probes are cooperative tool for precise detection of different mRNA targets. With assistance of a complementary fluorophore-quencher labeled substrate, the DNAzymes provide a beneficial strategy for simultaneous tracking readily accomplished by multicolor imaging with diverse fluorescent tags. The third method in this thesis will demonstrate the advantage of DNAzymes probes amplification, and offers potential strategy to monitor miRNAs in mammalian live cells.

Assessing the Diversity of Rodent-Borne Viruses: Exploring of High-Throughput Sequencing and Classical Amplification/Sequencing Approaches.

PubMed

Drewes, Stephan; Straková, Petra; Drexler, Jan F; Jacob, Jens; Ulrich, Rainer G

2017-01-01

Rodents are distributed throughout the world and interact with humans in many ways. They provide vital ecosystem services, some species are useful models in biomedical research and some are held as pet animals. However, many rodent species can have adverse effects such as damage to crops and stored produce, and they are of health concern because of the transmission of pathogens to humans and livestock. The first rodent viruses were discovered by isolation approaches and resulted in break-through knowledge in immunology, molecular and cell biology, and cancer research. In addition to rodent-specific viruses, rodent-borne viruses are causing a large number of zoonotic diseases. Most prominent examples are reemerging outbreaks of human hemorrhagic fever disease cases caused by arena- and hantaviruses. In addition, rodents are reservoirs for vector-borne pathogens, such as tick-borne encephalitis virus and Borrelia spp., and may carry human pathogenic agents, but likely are not involved in their transmission to human. In our days, next-generation sequencing or high-throughput sequencing (HTS) is revolutionizing the speed of the discovery of novel viruses, but other molecular approaches, such as generic RT-PCR/PCR and rolling circle amplification techniques, contribute significantly to the rapidly ongoing process. However, the current knowledge still represents only the tip of the iceberg, when comparing the known human viruses to those known for rodents, the mammalian taxon with the largest species number. The diagnostic potential of HTS-based metagenomic approaches is illustrated by their use in the discovery and complete genome determination of novel borna- and adenoviruses as causative disease agents in squirrels. In conclusion, HTS, in combination with conventional RT-PCR/PCR-based approaches, resulted in a drastically increased knowledge of the diversity of rodent viruses. Future improvements of the used workflows, including bioinformatics analysis, will further enhance our knowledge and preparedness in case of the emergence of novel viruses. Classical virological and additional molecular approaches are needed for genome annotation and functional characterization of novel viruses, discovered by these technologies, and evaluation of their zoonotic potential. © 2017 Elsevier Inc. All rights reserved.

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

Reactive oxygen species regulate DNA copy number in isolated yeast mitochondria by triggering recombination-mediated replication.

PubMed

Hori, Akiko; Yoshida, Minoru; Shibata, Takehiko; Ling, Feng

2009-02-01

Mitochondrial DNA (mtDNA) encodes proteins that are essential for cellular ATP production. Reactive oxygen species (ROS) are respiratory byproducts that damage mtDNA and other cellular components. In Saccharomyces cerevisiae, the oxidized base excision-repair enzyme Ntg1 introduces a double-stranded break (DSB) at the mtDNA replication origin ori5; this DSB initiates the rolling-circle mtDNA replication mediated by the homologous DNA pairing protein Mhr1. Thus, ROS may play a role in the regulation of mtDNA copy number. Here, we show that the treatment of isolated mitochondria with low concentrations of hydrogen peroxide increased mtDNA copy number in an Ntg1- and Mhr1-dependent manner. This treatment elevated the DSB levels at ori5 of hypersuppressive [rho(-)] mtDNA only if Ntg1 was active. In vitro Ntg1-treatment of hypersuppressive [rho(-)] mtDNA extracted from hydrogen peroxide-treated mitochondria revealed increased oxidative modifications at ori5 loci. We also observed that purified Ntg1 created breaks in single-stranded DNA harboring oxidized bases, and that ori5 loci have single-stranded character. Furthermore, chronic low levels of hydrogen peroxide increased in vivo mtDNA copy number. We therefore propose that ROS act as a regulator of mtDNA copy number, acting through the Mhr1-dependent initiation of rolling-circle replication promoted by Ntg1-induced DSB in the single-stranded regions at ori5.

Precise determination, cross-recognition, and functional analysis of the double-strand origins of the rolling-circle replication plasmids in haloarchaea.

PubMed

Zhou, Ligang; Zhou, Meixian; Sun, Chaomin; Han, Jing; Lu, Qiuhe; Zhou, Jian; Xiang, Hua

2008-08-01

The precise nick site in the double-strand origin (DSO) of pZMX201, a 1,668-bp rolling-circle replication (RCR) plasmid from the haloarchaeon Natrinema sp. CX2021, was determined by electron microscopy and DSO mapping. In this plasmid, DSO nicking occurred between residues C404 and G405 within a heptanucleotide sequence (TCTC/GGC) located in the stem region of an imperfect hairpin structure. This nick site sequence was conserved among the haloarchaeal RCR plasmids, including pNB101, suggesting that the DSO nick site might be the same for all members of this plasmid family. Interestingly, the DSOs of pZMX201 and pNB101 were found to be cross-recognized in RCR initiation and termination in a hybrid plasmid system. Mutation analysis of the DSO from pZMX201 (DSO(Z)) in this hybrid plasmid system revealed that: (i) the nucleotides in the middle of the conserved TCTCGGC sequence play more-important roles in the initiation and termination process; (ii) the left half of the hairpin structure is required for initiation but not for termination; and (iii) a 36-bp sequence containing TCTCGGC and the downstream sequence is essential and sufficient for termination. In conclusion, these haloarchaeal plasmids, with novel features that are different from the characteristics of both single-stranded DNA phages and bacterial RCR plasmids, might serve as a good model for studying the evolution of RCR replicons.

Novel papillomavirus isolated from the oral mucosa of a polar bear does not cluster with other papillomaviruses of carnivores.

PubMed

Stevens, Hans; Rector, Annabel; Bertelsen, Mads F; Leifsson, Pall S; Van Ranst, Marc

2008-05-25

Papillomatosis has been documented in several carnivores, and papillomavirus (PV) types have been characterized from lesions in a number of carnivore species: the canine oral PV (COPV), the Felis domesticus PV type 1 (FdPV-1) isolated from a Persian cat, the Procyon lotor PV type 1 (PlPV-1) isolated from a raccoon, the canine PV type 2 (CPV-2) from a dog's foot pad lesion and the canine PV type 3 (CPV-3) associated with a canine epidermodysplasia verruciformis - like disease. A tissue sample was taken from a papillomatous lesion on the oral mucosa of a polar bear (Ursus maritimus). Extracted DNA was used as a template for multiply primed rolling-circle amplification (RCA), and restriction enzyme analysis of the RCA product indicated the presence of papillomaviral DNA. The genome of this PV was cloned and the complete genomic sequence was determined. The Ursus maritimus PV type 1 (UmPV-1) genome counts 7582 basepairs and is smaller than that of other papillomaviruses from carnivore species. UmPV-1 contains the typical noncoding region NCR1, but unlike the carnivore PVs of the Lambda genus, UmPV-1 does not possess a second noncoding region NCR2. Phylogenetic analysis based on a nucleotide sequence alignment of the L1 ORF of UmPV-1 and 51 other PV types indicates that UmPV-1 does not cluster with any of the other carnivore PVs, but branches off near the root of the common branch of the genus Alphapapillomavirus.

Molecular characterization of a distinct begomovirus species from Vernonia cinerea and its associated DNA-beta using the bacteriophage Phi 29 DNA polymerase.

PubMed

Packialakshmi, R M; Srivastava, N; Girish, K R; Usha, R

2010-08-01

Vernonia cinerea plants with yellow vein symptoms were collected around crop fields in Madurai. A portion (550 bp) of the AV1 gene amplified using degenerate primers from the total DNA purified from diseased leaf sample was cloned and sequenced. Specific primers derived from the above sequence were used to amplify 2,745 nucleotides with the typical genome organization of begomoviral DNA A (EMBL Accession No. AM182232). Sequence comparison with other begomoviruses revealed the greatest identity (82.4%) with Emilia yellow vein virus (EmYVV-[Fz1]) from China and less than 80% with all other known begomoviruses. The International Committee on Taxonomy of Viruses (ICTV) has therefore recognized Vernonia yellow vein virus (VeYVV) as a distinct begomovirus species. Conventional PCR could not amplify the DNA B or DNA beta from the diseased tissue. However, the beta DNA (1364 bp) associated with the disease was obtained (Accession No. FN435836) by the rolling circle amplification-restriction fragment length polymorphism method (RCA-RFLP) using Phi 29 DNA polymerase. Sequence analysis shows that DNA beta of VeYVV has the highest identity (56.8%) with DNA beta of Sigesbeckia yellow vein Guangxi betasatellite (SibYVGxB-[CN: Gx111:05]) and 56-53% with DNA beta associated with other begomoviruses. This is the first report of the molecular characterization of VeYVV from V. cinerea in India. The complete molecular characterization, phylogenetic analysis, and putative recombination events in VeYVV are reported.

A Novel Virus Detected in Papillomas and Carcinomas of the Endangered Western Barred Bandicoot (Perameles bougainville) Exhibits Genomic Features of both the Papillomaviridae and Polyomaviridae▿

PubMed Central

Woolford, Lucy; Rector, Annabel; Van Ranst, Marc; Ducki, Andrea; Bennett, Mark D.; Nicholls, Philip K.; Warren, Kristin S.; Swan, Ralph A.; Wilcox, Graham E.; O'Hara, Amanda J.

2007-01-01

Conservation efforts to prevent the extinction of the endangered western barred bandicoot (Perameles bougainville) are currently hindered by a progressively debilitating cutaneous and mucocutaneous papillomatosis and carcinomatosis syndrome observed in captive and wild populations. In this study, we detected a novel virus, designated the bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1), in lesional tissue from affected western barred bandicoots using multiply primed rolling-circle amplification and PCR with the cutaneotropic papillomavirus primer pairs FAP59/FAP64 and AR-L1F8/AR-L1R9. Sequencing of the BPCV1 genome revealed a novel prototype virus exhibiting genomic properties of both the Papillomaviridae and the Polyomaviridae. Papillomaviral properties included a large genome size (∼7.3 kb) and the presence of open reading frames (ORFs) encoding canonical L1 and L2 structural proteins. The genomic organization in which structural and nonstructural proteins were encoded on different strands of the double-stranded genome and the presence of ORFs encoding the nonstructural proteins large T and small t antigens were, on the other hand, typical polyomaviral features. BPCV1 may represent the first member of a novel virus family, descended from a common ancestor of the papillomaviruses and polyomaviruses recognized today. Alternatively, it may represent the product of ancient recombination between members of these two virus families. The discovery of this virus could have implications for the current taxonomic classification of Papillomaviridae and Polyomaviridae and can provide further insight into the evolution of these ancient virus families. PMID:17898069

A fosmid cloning strategy for detecting the widest possible spectrum of microbes from the international space station drinking water system.

PubMed

Choi, Sangdun; Chang, Mi Sook; Stuecker, Tara; Chung, Christine; Newcombe, David A; Venkateswaran, Kasthuri

2012-12-01

In this study, fosmid cloning strategies were used to assess the microbial populations in water from the International Space Station (ISS) drinking water system (henceforth referred to as Prebiocide and Tank A water samples). The goals of this study were: to compare the sensitivity of the fosmid cloning strategy with that of traditional culture-based and 16S rRNA-based approaches and to detect the widest possible spectrum of microbial populations during the water purification process. Initially, microbes could not be cultivated, and conventional PCR failed to amplify 16S rDNA fragments from these low biomass samples. Therefore, randomly primed rolling-circle amplification was used to amplify any DNA that might be present in the samples, followed by size selection by using pulsed-field gel electrophoresis. The amplified high-molecular-weight DNA from both samples was cloned into fosmid vectors. Several hundred clones were randomly selected for sequencing, followed by Blastn/Blastx searches. Sequences encoding specific genes from Burkholderia, a species abundant in the soil and groundwater, were found in both samples. Bradyrhizobium and Mesorhizobium, which belong to rhizobia, a large community of nitrogen fixers often found in association with plant roots, were present in the Prebiocide samples. Ralstonia, which is prevalent in soils with a high heavy metal content, was detected in the Tank A samples. The detection of many unidentified sequences suggests the presence of potentially novel microbial fingerprints. The bacterial diversity detected in this pilot study using a fosmid vector approach was higher than that detected by conventional 16S rRNA gene sequencing.

Impact-based piezoelectric energy harvester for multidimensional, low-level, broadband, and low-frequency vibrations

NASA Astrophysics Data System (ADS)

Zhang, Hongjiang; Jiang, Senlin; He, Xuefeng

2017-05-01

This letter proposes an impact-based piezoelectric energy harvester that uses a rolling bead contained in a bracket that is supported by a spring. Under either translational or rotational base excitation, the bead moves within the bracket and collides with piezoelectric cantilevers that are located around the bracket; these collisions cause the piezoelectric beams to vibrate and thus produce electrical outputs. The low rolling friction and the motion amplification effect of the spring make the resulting device suitable for collection of low-level vibration energy. Experiments show that the proposed harvester is promising for use in scavenging of energy from the multidimensional, low-level, broadband, and low-frequency vibrations that occur in natural environments.

31. RW Meyer Sugar Mill: 18761889. Threeroll sugar mill: oneton ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

31. RW Meyer Sugar Mill: 1876-1889. Three-roll sugar mill: one-ton daily processing capacity. Manufactured by Edwin Maw, Liverpool, England, ca. 1855-1870. View: View down at the mill from top of the mill's circular masonry enclosure. Mill animals circling above the mill, on top of the enclosure, dragged booms radiating from the drive shaft to power the mill. The drive-shaft is no longer in its upright positon but is lying next to the mill in the foreground. - R. W. Meyer Sugar Mill, State Route 47, Kualapuu, Maui County, HI

30. RW Meyer Sugar Mill: 18761889. Threeroll sugar mill: oneton ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

30. RW Meyer Sugar Mill: 1876-1889. Three-roll sugar mill: one-ton daily processing capacity. Manufactured by Edwin Maw, Liverpool, England, ca. 1885-1870. View: Masonry-lined passage-way leading to the mill at the center of its circular masonry enclosure. The passageway permitted cane to be carried to the mill and cane trash (bagasse) to be carried away. Bridges over the passageways, no longer in place, permitted the mill animals to circle and power the mill from above. - R. W. Meyer Sugar Mill, State Route 47, Kualapuu, Maui County, HI

Advanced Response Surface Modeling of Ares I Roll Control Jet Aerodynamic Interactions

NASA Technical Reports Server (NTRS)

Favaregh, Noah M.

2010-01-01

The Ares I rocket uses roll control jets. These jets have aerodynamic implications as they impinge on the surface and protuberances of the vehicle. The jet interaction on the body can cause an amplification or a reduction of the rolling moment produced by the jet itself, either increasing the jet effectiveness or creating an adverse effect. A design of experiments test was planned and carried out using computation fluid dynamics, and a subsequent response surface analysis ensued on the available data to characterize the jet interaction across the ascent portion of the Ares I flight envelope. Four response surface schemes were compared including a single response surface covering the entire design space, separate sector responses that did not overlap, continuously overlapping surfaces, and recursive weighted response surfaces. These surfaces were evaluated on traditional statistical metrics as well as visual inspection. Validation of the recursive weighted response surface was performed using additionally available data at off-design point locations.

Vortex shedding within laminar separation bubbles forming over an airfoil

NASA Astrophysics Data System (ADS)

Kirk, Thomas M.; Yarusevych, Serhiy

2017-05-01

Vortex shedding within laminar separation bubbles forming over the suction side of a NACA 0018 airfoil is studied through a combination of high-speed flow visualization and boundary layer measurements. Wind tunnel experiments are performed at a chord-based Reynolds number of 100,000 and four angles of attack. The high-speed flow visualization is complemented by quantitative velocity and surface pressure measurements. The structures are shown to originate from the natural amplification of small-amplitude disturbances, and the shear layer roll-up is found to occur coherently across the span. However, significant cycle-to-cycle variations are observed in vortex characteristics, including shedding period and roll-up location. The formation of the roll-up vortices precedes the later stages of transition, during which these structures undergo significant deformations and breakdown to smaller scales. During this stage of flow development, vortex merging is also observed. The results provide new insight into the development of coherent structures in separation bubbles and their relation to the overall bubble dynamics and mean bubble topology.

Application of a modified complementary filtering technique for increased aircraft control system frequency bandwidth in high vibration environment

NASA Technical Reports Server (NTRS)

Garren, J. F., Jr.; Niessen, F. R.; Abbott, T. S.; Yenni, K. R.

1977-01-01

A modified complementary filtering technique for estimating aircraft roll rate was developed and flown in a research helicopter to determine whether higher gains could be achieved. Use of this technique did, in fact, permit a substantial increase in system frequency bandwidth because, in comparison with first-order filtering, it reduced both noise amplification and control limit-cycle tendencies.

Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major

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

Petrillo-Peixoto, M.L.; Beverley, S.M.

1988-12-01

We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasite Leishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8- to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistant Leishmania major. Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-headmore » configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselected L. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and other Leishmania lines suggests that the study of gene amplification in Leishmania spp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis.« less

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

DNA recombination-initiation plays a role in the extremely biased inheritance of yeast [rho-] mitochondrial DNA that contains the replication origin ori5.

PubMed

Ling, Feng; Hori, Akiko; Shibata, Takehiko

2007-02-01

Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance of a small mitochondrial DNA (mtDNA) fragment that contains a replication origin (HS [rho(-)] mtDNA). Our previous studies showed that concatemers (linear head-to-tail multimers) are obligatory intermediates for mtDNA partitioning and are primarily formed by rolling-circle replication mediated by Mhr1, a protein required for homologous mtDNA recombination. In this study, we found that Mhr1 is required for the hypersuppressiveness of HS [ori5] [rho(-)] mtDNA harboring ori5, one of the replication origins of normal ([rho(+)]) mtDNA. In addition, we detected an Ntg1-stimulated double-strand break at the ori5 locus. Purified Ntg1, a base excision repair enzyme, introduced a double-stranded break by itself into HS [ori5] [rho(-)] mtDNA at ori5 isolated from yeast cells. Both hypersuppressiveness and concatemer formation of HS [ori5] [rho(-)] mtDNA are simultaneously suppressed by the ntg1 null mutation. These results support a model in which, like homologous recombination, rolling-circle HS [ori5] [rho(-)] mtDNA replication is initiated by double-stranded breakage in ori5, followed by Mhr1-mediated homologous pairing of the processed nascent DNA ends with circular mtDNA. The hypersuppressiveness of HS [ori5] [rho(-)] mtDNA depends on a replication advantage furnished by the higher density of ori5 sequences and on a segregation advantage furnished by the higher genome copy number on transmitted concatemers.

The first complete chloroplast genome sequence of a lycophyte,Huperzia lucidula (Lycopodiaceae)

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

Wolf, Paul G.; Karol, Kenneth G.; Mandoli, Dina F.

2005-02-01

We used a unique combination of techniques to sequence the first complete chloroplast genome of a lycophyte, Huperzia lucidula. This plant belongs to a significant clade hypothesized to represent the sister group to all other vascular plants. We used fluorescence-activated cell sorting (FACS) to isolate the organelles, rolling circle amplification (RCA) to amplify the genome, and shotgun sequencing to 8x depth coverage to obtain the complete chloroplast genome sequence. The genome is 154,373bp, containing inverted repeats of 15,314 bp each, a large single-copy region of 104,088 bp, and a small single-copy region of 19,671 bp. Gene order is more similarmore » to those of mosses, liverworts, and hornworts than to gene order for other vascular plants. For example, the Huperziachloroplast genome possesses the bryophyte gene order for a previously characterized 30 kb inversion, thus supporting the hypothesis that lycophytes are sister to all other extant vascular plants. The lycophytechloroplast genome data also enable a better reconstruction of the basaltracheophyte genome, which is useful for inferring relationships among bryophyte lineages. Several unique characters are observed in Huperzia, such as movement of the gene ndhF from the small single copy region into the inverted repeat. We present several analyses of evolutionary relationships among land plants by using nucleotide data, amino acid sequences, and by comparing gene arrangements from chloroplast genomes. The results, while still tentative pending the large number of chloroplast genomes from other key lineages that are soon to be sequenced, are intriguing in themselves, and contribute to a growing comparative database of genomic and morphological data across the green plants.« less

Molecular characterization of a new begomovirus associated with leaf yellow mosaic disease of Jatropha curcas in India.

PubMed

Srivastava, Ashish; Kumar, S; Jaidi, Meraj; Raj, S K

2015-05-01

During a survey in June 2011, severe leaf yellow mosaic disease was observed on about 45 % plants of Jatropha curcas growing in the Katerniaghat wildlife sanctuary in India. An association of a begomovirus with disease was detected in 15 out of 20 samples by PCR using begomovirus genus-specific primers and total DNA isolated from symptomatic leaf samples. For identification of the begomovirus, the complete genome was amplified using a Phi-29 DNA-polymerase-based rolling-circle amplification kit and total DNA from five representative samples and then digested with BamHI. The linearized RCA products were cloned and sequenced. Their GenBank accession numbers are JN698954 (SKRK1) and JN135236 (SKRK2). The sequences of the two begomovirus isolates were 97 % identical to each other and no more than 86 % to those of jatropha mosaic India virus (JMIV, HM230683) and other begomoviruses reported worldwide. In phylogenetic analysis, SKRK1 and SKRK2 clustered together and showed distant relationships to jatropha mosaic India virus, Jatropha curcas mosaic virus, Indian cassava mosaic virus, Sri Lankan cassava mosaic virus and other begomoviruses. Based on 86 % sequence identities and distant phylogenetic relationships to JMIV and other begomoviruses and the begomovirus species demarcation criteria of the ICTV (<89 % sequence identity of complete DNA-A genome), the begomovirus isolates associated with leaf yellow mosaic disease of J. curcas were identified as members of a new begomovirus species and provisionally designated as jatropha leaf yellow mosaic Katerniaghat virus (JLYMKV). Agroinfectious clones of the DNA molecule of the begomovirus isolate were also generated, and the fulfillment of Koch's postulates was demonstrated in J. curcas plants.

A Fosmid Cloning Strategy for Detecting the Widest Possible Spectrum of Microbes from the International Space Station Drinking Water System

PubMed Central

Choi, Sangdun; Chang, Mi Sook; Stuecker, Tara; Chung, Christine; Newcombe, David A.; Venkateswaran, Kasthuri

2012-01-01

In this study, fosmid cloning strategies were used to assess the microbial populations in water from the International Space Station (ISS) drinking water system (henceforth referred to as Prebiocide and Tank A water samples). The goals of this study were: to compare the sensitivity of the fosmid cloning strategy with that of traditional culture-based and 16S rRNA-based approaches and to detect the widest possible spectrum of microbial populations during the water purification process. Initially, microbes could not be cultivated, and conventional PCR failed to amplify 16S rDNA fragments from these low biomass samples. Therefore, randomly primed rolling-circle amplification was used to amplify any DNA that might be present in the samples, followed by size selection by using pulsed-field gel electrophoresis. The amplified high-molecular-weight DNA from both samples was cloned into fosmid vectors. Several hundred clones were randomly selected for sequencing, followed by Blastn/Blastx searches. Sequences encoding specific genes from Burkholderia, a species abundant in the soil and groundwater, were found in both samples. Bradyrhizobium and Mesorhizobium, which belong to rhizobia, a large community of nitrogen fixers often found in association with plant roots, were present in the Prebiocide samples. Ralstonia, which is prevalent in soils with a high heavy metal content, was detected in the Tank A samples. The detection of many unidentified sequences suggests the presence of potentially novel microbial fingerprints. The bacterial diversity detected in this pilot study using a fosmid vector approach was higher than that detected by conventional 16S rRNA gene sequencing. PMID:23346038

Molecular Evidence for Occurrence of Tomato leaf curl New Delhi virus in Ash Gourd (Benincasa hispida) Germplasm Showing a Severe Yellow Stunt Disease in India.

PubMed

Roy, Anirban; Spoorthi, P; Panwar, G; Bag, Manas Kumar; Prasad, T V; Kumar, Gunjeet; Gangopadhyay, K K; Dutta, M

2013-06-01

An evaluation of 70 accessions of ash gourd germplasm grown at National Bureau of Plant Genetic Resources, New Delhi, India during Kharif season (2010) showed natural occurrence of a yellow stunt disease in three accessions (IC554690, IC036330 and Pusa Ujjwal). A set of begomovirus specific primers used in PCR gave expected amplicon from all the symptomatic plants; however no betasatellite was detected. Complete genome of the begomovirus (DNA-A and DNA-B), amplified through rolling circle amplification, was cloned and sequenced. The begomovirus under study shared high sequence identities to different isolates of Tomato leaf curl New Delhi virus (ToLCNDV) and clustered with them. Among those isolates, the DNA-A and DNA-B of the present begomovirus isolate showed highest 99.6 and 96.8 % sequence identities, respectively with an isolate reported on pumpkin from India (DNA-A: AM286433, DNA-B: AM286435). Based on the sequence analysis, the begomovirus obtained from ash gourd was considered as an isolate of ToLCNDV. Thus, the present findings constitute the first report of occurrence of a new yellow stunt disease in ash gourd from India and demonstrated the association of ToLCNDV with the symptomatic samples. Occurrence of ToLCNDV in ash gourd germplasm not only adds up a new cucurbitaceous host of this virus but also raises the concern about the perpetuation of this virus in absence of its main host tomato and thus has an epidemiological relevance for understanding the rapid spread of this virus in tomato and other hosts in Indian sub-continent.

Discovery of a New Human Polyomavirus Associated with Trichodysplasia Spinulosa in an Immunocompromized Patient

PubMed Central

van der Meijden, Els; Janssens, René W. A.; Lauber, Chris; Bouwes Bavinck, Jan Nico; Gorbalenya, Alexander E.; Feltkamp, Mariet C. W.

2010-01-01

The Polyomaviridae constitute a family of small DNA viruses infecting a variety of hosts. In humans, polyomaviruses can cause infections of the central nervous system, urinary tract, skin, and possibly the respiratory tract. Here we report the identification of a new human polyomavirus in plucked facial spines of a heart transplant patient with trichodysplasia spinulosa, a rare skin disease exclusively seen in immunocompromized patients. The trichodysplasia spinulosa-associated polyomavirus (TSV) genome was amplified through rolling-circle amplification and consists of a 5232-nucleotide circular DNA organized similarly to known polyomaviruses. Two putative “early” (small and large T antigen) and three putative “late” (VP1, VP2, VP3) genes were identified. The TSV large T antigen contains several domains (e.g. J-domain) and motifs (e.g. HPDKGG, pRb family-binding, zinc finger) described for other polyomaviruses and potentially involved in cellular transformation. Phylogenetic analysis revealed a close relationship of TSV with the Bornean orangutan polyomavirus and, more distantly, the Merkel cell polyomavirus that is found integrated in Merkel cell carcinomas of the skin. The presence of TSV in the affected patient's skin was confirmed by newly designed quantitative TSV-specific PCR, indicative of a viral load of 105 copies per cell. After topical cidofovir treatment, the lesions largely resolved coinciding with a reduction in TSV load. PCR screening demonstrated a 4% prevalence of TSV in an unrelated group of immunosuppressed transplant recipients without apparent disease. In conclusion, a new human polyomavirus was discovered and identified as the possible cause of trichodysplasia spinulosa in immunocompromized patients. The presence of TSV also in clinically unaffected individuals suggests frequent virus transmission causing subclinical, probably latent infections. Further studies have to reveal the impact of TSV infection in relation to other populations and diseases. PMID:20686659

ICTV Virus Taxonomy Profile: Avsunviroidae.

PubMed

Di Serio, Francesco; Li, Shi-Fang; Matoušek, Jaroslav; Owens, Robert A; Pallás, Vicente; Randles, John W; Sano, Teruo; Verhoeven, Jacobus Th J; Vidalakis, Georgios; Flores, Ricardo; Ictv Report Consortium

2018-05-01

Members of the family Avsunviroidae have a single-stranded circular RNA genome that adopts a rod-like or branched conformation and can form, in the strands of either polarity, hammerhead ribozymes involved in their replication in plastids through a symmetrical RNA-RNA rolling-circle mechanism. These viroids lack the central conserved region typical of members of the family Pospiviroidae. The family Avsunviroidae includes three genera, Avsunviroid, Pelamoviroid and Elaviroid, with a total of four species. This is a summary of the ICTV Report on the taxonomy of the family Avsunviroidae, which is available at http://www.ictv.global/report/avsunviroidae.

Dynamic Measurement for the Diameter of A Train Wheel Based on Structured-Light Vision

PubMed Central

Gong, Zheng; Sun, Junhua; Zhang, Guangjun

2016-01-01

Wheels are very important for the safety of a train. The diameter of the wheel is a significant parameter that needs regular inspection. Traditional methods only use the contact points of the wheel tread to fit the rolling round. However, the wheel tread is easily influenced by peeling or scraping. Meanwhile, the circle fitting algorithm is sensitive to noise when only three points are used. This paper proposes a dynamic measurement method based on structured-light vision. The axle of the wheelset and the tread are both employed. The center of the rolling round is determined by the axle rather than the tread only. Then, the diameter is calculated using the center and the contact points together. Simulations are performed to help design the layout of the sensors, and the influences of different noise sources are also analyzed. Static and field experiments are both performed, and the results show it to be quite stable and accurate. PMID:27104543

KSC-99pp0539

NASA Image and Video Library

1999-05-16

KENNEDY SPACE CENTER, FLA. -- Inside High Bay 1 of the Vehicle Assembly Building (VAB) Mike Sestile, with United Space Alliance, draws circles around divots in the foam insulation on the top of the external tank of Space Shuttle Discovery. About 150 divots were caused by hail during recent storms. The Shuttle was rolled back from Pad 39B to the VAB for repairs because access to all of the damaged areas was not possible at the pad. The work is expected to take two to three days, allowing Discovery to roll back to the pad as early as May 20 for launch of mission STS-96, the 94th launch in the Space Shuttle Program. Liftoff will occur no earlier than May 27. STS-96 is a logistics and resupply mission for the International Space Station, carrying such payloads as a Russian crane, the Strela; a U.S.-built crane; the Spacehab Oceaneering Space System Box (SHOSS), a logistics items carrier; and STARSHINE, a student-shared experiment

Dynamic Measurement for the Diameter of A Train Wheel Based on Structured-Light Vision.

PubMed

Gong, Zheng; Sun, Junhua; Zhang, Guangjun

2016-04-20

Wheels are very important for the safety of a train. The diameter of the wheel is a significant parameter that needs regular inspection. Traditional methods only use the contact points of the wheel tread to fit the rolling round. However, the wheel tread is easily influenced by peeling or scraping. Meanwhile, the circle fitting algorithm is sensitive to noise when only three points are used. This paper proposes a dynamic measurement method based on structured-light vision. The axle of the wheelset and the tread are both employed. The center of the rolling round is determined by the axle rather than the tread only. Then, the diameter is calculated using the center and the contact points together. Simulations are performed to help design the layout of the sensors, and the influences of different noise sources are also analyzed. Static and field experiments are both performed, and the results show it to be quite stable and accurate.

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

Vladimir Larionov, Ph D

A special interest in the organization of human centromeric DNA was stimulated a few years ago when two independent groups succeeded in reconstituting a functional human centromere, using constructs carrying centromere-specific alphoid DNA arrays. This work demonstrated the importance of DNA components in mammalian centromeres and opened a way for studying the structural requirements for de novo kinetochore formation and for construction of human artificial chromosomes (HACs) with therapeutic potential. To elucidate the structural requirements for formation of HACs with a functional kinetochore, we developed a new method for cloning of large DNA fragments for human centromeric regions that canmore » be used as a substrate for HAC formation. This method exploits in vivo recombination in yeast (TAR cloning). In addition, a new strategy for the construction of alphoid DNA arrays was developed in our lab. The strategy involves the construction of uniform or hybrid synthetic alphoid DNA arrays by the RCA-TAR technique. This technique comprises two steps: rolling circle amplification of an alphoid DNA dimer and subsequent assembling of the amplified fragments by in vivo homologous recombination in yeast (Figure 1). Using this system, we constructed a set of different synthetic alphoid DNA arrays with a predetermined sequence varying in size from 30 to 140 kb and demonstrated that some of the arrays are competent in HAC formation. Because any nucleotide can be changed in a dimer before its amplification, this new technique is optimal for identifying the structural requirements for de novo kinetochore formation in HACs. Moreover, the technique makes possible to introduce into alphoid DNA arrays recognition sites for DNA-binding proteins. We have made the following progress on the studying of human centromeric regions using transformation-associated recombination cloning technology: i) minimal size of alphoid DNA array required for de novo kinetochore formation was estimated; ii) critical role of CENP-B binding site in do novo kinetochore formation was demonstrated; iii) role of gamma-satellite DNA in functional centromere was elucidated; iv) new generation of HAC with a conditional centromere was constructed for the study of epigenetic control of kinetochore function and for gene expression studies. These studies de novo kinetochore formation may thus provide both a fundamental knowledge and new points of intervention for therapy.« less

Mobilization Function of the pBHR1 Plasmid, a Derivative of the Broad-Host-Range Plasmid pBBR1

PubMed Central

Szpirer, Cédric Y.; Faelen, Michel; Couturier, Martine

2001-01-01

The pBHR1 plasmid is a derivative of the small (2.6-kb), mobilizable broad-host-range plasmid pBBR1, which was isolated from the gram-negative bacterium Bordetella bronchiseptica (R. Antoine and C. Locht, Mol. Microbiol. 6:1785–1799, 1992). Plasmid pBBR1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable transposons from gram-positive bacteria. We show that the Mob protein specifically recognizes a 52-bp sequence which contains, in addition to the transfer origin, the promoter of the mob gene. We demonstrate that this gene is autoregulated. The binding of the Mob protein to the 52-bp sequence could thus allow the formation of a protein-DNA complex with a double function: relaxosome formation and mob gene regulation. We show that the Mob protein is a relaxase, and we located the nic site position in vitro. After sequence alignment, the position of the nic site of pBBR1 corresponds with those of the nick sites of the Bacteroides mobilizable transposon Tn4555 and the streptococcal plasmid pMV158. The oriT of the latter is characteristic of a family of mobilizable plasmids that are found in gram-positive bacteria and that replicate by the rolling-circle mechanism. Plasmid pBBR1 thus appears to be a new member of this group, even though it resides in gram-negative bacteria and does not replicate via a rolling-circle mechanism. In addition, we identified two amino acids of the Mob protein necessary for its activity, and we discuss their involvement in the mobilization mechanism. PMID:11222611

High-mobility group 1/2 proteins are essential for initiating rolling-circle-type DNA replication at a parvovirus hairpin origin.

PubMed

Cotmore, S F; Tattersall, P

1998-11-01

Rolling-circle replication is initiated by a replicon-encoded endonuclease which introduces a single-strand nick into specific origin sequences, becoming covalently attached to the 5' end of the DNA at the nick and providing a 3' hydroxyl to prime unidirectional, leading-strand synthesis. Parvoviruses, such as minute virus of mice (MVM), have adapted this mechanism to amplify their linear single-stranded genomes by using hairpin telomeres which sequentially unfold and refold to shuttle the replication fork back and forth along the genome, creating a continuous, multimeric DNA strand. The viral initiator protein, NS1, then excises individual genomes from this continuum by nicking and reinitiating synthesis at specific origins present within the hairpin sequences. Using in vitro assays to study ATP-dependent initiation within the right-hand (5') MVM hairpin, we have characterized a HeLa cell factor which is absolutely required to allow NS1 to nick this origin. Unlike parvovirus initiation factor (PIF), the cellular complex which activates NS1 endonuclease activity at the left-hand (3') viral origin, the host factor which activates the right-hand hairpin elutes from phosphocellulose in high salt, has a molecular mass of around 25 kDa, and appears to bind preferentially to structured DNA, suggesting that it might be a member of the high-mobility group 1/2 (HMG1/2) protein family. This prediction was confirmed by showing that purified calf thymus HMG1 and recombinant human HMG1 or murine HMG2 could each substitute for the HeLa factor, activating the NS1 endonuclease in an origin-specific nicking reaction.

Self-assembly of free-standing RNA membranes

NASA Astrophysics Data System (ADS)

Han, Daehoon; Park, Yongkuk; Kim, Hyejin; Lee, Jong Bum

2014-07-01

RNA has emerged as a promising material for nanostructure and microstructure engineering. Although rare, some macroscopic RNA structures have also been constructed using lipid or polymer materials. Here, we report the first example of an enzymatically generated RNA membrane. This robust and free-standing RNA membrane has a macroscopic structure and is generated without any polymer support or complexation. Our RNA membrane is fabricated following two sequential processes, complementary rolling circle transcription and evaporation-induced self-assembly, and its structural and functional properties are rationally controlled by adjusting RNA base pairing. In this study, three types of RNA membranes are fabricated and are used to demonstrate potential applications.

Machine protection system for rotating equipment and method

DOEpatents

Lakshminarasimha, Arkalgud N.; Rucigay, Richard J.; Ozgur, Dincer

2003-01-01

A machine protection system and method for rotating equipment introduces new alarming features and makes use of full proximity probe sensor information, including amplitude and phase. Baseline vibration amplitude and phase data is estimated and tracked according to operating modes of the rotating equipment. Baseline vibration and phase data can be determined using a rolling average and variance and stored in a unit circle or tracked using short term average and long term average baselines. The sensed vibration amplitude and phase is compared with the baseline vibration amplitude and phase data. Operation of the rotating equipment can be controlled based on the vibration amplitude and phase.

Prediction of vortex shedding from circular and noncircular bodies in subsonic flow

NASA Technical Reports Server (NTRS)

Mendenhall, Michael R.; Lesieutre, Daniel J.

1987-01-01

An engineering prediction method and associated computer code VTXCLD are presented which predict nose vortex shedding from circular and noncircular bodies in subsonic flow at angles of attack and roll. The axisymmetric body is represented by point sources and doublets, and noncircular cross sections are transformed to a circle by either analytical or numerical conformal transformations. The leeward vortices are modeled by discrete vortices in crossflow planes along the body; thus, the three-dimensional steady flow problem is reduced to a two-dimensional, unsteady, separated flow problem for solution. Comparison of measured and predicted surface pressure distributions, flowfield surveys, and aerodynamic characteristics are presented for bodies with circular and noncircular cross sectional shapes.

Characterization of replication and conjugation of plasmid pWTY27 from a widely distributed Streptomyces species

PubMed Central

2012-01-01

Background Streptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments. Replication loci of several Streptomyces theta-type plasmids have been reported, but are not characterized in details. Conjugation loci of some Streptomyces rolling-circle-type plasmids are identified and mechanism of conjugal transferring are described. Results We report the detection of a widely distributed Streptomyces strain Y27 and its indigenous plasmid pWTY27 from fourteen plants and four soil samples cross China by both culturing and nonculturing methods. The complete nucleotide sequence of pWTY27 consisted of 14,288 bp. A basic locus for plasmid replication comprised repAB genes and an adjacent iteron sequence, to a long inverted-repeat (ca. 105 bp) of which the RepA protein bound specifically in vitro, suggesting that RepA may recognize a second structure (e.g. a long stem-loop) of the iteron DNA. A plasmid containing the locus propagated in linear mode when the telomeres of a linear plasmid were attached, indicating a bi-directional replication mode for pWTY27. As for rolling-circle plasmids, a single traA gene and a clt sequence (covering 16 bp within traA and its adjacent 159 bp) on pWTY27 were required for plasmid transfer. TraA recognized and bound specifically to the two regions of the clt sequence, one containing all the four DC1 of 7 bp (TGACACC) and one DC2 (CCCGCCC) and most of IC1, and another covering two DC2 and part of IC1, suggesting formation of a high-ordered DNA-protein complex. Conclusions This work (i) isolates a widespread Streptomyces strain Y27 and sequences its indigenous theta-type plasmid pWTY27; (ii) identifies the replication and conjugation loci of pWTY27 and; (iii) characterizes the binding sequences of the RepA and TraA proteins. PMID:23134842

Velvet bean severe mosaic virus: a distinct begomovirus species causing severe mosaic in Mucuna pruriens (L.) DC.

PubMed

Zaim, Mohammad; Kumar, Yogesh; Hallan, Vipin; Zaidi, A A

2011-08-01

Velvet bean [Mucuna pruriens (L.) DC] is one of the most important medicinal plants. It is used to treat many ailments, but is widely used for the treatment especially for Parkinson's disease because of the presence of 3,4-dihydroxyphenylalanine (L-dopa) in it. It was noticed in last 5 years that the plants in the field showed severe mosaic, downward curling of the leaves, stunting, etc. This is consistently observed over the years in India. The disease was transmitted by whiteflies and by grafting and the causal agent was found to be a bipartite begomovirus. The whole genome was amplified by rolling circle amplification (RCA) using ϕ-29 DNA polymerase and characterized. DNA-A and DNA-B shared a 124-nucleotide (nt) long highly conserved (98%) common region (CR). Comparisons with other begomovirus showed that DNA-A sequence has highest identity (76%) with an isolate of Mungbean yellow mosaic India virus (MYMIV; AY937195) reported from India. This data suggested that the present isolate is a new species of genus Begomovirus for which the name "Velvet bean severe mosaic virus" (VbSMV) is proposed. DNA-B has a maximum sequence identity of 49% with an isolate of Horsegram yellow mosaic virus (HgYMV; AM932426) reported from India. Infectious clones consisting of a 1.7 mer partial tandem repeat of DNA-A and a dimer of DNB-B were constructed and agro-inoculated to Macuna pruriens (L.) DC plants, which showed field observed symptoms 24 days post-infiltration (dpi). In phylogenetic analysis, DNA-A and DNA-B of the present isolate grouped with DNA-A of different begomoviruses reported from fabaceous crops. The study presents first ever molecular evidence of any disease in velvet bean and whole genome analysis of the causative virus which is a distinct bipartite species of Begomovirus.

Detection and genome characterization of bovine polyomaviruses in beef muscle and ground beef samples from Germany.

PubMed

Gräfe, Donina; Ehlers, Bernhard; Mäde, Dietrich; Ellerbroek, Lüppo; Seidler, Tassilo; Johne, Reimar

2017-01-16

Polyomaviruses are small, non-enveloped, circular double-stranded DNA viruses. Some polyomaviruses can induce tumors and cancer under certain circumstances. The bovine polyomaviruses (BPyV) 1-3 have been only scarcely analyzed so far. It was hypothesized that the consumption of beef meat containing polyomaviruses could contribute to the development of cancer in humans. In order to assess the distribution of the BPyV genome in meat from Germany, 101 beef muscle samples and 10 ground beef samples were analyzed here. A specific sample preparation method combined with or without rolling circle amplification (RCA), and BPyV-specific PCRs were developed and applied. BPyV-1 DNA was detected in 1/101 (1%) samples from beef meat and in 2/10 (20%) ground beef samples. BPyV-2 DNA was detected in 3/10 (30%) ground beef samples, whereas BPyV-3 was not detected in the samples. Application of RCA did not increase the detection rate in ground beef samples. Sequence analysis of the PCR products indicated the presence of BPyV-1, BPyV-2a and BPyV-2b. The whole genome of a BPyV-1 strain from ground beef meat showed 97.8% sequence identity to the BPyV-1 reference strain and that of a BPyV-2a strain from ground beef meet showed 99.9% sequence identity to strain 2aS11. It can be concluded that BPyV genomes can be frequently detected in ground beef samples, although higher sample numbers should be investigated in future to confirm this finding. Further studies should focus on the infectivity, tumorigenicity and heat resistance of the contained viruses in order to assess the risk of cancer induction through consumption of BPyVs present in beef products. Copyright © 2016 Elsevier B.V. All rights reserved.

12. RW Meyer Sugar Mill: 18761889. Threeroll sugar mill: oneton ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

12. RW Meyer Sugar Mill: 1876-1889. Three-roll sugar mill: one-ton daily processing capacity. Manufactured by Edwin Maw, Liverpool, England, ca. 1855-1870. View: Historical view, 1934, T.T. Waterman Collection, Hawaiian Sugar Planters' Association, Oahu, Hawaii. Masonry-lined passageway leading to the mill at the center of its circular masonry enclosure. The passageway permitted cane to be carried to the mill and cane trash (bagasse) to be carried away after milling. Bridges over the passageways, not in place, permitted the mill animals to circle and power the mill from above. View shows area prior to substantial overgrowth existing in 1978 views of the area. - R. W. Meyer Sugar Mill, State Route 47, Kualapuu, Maui County, HI

Prediction of subsonic vortex shedding from forebodies with chines

NASA Technical Reports Server (NTRS)

Mendenhall, Michael R.; Lesieutre, Daniel J.

1990-01-01

An engineering prediction method and associated computer code VTXCHN to predict nose vortex shedding from circular and noncircular forebodies with sharp chine edges in subsonic flow at angles of attack and roll are presented. Axisymmetric bodies are represented by point sources and doublets, and noncircular cross sections are transformed to a circle by either analytical or numerical conformal transformations. The lee side vortex wake is modeled by discrete vortices in crossflow planes along the body; thus the three-dimensional steady flow problem is reduced to a two-dimensional, unsteady, separated flow problem for solution. Comparison of measured and predicted surface pressure distributions, flow field surveys, and aerodynamic characteristics are presented for noncircular bodies alone and forebodies with sharp chines.

Ultrasensitive electrochemical detection of avian influenza A (H7N9) virus DNA based on isothermal exponential amplification coupled with hybridization chain reaction of DNAzyme nanowires.

PubMed

Yu, Yanyan; Chen, Zuanguang; Jian, Wensi; Sun, Duanping; Zhang, Beibei; Li, Xinchun; Yao, Meicun

2015-02-15

In this work, a simple and label-free electrochemical biosensor with duel amplification strategy was developed for DNA detection based on isothermal exponential amplification (EXPAR) coupled with hybridization chain reaction (HCR) of DNAzymes nanowires. Through rational design, neither the primer nor the DNAzymes containing molecular beacons (MBs) could react with the duplex probe which were fixed on the electrode surface. Once challenged with target, the duplex probe cleaved and triggered the EXPAR mediated target recycle and regeneration circles as well as the HCR process. As a result, a greater amount of targets were generated to cleave the duplex probes. Subsequently, the nanowires consisting of the G-quadruplex units were self-assembled through hybridization with the strand fixed on the electrode surface. In the presence of hemin, the resulting catalytic G-quadruplex-hemin HRP-mimicking DNAzymes were formed. Electrochemical signals can be obtained by measuring the increase in reduction current of oxidized 3.3',5.5'-tetramethylbenzidine sulfate (TMB), which was generated by DNAzyme in the presence of H2O2. This method exhibited ultrahigh sensitivity towards avian influenza A (H7N9) virus DNA sequence with detection limits of 9.4 fM and a detection range of 4 orders of magnitude. The biosensor was also capable of discriminating single-nucleotide difference among concomitant DNA sequences and performed well in spiked cell lysates. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Enzymatic Synthesis of Self-assembled Dicer Substrate RNA Nanostructures for Programmable Gene Silencing.

PubMed

Jang, Bora; Kim, Boyoung; Kim, Hyunsook; Kwon, Hyokyoung; Kim, Minjeong; Seo, Yunmi; Colas, Marion; Jeong, Hansaem; Jeong, Eun Hye; Lee, Kyuri; Lee, Hyukjin

2018-06-08

Enzymatic synthesis of RNA nanostructures is achieved by isothermal rolling circle transcription (RCT). Each arm of RNA nanostructures provides a functional role of Dicer substrate RNA inducing sequence specific RNA interference (RNAi). Three different RNAi sequences (GFP, RFP, and BFP) are incorporated within the three-arm junction RNA nanostructures (Y-RNA). The template and helper DNA strands are designed for the large-scale in vitro synthesis of RNA strands to prepare self-assembled Y-RNA. Interestingly, Dicer processing of Y-RNA is highly influenced by its physical structure and different gene silencing activity is achieved depending on its arm length and overhang. In addition, enzymatic synthesis allows the preparation of various Y-RNA structures using a single DNA template offering on demand regulation of multiple target genes.

Acoustic Guided Wave Testing of Pipes of Small Diameters

NASA Astrophysics Data System (ADS)

Muravev, V. V.; Muraveva, O. V.; Strizhak, V. A.; Myshkin, Y. V.

2017-10-01

Acoustic path is analyzed and main parameters of guided wave testing are substanti- ated applied to pipes of small diameters. The method is implemented using longitudinal L(0,1) and torsional T(0,1) waves based on electromagnetic-acoustic (EMA) transducers. The method of multiple reflections (MMR) combines echo-through, amplitude-shadow and time-shadow methods. Due to the effect of coherent amplification of echo-pulses from defects the sensitivity to the defects of small sizes at the signal analysis on the far reflections is increased. An oppor- tunity of detection of both local defects (dents, corrosion damages, rolling features, pitting, cracks) and defects extended along the pipe is shown.

Development of mobile laboratory for viral haemorrhagic fever detection in Africa.

PubMed

Weidmann, Manfred; Faye, Ousmane; Faye, Oumar; Abd El Wahed, Ahmed; Patel, Pranav; Batejat, Christophe; Manugerra, Jean Claude; Adjami, Aimee; Niedrig, Matthias; Hufert, Frank T; Sall, Amadou A

2018-06-15

In order to enable local response to viral haemorrhagic fever outbreaks a mobile laboratory transportable on commercial flights was developed. The development progressed from use of mobile real time RT-PCR to mobile Recombinase Polymerase Amplification (RT-RPA). The various stages of the mobile laboratory development are described. A brief overview of its deployments, which culminated in the first on site detection of Ebola virus disease (EVD) in March 2014 and a successful use in a campaign to roll back EVD cases in Conakry in the West-Africa Ebola virus outbreak are described. The developed mobile laboratory successfully enabled local teams to perform rapid viral haemorrhagic fever disgnostics.

Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions.

PubMed

Ponchel, Frederique; Toomes, Carmel; Bransfield, Kieran; Leong, Fong T; Douglas, Susan H; Field, Sarah L; Bell, Sandra M; Combaret, Valerie; Puisieux, Alain; Mighell, Alan J; Robinson, Philip A; Inglehearn, Chris F; Isaacs, John D; Markham, Alex F

2003-10-13

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

Click nucleic acid ligation: applications in biology and nanotechnology.

PubMed

El-Sagheer, Afaf H; Brown, Tom

2012-08-21

Biochemical strategies that use a combination of synthetic oligonucleotides, thermostable DNA polymerases, and DNA ligases can produce large DNA constructs up to 1 megabase in length. Although these ambitious targets are feasible biochemically, comparable technologies for the chemical synthesis of long DNA strands lag far behind. The best available chemical approach is the solid-phase phosphoramidite method, which can be used to assemble DNA strands up to 150 bases in length. Beyond this point, deficiencies in the chemistry make it impossible to produce pure DNA. A possible alternative approach to the chemical synthesis of large DNA strands is to join together carefully purified synthetic oligonucleotides by chemical methods. Click ligation by the copper-catalyzed azide-alkyne (CuAAC) reaction could facilitate this process. In this Account, we describe the synthesis, characterization, and applications of oligonucleotides prepared by click ligation. The alkyne and azide oligonucleotide strands can be prepared by standard protocols, and the ligation reaction is compatible with a wide range of chemical modifications to DNA and RNA. We have employed click ligation to synthesize DNA constructs up to 300 bases in length and much longer sequences are feasible. When the resulting triazole linkage is placed in a PCR template, various DNA polymerases correctly copy the entire base sequence. We have also successfully demonstrated both in vitro transcription and rolling circle amplification through the modified linkage. This linkage has shown in vivo biocompatibility: an antibiotic resistance gene containing triazole linkages functions in E. coli . Using click ligation, we have synthesized hairpin ribozymes up to 100 nucleotides in length and a hammerhead ribozyme with the triazole linkage located at the substrate cleavage site. At the opposite end of the length scale, click-ligated, cyclic mini-DNA duplexes have been used as models to study base pairing. Cyclic duplexes have potential therapeutic applications. They have extremely high thermodynamic stability, have increased resistance to enzymatic degradation, and have been investigated as decoys for regulatory proteins. For potential nanotechnology applications, we have synthesized double stranded DNA catenanes by click ligation. Other researchers have studied covalently fixed multistranded DNA constructs including triplexes and quadruplexes.

Fangorn Forest (F2): a machine learning approach to classify genes and genera in the family Geminiviridae.

PubMed

Silva, José Cleydson F; Carvalho, Thales F M; Fontes, Elizabeth P B; Cerqueira, Fabio R

2017-09-30

Geminiviruses infect a broad range of cultivated and non-cultivated plants, causing significant economic losses worldwide. The studies of the diversity of species, taxonomy, mechanisms of evolution, geographic distribution, and mechanisms of interaction of these pathogens with the host have greatly increased in recent years. Furthermore, the use of rolling circle amplification (RCA) and advanced metagenomics approaches have enabled the elucidation of viromes and the identification of many viral agents in a large number of plant species. As a result, determining the nomenclature and taxonomically classifying geminiviruses turned into complex tasks. In addition, the gene responsible for viral replication (particularly, the viruses belonging to the genus Mastrevirus) may be spliced due to the use of the transcriptional/splicing machinery in the host cells. However, the current tools have limitations concerning the identification of introns. This study proposes a new method, designated Fangorn Forest (F2), based on machine learning approaches to classify genera using an ab initio approach, i.e., using only the genomic sequence, as well as to predict and classify genes in the family Geminiviridae. In this investigation, nine genera of the family Geminiviridae and their related satellite DNAs were selected. We obtained two training sets, one for genus classification, containing attributes extracted from the complete genome of geminiviruses, while the other was made up to classify geminivirus genes, containing attributes extracted from ORFs taken from the complete genomes cited above. Three ML algorithms were applied on those datasets to build the predictive models: support vector machines, using the sequential minimal optimization training approach, random forest (RF), and multilayer perceptron. RF demonstrated a very high predictive power, achieving 0.966, 0.964, and 0.995 of precision, recall, and area under the curve (AUC), respectively, for genus classification. For gene classification, RF could reach 0.983, 0.983, and 0.998 of precision, recall, and AUC, respectively. Therefore, Fangorn Forest is proven to be an efficient method for classifying genera of the family Geminiviridae with high precision and effective gene prediction and classification. The method is freely accessible at www.geminivirus.org:8080/geminivirusdw/discoveryGeminivirus.jsp .

High-Performance Flexible Thin-Film Transistors Based on Single-Crystal-like Silicon Epitaxially Grown on Metal Tape by Roll-to-Roll Continuous Deposition Process.

PubMed

Gao, Ying; Asadirad, Mojtaba; Yao, Yao; Dutta, Pavel; Galstyan, Eduard; Shervin, Shahab; Lee, Keon-Hwa; Pouladi, Sara; Sun, Sicong; Li, Yongkuan; Rathi, Monika; Ryou, Jae-Hyun; Selvamanickam, Venkat

2016-11-02

Single-crystal-like silicon (Si) thin films on bendable and scalable substrates via direct deposition are a promising material platform for high-performance and cost-effective devices of flexible electronics. However, due to the thick and unintentionally highly doped semiconductor layer, the operation of transistors has been hampered. We report the first demonstration of high-performance flexible thin-film transistors (TFTs) using single-crystal-like Si thin films with a field-effect mobility of ∼200 cm 2 /V·s and saturation current, I/l W > 50 μA/μm, which are orders-of-magnitude higher than the device characteristics of conventional flexible TFTs. The Si thin films with a (001) plane grown on a metal tape by a "seed and epitaxy" technique show nearly single-crystalline properties characterized by X-ray diffraction, Raman spectroscopy, reflection high-energy electron diffraction, and transmission electron microscopy. The realization of flexible and high-performance Si TFTs can establish a new pathway for extended applications of flexible electronics such as amplification and digital circuits, more than currently dominant display switches.

Evolution of eukaryotic single-stranded DNA viruses of the Bidnaviridae family from genes of four other groups of widely different viruses

NASA Astrophysics Data System (ADS)

Krupovic, Mart; Koonin, Eugene V.

2014-06-01

Single-stranded (ss)DNA viruses are extremely widespread, infect diverse hosts from all three domains of life and include important pathogens. Most ssDNA viruses possess small genomes that replicate by the rolling-circle-like mechanism initiated by a distinct virus-encoded endonuclease. However, viruses of the family Bidnaviridae, instead of the endonuclease, encode a protein-primed type B DNA polymerase (PolB) and hence break this pattern. We investigated the provenance of all bidnavirus genes and uncover an unexpected turbulent evolutionary history of these unique viruses. Our analysis strongly suggests that bidnaviruses evolved from a parvovirus ancestor from which they inherit a jelly-roll capsid protein and a superfamily 3 helicase. The radiation of bidnaviruses from parvoviruses was probably triggered by integration of the ancestral parvovirus genome into a large virus-derived DNA transposon of the Polinton (polintovirus) family resulting in the acquisition of the polintovirus PolB gene along with terminal inverted repeats. Bidnavirus genes for a receptor-binding protein and a potential novel antiviral defense modulator are derived from dsRNA viruses (Reoviridae) and dsDNA viruses (Baculoviridae), respectively. The unusual evolutionary history of bidnaviruses emphasizes the key role of horizontal gene transfer, sometimes between viruses with completely different genomes but occupying the same niche, in the emergence of new viral types.

Roles of the 2 microns gene products in stable maintenance of the 2 microns plasmid of Saccharomyces cerevisiae.

PubMed Central

Reynolds, A E; Murray, A W; Szostak, J W

1987-01-01

We have examined the replication and segregation of the Saccharomyces cerevisiae 2 microns circle. The amplification of the plasmid at low copy numbers requires site-specific recombination between the 2 microns inverted repeat sequences catalyzed by the plasmid-encoded FLP gene. No other 2 microns gene products are required. The overexpression of FLP in a strain carrying endogenous 2 microns leads to uncontrolled plasmid replication, longer cell cycles, and cell death. Two different assays show that the level of Flp activity decreases with increasing 2 microns copy number. This regulation requires the products of the REP1 and REP2 genes. These gene products also act together to ensure that 2 microns molecules are randomly segregated between mother and daughter cells at cell division. Images PMID:3316982

Car suspension system monitoring under road conditions

NASA Astrophysics Data System (ADS)

Fedotov, A. I.; Kuznetsov, N. Y.; Lysenko, A. V.; Vlasov, V. G.

2017-12-01

The paper describes an advanced gyro-based measuring system comprising a CGV-4K central vertical gyro and a G-3M gyrocompass. The advanced system provides additional functions that help measure unsprung mass rotation angles about a vertical axis, rolling angles, trim angles and movements of the unsprung masses of the front (ap and al) and rear b axes when a car wheel hits a single obstruction. The paper also describes the operation of the system, which measures movements of unsprung masses about the body of a car when it hits a single obstruction. The paper presents the dependency diagrams ap = f(t) and al = f(t) for front and rear wheels respectively, as well as b = f(t) for a rear left wheel, which were determined experimentally. Test results for a car equipped with an advanced gyro-based measuring system moving around a circle can form a basis for developing a mathematical model of the process.

RNA-primed complementary-sense DNA synthesis of the geminivirus African cassava mosaic virus.

PubMed Central

Saunders, K; Lucy, A; Stanley, J

1992-01-01

The plant DNA virus African cassava mosaic virus (ACMV) is believed to replicate by a rolling circle mechanism. To investigate complementary-sense DNA (lagging strand) synthesis, we have analysed the heterogenous form of complementary-sense DNA (H3 DNA) from infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and blot hybridisation. The presence of an RNA moeity is demonstrated by comparison of results for nucleic acids resolved on neutral/alkaline and neutral/formamide gels, suggesting that complementary-sense DNA synthesis on the virus-sense single-stranded DNA template is preceded by the synthesis of an RNA primer. Hybridisation with probes to specific parts of ACMV DNA A genome indicates that synthesis of the putative RNA primer initiates between nucleotides 2581-221, a region that includes intergenic sequences that have been implicated in geminivirus DNA replication and the control of gene expression. Images PMID:1475192

Preparation and biomedical applications of programmable and multifunctional DNA nanoflowers

PubMed Central

Lv, Yifan; Hu, Rong; Zhu, Guizhi; Zhang, Xiaobing; Mei, Lei; Liu, Qiaoling; Qiu, Liping; Wu, Cuichen; Tan, Weihong

2016-01-01

We describe a comprehensive protocol for the preparation of multifunctional DNA nanostructures termed nanoflowers (NFs), which are self-assembled from long DNA building blocks generated via rolling-circle replication (RCR) of a designed template. NF assembly is driven by liquid crystallization and dense packaging of building blocks, which eliminates the need for conventional Watson-Crick base pairing. As a result of dense DNA packaging, NFs are resistant to nuclease degradation, denaturation or dissociation at extremely low concentrations. By manually changing the template sequence, many different functional moieties including aptamers, bioimaging agents and drug-loading sites could be easily integrated into NF particles, making NFs ideal candidates for a variety of applications in biomedicine. In this protocol, the preparation of multifunctional DNA NFs with highly tunable sizes is described for applications in cell targeting, intracellular imaging and drug delivery. Preparation and characterization of functional DNA NFs takes ~5 d; the following biomedical applications take ~10 d. PMID:26357007

A study of the nonlinear aerodynamics of bodies in nonplanar motion. Ph.D. Thesis - Stanford Univ., Calif.; [numerical analysis of aerodynamic force and moment systems during large amplitude, arbitrary motions

NASA Technical Reports Server (NTRS)

Schiff, L. B.

1974-01-01

Concepts from the theory of functionals are used to develop nonlinear formulations of the aerodynamic force and moment systems acting on bodies in large-amplitude, arbitrary motions. The analysis, which proceeds formally once the functional dependence of the aerodynamic reactions upon the motion variables is established, ensures the inclusion, within the resulting formulation, of pertinent aerodynamic terms that normally are excluded in the classical treatment. Applied to the large-amplitude, slowly varying, nonplanar motion of a body, the formulation suggests that the aerodynamic moment can be compounded of the moments acting on the body in four basic motions: steady angle of attack, pitch oscillations, either roll or yaw oscillations, and coning motion. Coning, where the nose of the body describes a circle around the velocity vector, characterizes the nonplanar nature of the general motion.

Electrochemical DNA probe for Hg(2+) detection based on a triple-helix DNA and Multistage Signal Amplification Strategy.

PubMed

Wang, Huan; Zhang, Yihe; Ma, Hongmin; Ren, Xiang; Wang, Yaoguang; Zhang, Yong; Wei, Qin

2016-12-15

In this work, an ultrasensitive electrochemical sensor was developed for detection of Hg(2+). Gold nanoparticles decorated bovine serum albumin reduction of graphene oxide (AuNP-BSA-rGO) were used as subsurface material for the immobilization of triple-helix DNA. The triple-helix DNA containing a thiol labelled single-stranded DNA (sDNA) and a thymine-rich DNA (T-rich DNA), which could be unwinded in the present of Hg(2+) to form more stable thymine-Hg(2+)-thymine (T-Hg(2+)-T) complex. T-Hg(2+)-T complex was then removed and the sDNA was left on the electrode. At this time, gold nanoparticle carrying thiol labelled cytosine-rich complementary DNA (cDNA-AuNP) could bind with the free sDNA. Meanwhile, the other free cDNA on AuNP could bind with each other in the present of Ag(+) to form the stable cytosine-Ag(+)-cytosine (C-Ag(+)-C) complex and circle amplification. Plenty of C-Ag(+)-C could form silver nanoclusters by electrochemical reduction and the striping signal of Ag could be measured for purpose of the final electrochemical detection of Hg(2+). This sensor could detect Hg(2+) over a wide concentration range from 0.1 to 130nM with a detection limit of 0.03nM. Copyright © 2016 Elsevier B.V. All rights reserved.

Recent progress in developing durable and permanent impeller pump.

PubMed

Qian, K X; Zeng, P; Ru, W M; Yuan, H Y

2002-04-01

Since 1980s, the author's impeller pump has successively achieved the device implantability, blood compatibility and flow pulsatility. In order to realize a performance durability, the author has concentrated in past years on solving the bearing problems of the impeller pump. Recent progress has been obtained in developing durable and permanent impeller blood pumps. At first, a durable impeller pump with rolling bearing and purge system has been developed, in which the wear-less rollers made of super-high-molecular weight polythene make the pump to work for years without mechanical wear; and the purge system enables the bearing to work in saline and heparin, and no thrombus therefore could be formed. Secondly, a durable centrifugal pump with rolling bearing and axially reciprocating impeller has been developed, the axial reciprocation of rotating impeller makes the fresh blood in and out of the bearing and to wash the rollers once a circle; in such way, no thrombus could be formed and no fluid infusion is necessary, which may bring inconvenience and discomfort to the receptors. Finally, a permanent maglev impeller pump has been developed, its rotor is suspended and floating in the blood under the action of permanent magnetic force and nonmagnetic forces, without need for position measurement and feed-back control. In conclusion, an implantable, pulsatile, and blood compatible impeller pump with durability may have more extensive applications than ever before and could replace the donor heart for transplantation in the future.

Non-singular bounce transitions in the multiverse

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

Garriga, Jaume; Vilenkin, Alexander; Zhang, Jun, E-mail: jaume.garriga@ub.edu, E-mail: vilenkin@cosmos.phy.tufts.edu, E-mail: jun.zhang@tufts.edu

2013-11-01

According to classical GR, negative-energy (AdS) bubbles in the multiverse terminate in big crunch singularities. It has been conjectured, however, that the fundamental theory may resolve these singularities and replace them by non-singular bounces. Here we explore possible dynamics of such bounces using a simple modification of the Friedmann equation, which ensures that the scale factor bounces when the matter density reaches some critical value ρ{sub c}. This is combined with a simple scalar field 'landscape', where the energy barriers between different vacua are small compared to ρ{sub c}. We find that the bounce typically results in a transition tomore » another vacuum, with a scalar field displacement Δφ ∼ 1 in Planck units. If the new vacuum is AdS, we have another bounce, and so on, until the field finally transits to a positive-energy (de Sitter) vacuum. We also consider perturbations about the homogeneous solution and discuss some of their amplification mechanisms (e.g., tachyonic instability and parametric resonance). For a generic potential, these mechanisms are much less efficient than in models of slow-roll inflation. But the amplification may still be strong enough to cause the bubble to fragment into a mosaic of different vacua.« less

Experiments Testing the Causes of Namibian Fairy Circles.

PubMed

Tschinkel, Walter R

2015-01-01

The grasslands on the sandy soils of the eastern edge of the Namib Desert of Namibia are strikingly punctuated by millions of mostly regularly-spaced circular bare spots 2 to 10 m or more in diameter, generally with a margin of taller grasses. The causes of these so called fairy circles are unknown, but several hypotheses have been advanced. In October 2009, we set up experiments that specifically tested four hypothesized causes, and monitored these 5 times between 2009 and 2015. Grass exclusion in circles due to seepage of subterranean vapors or gases was tested by burying an impermeable barrier beneath fairy circles, but seedling density and growth did not differ from barrier-less controls. Plant germination and growth inhibition by allelochemicals or nutrient deficiencies in fairy circle soils were tested by transferring fairy circle soil to artificially cleared circles in the grassy matrix, and matrix soil to fairy circles (along with circle to circle and matrix to matrix controls). None of the transfers changed the seedling density and growth from the control reference conditions. Limitation of plant growth due to micronutrient depletion within fairy circles was tested by supplementing circles with a micronutrient mixture, but did not result in differences in plant seedling density and growth. Short-range vegetation competitive feedbacks were tested by creating artificially-cleared circles of 2 or 4 m diameter located 2 or 6 m from a natural fairy circle. The natural circles remained bare and the artificial circles revegetated. These four experiments provided evidence that fairy circles were not caused by subterranean vapors, that fairy circle soil per se did not inhibit plant growth, and that the circles were not caused by micronutrient deficiency. There was also no evidence that vegetative feedbacks affected fairy circles on a 2 to 10 m scale. Landscape-scale vegetative self-organization is discussed as a more likely cause of fairy circles.

Self-assembly Controls Self-cleavage of HHR from ASBVd (-): a Combined SANS and Modeling Study

NASA Astrophysics Data System (ADS)

Leclerc, Fabrice; Zaccai, Giuseppe; Vergne, Jacques; Řìhovà, Martina; Martel, Anne; Maurel, Marie-Christine

2016-07-01

In the Avocado Sunblotch Viroid (ASBVd: 249-nt) from the Avsunviroidae family, a symmetric rolling-circle replication operates through an autocatalytic mechanism mediated by hammerhead ribozymes (HHR) embedded in both polarity strands. The concatenated multimeric ASBVd (+) and ASBVd (-) RNAs thus generated are processed by cleavage to unit-length where ASBVd (-) self-cleaves with more efficiency. Absolute scale small angle neutron scattering (SANS) revealed a temperature-dependent dimer association in both ASBVd (-) and its derived 79-nt HHR (-). A joint thermodynamic analysis of SANS and catalytic data indicates the rate-determining step corresponds to the dimer/monomer transition. 2D and 3D models of monomeric and dimeric HHR (-) suggest that the inter-molecular contacts stabilizing the dimer (between HI and HII domains) compete with the intra-molecular ones stabilizing the active conformation of the full-length HHR required for an efficient self-cleavage. Similar competing intra- and inter-molecular contacts are proposed in ASBVd (-) though with a remoter region from an extension of the HI domain.

Pilot evaluation of sailplane handling qualities

NASA Technical Reports Server (NTRS)

Bennett, A. G., Jr.

1978-01-01

The evaluation sailplanes were found generally deficient in the area of cockpit layout. The pilots indicated general dissatisfaction with high pitch sensitivity especially when coupled with inertially induced stick forces. While all sailplanes were judged satisfactory for centering thermals and in the ease of speed control in circling flight, pilot opinions diverged on the maneuvering response, pull-out characteristics from a dive, and on phugoid damping. Lateral-directional control problems were noted mainly during takeoff and landing for most sailplanes with the landing wheel ahead of center of gravity. Pilot opinion of in-flight lateral-directional stability and control was generally satisfactory. Five of the evaluation sailplanes exhibited a very narrow airspeed band in which perceptible stall warning buffet occurred. However, this characteristic was considered not objectionable when stall recovery was easy. The pilots objected to the characteristics of a wide airspeed band of stall warning followed by a stall with yawing and rolling tendency and substantial loss of altitude during the stall. Glide path control for the evaluation sailplanes was found to be generally objectionable.

Active Site Sharing and Subterminal Hairpin Recognition in a New Class of DNA Transposases

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

Ronning, Donald R.; Guynet, Catherine; Ton-Hoang, Bao

2010-07-20

Many bacteria harbor simple transposable elements termed insertion sequences (IS). In Helicobacter pylori, the chimeric IS605 family elements are particularly interesting due to their proximity to genes encoding gastric epithelial invasion factors. Protein sequences of IS605 transposases do not bear the hallmarks of other well-characterized transposases. We have solved the crystal structure of full-length transposase (TnpA) of a representative member, ISHp608. Structurally, TnpA does not resemble any characterized transposase; rather, it is related to rolling circle replication (RCR) proteins. Consistent with RCR, Mg{sup 2+} and a conserved tyrosine, Tyr127, are essential for DNA nicking and the formation of a covalentmore » intermediate between TnpA and DNA. TnpA is dimeric, contains two shared active sites, and binds two DNA stem loops representing the conserved inverted repeats near each end of ISHp608. The cocrystal structure with stem-loop DNA illustrates how this family of transposases specifically recognizes and pairs ends, necessary steps during transposition.« less

Animal Mitochondrial DNA Replication

PubMed Central

Ciesielski, Grzegorz L.; Oliveira, Marcos T.; Kaguni, Laurie S.

2016-01-01

Recent advances in the field of mitochondrial DNA (mtDNA) replication highlight the diversity of both the mechanisms utilized and the structural and functional organization of the proteins at mtDNA replication fork, despite the simplicity of the animal mtDNA genome. DNA polymerase γ, mtDNA helicase and mitochondrial single-stranded DNA-binding protein- the key replisome proteins, have evolved distinct structural features and biochemical properties. These appear to be correlated with mtDNA genomic features in different metazoan taxa and with their modes of DNA replication, although a substantial integrative research is warranted to establish firmly these links. To date, several modes of mtDNA replication have been described for animals: rolling circle, theta, strand-displacement, and RITOLS/bootlace. Resolution of a continuing controversy relevant to mtDNA replication in mammals/vertebrates will have a direct impact on the mechanistic interpretation of mtDNA-related human diseases. Here we review these subjects, integrating earlier and recent data to provide a perspective on the major challenges for future research. PMID:27241933

Bypass of a Nick by the Replisome of Bacteriophage T7*

PubMed Central

Zhu, Bin; Lee, Seung-Joo; Richardson, Charles C.

2011-01-01

DNA polymerase and DNA helicase are essential components of DNA replication. The helicase unwinds duplex DNA to provide single-stranded templates for DNA synthesis by the DNA polymerase. In bacteriophage T7, movement of either the DNA helicase or the DNA polymerase alone terminates upon encountering a nick in duplex DNA. Using a minicircular DNA, we show that the helicase·polymerase complex can bypass a nick, albeit at reduced efficiency of 7%, on the non-template strand to continue rolling circle DNA synthesis. A gap in the non-template strand cannot be bypassed. The efficiency of bypass synthesis depends on the DNA sequence downstream of the nick. A nick on the template strand cannot be bypassed. Addition of T7 single-stranded DNA-binding protein to the complex stimulates nick bypass 2-fold. We propose that the association of helicase with the polymerase prevents dissociation of the helicase upon encountering a nick, allowing the helicase to continue unwinding of the duplex downstream of the nick. PMID:21701044

Bypass of a nick by the replisome of bacteriophage T7.

PubMed

Zhu, Bin; Lee, Seung-Joo; Richardson, Charles C

2011-08-12

DNA polymerase and DNA helicase are essential components of DNA replication. The helicase unwinds duplex DNA to provide single-stranded templates for DNA synthesis by the DNA polymerase. In bacteriophage T7, movement of either the DNA helicase or the DNA polymerase alone terminates upon encountering a nick in duplex DNA. Using a minicircular DNA, we show that the helicase · polymerase complex can bypass a nick, albeit at reduced efficiency of 7%, on the non-template strand to continue rolling circle DNA synthesis. A gap in the non-template strand cannot be bypassed. The efficiency of bypass synthesis depends on the DNA sequence downstream of the nick. A nick on the template strand cannot be bypassed. Addition of T7 single-stranded DNA-binding protein to the complex stimulates nick bypass 2-fold. We propose that the association of helicase with the polymerase prevents dissociation of the helicase upon encountering a nick, allowing the helicase to continue unwinding of the duplex downstream of the nick.

Characterization of a cryptic plasmid from an alpha-proteobacterial endosymbiont of Amoeba proteus.

PubMed

Park, Miey; Kim, Min-Soo; Lee, Kyung-Min; Hwang, Sue-Yun; Ahn, Tae In

2009-01-01

A new cryptic plasmid pAP3.9 was discovered in symbiotic alpha-proteobacteria present in the cytoplasm of Amoeba proteus. The plasmid is 3869bp with a GC content of 34.66% and contains replication origins for both double-strand (dso) and single-strand (sso). It has three putative ORFs encoding Mob, Rep and phosphoglycolate phosphatase (PGPase). The pAP3.9 plasmid appears to propagate by the conjugative rolling-circle replication (RCR), since it contains all required factors such as Rep, sso and dso. Mob and Rep showed highest similarities to those of the cryptic plasmid pBMYdx in Bacillus mycoides. The PGPase was homologous to that of Bacillus cereus and formed a clade with those of Bacillus sp. in molecular phylogeny. These results imply that the pAP3.9 plasmid evolved by the passage through Bacillus species. We hypothesize that the plasmid-encoded PGPase may have contributed to the establishment of bacterial symbiosis within the hostile environment of amoeba cytoplasm.

High-throughput multiplexed T-cell-receptor excision circle quantitative PCR assay with internal controls for detection of severe combined immunodeficiency in population-based newborn screening.

PubMed

Gerstel-Thompson, Jacalyn L; Wilkey, Jonathan F; Baptiste, Jennifer C; Navas, Jennifer S; Pai, Sung-Yun; Pass, Kenneth A; Eaton, Roger B; Comeau, Anne Marie

2010-09-01

Real-time quantitative PCR (qPCR) targeting a specific marker of functional T cells, the T-cell-receptor excision circle (TREC), detects the absence of functional T cells and has a demonstrated clinical validity for detecting severe combined immunodeficiency (SCID) in infants. There is need for a qPCR TREC assay with an internal control to monitor DNA quality and the relative cellular content of the particular dried blood spot punch sampled in each reaction. The utility of the qPCR TREC assay would also be far improved if more tests could be performed on the same newborn screening sample. We approached the multiplexing of qPCR for TREC by attenuating the reaction for the reference gene, with focus on maintaining tight quality assurance for reproducible slopes and for prevention of sample-to-sample cross contamination. Statewide newborn screening for SCID using the multiplexed assay was implemented, and quality-assurance data were recorded. The multiplex qPCR TREC assay showed nearly 100% amplification efficiency for each of the TREC and reference sequences, clinical validity for multiple forms of SCID, and an analytic limit of detection consistent with prevention of contamination. The eluate and residual ghost from a 3.2-mm dried blood spot could be used as source material for multiplexed immunoassays and multiplexed DNA tests (Multiplex Plus), with no disruption to the multiplex TREC qPCR. Population-based SCID newborn screening programs should consider multiplexing for quality assurance purposes. Potential benefits of using Multiplex Plus include the ability to perform multianalyte profiling.

Introducing Nine-Point Circle to Junior High School Students

NASA Astrophysics Data System (ADS)

Fiangga, S.; Azizah, M. A. N.; Rini, R. N. K.; Hidayanti, A. N.

2018-01-01

The concept of circles is an ancient concept that has appeared since Ancient Egypt from which this concept gives many significant contributions in mathematics’ development until now. Nevertheless, the concept of circles hides many uncover mysterious features that are of applications in mathematics. One of the mysterious features is the Nine-Point Circle. This Nine-point circle is also known as Euler’s circle, six-point circle, Feuerbach’s circle, the twelve-point circle, and many others. Because of these different names, there have been misunderstand among mathematicians about the Nine-Point Circle’s history. Besides, the discussion of Nine-Point Circle can be used to be an initial material to explain elementary geometry topic in junior high school’s level curriculum of 2013. Therefore, this concept needs to be delivered to the students as a geometry introduction. A possible form of the integration historical aspect of Nine-point circle is suggested in this paper as well as its importance in the curriculum of 2013.

The Mediation of Tools in the Development of Formal Mathematical Concepts: The Compass and the Circle as an Example.

ERIC Educational Resources Information Center

Chassapis, Dimitris

1999-01-01

Focuses on the process by which children develop a formal mathematical concept of the circle by using various instruments to draw circles within the context of a goal-directed drawing task. Concludes that the use of the compass in circle drawing structures the circle-drawing operation in a radically different fashion than circle tracers and…

Circling motion and screen edges as an alternative input method for on-screen target manipulation.

PubMed

Ka, Hyun W; Simpson, Richard C

2017-04-01

To investigate a new alternative interaction method, called circling interface, for manipulating on-screen objects. To specify a target, the user makes a circling motion around the target. To specify a desired pointing command with the circling interface, each edge of the screen is used. The user selects a command before circling the target. To evaluate the circling interface, we conducted an experiment with 16 participants, comparing the performance on pointing tasks with different combinations of selection method (circling interface, physical mouse and dwelling interface) and input device (normal computer mouse, head pointer and joystick mouse emulator). A circling interface is compatible with many types of pointing devices, not requiring physical activation of mouse buttons, and is more efficient than dwell-clicking. Across all common pointing operations, the circling interface had a tendency to produce faster performance with a head-mounted mouse emulator than with a joystick mouse. The performance accuracy of the circling interface outperformed the dwelling interface. It was demonstrated that the circling interface has the potential as another alternative pointing method for selecting and manipulating objects in a graphical user interface. Implications for Rehabilitation A circling interface will improve clinical practice by providing an alternative pointing method that does not require physically activating mouse buttons and is more efficient than dwell-clicking. The Circling interface can also work with AAC devices.

A localization algorithm of adaptively determining the ROI of the reference circle in image

NASA Astrophysics Data System (ADS)

Xu, Zeen; Zhang, Jun; Zhang, Daimeng; Liu, Xiaomao; Tian, Jinwen

2018-03-01

Aiming at solving the problem of accurately positioning the detection probes underwater, this paper proposed a method based on computer vision which can effectively solve this problem. The theory of this method is that: First, because the shape information of the heat tube is similar to a circle in the image, we can find a circle which physical location is well known in the image, we set this circle as the reference circle. Second, we calculate the pixel offset between the reference circle and the probes in the picture, and adjust the steering gear through the offset. As a result, we can accurately measure the physical distance between the probes and the under test heat tubes, then we can know the precise location of the probes underwater. However, how to choose reference circle in image is a difficult problem. In this paper, we propose an algorithm that can adaptively confirm the area of reference circle. In this area, there will be only one circle, and the circle is the reference circle. The test results show that the accuracy of the algorithm of extracting the reference circle in the whole picture without using ROI (region of interest) of the reference circle is only 58.76% and the proposed algorithm is 95.88%. The experimental results indicate that the proposed algorithm can effectively improve the efficiency of the tubes detection.

Are Namibian “Fairy Circles” the Consequence of Self-Organizing Spatial Vegetation Patterning?

PubMed Central

Cramer, Michael D.; Barger, Nichole N.

2013-01-01

Causes of over-dispersed barren “fairy circles” that are often surrounded by ca. 0.5 m tall peripheral grasses in a matrix of shorter (ca. 0.2 m tall) grasses in Namibian grasslands remain mysterious. It was hypothesized that the fairy circles are the consequence of self-organizing spatial vegetation patterning arising from resource competition and facilitation. We examined the edaphic properties of fairy circles and variation in fairy circle size, density and landscape occupancy (% land surface) with edaphic properties and water availability at a local scale (<50 km) and with climate and vegetation characteristics at a regional scale. Soil moisture in the barren fairy circles declines from the center towards the periphery and is inversely correlated with soil organic carbon, possibly indicating that the peripheral grass roots access soil moisture that persists into the dry season within fairy circles. Fairy circle landscape occupancy is negatively correlated with precipitation and soil [N], consistent with fairy circles being the product of resource-competition. Regional fairy circle presence/absence is highly predictable using an empirical model that includes narrow ranges of vegetation biomass, precipitation and temperature seasonality as predictor variables, indicating that fairy circles are likely a climate-dependent emergent phenomenon. This dependence of fairy circle occurrence on climate explains why fairy circles in some locations may appear and disappear over time. Fairy circles are only over-dispersed at high landscape occupancies, indicating that inter-circle competition may determine their spacing. We conclude that fairy circles are likely to be an emergent arid-grassland phenomenon that forms as a consequence of peripheral grass resource-competition and that the consequent barren circle may provide a resource-reservoir essential for the survival of the larger peripheral grasses and provides a habitat for fossicking fauna. PMID:23976962

Procedure for normalization of cDNA libraries

DOEpatents

Bonaldo, Maria DeFatima; Soares, Marcelo Bento

1997-01-01

This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

The newest miracle drug: quality circles in hospitals.

PubMed

McKinney, M M

1984-01-01

In recent years, a number of hospitals throughout the United States have been exploring the use of Japanese-style quality circles to reduce their operating expenses, improve productivity, and enhance the quality of work life for hospital employees. This article examines the organizational climate necessary for quality circles, methods used to implement quality circles, and management's role in guiding and responding to circle activities. Ideas for building and maintaining staff support are presented along with a cost/benefit analysis of quality circle programs. The author concludes that quality circles are most successful in hospitals where they are part of a larger organizational development effort. When administrators believe in their employees' ability to contribute to the institution and are willing to invest necessary time and resources in employee education and the measurement of quality circle achievements, quality circles can produce creative solutions to perplexing institutional problems.

Plume-induced roll back subduction around Venus large coronae

NASA Astrophysics Data System (ADS)

Davaille, A.; Smrekar, S. E.; Tomlinson, S. M.

2016-12-01

On Venus, possible subduction trenches are mainly associated with large coronae, eventhough the latter are thought to be produced by hot mantle plumes. The mechanism of assocation between subduction and plume has long remained elusive. However, we recently observe the same association in laboratory experiments on thermal convection in colloidal aqueous dispersions of silica nanoparticles, which deform in the Newtonian regime at low solid particle fraction φp, and transition to strain-rate weakening, plasticity, elasticity, and brittle properties as φp increases. Hence, a dense skin akin to a planetary lithosphere grows on the surface when the system is dried from above. When a hot plume rises under the skin, the latter undergoes a flexural deformation which puts it under tension. Cracks then develop, sometimes using pre-existing weaknesses. Plume material (being more buoyant that the laboratory lithosphere) upwells through the cracks and spreads as a axisymmetric gravity current above the broken denser skin. The latter bends and sinks under the conjugate action of its own weight and the plume gravity current. The brittle character of the top experimental lithosphere forbids it to deform viscously to accomodate the sinking motions. Instead, the plate continues to tear as a sheet of paper would do upon intrusion. Several slabs are therefore produced, associated with trenches localized along partial circles on the plume, and strong roll-back is always observed. Depending on the lithospheric strength, roll-back can continue and triggers a complete resurfacing, or it stops when the plume stops spreading. Scalings derived from the experiments suggest that a weaker lithosphere than that present on Earth today is required for such a convective regime. We identified two candidates on Venus. At Artemis and Quetzelpetlatl Coronae, the radar image observations and subsurface density variations inferred from modeling the gravity and topography agree with the predictions from the laboratory. Evidence for geologically recent volcanism at Quetzelpetlatl further suggests that subduction may be currently active on Venus. However, a more complete appraisal of this mechanism for Venus resurfacing will require a better knowledge of Venus topography and gravity field, such as proposed by the future mission VERITAS.

A Cross-Sectional Study of the Effect of Quality Circles on Twelve Attitudinal Variables.

DTIC Science & Technology

1985-09-01

task of rebuilding its industrial capabilities which had been largely destroyed during the war. Japanese leaders had a goal of making their country an...members, providing support when necessary, and acting as a liaison between the circles and other organizations. The circle leader or supervisor...34" " .-.-- • .- .".-",,-, . -.- -,: ".’-"’ -. -. .,-’,: Top & Middle - Management Steering Committee Supervisors . . . ....- Circle Circle Leaders Facilitator Employees Circle Members Source

Circles of support and accountability: The characteristics of core members in England and Wales.

PubMed

Clarke, Martin; Warwick, Leah; Völlm, Birgit

2017-04-01

Circles of support and accountability, or Circles, use community volunteers to help reintegrate sex offenders at risk of reoffending in the community. The aims of this study are to describe the first 275 male sex offenders ('core members') in England and Wales supported by a Circle and to compare those attending the five largest Circles. As part of their monitoring activity, 10 Circles extracted data from case files, anonymised it and submitted it to Circles UK, the national oversight body. Circles have expanded rapidly with 165 (60%) of Circles commencing in the three years 2011-2013 compared with 110 in the nine years 2002-2010. Most core members were referred from the Probation Service (82%). Circles were provided to men with a range of predicted risks of reoffending - from low (26%) to very high (12%). There were some positive changes between the beginning and end of Circles, such as fewer men being unemployed and more living in their own chosen accommodation. Circles have been used to support the reintegration of a wide range of sex offenders. Given their rapid growth and flexibility, consistent recording standards are required across. These standards should be reviewed periodically to ensure all important fields of change are captured, including frequency of attendance, length per session and quality of engagement in the work. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Three Concentric Circles: Young Chinese English Learners' Perceptions of Purposeful Audiences

ERIC Educational Resources Information Center

Liu, Jack Jinghui

2015-01-01

English learners have more access to communicate with different purposeful audiences across the Three Concentric Circles of English (Kachu, 1985): the Inner Circle, the Outer Circle and the Expanding Circle. However, young language learners' purposeful audience as a focus of communication has not been emphasized as much as other linguistic…

Procedure for normalization of cDNA libraries

DOEpatents

Bonaldo, M.D.; Soares, M.B.

1997-12-30

This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library. 1 fig.

Closed Circular Chains

ERIC Educational Resources Information Center

Caglayan, Günhan

2016-01-01

A Steiner chain is defined as the sequence of n circles that are all tangent to two given non-intersecting circles. A closed chain, in particular, is one in which every circle in the sequence is tangent to the previous and next circles of the chain. In a closed Steiner chain the first and the "n"th circles of the chain are also tangent…

The Director Circle of a Central Conic Section

ERIC Educational Resources Information Center

Ayoub, Ayoub B.

2007-01-01

Each ellipse and hyperbola has a circle associated with it called the director circle. In this article, the author derives the equations of the circle for the ellipse and hyperbola through a different approach. Then the author concentrates on the director circle of the central conic given by the general quadratic equation. The content of this…

Dark Circles under Eyes

MedlinePlus

Symptoms Dark circles under eyes By Mayo Clinic Staff Dark circles under your eyes generally implies that the darkening ... eye. Fatigue is the most common cause of dark circles under your eyes. Sometimes, what appear to ...

Study of the surface wave off-great-circle propagation based on dense seismic array: a case study in Northeast China

NASA Astrophysics Data System (ADS)

Chen, H.; Chong, J.

2016-12-01

The traditional surface wave tomography is based on the ray theory, which assumes that surface wave propagates along the great-circle. The great-circle assumption is valid only when the size of the anomaly is larger than the width of the Fresnel zone and the lateral variation is relatively smooth. However, off-great-circle propagation may occur when the surface wave travels across tectonic boundaries with strong heterogeneity and sharp velocity change, e.g., continental margin, mid-ridge and sea trench, resulting in arrival angle anomaly and multi-pathing effect. The off-great-circle propagation may deviate the result of surface wave tomography based on great-circle approximation, so it is of great importance to study the off-great-circle propagation. In this study, we used the teleseismic waveforms from September 2009 to August 2011, recorded by the NECESSArray in Northeast China, to study the off-great-circle propagation of Rayleigh wave by the Beamforming method. Our results show that the off-great-circle effect increases with decreasing period. At the period of 60 s, the off-great-circle effect is relatively weak and the Rayleigh wave propagates approximately along the great-circle. While at the period of 20 s, the off-great-circle effect becomes strong, the arrival angle anomaly measured from some events can be as large as 20º, and obvious multi-pathing effect is also observed. Lateral variations of the arrival angle anomaly and phase velocity have also been found in the study region, which may be correlated with the lithosphere heterogeneity in Northeast China. Our results demonstrate the necessity to study the surface wave off-great-circle propagation. Acknowledgement: This study is financially supported by National Natural Science Foundation of China under Grant No. 41590854.

Classification by causes of dark circles and appropriate evaluation method of dark circles.

PubMed

Park, S R; Kim, H J; Park, H K; Kim, J Y; Kim, N S; Byun, K S; Moon, T K; Byun, J W; Moon, J H; Choi, G S

2016-08-01

Dark circles refer to a symptom that present darkness under the eyes. Because of improvement in the quality of life, the dark circles have been recognized as one of major cosmetic concerns. However, it is not easy to classify the dark circles because they have various causes. To select suitable instruments and detailed evaluation items, the dark circles were classified according to the causes through visual assessment, Wood's lamp test, and medical history survey for 100 subjects with dark circles. After the classification, were newly recruited for instrument conformity assessment. Through this, suitable instruments for dark circle evaluation were selected. We performed a randomized clinical trial for dark circles, a placebo-controlled double-blind study, using effective parameters of the instruments selected from the preliminary test. Dark circles of vascular type (35%) and mixed type (54%), a combination of pigmented and vascular types, were the most common. Twenty four subjects with the mixed type dark circles applied the test product (Vitamin C 3%, Vitamin A 0.1%, Vitamin E 0.5%) and placebo on randomized split-face for 8 weeks. The effective parameters (L*, a, M.I., E.I., quasi L*, quasi a* and dermal thickness) were measured during the study period. Result showed that the L* value of Chromameter(®) , Melanin index (M.I.) of Mexameter(®) and quasi L* value obtained by image analysis improved with statistical significance after applying the test product compared with the placebo product. We classified the dark circles according to the causes of the dark circles and verified the reliability of the parameter obtained by the instrument conformity assessment used in this study through the efficacy evaluation. Also based on this study, we were to suggest newly established methods which can be applied to the evaluation of efficacy of functional cosmetics for dark circles. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Quality circles: Organizational adaptations, improvements and results

NASA Technical Reports Server (NTRS)

Tortorich, R.

1985-01-01

The effective application in industry and government of quality circles work was demonstrated. The results achieved in quality and productivity improvements and cost savings are impressive. The circle process should be institutionalized within industry and government. The stages of circle program growth, innovations that help achieve circle process institutionalization, and the result achieved at Martin Marietta's Michoud Division and within the National Aeronautics and Space Administration (NASA) are addressed.

Participant Experiences of Talking Circles on Type 2 Diabetes in Two Northern Plains American Indian Tribes

PubMed Central

Struthers, Roxanne; Hodge, Felicia Schanche; Geishirt-Cantrell, Betty; De Cora, Lorelei

2011-01-01

The Talking Circle, a culturally appropriate, 12-week educational intervention, was employed on two Northern Plains American Indian reservations to provide information on type 2 diabetes. In a phenomenological study, funded as a minority supplement to the Talking Circle intervention, the authors asked 8 American Indian participants of the Talking Circle to describe their experience of being an American Indian Talking Circle participant. Seven common themes describe the phenomenon of participating in a Talking Circle diabetic intervention. The Talking Circle technique was effective in providing information on type 2 diabetes through culturally appropriate community sharing. Type 2 diabetes is viewed by both outsiders and those involved as a chronic disease of the utmost concern in American Indian communities. PMID:14556421

[Bioinformatics analysis of mosquito densovirus nostructure protein NS1].

PubMed

Dong, Yun-qiao; Ma, Wen-li; Gu, Jin-bao; Zheng, Wen-ling

2009-12-01

To analyze and predict the structure and function of mosquito densovirus (MDV) nostructual protein1 (NS1). Using different bioinformatics software, the EXPASY pmtparam tool, ClustalX1.83, Bioedit, MEGA3.1, ScanProsite, and Motifscan, respectively to comparatively analyze and predict the physic-chemical parameters, homology, evolutionary relation, secondary structure and main functional motifs of NS1. MDV NS1 protein was a unstable hydrophilic protein and the amino acid sequence was highly conserved which had a relatively closer evolutionary distance with infectious hypodermal and hematopoietic necrosis virus (IHHNV). MDV NS1 has a specific domain of superfamily 3 helicase of small DNA viruses. This domain contains the NTP-binding region with a metal ion-dependent ATPase activity. A virus replication roller rolling-circle replication(RCR) initiation domain was found near the N terminal of this protein. This protien has the biological function of single stranded incision enzyme. The bioinformatics prediction results suggest that MDV NS1 protein plays a key role in viral replication, packaging, and the other stages of viral life.

Acinetobacter phage genome is similar to Sphinx 2.36, the circular DNA copurified with TSE infected particles.

PubMed

Longkumer, Toshisangba; Kamireddy, Swetha; Muthyala, Venkateswar Reddy; Akbarpasha, Shaikh; Pitchika, Gopi Krishna; Kodetham, Gopinath; Ayaluru, Murali; Siddavattam, Dayananda

2013-01-01

While analyzing plasmids of Acinetobacter sp. DS002 we have detected a circular DNA molecule pTS236, which upon further investigation is identified as the genome of a phage. The phage genome has shown sequence similarity to the recently discovered Sphinx 2.36 DNA sequence co-purified with the Transmissible Spongiform Encephalopathy (TSE) particles isolated from infected brain samples collected from diverse geographical regions. As in Sphinx 2.36, the phage genome also codes for three proteins. One of them codes for RepA and is shown to be involved in replication of pTS236 through rolling circle (RC) mode. The other two translationally coupled ORFs, orf106 and orf96, code for coat proteins of the phage. Although an orf96 homologue was not previously reported in Sphinx 2.36, a closer examination of DNA sequence of Sphinx 2.36 revealed its presence downstream of orf106 homologue. TEM images and infection assays revealed existence of phage AbDs1 in Acinetobacter sp. DS002.

Acinetobacter phage genome is similar to Sphinx 2.36, the circular DNA copurified with TSE infected particles

PubMed Central

Longkumer, Toshisangba; Kamireddy, Swetha; Muthyala, Venkateswar Reddy; Akbarpasha, Shaikh; Pitchika, Gopi Krishna; Kodetham, Gopinath; Ayaluru, Murali; Siddavattam, Dayananda

2013-01-01

While analyzing plasmids of Acinetobacter sp. DS002 we have detected a circular DNA molecule pTS236, which upon further investigation is identified as the genome of a phage. The phage genome has shown sequence similarity to the recently discovered Sphinx 2.36 DNA sequence co-purified with the Transmissible Spongiform Encephalopathy (TSE) particles isolated from infected brain samples collected from diverse geographical regions. As in Sphinx 2.36, the phage genome also codes for three proteins. One of them codes for RepA and is shown to be involved in replication of pTS236 through rolling circle (RC) mode. The other two translationally coupled ORFs, orf106 and orf96, code for coat proteins of the phage. Although an orf96 homologue was not previously reported in Sphinx 2.36, a closer examination of DNA sequence of Sphinx 2.36 revealed its presence downstream of orf106 homologue. TEM images and infection assays revealed existence of phage AbDs1 in Acinetobacter sp. DS002. PMID:23867905

Real-space transmission electron microscopy investigations of attachment of functionalized magnetic nanoparticles to DNA-coils acting as a biosensor.

PubMed

Akhtar, Sultan; Strömberg, Mattias; Zardán Gómez de la Torre, Teresa; Russell, Camilla; Gunnarsson, Klas; Nilsson, Mats; Svedlindh, Peter; Strømme, Maria; Leifer, Klaus

2010-10-21

The present work provides the first real-space analysis of nanobead-DNA coil interactions. Immobilization of oligonucleotide-functionalized magnetic nanobeads in rolling circle amplified DNA-coils was studied by complex magnetization measurements and transmission electron microscopy (TEM), and a statistical analysis of the number of beads hybridized to the DNA-coils was performed. The average number of beads per DNA-coil using the results from both methods was found to be around 6 and slightly above 2 for samples with 40 and 130 nm beads, respectively. The TEM analysis supported an earlier hypothesis that 40 nm beads are preferably immobilized in the interior of DNA-coils whereas 130 nm beads, to a larger extent, are immobilized closer to the exterior of the coils. The methodology demonstrated in the present work should open up new possibilities for characterization of interactions of a large variety of functionalized nanoparticles with macromolecules, useful for gaining more fundamental understanding of such interactions as well as for optimizing a number of biosensor applications.

Crosstalk between vertical and horizontal gene transfer: plasmid replication control by a conjugative relaxase

PubMed Central

Lorenzo-Díaz, Fabián; Fernández-López, Cris; Lurz, Rudi

2017-01-01

Abstract Horizontal gene transfer is a key process in the evolution of bacteria and also represents a source of genetic variation in eukaryotes. Among elements participating in gene transfer, thousands of small (<10 kb) mobile bacterial plasmids that replicate by the rolling circle mechanism represent a driving force in the spread of antibiotic resistances. In general, these plasmids are built as genetic modules that encode a replicase, an antibiotic-resistance determinant, and a relaxase that participates in their conjugative mobilization. Further, they control their relatively high copy number (∼30 copies per genome equivalent) by antisense RNAs alone or combined with a repressor protein. We report here that the MobM conjugative relaxase encoded by the promiscuous plasmid pMV158 participates in regulation of the plasmid copy number by transcriptional repression of the antisense RNA, thus increasing the number of plasmid molecules ready to be horizontally transferred (mobilization) and/or vertically inherited (replication). This type of crosstalk between genetic modules involved in vertical and horizontal gene flow has not been reported before. PMID:28525572

SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes

PubMed Central

Shen, Yue; Stracquadanio, Giovanni; Wang, Yun; Yang, Kun; Mitchell, Leslie A.; Xue, Yaxin; Cai, Yizhi; Chen, Tai; Dymond, Jessica S.; Kang, Kang; Gong, Jianhui; Zeng, Xiaofan; Zhang, Yongfen; Li, Yingrui; Feng, Qiang; Xu, Xun; Wang, Jun; Wang, Jian; Yang, Huanming; Boeke, Jef D.; Bader, Joel S.

2016-01-01

Synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) generates combinatorial genomic diversity through rearrangements at designed recombinase sites. We applied SCRaMbLE to yeast synthetic chromosome arm synIXR (43 recombinase sites) and then used a computational pipeline to infer or unscramble the sequence of recombinations that created the observed genomes. Deep sequencing of 64 synIXR SCRaMbLE strains revealed 156 deletions, 89 inversions, 94 duplications, and 55 additional complex rearrangements; several duplications are consistent with a double rolling circle mechanism. Every SCRaMbLE strain was unique, validating the capability of SCRaMbLE to explore a diverse space of genomes. Rearrangements occurred exclusively at designed loxPsym sites, with no significant evidence for ectopic rearrangements or mutations involving synthetic regions, the 99% nonsynthetic nuclear genome, or the mitochondrial genome. Deletion frequencies identified genes required for viability or fast growth. Replacement of 3′ UTR by non-UTR sequence had surprisingly little effect on fitness. SCRaMbLE generates genome diversity in designated regions, reveals fitness constraints, and should scale to simultaneous evolution of multiple synthetic chromosomes. PMID:26566658

Rapidly expanding genetic diversity and host range of the Circoviridae viral family and other Rep encoding small circular ssDNA genomes

PubMed Central

Delwart, Eric; Li, Linlin

2011-01-01

The genomes of numerous circoviruses and distantly related circular DNA viruses encoding a rolling circle replication initiator protein (Rep) have been characterized from the tissues of mammals, fish, insects, and plants (geminivirus and nanovirus), human and animal feces, in an algae cell, and in diverse environmental samples. We review the genome organization, phylogenetic relationships and initial prevalence studies of cycloviruses, a proposed new genus in the Circoviridae family. Viral fossil rep sequences were also identified integrated on the chromosomes of mammals, frogs, lancelets, crustaceans, mites, gastropods, roundworms, placozoans, hydrozoans, protozoans, land plants, fungi, algae, and phytoplasma bacterias and their plasmids, reflecting their past host range. An ancient origin for viruses with rep-encoding single stranded small circular genomes, predating the diversification of eukaryotes, is discussed. The cellular hosts and pathogenicity of many recently described rep-containing circular genomes remain to be determined. Future studies of the virome of single cell and multi-cellular eukaryotes are likely to further extend the known diversity and host-range of small rep-containing circular viral genomes. PMID:22155583

Intra- and intermolecular nonenzymatic ligations occur within transcripts derived from the peach latent mosaic viroid.

PubMed

Lafontaine, D; Beaudry, D; Marquis, P; Perreault, J P

1995-10-01

We report here the nonenzymatic self-ligation of transcripts corresponding to the peach latent mosaic viroid (PLMVd). This is the first description of this process with viroid sequences, although it has been reported to occur with human hepatitis delta virus RNA. Self-ligation occurs when the 5'-hydroxyl and the 2',3'-cyclic phosphate termini produced by the hammerhead self-cleavage of the viroid RNA are juxtaposed by the viroid rod-like structure, and a phosphodiester bond is formed between the two following hydrolysis of the cyclic phosphate. Unit-length transcripts undergo intramolecular folding, and their subsequent self-ligation produces circular molecules. The self-ligation observed in vitro may contribute to PLMVd circularization during rolling circle replication; however, this does not exclude the possibility that a host RNA ligase catalyzes the ligation steps in vivo. Like self-cleavage, self-ligation is probably an ancestral reaction, and the enzyme-catalyzed ligation most likely evolved from this primitive mechanism. Furthermore, the intermolecular self-ligation of annealed transcripts derived from PLMVd is demonstrated, suggesting a possible mechanism for sequence reassortment in viroids.

KSC-08pd1812

NASA Image and Video Library

2008-06-19

VANDENBERG AIR FORCE BASE, Calif. – The mobile service tower on Space Launch Complex 2 begins to roll back from the Delta II launch vehicle in preparation for launch of the Ocean Surface Topography Mission, or OSTM/Jason 2. The OSTM/Jason 2 satellite will embark on a globe-circling voyage to continue charting sea level, a vital indicator of global climate change. The mission will return a vast amount of new data that will improve weather, climate and ocean forecasts. OSTM/Jason 2's expected lifetime of at least three years will extend into the next decade the continuous record of these data started in 1992 by NASA and the French space agency Centre National d'Etudes Spatiales, or CNES, with the TOPEX/Poseidon mission. The data collection was continued by the two agencies on Jason-1 in 2001. The launch window extends from 12:46 a.m. to 12:55 a.m. PDT. The satellite will be placed in an 830-mile-high orbit at an inclination of 66 degrees after separating from the Delta II 55 minutes after liftoff. Photo credit: Photograph by Carleton Bailie for United Launch Alliance

Made-in-USA Quality Circles Become People-Building Tool.

ERIC Educational Resources Information Center

Cohen, Larry

1983-01-01

Discusses the use of quality circles as a human resources development tool in Middlesex Community College's Career-Oriented Peer Services tutoring center. Delineates rules for circle participants and follows the activities of two circles comprised of business-oriented and engineering-oriented students. (DMM)

Operational improvements at traffic circles : safety analysis, final report, December 2008.

DOT National Transportation Integrated Search

2008-12-01

The purpose of this study was to improve the safety and operation at three traffic circles in New : Jersey. To do this, data were collected at the traffic circles to allow researchers to model the : circles using the PARAMICS software simulation pack...

Soil Biota and Litter Decay in High Arctic Ecosystems

NASA Astrophysics Data System (ADS)

González, G.; Rivera, F.; Makarova, O.; Gould, W. A.

2006-12-01

Frost heave action contributes to the formation of non-sorted circles in the High Arctic. Non-sorted circles tend to heave more than the surrounding tundra due to deeper thaw and the formation of ice lenses. Thus, the geomorphology, soils and vegetation on the centers of the patterned-ground feature (non-sorted circles) as compared to the surrounding soils (inter-circles) can be different. We established a decomposition experiment to look at in situ decay rates of the most dominant graminoid species on non-sorted circles and adjacent inter-circle soils along a climatic gradient in the Canadian High Arctic as a component of a larger study looking at the biocomplexity of small-featured patterned ground ecosystems. Additionally, we investigated variation in soil chemical properties and biota, including soil microarthropods and microbial composition and biomass, as they relate to climate, topographic position, and litter decay rates. Our three sites locations, from coldest to warmest, are Isachsen, Ellef Ringnes Island (ER), NU (bioclimatic subzone A); Mould Bay (MB), Prince Patrick Island, NT (bioclimatic subzone B), and Green Cabin (GC), Aulavik National Park, Thomsen River, Banks Island, NT (bioclimatic subzone C). Our sample design included the selection of 15 non-sorted circles and adjacent inter-circle areas within the zonal vegetation at each site (a total of 90 sites), and a second set of 3 non-sorted circles and adjacent inter-circle areas in dry, mesic and wet tundra at each of the sites. Soil invertebrates were sampled at each site using both pitfall traps, soil microbial biomass was determined using substrate induced respiration and bacterial populations were determined using the most probable number method. Decomposition rates were measured using litterbags and as the percent of mass remaining of Carex misandra, Luzula nivalis and Alopecuris alpinus in GC, MB and ER, respectively. Our findings indicate these graminoid species decayed significantly over time at a rate of 10-15 % mass loss / yr during the first year of decay. Decay rates are different in non-sorted circles vs. inter-circle soils along the climatic gradient. In MB, L. nivalis seems to decay faster in the inter-circle soils than in non-sorted circles (0.05

Quality/Performance Circles Three Years after Implementation.

ERIC Educational Resources Information Center

Ladwig, Dennis J.

An overview is provided of the development of quality/performance circles at Lakeshore Technical Institute (LTI), Wisconsin, and of the projects undertaken through the quality/performance circle program during its 3-year history. First, background information is provided on the use of quality circles in Japan and the United States, including…

Fostering Classroom Communities through Circling with Teacher Candidates

ERIC Educational Resources Information Center

Bouchard, Karen L.; Hollweck, Trista; Smith, J. David

2016-01-01

Classroom circles have been recognized as a valuable pedagogical approach to develop students' social-emotional learning and to establish a sense of community within a classroom. Until recently, there has been little consideration that teachers, themselves, may benefit from circling experiences. To garner a deeper understanding of circling for…

Making Morning Circle Meaningful

ERIC Educational Resources Information Center

Bruce, Susan; Fasy, Cara; Gulick, Jessica; Jones, Jill; Pike, Elizabeth

2006-01-01

Morning Circle, also known as Morning Meeting, is often a daily lesson in both general education and special education classrooms. The primary purpose of the Circle is to support each child to establish membership in the class while developing a classroom community and culture. The Responsive Classroom Approach recommends four Circle components:…

Transaction Circles with Digital Texts as a Foundation for Democratic Practices

ERIC Educational Resources Information Center

Brown, Sally

2015-01-01

Transaction circles weave together elements of guided reading and literature circles in an open conversational structure that supports students as agentive learners. Discourse within these circles utilizing digital informational texts assist in the development of democratic practices even in a time when federal mandates limit curricula and…

Perceptual Differences in Attitudes on Quality Circles.

ERIC Educational Resources Information Center

Holcomb, Lynn; Berger, Leonard

1986-01-01

A study was conducted to determine any perceptual differences toward quality circles in a chemical plant. It also tried to determine if any perceptual differences that might be found could be related to attitudes toward the circles themselves or the attitudes toward circle members. Length of service was also a factor. (CT)

Quality Circles: Determination of Significant Factors for Success an a General Model for Implementing a Quality Circle Process.

DTIC Science & Technology

1981-06-01

receive for contributions to quality and productivity /4:Apdx-47. The initiative for the Quality Circle concept came from Dr Kaoru Ishikawa , a...eloquently summarized by the "Father of Quality Circles", Dr Kaoru Ishikawa . He sees that, although Japan started with the worst quality reputation among...Perceptions of Influence, Academy of Management Journal. December 1974, pp 649-bz. 49. Ishikawa , Kaoru . S.C. Circle Activities. Union of Japanese Scientists and

GENERATING FRACTAL PATTERNS BY USING p-CIRCLE INVERSION

NASA Astrophysics Data System (ADS)

Ramírez, José L.; Rubiano, Gustavo N.; Zlobec, Borut Jurčič

2015-10-01

In this paper, we introduce the p-circle inversion which generalizes the classical inversion with respect to a circle (p = 2) and the taxicab inversion (p = 1). We study some basic properties and we also show the inversive images of some basic curves. We apply this new transformation to well-known fractals such as Sierpinski triangle, Koch curve, dragon curve, Fibonacci fractal, among others. Then we obtain new fractal patterns. Moreover, we generalize the method called circle inversion fractal be means of the p-circle inversion.

Native American Values and Management Education: Envisioning an Inclusive Virtuous Circle

ERIC Educational Resources Information Center

Verbos, Amy Klemm; Gladstone, Joe S.; Kennedy, Deanna M.

2011-01-01

Circles are symbols of interconnectedness. Behavioral circles can be vicious or virtuous. Many American Indians are caught in a vicious circle of exclusion from the purported benefits of Westernization, entrapment in its negative elements, and the ongoing undermining of their culture and thus their identities. Yet Native Americans, along with many…

The Value of the Math Circle for Gifted Middle School Students

ERIC Educational Resources Information Center

Burns, Barbara; Henry, Julie; McCarthy, Dianne; Tripp, Jennifer

2017-01-01

Math Circles are designed to allow students to explore mathematics using a problem-solving/inquiry approach. Many of the students attending our Math Circle are mathematically talented and curious. This study examines the perspectives of the students and their families in determining why students attend Math Circle, what they enjoy about Math…

Expanding the Reach of Extension to Underserved Audiences through Study Circles in Rural Idaho

ERIC Educational Resources Information Center

Cummins, Melissa; Petty, Barbara; Hansen, Lyle; Hoffman, Katie; Wittman, Grace

2012-01-01

Extension educators expanded the reach of their programming to underserved audiences through the implementation of Study Circles in rural Southern Idaho. Study Circles gave educators entry into communities by establishing relationships necessary for long-term change. Study Circle discussions in rural Southern Idaho led to stronger relationships…

The Acoustic Reality of the Kachruvian Circles: A Rhythmic Perspective

ERIC Educational Resources Information Center

Low, Ee Ling

2010-01-01

This paper investigates whether the rhythmic properties of varieties of English found in each of the concentric circles of Kachru's model can, in any way, be elucidated by the "Three Circles" model. A measurement and comparison of the rhythm of three varieties of English: British English (from the Inner Circle), Singapore English (from…

The Relationship of Communication to Productivity: Quality Circles as a Mediating Variable.

ERIC Educational Resources Information Center

Creagh, Sara; Smeltzer, Larry

Quality circles, small groups of employees working voluntarily toward performance improvement, have become a popular business strategy in the past decade. When effective, the quality circle may be linked directly to the increased productivity of the work group. The quality circle process may be divided into four components: identification and…

Quality Circles: Involvement, Problem-Solving, and Recognition.

ERIC Educational Resources Information Center

Moretz, H. Lynn

1983-01-01

The media production department of Central Piedmont Community College (CPCC) began quality circle meetings in January 1981 after studying the process of quality circles and obtaining the understanding and support of the college administration. A quality circle is a small group of people doing similar work who voluntarily meet on a regular basis to…

The "Us" in Discuss: Grouping in Literature Circles

ERIC Educational Resources Information Center

Batchelor, Katherine

2012-01-01

This article describes one middle school teacher's use of literature circles using heterogeneous grouping. It begins with a brief rationale for using literature circles in the language arts classroom. Next, it describes techniques to form literature circles. Then, it shares how to build and establish a supportive environment within each group. It…

A Round Is a Circle...

ERIC Educational Resources Information Center

Boyarsky, Terry L.

2006-01-01

Circles are everywhere and endlessly intriguing. We know, sense, and feel them in time, space, sound, and the cycles of nature. A person's life contains spirals, repetitions, going out and coming back. Poets write of circles; composers write song cycles. Circles are at the root of the curriculum, concepts deep yet accessible, infinite in their…

Accuracy of tree diameter estimation from terrestrial laser scanning by circle-fitting methods

NASA Astrophysics Data System (ADS)

Koreň, Milan; Mokroš, Martin; Bucha, Tomáš

2017-12-01

This study compares the accuracies of diameter at breast height (DBH) estimations by three initial (minimum bounding box, centroid, and maximum distance) and two refining (Monte Carlo and optimal circle) circle-fitting methods The circle-fitting algorithms were evaluated in multi-scan mode and a simulated single-scan mode on 157 European beech trees (Fagus sylvatica L.). DBH measured by a calliper was used as reference data. Most of the studied circle-fitting algorithms significantly underestimated the mean DBH in both scanning modes. Only the Monte Carlo method in the single-scan mode significantly overestimated the mean DBH. The centroid method proved to be the least suitable and showed significantly different results from the other circle-fitting methods in both scanning modes. In multi-scan mode, the accuracy of the minimum bounding box method was not significantly different from the accuracies of the refining methods The accuracy of the maximum distance method was significantly different from the accuracies of the refining methods in both scanning modes. The accuracy of the Monte Carlo method was significantly different from the accuracy of the optimal circle method in only single-scan mode. The optimal circle method proved to be the most accurate circle-fitting method for DBH estimation from point clouds in both scanning modes.

COMPLETENESS OF CIRCLE OF WILLIS IN ASYMPTOMATIC AND SYMPTOMATIC EXTRACRANIAL CAROTID DISEASE.

PubMed

Manojlovic, Vladimir; Popovic, Vlandan; Nikoloc, Dragan; Milosevic, Dorde; Pasternak, Janko; Budakov, Nebojsa

2016-11-01

This research has been aimed at determining whether incomplete Circle of Willis in patients with significant extracranial carotid stenosis is associated with a higher incidence of neurological symptomatology and/or ischemnic cerebral lesions. The research was conducted as a prospective study which comprised 211 patients who underwent surgical treatment of extracranial carotid disease at the Department of Vascular Surgery in Novi Sad and 102 patients in the control group. Each patient underwent preoperative magnetic resonance imaging and magnetic resonance angiography with visualization of cerebral parenchyma. extracranial and intracranial cerebral circulation. Assessment of Circle of Willis morphology was performed by 3D time-of-fight magnetic resonance angiogram sequence analysis. The patients were divided into two groups: group I - the patients with'complete Circle of Willis and group II - the patients with incomplete Circle of Willis i.e. with the disruption of anterior and/ or ipsilateral posterior circulation - regarding the side of signif icant carotid stenosis. Out of 211 patients who -were operated during a two-year period, 133 had the complete Circle of Willis. while 78 patients had the incomplete Circle of Willis. Out of 111 patients with symptomatic carotid disease or silent cerebral infarction, 52.5% (58) had the complete Circle of' Willis and 47.5% (53) had the incomplete Circle of Willis. It was shown to be statistically different (P = 0.0146) in relation with the asymptomatic group of patients (100), where the frequency of the complete Circle of Willis was 75% (75) while the insufficiency of anterior or ipsilateral posterior collateral ization was found in 25% (25). In the control group there were significantly fewer cases of developed collateral flow and the complete Circle of Willis (41%) compared to the operated patients with extracranial carotid stenosis (63%) (P= 0.0003). Incompleteness of Circle of Willis is associated with more frequent occurrence of neurological symptomatology or ischemic lesions of brain parenchyma in operated patients with significant extracranial carotid stenosis. The control group without extracranial carotid stenosis had less developed collaterals of Circle of Willis compared to extracranial carotid patients.

Correlation Between the Integrity of the Circle of Willis and the Severity of Initial Noncardiac Cerebral Infarction and Clinical Prognosis

PubMed Central

Zhou, Houshi; Sun, Jian; Ji, Xiaotan; Lin, Jing; Tang, Shujin; Zeng, Jinsheng; Fan, Yu-hua

2016-01-01

Abstract The quality of collateral circulation affects the severity and prognosis of stroke patients. The effect of the circle of Willis, which is the primary collateral circulation, on ischemic stroke has attracted significant attention. This study was designed to investigate the effect of different circles of Willis types on stroke severity and prognosis in patients with noncardiac stroke. A total of 376 patients with noncardiac ischemic stroke, who were treated by the specialty team of cerebrovascular diseases at the First Affiliated Hospital of Sun Yat-sen Hospital, were successively enrolled in this study. The detailed clinical characteristics of the patients were recorded upon admission, including risk factors of vascular disease and National Institutes of Health Stroke Scale (NIHSS) scores. The patients were divided into groups of different circles of Willis types based on magnetic resonance angiography (MRA) that was performed within 3 days of admission—type I: complete circle of Willis; type II: complete anterior half of the circle of Willis and incomplete posterior half of the circle of Willis; type III: incomplete anterior half of the circle of Willis and complete posterior half of the circle of Willis; and type IV: incomplete anterior and posterior halves of the circle of Willis. Patients were re-evaluated for NIHSS scores at discharge and after discharge. The modified Rankin score (mRS) was recorded for 90 days, and stroke recurrence and death after 90 days were also recorded until the end of the study. The 376 patients were divided into 4 groups based on the MRA—type I group: 92 patients (24.5%); type II group: 215 patients (57.2%); type III group: 12 patients (3.2%), and type IV group: 57 patients (15.2%). NIHSS scores at admission and discharge were significantly lower for the type I group compared with those for the type II and type IV groups (P < 0.05). NIHSS scores were higher in the groups with an incomplete circle of Willis compared with the group with a complete circle of Willis. A poor recovery rate was highest for the type IV group, whereas a good recovery rate was highest for the type I group. The logistic regression analysis showed that a complete circle of Willis was one of the predictors of suitable recovery (odds ratio [OR] = 0.708, 95% confidence interval [CI]: 0.554–0.906). Circle of Willis type was associated with stroke severity and patient prognosis, whereas an incomplete circle of Willis was associated with more severe conditions and a higher 90-day poor diagnosis rate. A complete circle of Willis was an independent predictor of good prognosis. PMID:26962785

Analytical approximations to the dynamics of an array of coupled DC SQUIDs

NASA Astrophysics Data System (ADS)

Berggren, Susan; Palacios, Antonio

2014-04-01

Coupled dynamical systems that operate near the onset of a bifurcation can lead, under certain conditions, to strong signal amplification effects. Over the past years we have studied this generic feature on a wide range of systems, including: magnetic and electric fields sensors, gyroscopic devices, and arrays of loops of superconducting quantum interference devices, also known as SQUIDs. In this work, we consider an array of SQUID loops connected in series as a case study to derive asymptotic analytical approximations to the exact solutions through perturbation analysis. Two approaches are considered. First, a straightforward expansion in which the non-linear parameter related to the inductance of the DC SQUID is treated as the small perturbation parameter. Second, a more accurate procedure that considers the SQUID phase dynamics as non-uniform motion on a circle. This second procedure is readily extended to the series array and it could serve as a mathematical framework to find approximate solutions to related complex systems with high-dimensionality. To the best of our knowledge, an approximate analytical solutions to an array of SQUIDs has not been reported yet in the literature.

What Works: Study Circles in the Real World. Best Practices for Producing Community-Wide Study Circles.

ERIC Educational Resources Information Center

Mengual, Gloria

This document, which is based on information gathered during a 1998 study of how study circle programs contribute to community problem solving, presents best practices for producing community-wide study circles. The best practices are illustrated through stories that are grouped into five sections on the following themes: (1) creating a…

"Quality Circles": A Strategy for Personal and Curriculum Development. Coombe Lodge Working Paper. Information Bank Number 1803.

ERIC Educational Resources Information Center

Field, M. J.; Harrison, A. B.

Quality circles attempt to satisfy both task and personal needs through staff involvement in solving work-related problems. This paper summarizes quality circle theory, applies it to school settings, and suggests a framework for introducing the process to educational institutions. After briefly defining quality circles, the article presents two…

Mismanagement and Quality Circles: How Middle Managers Influence Direct Participation.

ERIC Educational Resources Information Center

Brennan, Maire

1991-01-01

Case studies of five Scottish companies found that four of their quality circles programs had ceased. Essential to the success of quality circles were changes in the systems of reward, communication, and decision making and the cooperation and support of middle managers, who may see quality circles as a threat and who control the resources they…

Expanding-Circle Students Learning "Standard English" in the Outer-Circle Asia

ERIC Educational Resources Information Center

Kobayashi, Yoko

2011-01-01

Drawing upon Kachru's concentric circles of English, the present study explores whether middle-class Japanese students who chose to study English solo at private language schools in Singapore diverge from many others who (wish to) study inner-circle English. The study is stimulated by the repeated interdisciplinary findings that, in spite of the…

Circles of Support and Personalization: Exploring the Economic Case

ERIC Educational Resources Information Center

Wistow, Gerald; Perkins, Margaret; Knapp, Martin; Bauer, Annette; Bonin, Eva-Maria

2016-01-01

Circles of Support aim to enable people with learning disabilities (and others) to live full lives as part of their communities. As part of a wider study of the economic case for community capacity building conducted from 2012 to 2014, we conducted a mixed methods study of five Circles in North West England. Members of these Circles were…

Children with Autism and Peer Group Support: "Using Circles of Friends".

ERIC Educational Resources Information Center

Whitaker, Philip; Barratt, Penny; Joy, Helen; Potter, Mo; Thomas, George

1998-01-01

Explains the "circle of friends approach" as one strategy aimed at using peer group support to promote the inclusion of children with autism into mainstream schools. It describes use of the approach with six autistic children in years 3 to 10 of British mainstream schools. Evaluation comments by the circle leaders, circle members, and…

The Chicken and the Egg: Inviting Response and Talk through Socratic Circles

ERIC Educational Resources Information Center

Styslinger, Mary E.; Pollock, Timothy

2010-01-01

This collaborative inquiry answers the following questions: 1) What is the nature of talk during Socratic Circles? 2) What is student response to talk? 3) How might knowing more about student response to talk and the nature of talk improve teaching during Socratic Circles? The article first describes the process of implementing Socratic Circles,…

Getting Started with Literature Circles. The Bill Harp Professional Teachers Library Series.

ERIC Educational Resources Information Center

Noe, Katherine L. Schlick; Johnson, Nancy J.

Designed to help teachers get started using literature circles in their classrooms, this book gives teachers a boost to begin, offers some insights from other teachers, and helps teachers clarify where to go next. It notes that literature circles (or literature study groups, book clubs, or discussion circles) take many forms and engage students in…

Guided Discovery of the Nine-Point Circle Theorem and Its Proof

ERIC Educational Resources Information Center

Buchbinder, Orly

2018-01-01

The nine-point circle theorem is one of the most beautiful and surprising theorems in Euclidean geometry. It establishes an existence of a circle passing through nine points, all of which are related to a single triangle. This paper describes a set of instructional activities that can help students discover the nine-point circle theorem through…

Ecohydrological interactions within "fairy circles" in the Namib Desert: Revisiting the self-organization hypothesis

NASA Astrophysics Data System (ADS)

Ravi, Sujith; Wang, Lixin; Kaseke, Kudzai Farai; Buynevich, Ilya V.; Marais, Eugene

2017-02-01

Vegetation patterns such as rings, bands, and spots are recurrent characteristics of resource-limited arid and semiarid ecosystems. One of the most recognizable vegetation patterns is the millions of circular patches, often referred to as "fairy circles," within the arid grassland matrix extending over hundreds of kilometers in the Namib Desert. Several modeling studies have highlighted the role of plant-soil interactions in the formation of these fairy circles. However, little is known about the spatial and temporal variabilities of hydrological processes inside a fairy circle. In particular, a detailed field assessment of hydrological and soil properties inside and outside the fairy circles is limited. We conducted extensive measurements of infiltration rate, soil moisture, grass biometric, and sediment grain-size distribution from multiple circles and interspaces in the Namib Desert. Our results indicate that considerable heterogeneity in hydrological processes exists within the fairy circles, resulting from the presence of coarser particles in the inner bare soil areas, whereas concentration of fine soil occurs on the vegetated edges. The trapping of aeolian and water-borne sediments by plants may result in the observed soil textural changes beneath the vegetation, which in turn, explains the heterogeneity in hydrological processes such as infiltration and runoff. Our investigation provides new insights and experimental data on the ecohydrological processes associated with fairy circles, from a less studied location devoid of sand termite activity within the circles. The results seem to provide support to the "self-organization hypothesis" of fairy circle formation attributed to the antiphase spatial biomass-water distributions.

A Spring Playscape Project: Building a Tree Circle

ERIC Educational Resources Information Center

Keeler, Rusty

2009-01-01

The Tree Circle is a green gathering area for children made by planting trees in a circle. For children, the Tree Circle becomes a magical place for dramatic play, quiet retreat, or lively nature exploration. For teachers and parents it becomes a shady grove for snacks and stories. The trees create a sweet spot that changes during the seasons and…

National Forest System working circles: a question of size and ownership composition

Treesearch

Robert J. Hrubes

1976-01-01

Allowable-cut (potential yield) levels on National Forest land are determined for planning units called working circles. The size of working circles has been increased over the past 30 years to the present scale which is often coincident with National Forest boundaries. Larger working circles have recently been considered because of the anticipated impacts on timber...

The combination of circle topology and leaky integrator neurons remarkably improves the performance of echo state network on time series prediction.

PubMed

Xue, Fangzheng; Li, Qian; Li, Xiumin

2017-01-01

Recently, echo state network (ESN) has attracted a great deal of attention due to its high accuracy and efficient learning performance. Compared with the traditional random structure and classical sigmoid units, simple circle topology and leaky integrator neurons have more advantages on reservoir computing of ESN. In this paper, we propose a new model of ESN with both circle reservoir structure and leaky integrator units. By comparing the prediction capability on Mackey-Glass chaotic time series of four ESN models: classical ESN, circle ESN, traditional leaky integrator ESN, circle leaky integrator ESN, we find that our circle leaky integrator ESN shows significantly better performance than other ESNs with roughly 2 orders of magnitude reduction of the predictive error. Moreover, this model has stronger ability to approximate nonlinear dynamics and resist noise than conventional ESN and ESN with only simple circle structure or leaky integrator neurons. Our results show that the combination of circle topology and leaky integrator neurons can remarkably increase dynamical diversity and meanwhile decrease the correlation of reservoir states, which contribute to the significant improvement of computational performance of Echo state network on time series prediction.

Fabrication and characteristics of thin disc piezoelectric transformers based on piezoelectric buzzers with gap circles.

PubMed

Chang, Kuo-Tsai; Lee, Chun-Wei

2008-04-01

This paper investigates design, fabrication and test of thin disc piezoelectric transformers (PTs) based on piezoelectric buzzers with gap circles at different diameters of the gap circles. The performance test is focused on characteristics of voltage gains, including maximum voltage gains and maximum-gain frequencies, for each piezoelectric transformer under different load conditions. Both a piezoelectric buzzer and a gap circle on a silver electrode of the buzzer are needed to build any type of the PTs. Here, the gap circle is used to form a ring-shaped input electrode and a circle-shaped output electrode for each piezoelectric transformer. To do so, both structure and connection of a PT are first expressed. Then, operating principle of a PT and its related vibration mode observed by a carbon-power imaging technique are described. Moreover, an experimental setup for characterizing each piezoelectric transformer is constructed. Finally, effects of diameters of the gap circles on characteristics of voltage gains at different load resistances are discussed.

A programmable systolic array correlator as a trigger processor for electron pairs in rich (ring image Cherenkov) counters

NASA Astrophysics Data System (ADS)

Männer, R.

1989-12-01

This paper describes a systolic array processor for a ring image Cherenkov counter which is capable of identifying pairs of electron circles with a known radius and a certain minimum distance within 15 μs. The processor is a very flexible and fast device. It consists of 128 x 128 processing elements (PEs), where one PE is assigned to each pixel of the image. All PEs run synchronously at 40 MHz. The identification of electron circles is done by correlating the detector image with the proper circle circumference. Circle centers are found by peak detection in the correlation result. A second correlation with a circle disc allows circles of closed electron pairs to be rejected. The trigger decision is generated if a pseudo adder detects at least two remaining circles. The device is controlled by a freely programmable sequencer. A VLSI chip containing 8 x 8 PEs is being developed using a VENUS design system and will be produced in 2μ CMOS technology.

Circles South East: the first 10 years 2002-2012.

PubMed

Bates, Andrew; Williams, Dominic; Wilson, Chris; Wilson, Robin J

2014-07-01

This article describes the first 10 years of the implementation of Circles of Support and Accountability (Circles) in the management of sexual offenders in South-East England by Circles South East (CSE). The Circles of 71 core members are reviewed in detail, with reference to demographic data, offense and sentencing histories, risk assessment data, and considerations regarding Multi-Agency Public Protection Arrangements. A group of 71 comparison subjects who were referred to CSE and deemed suitable for but did not receive the service was identified. Follow-up behaviors of both groups are examined (including all forms of reconviction, breach of orders, and prison recall). Over a comparable follow-up period of 55 months, the incidence of violent and contact sexual reconviction in the comparison group was significantly higher than for the Circles cohort. Comparisons are made between expected and actual levels of sexual reconviction, with the Circles cohort showing lower than expected rate of sexual reconviction but not to a statistically significant degree. © The Author(s) 2013.

Wear simulation of apex seal in rotary engine under mixed lubrication

NASA Astrophysics Data System (ADS)

Jiang, Hanying; Zuo, Zhengxing; Liu, Jinxiang

2018-05-01

In this work, the wear of apex seal's running face under mixed lubrication is studied. Numerical simulation is carried out by employing the couple model of Reynolds equation, Greenwood and Tripp model and Archard's wear law. The simulation is performed both for one circle and multi circle. In the multi circle simulation, the change of contact position due to wear is considered. A method that is able to find the new contact position based on the updated apex seal's contour profile is proposed, validated and used. The result of multi circle simulation indicates that contact position changes obviously around the maximum swing angles both on leading and trailing sides with the increase number of circles. The wear depth distribution becomes more uniform with the increase of operation circle number.

Algebraic criteria for positive realness relative to the unit circle.

NASA Technical Reports Server (NTRS)

Siljak, D. D.

1973-01-01

A definition is presented of the circle positive realness of real rational functions relative to the unit circle in the complex variable plane. The problem of testing this kind of positive reality is reduced to the algebraic problem of determining the distribution of zeros of a real polynomial with respect to and on the unit circle. Such reformulation of the problem avoids the search for explicit information about imaginary poles of rational functions. The stated algebraic problem is solved by applying the polynomial criteria of Marden (1966) and Jury (1964), and a completely recursive algorithm for circle positive realness is obtained.

Influence of off-great-circle propagation of Rayleigh waves on event-based surface wave tomography in Northeast China

NASA Astrophysics Data System (ADS)

Chen, Haopeng; Ni, Sidao; Chu, Risheng; Chong, Jiajun; Liu, Zhikun; Zhu, Liangbao

2018-05-01

Surface waves are generally assumed to propagate along great-circle paths in most surface-wave tomography. However, when lateral heterogeneity is strong, off-great-circle propagation may occur and deteriorate surface wave tomography results based on the great-circle assumption. In this study, we used teleseismic waveforms recorded by the NECESSArray in Northeast China to study off-great-circle propagation of Rayleigh waves using the beamforming method and evaluated the influence of off-great-circle propagation on event-based surface wave tomography. The results show that arrival angle anomalies generally increase with decreasing period. The arrival angle anomalies at 60 and 50 s periods are smaller than that at 40 and 30 s periods, which indicates that the off-great-circle propagation is relatively weak for longer periods. At 30 s period, the arrival angle anomalies are relatively larger and some of the measurements can exceed 20°, which represents a strong off-great-circle propagation effect. In some areas, the arrival angle anomalies of adjacent events differ significantly, which may be attributed to multipathing propagation of surface waves. To evaluate the influence of off-great-circle propagation on event-based surface wave tomography, we used measured arrival angle anomalies to correct two-station phase velocity measurements, and performed azimuthal anisotropy tomography using dispersion datasets with and without the arrival angle correction. At longer periods, such as 60 s, the influence of off-great-circle propagation on surface wave tomography is weak even though the corrected model has better data fit than the uncorrected model. However, the influence of off-great-circle propagation is non-negligible at short periods. The tomography results at 30 s period show that the differences in phase velocity, the strength of anisotropy and the fast direction can be as large as 1.5 per cent, 1.0 per cent and 30°, respectively. Furthermore, the corrected phase velocity is systematically lower than that without correction. This study illustrates the necessity of studying the off-great-circle propagation of surface waves to improve the accuracy of event-based surface wave tomography, especially for shorter periods.

Continuous-flow, microfluidic, qRT-PCR system for RNA virus detection.

PubMed

Fernández-Carballo, B Leticia; McBeth, Christine; McGuiness, Ian; Kalashnikov, Maxim; Baum, Christoph; Borrós, Salvador; Sharon, Andre; Sauer-Budge, Alexis F

2018-01-01

One of the main challenges in the diagnosis of infectious diseases is the need for rapid and accurate detection of the causative pathogen in any setting. Rapid diagnosis is key to avoiding the spread of the disease, to allow proper clinical decisions to be made in terms of patient treatment, and to mitigate the rise of drug-resistant pathogens. In the last decade, significant interest has been devoted to the development of point-of-care reverse transcription polymerase chain reaction (PCR) platforms for the detection of RNA-based viral pathogens. We present the development of a microfluidic, real-time, fluorescence-based, continuous-flow reverse transcription PCR system. The system incorporates a disposable microfluidic chip designed to be produced industrially with cost-effective roll-to-roll embossing methods. The chip has a long microfluidic channel that directs the PCR solution through areas heated to different temperatures. The solution first travels through a reverse transcription zone where RNA is converted to complementary DNA, which is later amplified and detected in real time as it travels through the thermal cycling area. As a proof of concept, the system was tested for Ebola virus detection. Two different master mixes were tested, and the limit of detection of the system was determined, as was the maximum speed at which amplification occurred. Our results and the versatility of our system suggest its promise for the detection of other RNA-based viruses such as Zika virus or chikungunya virus, which constitute global health threats worldwide. Graphical abstract Photograph of the RT-PCR thermoplastic chip.

Quality Control in construction.

DTIC Science & Technology

1984-01-01

behavioral scientists. In 1962, Dr. Kaoru Ishikawa gave shape to the form of training which featured intradepartmental groups of ten or so workers seated...and Japanese circles bears closer scrutiny. 4.3.1 Japanese Ingredients of Quality The founder of quality circles, Dr. Kaoru Ishikawa , gives six...around 51 a table; hence the name Quality Control Circle. 4 Dr. 0 Ishikawa was an engineering professor at Tokyo University, and the circles were

Circle of influence

NASA Astrophysics Data System (ADS)

Robinson, Andrew

2018-04-01

The founder of the Vienna Circle – a polymathic and influential group of intellectuals dedicated to the philosophy of science from the late 1920s until the Nazi takeover of Austria in 1938 – was German philosopher and physicist Moritz Schlick. Karl Sigmund's latest book – Exact Thinking in Demented Times: the Vienna Circle and the Epic Quest for the Foundations of Science – tells the story of the Vienna Circle's ideas and personalities.

Calculation of Latitude and Longitude for Points on Perimeter of a Circle on a Sphere

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

Morris, Heidi E.

2015-08-14

This document describes the calculation of the Earth-Centered Earth Fixed (ECEF) coordinates for points lying on the perimeter of a circle. Here, the perimeter of the circle lies on the surface of the sphere and the center of the planar circle is below the surface. These coordinates are converted to latitude and longitude for mapping fields on the surface of the earth.

Empirical Evaluation of Different Feature Representations for Social Circles Detection

DTIC Science & Technology

2015-06-16

study and compare the performance on the available labelled Facebook data from the Kaggle competition on learning social circles in networks . We...Kaggle competition on learning social circles in networks [5]. The data consist of hand- labelled friendship egonets from Facebook and a set of 57...16. SECURITY CLASSIFICATION OF: Social circles detection is a special case of community detection in social network that is currently attracting a

Practical Environmental Education and Local Contribution in the Environmental Science Laboratory Circle in the College of Science and Technology in Nihon University

NASA Astrophysics Data System (ADS)

Taniai, Tetsuyuki; Ito, Ken-Ichi; Sakamaki, Hiroshi

In this paper, we presented a method and knowledge about a practical and project management education and local contribution obtained through the student activities of “Environmental science laboratory circle in the College of Science and technology in Nihon University” from 1991 to 2001. In this circle, four major projects were acted such as research, protection, clean up and enlightenment projects. Due to some problems from inside or outside of this circle, this circle projects have been stopped. The diffusion and popularization of the internet technology will help to resolve some of these problems.

Numerical Grid Generation. Proceedings of a Symposium on the Numerical Generation of Curvilinear Coordinate Systems and their Use in the Numerical Solution of Partial Differential Equations, held April 1982, Nashville, Tennessee

DTIC Science & Technology

1982-04-01

the transformation 2 1 V I " |€ , (22) 121 I which transforms a unit circle in the Z plane to a unit upper half ...circle (and to the real axis from -1. to 1.) in the 4 plane as illustrated in Fig. 6. I Zpbns rpIUII A A , FIGURE 6. , CIRCLE TO HALF CIRCLE MAPPING The ...an airfoil, or inlet, etc. ... ) to a rectangle (or equivalently to a circle or half plane ). The cumputing time required for this step is

Component analysis and synthesis of dark circles under the eyes using a spectral image

NASA Astrophysics Data System (ADS)

Akaho, Rina; Hirose, Misa; Ojima, Nobutoshi; Igarashi, Takanori; Tsumura, Norimichi

2017-02-01

This paper proposes to apply nonlinear estimation of chromophore concentrations: melanin, oxy-hemoglobin, deoxyhemoglobin and shading to the real hyperspectral image of skin. Skin reflectance is captured in the wavelengths between 400nm and 700nm by hyperspectral scanner. Five-band wavelengths data are selected from skin reflectance. By using the cubic function which obtained by Monte Carlo simulation of light transport in multi-layered tissue, chromophore concentrations and shading are determined by minimize residual sum of squares of reflectance. When dark circles are appeared under the eyes, the subject looks tired and older. Therefore, woman apply cosmetic cares to remove dark circles. It is not clear about the relationship between color and chromophores distribution in the dark circles. Here, we applied the separation method of the skin four components to hyperspectral image of dark circle, and the separated components are modulated and synthesized. The synthesized images are evaluated to know which components are contributed into the appearance of dark circles. Result of the evaluation shows that the cause of dark circles for the one subject was mainly melanin pigmentation.

Nonlinear Dynamics of Non-uniform Current-Vortex Sheets in Magnetohydrodynamic Flows

NASA Astrophysics Data System (ADS)

Matsuoka, C.; Nishihara, K.; Sano, T.

2017-04-01

A theoretical model is proposed to describe fully nonlinear dynamics of interfaces in two-dimensional MHD flows based on an idea of non-uniform current-vortex sheet. Application of vortex sheet model to MHD flows has a crucial difficulty because of non-conservative nature of magnetic tension. However, it is shown that when a magnetic field is initially parallel to an interface, the concept of vortex sheet can be extended to MHD flows (current-vortex sheet). Two-dimensional MHD flows are then described only by a one-dimensional Lagrange parameter on the sheet. It is also shown that bulk magnetic field and velocity can be calculated from their values on the sheet. The model is tested by MHD Richtmyer-Meshkov instability with sinusoidal vortex sheet strength. Two-dimensional ideal MHD simulations show that the nonlinear dynamics of a shocked interface with density stratification agrees fairly well with that for its corresponding potential flow. Numerical solutions of the model reproduce properly the results of the ideal MHD simulations, such as the roll-up of spike, exponential growth of magnetic field, and its saturation and oscillation. Nonlinear evolution of the interface is found to be determined by the Alfvén and Atwood numbers. Some of their dependence on the sheet dynamics and magnetic field amplification are discussed. It is shown by the model that the magnetic field amplification occurs locally associated with the nonlinear dynamics of the current-vortex sheet. We expect that our model can be applicable to a wide variety of MHD shear flows.

Cross-directional interlocking of rolls in an air press of a papermaking machine

DOEpatents

Beck, David A.; Gorshe, Thomas

2003-05-13

An air press for pressing a paper web is composed of a plurality of rolls including at least a first roll and a second roll. The first roll and the second roll are positioned adjacent one another and form a first nip therebetween. Further, the first roll and the second roll each have a roll end, the roll end of the first roll adjoining the roll end of the second roll. A bevel plate is attached to the roll end of the first roll, the bevel plate having at least a first angled plate face. A seal ring is positioned adjacent the roll end of the second roll, the seal ring being juxtaposed to the bevel plate. The seal ring has at least a first angled ring face, and the first angled ring face mates with the first angled plate face.

Traffic Circle Model

DOT National Transportation Integrated Search

1971-05-01

The report describes a dynamic model of a traffic circle which has been implemented on a CRT display terminal. The model includes sufficient parameters to allow changes in the structure of the traffic circle, the frequency of traffic introduced to th...

Can the Expanding Circle Own English? Comments on Yoo's "Nonnative Teachers in the Expanding Circle and the Ownership of English"

ERIC Educational Resources Information Center

Ren, Wei

2014-01-01

Yoo's (2014) article raises a number of questions concerning local teachers' status and the ownership of English in the Expanding Circle. In this article, I address five issues that I see as most important relating to the ownership of English and empowering local teachers in the Expanding Circle. I provide up-to-date evidence of World…

Circles of Support and Accountability for Sex Offenders: A Systematic Review of Outcomes.

PubMed

Clarke, Martin; Brown, Susan; Völlm, Birgit

2017-08-01

We conducted a systematic review of studies reporting on the effectiveness of Circles of Support and Accountability (Circles). Circles use volunteers to provide support for sex offenders living in the community. We searched 10 databases up to the end of 2013 and identified 3 relevant outcome studies. An additional 12 papers or reports were identified by searching reference lists, Google, and contacting key authors and Circles providers to obtain unpublished data. These 15 studies comprised one randomized controlled trial, three retrospective cohorts with matched controls, and 11 case series. The majority reported measures of recidivism, particularly reconviction. The 4 studies with controls generally reported that participation in Circles was associated with lower recidivism although there were few statistically significant differences. Few studies examined changes in risk or psychosocial outcomes. A number of methodological issues are discussed. Longer term, prospective follow-up studies with control groups are required to address these issues.

Fine-scale Patterns in Thaw Depth, Micro-relief, and Ground Cover on Non-sorted Circles and Small Patterned Ground Features Along a Climatic Gradient From Low to High Arctic

NASA Astrophysics Data System (ADS)

Okie, J.; Gould, W. A.; González, G.

2006-12-01

Patterned ground is a ubiquitous feature in the Arctic and the related variation in microtopographic relief strongly affects biotic and abiotic patterns and processes. Patterned ground features are polygenic in origin and are often found superimposed in a complex pattern of multiple features. We investigated the relationship between thaw depth, micro-relief, the cover of vascular, bryophyte, cryptogamic crust and bare ground along transects traversing non-sorted circles and small non-sorted polygons at 8 research sites along a climatic gradient in bioclimatic subzones A-E in the North American Arctic. Non-sorted circles are the result of differential frost heave with circle centers typically showing greater heave during freezing than inter circle areas. Differential heave is a function of climate, soil texture, soil moisture, and vegetation cover. Differential heave and subsidence creates fine-scale gradients in microtopography that affect soil moisture, exposure to winds, and development of vegetation and soils. Non-sorted circles typically range from 20 to 200 cm in diameter and are most common in subzones C-E. Often superimposed on these features are the development of small non-sorted polygons 10-30 cm in diameter, and fine-scale desiccation cracking at a scale of less than 10 cm. These are most common in subzones A-C. We established three 20 m transects in zonal vegetation at each site. Thaw depth, micro-relief, and ground cover were measured at 10 cm intervals along each transect. Additionally, we measured vascular plant beta diversity in a set of 25 x 25 cm quadrates on 15 circles and 15 inter circles at each site. The resulting pattern of thaw depth and micro-relief is correlated with both summer temperatures and vegetation cover. The variability and degree of micro-relief decrease from the Low to the High Arctic. Non-sorted circle centers had deeper active layer than inter circle areas along the gradient. Thaw depths increase linearly with the degree of bare ground and nonlinearly with summer warmth. This unimodal pattern of shallower active layer at the warmest and coldest sites is due to the interaction of climate and the insulating vegetation layer. Greatest thaw depths are found on bare non-sorted circles in subzone C. Beta diversity is greatest in subzone D, where vegetated inter circle areas differ markedly from more barren non- sorted circles.

Controlled-force end seal arrangement for an air press of a papermaking machine

DOEpatents

Beck, David A.

2003-07-08

An air press for pressing a fiber web includes a plurality of rolls and a pair of end seal arrangements. Of the plurality of rolls, each pair of adjacent rolls forms a nip therebetween. Further, each roll has a pair of roll ends, the plurality of rolls together forming two sets of roll ends. Each end seal arrangement coacts with one set of roll ends, the plurality of rolls and the pair of end seal arrangements together defining an air press chamber having an air chamber pressure. Each end seal arrangement is composed of at least one roll seal, including a first roll seal, and an adjustable bias mechanism. Each roll seal forms a seal with at least one roll end, and one side of the first roll seal being exposed to the air chamber pressure. The adjustable bias mechanism is configured for controlling a position of each roll seal relative to a respective at least one roll end and for adjusting a seal force between the roll seal and the respective at least one roll end.

DNA Packaging Specificity of Bacteriophage N15 with an Excursion into the Genetics of a Cohesive End Mismatch

PubMed Central

Feiss, Michael; Young Min, Jea; Sultana, Sawsan; Patel, Priyal; Sippy, Jean

2015-01-01

During DNA replication by the λ-like bacteriophages, immature concatemeric DNA is produced by rolling circle replication. The concatemers are processed into mature chromosomes with cohesive ends, and packaged into prohead shells, during virion assembly. Cohesive ends are generated by the viral enzyme terminase, which introduces staggered nicks at cos, an approx. 200 bp-long sequence containing subsites cosQ, cosN and cosB. Interactions of cos subsites of immature concatemeric DNA with terminase orchestrate DNA processing and packaging. To initiate DNA packaging, terminase interacts with cosB and nicks cosN. The cohesive ends of N15 DNA differ from those of λ at 2/12 positions. Genetic experiments show that phages with chromosomes containing mismatched cohesive ends are functional. In at least some infections, the cohesive end mismatch persists through cyclization and replication, so that progeny phages of both allelic types are produced in the infected cell. N15 possesses an asymmetric packaging specificity: N15 DNA is not packaged by phages λ or 21, but surprisingly, N15-specific terminase packages λ DNA. Implications for genetic interactions among λ-like bacteriophages are discussed. PMID:26633301

Plasmid replication initiator RepB forms a hexamer reminiscent of ring helicases and has mobile nuclease domains

PubMed Central

Boer, D Roeland; Ruíz-Masó, José A; López-Blanco, José R; Blanco, Alexander G; Vives-Llàcer, Mireia; Chacón, Pablo; Usón, Isabel; Gomis-Rüth, F Xavier; Espinosa, Manuel; Llorca, Oscar; del Solar, Gloria; Coll, Miquel

2009-01-01

RepB initiates plasmid rolling-circle replication by binding to a triple 11-bp direct repeat (bind locus) and cleaving the DNA at a specific distant site located in a hairpin loop within the nic locus of the origin. The structure of native full-length RepB reveals a hexameric ring molecule, where each protomer has two domains. The origin-binding and catalytic domains show a three-layer α–β–α sandwich fold. The active site is positioned at one of the faces of the β-sheet and coordinates a Mn2+ ion at short distance from the essential nucleophilic Y99. The oligomerization domains (ODs), each consisting of four α-helices, together define a compact ring with a central channel, a feature found in ring helicases. The toroidal arrangement of RepB suggests that, similar to ring helicases, it encircles one of the DNA strands during replication to confer processivity to the replisome complex. The catalytic domains appear to be highly mobile with respect to ODs. This mobility may account for the adaptation of the protein to two distinct DNA recognition sites. PMID:19440202

Direct observation of single flexible polymers using single stranded DNA†

PubMed Central

Brockman, Christopher; Kim, Sun Ju

2012-01-01

Over the last 15 years, double stranded DNA (dsDNA) has been used as a model polymeric system for nearly all single polymer dynamics studies. However, dsDNA is a semiflexible polymer with markedly different molecular properties compared to flexible chains, including synthetic organic polymers. In this work, we report a new system for single polymer studies of flexible chains based on single stranded DNA (ssDNA). We developed a method to synthesize ssDNA for fluorescence microscopy based on rolling circle replication, which generates long strands (>65 kb) of ssDNA containing “designer” sequences, thereby preventing intramolecular base pair interactions. Polymers are synthesized to contain amine-modified bases randomly distributed along the backbone, which enables uniform labelling of polymer chains with a fluorescent dye to facilitate fluorescence microscopy and imaging. Using this approach, we synthesized ssDNA chains with long contour lengths (>30 μm) and relatively low dye loading ratios (~1 dye per 100 bases). In addition, we used epifluorescence microscopy to image single ssDNA polymer molecules stretching in flow in a microfluidic device. Overall, we anticipate that ssDNA will serve as a useful model system to probe the dynamics of polymeric materials at the molecular level. PMID:22956981

Rapidly expanding genetic diversity and host range of the Circoviridae viral family and other Rep encoding small circular ssDNA genomes.

PubMed

Delwart, Eric; Li, Linlin

2012-03-01

The genomes of numerous circoviruses and distantly related circular ssDNA viruses encoding a rolling circle replication initiator protein (Rep) have been characterized from the tissues of mammals, fish, insects, plants (geminivirus and nanovirus), in human and animal feces, in an algae cell, and in diverse environmental samples. We review the genome organization, phylogenetic relationships and initial prevalence studies of cycloviruses, a proposed new genus in the Circoviridae family. Viral fossil rep sequences were also recently identified integrated on the chromosomes of mammals, frogs, lancelets, crustaceans, mites, gastropods, roundworms, placozoans, hydrozoans, protozoans, land plants, fungi, algae, and phytoplasma bacterias and their plasmids, reflecting the very wide past host range of rep bearing viruses. An ancient origin for viruses with Rep-encoding small circular ssDNA genomes, predating the diversification of eukaryotes, is discussed. The cellular hosts and pathogenicity of many recently described rep-containing circular ssDNA genomes remain to be determined. Future studies of the virome of single cell and multi-cellular eukaryotes are likely to further extend the known diversity and host-range of small rep-containing circular ssDNA viral genomes. Copyright © 2011 Elsevier B.V. All rights reserved.

Classification of select category A and B bacteria by Fourier transform infrared spectroscopy

NASA Astrophysics Data System (ADS)

Samuels, Alan C.; Snyder, A. Peter; St. Amant, Diane; Emge, Darren K.; Minter, Jennifer; Campbell, Mark; Tripathi, Ashish

2008-04-01

Relatively few reports have investigated the determination and classification of pathogens such as the National Institute of Allergy and Infectious Diseases (NIAID) Category A Bacillus anthracis spores and cells (BA), Yersinia species, Francisella tularensis (FT), and Category B Brucella species from FTIR spectra. We investigated the classification ability of the Fourier transform infrared (FTIR) spectra of viable pathogenic and non-pathogenic NIAID Category A and B bacteria. The impact of different growth media, growth time and temperature, rolling circle filter of the data, and wavelength range were investigated for their microorganism differentiation. Various 2-D PC plots provided differential degrees of separation with respect to the four viable, bacterial genera including the BA sub-categories of pathogenic spores, vegetative cells, and nonpathogenic vegetative cells. FT spectra were separated from that of the three other genera. The BA pathogenic spore strains 1029, LA1, and Ames were clearly differentiated from the rest of the dataset. Yersinia species were distinctly separated from the remaining dataset and could also be classified by growth media. This work provided evidence that FTIR spectroscopy can separate the four major pathogenic bacterial genera of NIAID Category A and B biological threat agents.

SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes.

PubMed

Shen, Yue; Stracquadanio, Giovanni; Wang, Yun; Yang, Kun; Mitchell, Leslie A; Xue, Yaxin; Cai, Yizhi; Chen, Tai; Dymond, Jessica S; Kang, Kang; Gong, Jianhui; Zeng, Xiaofan; Zhang, Yongfen; Li, Yingrui; Feng, Qiang; Xu, Xun; Wang, Jun; Wang, Jian; Yang, Huanming; Boeke, Jef D; Bader, Joel S

2016-01-01

Synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) generates combinatorial genomic diversity through rearrangements at designed recombinase sites. We applied SCRaMbLE to yeast synthetic chromosome arm synIXR (43 recombinase sites) and then used a computational pipeline to infer or unscramble the sequence of recombinations that created the observed genomes. Deep sequencing of 64 synIXR SCRaMbLE strains revealed 156 deletions, 89 inversions, 94 duplications, and 55 additional complex rearrangements; several duplications are consistent with a double rolling circle mechanism. Every SCRaMbLE strain was unique, validating the capability of SCRaMbLE to explore a diverse space of genomes. Rearrangements occurred exclusively at designed loxPsym sites, with no significant evidence for ectopic rearrangements or mutations involving synthetic regions, the 99% nonsynthetic nuclear genome, or the mitochondrial genome. Deletion frequencies identified genes required for viability or fast growth. Replacement of 3' UTR by non-UTR sequence had surprisingly little effect on fitness. SCRaMbLE generates genome diversity in designated regions, reveals fitness constraints, and should scale to simultaneous evolution of multiple synthetic chromosomes. © 2016 Shen et al.; Published by Cold Spring Harbor Laboratory Press.

A hint of Poincaré dodecahedral topology in the WMAP first year sky map

NASA Astrophysics Data System (ADS)

Roukema, B. F.; Lew, B.; Cechowska, M.; Marecki, A.; Bajtlik, S.

2004-09-01

It has recently been suggested by Luminet et al. (\\cite{LumNat03}) that the WMAP data are better matched by a geometry in which the topology is that of a Poincaré dodecahedral model and the curvature is ``slightly'' spherical, rather than by an (effectively) infinite flat model. A general back-to-back matched circles analysis by Cornish et al. (\\cite{CSSK03}) for angular radii in the range 25-90 °, using a correlation statistic for signal detection, failed to support this. In this paper, a matched circles analysis specifically designed to detect dodecahedral patterns of matched circles is performed over angular radii in the range 1-40\\ddeg on the one-year WMAP data. Signal detection is attempted via a correlation statistic and an rms difference statistic. Extreme value distributions of these statistics are calculated for one orientation of the 36\\ddeg ``screw motion'' (Clifford translation) when matching circles, for the opposite screw motion, and for a zero (unphysical) rotation. The most correlated circles appear for circle radii of \\alpha =11 ± 1 \\ddeg, for the left-handed screw motion, but not for the right-handed one, nor for the zero rotation. The favoured six dodecahedral face centres in galactic coordinates are (\\lII,\\bII) ≈ (252\\ddeg,+65\\ddeg), (51\\ddeg,+51\\ddeg), (144\\ddeg,+38\\ddeg), (207\\ddeg,+10\\ddeg), (271\\ddeg,+3\\ddeg), (332\\ddeg,+25\\ddeg) and their opposites. The six pairs of circles independently each favour a circle angular radius of 11 ± 1\\ddeg. The temperature fluctuations along the matched circles are plotted and are clearly highly correlated. Whether or not these six circle pairs centred on dodecahedral faces match via a 36\\ddeg rotation only due to unexpected statistical properties of the WMAP ILC map, or whether they match due to global geometry, it is clear that the WMAP ILC map has some unusual statistical properties which mimic a potentially interesting cosmological signal.

[Comprehensive implementation of interprofessional quality circles regarding early prevention of childhood disadvantage in Baden Württemberg (Germany)].

PubMed

Siebolds, Marcus; Münzel, Brigitte; Müller, Roland; Häußermann, Sigrun; Paul, Mechthild; Kahl, Cornelia

2016-10-01

The integration of available early interventions and healthcare for families with children by practicing pediatricians has yet to be systematically established. For this reason, the Association of Statutory Health Insurance Physicians of Baden-Wuerttemberg established overarching, accredited, cross-system quality circles that serve to integrate all representatives of the healthcare system as well as child and youth welfare services. These quality circles are led by specially trained moderator tandems consisting of pediatricians and staff members from youth welfare services. The goal was to evaluate the endpoints of the regional implementation of cross-system quality circles for early interventions in the state of Baden-Wuerttemberg as well as the feasibility of establishing long-term training programs for cross-system moderator tandems. This was a noncontrolled, longitudinal study to prepare a yearly evaluation of the quality-circle assessments as well as to gather statistics on the training of the moderator tandems within the Association of Statutory Health Insurance Physicians of Baden-Wuerttemberg. A total of 59 moderator tandems were trained in nine separate training sessions within the project period from 2011 to 2015. Overall, 33 quality circles were founded. In 2015, 566 persons were participating in the respective circles. Over the course of the study between 26 and 33 of the 44 urban and rural districts in the state of Baden-Wuerttemberg had at least one quality circle dedicated to early interventions. Ten further circles are presently in the process of being founded; 29 moderators have yet to commence their activity or have withdrawn from the program. Between 59 and 81 % of the urban and rural districts implemented cross-system quality circles. The training of the moderator tandems proceeded without complications. Because of the dropout quota of the trained moderator tandems, systematic and continual training of new tandems proves to be necessary.

Models to support students’ understanding of measuring area of circles

NASA Astrophysics Data System (ADS)

Rejeki, S.; Putri, R. I. I.

2018-01-01

Many studies showed that enormous students got confused about the concepts of measuring area of circles. The main reason is because mathematics classroom practices emphasized on memorizing formulas rather than understanding concepts. Therefore, in this study, a set of learning activities were designed as an innovation in learning area measurement of circles. The activities involved two models namely grid paper and reshaping which are respectively as a means and a strategy to support students’ learning of area measurement of circles. Design research was used as the research approach to achieve the aim. Thirty-eight of 8th graders in Indonesia were involved in this study. In this study, together with the contextual problems, the grid paper and reshaping sectors, which used as the models in this learning, helped the students to gradually develop their understanding of the area measurement of circles. The grid papers plays important role in comparing and estimating areas. Whereas, the reshaping sectors might support students’ understanding of the circumference and the area measurement of circles. Those two models could be the tool for promoting the informal theory of area measurement. Besides, the whole activities gave important role on distinguishing the area and perimeter of circles.

Polar amplification of the early Eocene indicated by δ2H values of lignin methoxyl groups of mummified wood

NASA Astrophysics Data System (ADS)

Anhäuser, Tobias; Hook, Benjamin; Halfar, Jochen; Greule, Markus; Keppler, Frank

2017-04-01

A number of well-preserved mummified wood samples have been excavated during diamond mining operations in early Eocene (55-50 Ma) kimberlite deposits located near the Arctic Circle (64° N, 110° W) in the Canadian Northwest Territories. The preserved wood, containing multi-decadal length tree-ring information, therefore allows the reconstruction of an unprecedented snapshot of terrestrial high-latitude climate during the early Eocene. Here we used wood-derived stable hydrogen isotopes (δ2H) as proxy for paleoclimatic interpretations. While cellulose extractions are commonly used for the analysis of modern wood-derived δ2H values, the mummified wood samples had been affected by selective degradation leading to a strong or even complete loss of cellulose while leaving a lignin-rich material behind. We have therefore analyzed δ2H values of the lignin methoxyl groups that have previously been shown to reflect the δ2H values of the local precipitation and can thus be used to infer paleoclimate information such as temperature changes. We applied this proxy to specimens found in three adjacent kimberlite pipes (30 km apart) which represent a range of early Eocene ages (Rb/Sr dating: 55.5 ± 0.7, 55.2 ± 0.3 and 53.3 ± 0.6 Ma [2σ standard deviation]). The δ2H values were measured at annual resolution for the three mummified wood series (length of individual time series: 82, 62 and 40 years) and the mean δ2H value of precipitation for the three decadal-scale time slices was reconstructed. Finally, we used existing relationships between early Eocene temperatures and stable isotopes in precipitation to quantify temperature changes. Warming phases such as the one covered here (culminating in the Early Eocene Climatic Optimum [52 to 50 Ma]) are commonly accompanied by a stronger increase in arctic/subarctic surface air temperatures in comparison to the global average (the ratio of these temperature differences is referred to as the polar amplification). Our estimation shows for the period between 55.5 and 53.3 Ma a mean subarctic temperature increase of 7.3 ± 3.7 °C. Globally, for the same period, a mean temperature increase of 1.5 °C has previously been derived from the δ18O values of benthic foraminifera. This suggests a polar amplification with a magnitude of 5 (±3). Despite uncertainties regarding both the age determination and the temperature reconstructions, the magnitude of our estimation is broadly in line with polar amplifications estimated for other Cenozoic global warming intervals, which have shown magnitudes of 3 to 4. Even though occurring on a much shorter time scale, the current global warming shows a similar polar amplification pattern as post industrial revolution Arctic temperatures, which are projected to amplify significantly in the near future, have already increased up to 3 °C as compared to a lower latitude temperature increase of 1 °C.

Quality Circles: A Corporate Strategy Applied in a Student Services Setting.

ERIC Educational Resources Information Center

Steele, Brenton H.; And Others

1987-01-01

Discusses the historical and conceptual framework of quality circles, presents a brief case history of circles initiated by the University of Maryland Office of Admissions, and provides a summary of implications. Emphasizes implications for student affairs administrators. (Author/ABB)

Wind Factor Simulation Model: User’s Manual.

DTIC Science & Technology

1980-04-01

computer program documentation; com- puterized simulation; equivalent headwind technique; great circle; great circle distance; great circle equation ; great... equation of a great circle. Program listing and flow chart are included. iv UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGE(WIh.n Date EnItrd) USER’S...THE EQUATOR . 336 C 337 NTRIFG = 0 338 C 339 C END OF FUNCTION ICONV I 1. RETURN TO MAIN PROGRAM . 340 C 42 341 RETURN 34? C 343 C 344 C 345 C * PART II

Normalized cDNA libraries

DOEpatents

Soares, Marcelo B.; Efstratiadis, Argiris

1997-01-01

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

Normalized cDNA libraries

DOEpatents

Soares, M.B.; Efstratiadis, A.

1997-06-10

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3{prime} noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. 4 figs.

Developing learning trajectory on the circumference of a cycle with realistic mathematics education (RME)

NASA Astrophysics Data System (ADS)

Indriani, Novi; Julie, Hongki

2017-08-01

According Nuranisa, the students' difficulties on a lesson about the circumference of a circle were to understand the concept of what was the circumference of a circle and solve problems associated with the circumference of a circle [8]. According Nuranisa, this is because the teacher only gives a formula to find the circumference of a circle without helping students to construct the formula [8]. The purposes of this study were (1) to develop the hypothetical learning trajectory (HLT) for the cycle circumference material, (2) to describe the student results from the HLT implementation. The type of research used in this research was the design research developed by Gravemeijer and Cobb (2006). According Gravemeijer and Cobb, the design research was divided into three stages: (1) preparing for the experiment, (2) design experiments, and (3) retrospective analysis [6]. In this study, researchers developed the student learning trajectories which were help students to construct their knowledge about the understanding and how to calculate the circumference of a circle. The context used on the students' learning trajectory was sports and arts event. The learning trajectory has been tried out for 15 grade five students on the Budya Wacana elementary school in Yogyakarta. In this paper, researchers would describe how the learning trajectory traversed students to construct their knowledge about the understanding and how to calculate the circumference of the circle and the student thinking process on the understanding and how to calculate the circumference of a circle. The results showed that students could construct about the understanding of the circumference of a circle and how to calculate the circumference of a circle through the student learning trajectory.

The experience of meaning in circle dance

PubMed Central

Borges da Costa, Ana L.; Cox, Diane L.

2016-01-01

ABSTRACT Circle dance, which derives from the tradition of folk dances, is practised worldwide. This article explores the meanings participants attribute to it. In-depth interviews with 39 participants, teachers and coordinators of teacher training programmes from the circle dance network in the United Kingdom were undertaken. Applying a constructivist grounded theory approach, major categories, representing respectively the experiences of circle dance participants, teachers and coordinators, were developed. This article specifically focuses on the first major category, termed “I can't imagine life without it”, which relates to the experience of 22 dancers. From an occupational perspective, the study reveals how participants realise a sense of meaning and satisfaction through engagement in circle dance and the potential contribution of this occupation to well-being. PMID:27366111

Vicious Circles in Organizations.

ERIC Educational Resources Information Center

Masuch, Michael

1985-01-01

After examining some elementary notions of action theory and cybernetics, this article analyzes the dynamics, clustering, and survival chances of vicious circles. It argues that the action perspective implies that many structural suboptimalities of organizations are caused by vicious circles. Eleven figures and 105 references are provided. (DCS)

VIEW FROM EAST SIDE OF ELM DRIVE/BIRCH CIRCLE BLOCK, SHOWING ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

VIEW FROM EAST SIDE OF ELM DRIVE/BIRCH CIRCLE BLOCK, SHOWING SLOPING TOPOGRAPHY. VIEW FACING WEST. - Camp H.M. Smith and Navy Public Works Center Manana Title VII (Capehart) Housing, Intersection of Acacia Road and Brich Circle, Pearl City, Honolulu County, HI

The Exclusionary Circle Game: A Tool to Promote Critical Dialogue About HIV Stigma and Social Justice.

PubMed

Wong, Josephine Pui; Li, Alan Tai

2015-01-01

The Exclusionary Circle Game was a learning tool developed for an intervention study to address stigma associated with human immunodeficiency virus (HIV) infection and social exclusion. The objectives of The Exclusionary Circle Game were to enhance collective resonance and empathy, promote critical reflection and dialogue, and motivate collective action to address social exclusion. The game began with all participants being inside a circle. Each participant was randomly given one color-coded card. Each card color represented a character with a specific lived experience associated with racism, patriarchy, homophobia, transphobia, HIV stigma, and so on. Participants holding a marginalized status card were asked to leave the circle in sequence and go to designated spaces. Eventually, only one half of the participants were left in the circle. Participants then debriefed about their experiences within the entire group. The game has been used, beyond the intervention study, at research conferences with positive feedback. In this article, we detail the processes, strengths, and possibility of using this game for empowerment education.

Designing worked examples for learning tangent lines to circles

NASA Astrophysics Data System (ADS)

Retnowati, E.; Marissa

2018-03-01

Geometry is a branch of mathematics that deals with shape and space, including the circle. A difficult topic in the circle may be the tangent line to circle. This is considered a complex material since students have to simultaneously apply several principles to solve the problems, these are the property of circle, definition of the tangent, measurement and Pythagorean theorem. This paper discusses designs of worked examples for learning tangent line to circles and how to apply this design to an effective and efficient instructional activity. When students do not have sufficient prior knowledge, solving tangent problems might be clumsy, and as a consequence, the problem-solving activity hinders learning. According to a Cognitive Load Theory, learning occurs when students can construct new knowledge based on the relevant knowledge previously learned. When the relevant knowledge is unavailable, providing students with the worked example is suggested. Worked example may reduce unproductive process during learning that causes extraneous cognitive load. Nevertheless, worked examples must be created in such a way facilitate learning.

Improvement of rolling 6 mm thin plates in plate rolling mill PT. Krakatau Posco

NASA Astrophysics Data System (ADS)

Pujiyanto, Hamdani

2017-01-01

A 6-mm thin plate is difficult to produce especially if the product requires wide size and high strength. Flatness is the main quality issue in rolling 6-mm plate using a 4-high reversing mill which use ±1100-mm work roll. Thus some methods are applied to overcome such issue in order to comply to customer quality requirement. Pre-rolling, rolling, and post-rolling conditions have to be considered comprehensively. Roll unit management will be the key factor before rolling condition. The roll unit itself has a significant impact on work roll crown wearness in relation with work roll intial crown and thermal crown. Work roll crown along with the modification of hydraulic gap control (HGC) could directly alter the flatness of the plate.

Comfortable synchronization of cyclic drawing movements with a metronome.

PubMed

Repp, Bruno H

2011-02-01

Continuous circle drawing is considered a paragon of emergent timing, whereas the timing of finger tapping is said to be event-based. Synchronization with a metronome, however, must to some extent be event-based for both types of movement. Because the target events in the movement trajectory are more poorly defined in circle drawing than in tapping, circle drawing shows more variable asynchronies with a metronome than does tapping. One factor that may have contributed to high variability in past studies is that circle size, drawing direction, and target point were prescribed and perhaps outside the comfort range. In the present study, participants were free to choose most comfortable settings of these parameters for two continuously drawn shapes, circles and infinity signs, while synchronizing with a regular or intermittently perturbed metronome at four different tempi. Results showed that preferred circle sizes were generally smaller than in previous studies but tended to increase as tempo decreased. Synchronization results were similar for circles and infinity signs, and similar to earlier results for circles drawn within a fixed template (Repp & Steinman, 2010). Comparison with tapping data still showed drawing to exhibit much greater variability and persistence of asynchronies as well as slower phase correction in response to phase shifts in the metronome. With comfort level ruled out as a factor, these differences can now be attributed more confidently to differences in event definition and/or movement dynamics. Copyright © 2010 Elsevier B.V. All rights reserved.

What Goes Around Can Come Around: An Unexpected Deleterious Effect of Using Mouse Running Wheels for Environmental Enrichment

PubMed Central

Leduc, Renee Y M; Rauw, Gail; Baker, Glen B; McDermid, Heather E

2017-01-01

Environmental enrichment items such as running wheels can promote the wellbeing of laboratory mice. Growing evidence suggests that wheel running simulates exercise effects in many mouse models of human conditions, but this activity also might change other aspects of mouse behavior. In this case study, we show that the presence of running wheels leads to pronounced and permanent circling behavior with route-tracing in a proportion of the male mice of a genetically distinct cohort. The genetic background of this cohort includes a mutation in Arhgap19, but genetic crosses showed that an unknown second-site mutation likely caused the induced circling behavior. Behavioral tests for inner-ear function indicated a normal sense of gravity in the circling mice. However, the levels of dopamine, serotonin, and some dopamine metabolites were lower in the brains of circling male mice than in mice of the same genetic background that were weaned without wheels. Circling was seen in both singly and socially housed male mice. The additional stress of fighting may have exacerbated the predisposition to circling in the socially housed animals. Singly and socially housed male mice without wheels did not circle. Our current findings highlight the importance and possibly confounding nature of the environmental and genetic background in mouse behavioral studies, given that the circling behavior and alterations in dopamine and serotonin levels in this mouse cohort occurred only when the male mice were housed with running wheels. PMID:28315651

A Roll, Fin, and Fin Controller Prediction Computer Program.

DTIC Science & Technology

1980-06-01

IERATI *EQ. 03 WRITE16920301 ROLL 365 365 3R1TE(G. 26311 ROLL 366 no 505 ImU - 1,NNU ROLL 36? 50S WRITE(G.2011 3U(I’U),OAWPU(1PU,SIGLCfINU) ROLL 360...ROLL DAMPING WILL BE ONE**/$ ROLL 642 2016 FORMAT (/jIX,*LONGCRESTEO SVECTRA AND COMPONENTS WILL SE PRINTED. ROLL 44S 2’) ROLL 444 2019 FORMAT (1/19

Comprehensibility of Englishes within ASEAN: A Synopsis of Results

ERIC Educational Resources Information Center

Wilang, Jeffrey Dawala; Teo, Adisa

2012-01-01

The purpose of this study is to measure the comprehensibility of the Expanding Circle nations' citizens, namely Burmese, Cambodians, Indonesians, Laotians, Thais and Vietnamese towards the Outer Circle Englishes, namely Bruneian English, Malaysian English. Philippine English and Singaporean English. Ten universities in the Expanding Circle that…

CONTROL FOR ROLLING MILL

DOEpatents

Shuck, A.B.; Shaw, W.C.

1961-06-20

A plutonium-rolling apparatus is patented that has two sets of feed rolls, shaping rolls between the feed rolls, and grippers beyond the feed rolls, which ready a workpiece for a new pass through the shaping rolls by angularly shifting the workpiece about its axis or transversely moving it on a line parallel to the axes of the shaping rolls. Actuation of each gripper for gripping or releasing the workpiece is produced by the relative positions assumed by the feed rolls adjacent to the gripper as the workpiece enters or leaves the feed rolls.

An experimental study for determining human discomfort response to roll vibration

NASA Technical Reports Server (NTRS)

Leatherwood, J. D.; Dempsey, T. K.; Clevenson, S. A.

1976-01-01

An experimental study using a passenger ride quality apparatus (PRQA) was conducted to determine the subjective reactions of passengers to roll vibrations. The data obtained illustrate the effect upon human comfort of several roll-vibration parameters: namely, roll acceleration level, roll frequency, and seat location (i.e., distance from axis of rotation). Results of an analysis of variance indicated that seat location had no effect on discomfort ratings of roll vibrations. The effect of roll acceleration level was significant, and discomfort ratings increased markedly with increasing roll acceleration level at all roll frequencies investigated. Of particular interest, is the fact that the relationship between discomfort ratings and roll acceleration level was linear in nature. The effect of roll frequency also was significant as was the interaction between roll acceleration level and roll frequency.

Conceptions and Representations: The Circle as an Example.

ERIC Educational Resources Information Center

Janvier, Claude

This paper, which addresses the issue of representation as an internal construct corresponding to an external abstract configuration, attempts to extend A. A. DiSessa's phenomenological primitives to mathematics (particularly to the notion of circle). Various acceptations of the word representation are examined, using the notion of a circle as an…

Exploring Writing Circles as Innovative, Collaborative Writing Structures with Teacher Candidates

ERIC Educational Resources Information Center

Roberts, Sherron Killingsworth; Blanch, Norine; Gurjar, Nandita

2017-01-01

Writing circles are "small groups... meeting regularly to share drafts, choose common writing topics, practice positive response, and in general, help each other become better writers" (Vopat, 2009, p. 6). In this exploratory study, writing circles were employed with elementary teacher candidates in hopes of enhancing their perceptions…

The Effect of Literature Circles on Text Analysis and Reading Desire

ERIC Educational Resources Information Center

Karatay, Halit

2017-01-01

In order to make teaching activities more appealing, different techniques and strategies have been constantly employed. This study utilized the strategy of "literature circles" to improve the text-analysis skills, reading desires, and interests of prospective teachers of Turkish. "Literature circles" was not chosen to be used…

Adjusting an electron beam for drilling

NASA Technical Reports Server (NTRS)

Childress, C. L.

1980-01-01

Reticle contains two concentric circles: inner circle insures beam circularity and outer circle is guide to prevent beam from cutting workpiece clamp. Precise measurement of beam and clamp are required with old reticle. New reticle speeds up electron-beam drilling process by eliminating need to rotate eyepiece to make measurements against reticle scale.

Participation and Gender in Circle-Time Situations in Preschool

ERIC Educational Resources Information Center

Emilson, Anette; Johansson, Eva

2013-01-01

This study sought to investigate participatory values in relation to gender, as expressed in interactions between teachers and children in circle-time situations in Swedish and Norwegian preschools. This paper reports evidence from three research questions: How is children's participation conditioned in circle-time situations? How are…

An Effective Time and Management Strategy in Quality Circles.

ERIC Educational Resources Information Center

Halverson, Don E.

Contending that participation in quality circles enhances effective time management by school administrators and teachers, this guide provides both a theoretical briefing and practical recommendations for better time management. A pre- posttest prefaces a review of basic concepts of quality circles with reference to the work of Abraham Maslow,…

Quality Circles in Higher Education: Quality, Satisfaction, and Climate.

ERIC Educational Resources Information Center

Kay, Carol; Healy, Margaret

The effect of quality circles at Iowa State University on absenteeism, performance evaluation, perceptions of the organization climate, job satisfaction, and perceived opportunities for professional and personal growth was measured in this study. The process of quality circles is designed to promote job fulfillment and organizational productivity…

VIEW OF THE OUTER RING OF CENTER CIRCLE, LOOKING NORTH. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

VIEW OF THE OUTER RING OF CENTER CIRCLE, LOOKING NORTH. GRANITE COPING DEFINES THE SWAIN FAMILY PLOT, WHICH CONTAINS A CELTIC CROSS, ON WHICH THE CIRCLE REFERS TO ETERNAL LIFE, AND A RECLINING HUMAN FIGURE IN ETERNAL SLEEP - Woodlands Cemetery, 4000 Woodlands Avenue, Philadelphia, Philadelphia County, PA

Using Literature Circles to Enhance Student Knowledge of Nonfiction Text

ERIC Educational Resources Information Center

Whitworth, Amanda

2017-01-01

This mixed methods action research study explored how students reacted to using literature circles to enhance their knowledge and understanding of reading nonfiction text as compared to students using guided reading. This study showed a minimal improvement for students participating in the literature circle group in overall understanding of…

Literature Circles. ERIC Digest.

ERIC Educational Resources Information Center

Lin, Chia-Hui

The use of literature circles has been discussed in a variety of academic journals, conference papers, and workshops. Teachers at all grade levels use literature circles as a vehicle through which students learn to: think critically about literature; express their thoughts in oral and written forms; and better enjoy their literacy experiences.…

Developing Soft Skills Using "Literature Circles"

ERIC Educational Resources Information Center

Azmi, Mohd Nazri Bin Latiff

2013-01-01

This study investigates the impact of the implementation of "Literature Circles" in an Active Learning classroom in relations to developing soft skills among university students. The use of Literature Circles is a well-known strategy in teaching the students to be more creative, independent, and think out of the box. A group of…

Exploring Factors That Influence Quality Literature Circles

ERIC Educational Resources Information Center

Young, Chase; Mohr, Kathleen A. J.

2018-01-01

Research indicates that literature circles are an authentic means for literacy development that students typically enjoy. To better understand the potential value and to add to the research base regarding literature circles, this study, involving 17 fourth graders, explores factors that may influence the quality of literature discussions,…

Method for construction of normalized cDNA libraries

DOEpatents

Soares, Marcelo B.; Efstratiadis, Argiris

1996-01-01

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

Method for construction of normalized cDNA libraries

DOEpatents

Soares, M.B.; Efstratiadis, A.

1996-01-09

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form. The method comprises: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3` noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. 4 figs.

Sister Circles as a Culturally Relevant Intervention for Anxious African American Women

PubMed Central

Neal-Barnett, Angela; Stadulis, Robert; Murray, Marsheena; Payne, Margaret Ralston; Thomas, Anisha; Salley, Bernadette B.

2011-01-01

Research on anxiety treatment with African American women reveals a need to develop interventions that address factors relevant to their lives. Such factors include feelings of isolation, multiple roles undertaken by Black women, and faith. A recurrent theme across treatment studies is the importance of having support from other Black women. Sister circles are support groups that build upon existing friendships, fictive kin networks, and the sense of community found among African Americans females. Sister circles appear to offer many of the components Black women desire in an anxiety intervention. In this article, we explore sister circles as an intervention for anxious African American women. Culturally-infused aspects from our sister circle work with middle-class African American women are presented. Further research is needed. PMID:22081747

Age-related interference from irrelevant distracters in visual feature search among heterogeneous distracters.

PubMed

Merrill, Edward C; Conners, Frances A

2013-08-01

We evaluated age-related variations in the influence of heterogeneous distracters during feature target. Participants in three age groups-6-year-old children, 9-year-old children, and young adults-completed three conditions of search. In a singleton search condition, participants searched for a circle among squares of the same color. In two feature mode search conditions, participants searched for a gray circle or a black circle among gray and black squares. Singleton search was performed at the same level of efficiency for all age groups. In contrast, the two feature mode search conditions yielded age-related performance differences in both conditions. Younger children exhibited a steeper slope than young adults when searching for a gray or black circle. Older children exhibited a steeper slope than young adults when searching for a gray circle but not when searching for a black circle. We concluded that these differences revealed age-related improvements in the relative abilities of adults and children to execute attentional control processes during visual search. In particular, it appears that children found it more difficult to maintain the goal of searching for a circle target than adults and were distracted by the presence of the irrelevant feature dimension of color. Published by Elsevier Inc.

Experimental and Theoretical Study on Minimum Achievable Foil Thickness during Asymmetric Rolling

PubMed Central

Tang, Delin; Liu, Xianghua; Song, Meng; Yu, Hailiang

2014-01-01

Parts produced by microforming are becoming ever smaller. Similarly, the foils required in micro-machines are becoming ever thinner. The asymmetric rolling technique is capable of producing foils that are thinner than those produced by the conventional rolling technique. The difference between asymmetric rolling and conventional rolling is the ‘cross-shear’ zone. However, the influence of the cross-shear zone on the minimum achievable foil thickness during asymmetric rolling is still uncertain. In this paper, we report experiments designed to understand this critical influencing factor on the minimum achievable thickness in asymmetric rolling. Results showed that the minimum achievable thickness of rolled foils produced by asymmetric rolling with a rolling speed ratio of 1.3 can be reduced to about 30% of that possible by conventional rolling technique. Furthermore, the minimum achievable thickness during asymmetric rolling could be correlated to the cross-shear ratio, which, in turn, could be related to the rolling speed ratio. From the experimental results, a formula to calculate the minimum achievable thickness was established, considering the parameters cross-shear ratio, friction coefficient, work roll radius, etc. in asymmetric rolling. PMID:25203265

THE FORM AND STRUCTURE OF KINETOPLAST DNA OF CRITHIDIA

PubMed Central

Renger, Hartmut C.; Wolstenholme, David R.

1972-01-01

Cesium chloride centrifugation of each of the DNAs extracted from eight strains of Crithidia revealed a main band at ρ = 1.717 g/cm3 and a satellite band varying from ρ = 1.701 to 1.705 g/cm3 for the different strains By electron microscopy each DNA was shown to include circular molecules, 0.69–0.80 µ in mean contour length, and large, topologically two-dimensional masses of DNA in which the molecules appeared in the form of rosettes. DNA isolated from kinetoplast fractions of Crithidia acanthocephali was shown to consist of light satellite DNA and to be mainly in the form of large masses, 0.8 µ (mol wt = 1.54 x 106 daltons) circular molecules, and a few long, linear molecules. The results of experiments involving ultracentrifugation, heating, and quenching, sonication, and endodeoxyribonuclease digestion, combined with electron microscopy, are consistent with the following hypothesis. The large DNA masses are associations of 0.8 µ circles which are mainly covalently closed. The circles are held together in groups (the rosettes) of up to 46 by the topological interlocking of each circle with many other circles in the group. A group of circles is attached to an adjacent group by one or more circles, each interlocking with many circles of both groups. Each of the associations comprises, on the average, about 27,000 circles (total mol wt ≃ 41 x 109 daltons). A model is proposed for the in situ arrangement of the associations which takes into consideration their form and structure, and appearance in thin sections PMID:5040863

Flat panel detector-based cone beam computed tomography with a circle-plus-two-arcs data acquisition orbit: preliminary phantom study.

PubMed

Ning, Ruola; Tang, Xiangyang; Conover, David; Yu, Rongfeng

2003-07-01

Cone beam computed tomography (CBCT) has been investigated in the past two decades due to its potential advantages over a fan beam CT. These advantages include (a) great improvement in data acquisition efficiency, spatial resolution, and spatial resolution uniformity, (b) substantially better utilization of x-ray photons generated by the x-ray tube compared to a fan beam CT, and (c) significant advancement in clinical three-dimensional (3D) CT applications. However, most studies of CBCT in the past are focused on cone beam data acquisition theories and reconstruction algorithms. The recent development of x-ray flat panel detectors (FPD) has made CBCT imaging feasible and practical. This paper reports a newly built flat panel detector-based CBCT prototype scanner and presents the results of the preliminary evaluation of the prototype through a phantom study. The prototype consisted of an x-ray tube, a flat panel detector, a GE 8800 CT gantry, a patient table and a computer system. The prototype was constructed by modifying a GE 8800 CT gantry such that both a single-circle cone beam acquisition orbit and a circle-plus-two-arcs orbit can be achieved. With a circle-plus-two-arcs orbit, a complete set of cone beam projection data can be obtained, consisting of a set of circle projections and a set of arc projections. Using the prototype scanner, the set of circle projections were acquired by rotating the x-ray tube and the FPD together on the gantry, and the set of arc projections were obtained by tilting the gantry while the x-ray tube and detector were at the 12 and 6 o'clock positions, respectively. A filtered backprojection exact cone beam reconstruction algorithm based on a circle-plus-two-arcs orbit was used for cone beam reconstruction from both the circle and arc projections. The system was first characterized in terms of the linearity and dynamic range of the detector. Then the uniformity, spatial resolution and low contrast resolution were assessed using different phantoms mainly in the central plane of the cone beam reconstruction. Finally, the reconstruction accuracy of using the circle-plus-two-arcs orbit and its related filtered backprojection cone beam volume CT reconstruction algorithm was evaluated with a specially designed disk phantom. The results obtained using the new cone beam acquisition orbit and the related reconstruction algorithm were compared to those obtained using a single-circle cone beam geometry and Feldkamp's algorithm in terms of reconstruction accuracy. The results of the study demonstrate that the circle-plus-two-arcs cone beam orbit is achievable in practice. Also, the reconstruction accuracy of cone beam reconstruction is significantly improved with the circle-plus-two-arcs orbit and its related exact CB-FPB algorithm, as compared to using a single circle cone beam orbit and Feldkamp's algorithm.

Microstructural and mechanical responses to various rolling speeds determined in multi-pass break-down rolling of AZ31B alloy

NASA Astrophysics Data System (ADS)

Jia, Weitao; Tang, Yan; Ning, Fangkun; Le, Qichi; Cui, Jianzhong

2018-04-01

Different rolling operations of as-cast AZ31B alloy were performed under different rolling speed (18 ∼ 72 m min‑1) and rolling pass conditions at 400 °C. Microstructural studies, tensile testing and formability evaluation relevant to each rolling operation were investigated. For 1-pass rolling, coarse average grain size (CAGS) region gradually approached the center layer as the rolling speed increased. Moreover, twins, shear bands and coarse-grain structures were the dominant components in the microstructure of plates rolled at 18, 48 and 72 m min‑1, respectively, indicating the severe deformation inhomogeneity under the high reduction per pass condition. For 2-pass rolling and 4-pass rolling, dynamic recrystallization was observed to be well and CAGS region has substantially disappeared, indicating the significant improvement in deformation uniformity and further the grain homogenization under the conditions. Microstructure uniformity degree of 2-pass rolled plates did not vary much as the rolling speed varied. On this basis, shear band distribution dominated the deformation behavior during the uniaxial tension of the 2-pass rolled plates. However, microstructure uniformity accompanied by twin distribution played a leading role in stretching the 4-pass rolled plates.

Polygons and Their Circles

ERIC Educational Resources Information Center

Stephenson, Paul

2009-01-01

In order to find its circumference, Archimedes famously boxed the circle between two polygons. Ending the first of a series of articles (MT179) with an aside, Francis Lopez-Real reverses the situation to ask: Which polygons can be boxed between two circles? (The official term for such polygons is "bicentric".) The sides of these polygons are…

Empowerment of Children through Circle Time: Myth or Reality?

ERIC Educational Resources Information Center

Collins, Bernie

2013-01-01

The focus of this paper is circle time, a widely used method in primary schools in Ireland and elsewhere. It involves children sitting in a circle with their teacher using method-specific techniques and strategies for self-esteem enhancement, promoting positive relationships and development of social skills. Qualitative research was undertaken in…

78 FR 44119 - Circle Environmental #1 Superfund Site; Dawson, Terrell County, Georgia; Notice of Settlement

Federal Register 2010, 2011, 2012, 2013, 2014

2013-07-23

... ENVIRONMENTAL PROTECTION AGENCY [FRL-9837-3; CERCLA-04-2013-3760] Circle Environmental 1 Superfund... settlement with Walter G. Mercer, Jr. concerning the Circle Environmental 1 Superfund Site located in Dawson... methods: Internet: www.epa.gov/region4/superfund/programs/enforcement/enforcement.html U.S. Mail: U.S...

VIEW OF DATE DRIVE, FROM INTERSECTION WITH BIRCH CIRCLE, WITH ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

VIEW OF DATE DRIVE, FROM INTERSECTION WITH BIRCH CIRCLE, WITH FACILITY 809 ON LEFT, 816 ON RIGHT. NOTE THE MANY DATE PALMS. VIEW FACING NORTHWEST - Camp H.M. Smith and Navy Public Works Center Manana Title VII (Capehart) Housing, Intersection of Acacia Road and Brich Circle, Pearl City, Honolulu County, HI

From Silence to a Whisper to Active Participation: Using Literature Circles with ELL Students

ERIC Educational Resources Information Center

Carrison, Catherine; Ernst-Slavit, Gisela

2005-01-01

This article discusses benefits of using literature circles with ELL students to strengthen literacy skills and student confidence. Highlighting one teacher's implementation of literature circles, the authors present a candid examination of areas of initial weakness and describe strategies used for improvements in subsequent "rounds." A discussion…

Comparing Interactions in Literature Circles in Both Online and in Class Discussions

ERIC Educational Resources Information Center

Skeen, Christel Ghrist

2014-01-01

Discourse analysis of literature circles can lead educators to understand the different types of interactions taking place as students talk about text. Social and academic interactions exist in both face-to-face and online discussions of reading material. This study examines two different settings of literature circles and compares interactions of…

College Students' Science Societies and Special-Interest Circles

ERIC Educational Resources Information Center

Ivanov, A.

2005-01-01

From the point of view of their age, student science societies and special-interest circles are among the most venerable forms of corporate association among students in colleges and universities. In this article, the author traces the formation of different societies and special-interest circles by college students in different universities in…

Integrating Literature Circles into a Cotaught Inclusive Classroom

ERIC Educational Resources Information Center

Whittaker, Catharine R.

2012-01-01

Literature circles or book clubs are small, heterogeneous groups of students who have chosen to read and discuss the same book together. The research on literature circles suggests that they hold great promise for increasing students' enjoyment of reading and honing their literacy skills. When evidence-based strategies are embedded into a…

75 FR 19369 - Certain Hot-Rolled Flat-Rolled Carbon Quality Steel Products from Brazil: Preliminary Results of...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-14

...-Rolled Carbon Quality Steel Products from Brazil: Preliminary Results of Antidumping Duty Administrative... duty order on certain hot-rolled flat-rolled carbon quality steel products (hot-rolled steel) from... 1, 2008, through February 28, 2009. We preliminarily determine that the sale of hot-rolled steel...

Violence in the eye of adolescents: education intervention with Culture Circles.

PubMed

Brandão Neto, Waldemar; Silva, Marta Angélica Iossi; de Aquino, Jael Maria; de Lima, Luciane Soares; Monteiro, Estela Maria Leite Meirelles

2015-01-01

to apply the methodology of Culture Circles on adolescents as a strategy for health education of nurses in the construction of the collective knowledge of the thematic violence. action research type and qualitative study. Participants were 11 adolescents from a public school in Recife, PE, Brazil. Data production in Culture Circles included the participant observation with field diary, photographic recording and filming, as well as the photovoice technique. The analysis was performed by triangulating data in dialogue with the literature. it was shown that the problematizing action provided by the Culture Circle made possible to create situations in which adolescents felt invited to critically refl ect on the phenomenon of violence in all its complexity. the health education intervention, performed by Culture Circles, added learning and mutual growth subsidizing nursing care actions that excel at leadership and autonomy of adolescents.

Multimodal determination of Rayleigh dispersion and attenuation curves using the circle fit method

NASA Astrophysics Data System (ADS)

Verachtert, R.; Lombaert, G.; Degrande, G.

2018-03-01

This paper introduces the circle fit method for the determination of multi-modal Rayleigh dispersion and attenuation curves as part of a Multichannel Analysis of Surface Waves (MASW) experiment. The wave field is transformed to the frequency-wavenumber (fk) domain using a discretized Hankel transform. In a Nyquist plot of the fk-spectrum, displaying the imaginary part against the real part, the Rayleigh wave modes correspond to circles. The experimental Rayleigh dispersion and attenuation curves are derived from the angular sweep of the central angle of these circles. The method can also be applied to the analytical fk-spectrum of the Green's function of a layered half-space in order to compute dispersion and attenuation curves, as an alternative to solving an eigenvalue problem. A MASW experiment is subsequently simulated for a site with a regular velocity profile and a site with a soft layer trapped between two stiffer layers. The performance of the circle fit method to determine the dispersion and attenuation curves is compared with the peak picking method and the half-power bandwidth method. The circle fit method is found to be the most accurate and robust method for the determination of the dispersion curves. When determining attenuation curves, the circle fit method and half-power bandwidth method are accurate if the mode exhibits a sharp peak in the fk-spectrum. Furthermore, simulated and theoretical attenuation curves determined with the circle fit method agree very well. A similar correspondence is not obtained when using the half-power bandwidth method. Finally, the circle fit method is applied to measurement data obtained for a MASW experiment at a site in Heverlee, Belgium. In order to validate the soil profile obtained from the inversion procedure, force-velocity transfer functions were computed and found in good correspondence with the experimental transfer functions, especially in the frequency range between 5 and 80 Hz.

Guided discovery of the nine-point circle theorem and its proof

NASA Astrophysics Data System (ADS)

Buchbinder, Orly

2018-01-01

The nine-point circle theorem is one of the most beautiful and surprising theorems in Euclidean geometry. It establishes an existence of a circle passing through nine points, all of which are related to a single triangle. This paper describes a set of instructional activities that can help students discover the nine-point circle theorem through investigation in a dynamic geometry environment, and consequently prove it using a method of guided discovery. The paper concludes with a variety of suggestions for the ways in which the whole set of activities can be implemented in geometry classrooms.

Quality circles and their potential application to rural health care in Papua New Guinea.

PubMed

Cibulskis, R E; Edwards, K N

1993-06-01

A quality circle is a group of service providers who meet regularly to solve problems relating to the quality of their work. This is an example of bottom-up rather than top-down management which has found considerable success in the industries of the developed world. This article describes the principles which govern the operation of quality circles, the expected benefits and how best to introduce them. The problems relating to the provision of quality health care in rural areas and the potential application of the quality circle methodology are discussed.

75 FR 64246 - Certain Hot-Rolled Flat-Rolled Carbon-Quality Steel Products From Brazil: Correction to Notice of...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-19

...-Rolled Carbon-Quality Steel Products From Brazil: Correction to Notice of Antidumping Duty Order AGENCY... certain hot-rolled flat-rolled carbon-quality steel products from Brazil. See Antidumping Duty Order: Certain Hot-Rolled Flat-Rolled Carbon-Quality Steel Products From Brazil, 67 FR 11093 (March 12, 2002...

The Gasket of Circles: A Fractal of Circular Nature

ERIC Educational Resources Information Center

Haggar, Fred; Kricic, Senida

2017-01-01

Subdividing an equilateral triangle into four congruent triangles, then doing likewise to each of the three non-central triangles, and then again and again, leads to the Sierpinski gasket, from which the chaos game originated. An analogous procedure is hereforth applied to a circle, where a subdivision consists of two pairs of inscribed circles,…

Influence of a Mathematics Teachers' Circle on Elementary Teachers' Use of Problem Solving

ERIC Educational Resources Information Center

Garner, Mary L.; Watson, Virginia; Rogers, Beth; Head, Catherine

2017-01-01

Math teachers' circles are a form of professional development that is recommended by the Conference Board of the Mathematical Sciences in their publication Mathematical Education of Teachers II (2012). However, little research has been published on how effective math teachers' circles are in advancing the mathematical knowledge of teachers and…

Author Correction: Quantum Landauer erasure with a molecular nanomagnet

NASA Astrophysics Data System (ADS)

Gaudenzi, R.; Burzurí, E.; Maegawa, S.; van der Zant, H. S. J.; Luis, F.

2018-06-01

In the version of this Letter originally published, the key to the green and open circles in Fig. 4 was reversed; it should have shown that the green circles correspond to `From χ versus T' and the open circles correspond to `From χ versus Hy'. This has now been corrected in all versions of the Letter.

Growing a Circle of Courage Culture: One School's Journey

ERIC Educational Resources Information Center

Espiner, Deborah; Guild, Diane

2010-01-01

Mt. Richmond Special School is the first Circle of Courage school in New Zealand. The school reflects the richness of the cultural and learning diversity found in many New Zealand schools. Located in the heart of South Auckland, the school's 130 students represent a wide range of ethnic backgrounds. The universal values in the Circle of Courage…

Quality Circles: How Effective Are They in Improving Employee Performance and Attitudes?

ERIC Educational Resources Information Center

Buch, Kimberly; Raban, Amiram

1990-01-01

Used a quasi-experimental design to assess the effect of a quality circle intervention on behavior and attitudes of 88 employees at a large Midwestern organization. Results provide mixed support for the purported ability of circles to improve work behavior with no change for absenteeism and productivity but positive change for quality of work.…

Dynamic Investigation of Triangles Inscribed in a Circle, Which Tend to an Equilateral Triangle

ERIC Educational Resources Information Center

Stupel, Moshe; Oxman, Victor; Sigler, Avi

2017-01-01

We present a geometrical investigation of the process of creating an infinite sequence of triangles inscribed in a circle, whose areas, perimeters and lengths of radii of the inscribed circles tend to a limit in a monotonous manner. First, using geometrical software, we investigate four theorems that represent interesting geometrical properties,…

D'Nealian Handwriting versus Circle-Stick Print.

ERIC Educational Resources Information Center

Thurber, Donald N.

This paper argues against teaching children to make letters using circle-stick writing. It contends that the circle-stick method requires continued pen/pencil lifts hindering rhythm or flow in the writing process and that there is little carry-over value into cursive writing as the two scripts are totally different. D'Nealian print, one type of…

Unit Circles and Inverse Trigonometric Functions

ERIC Educational Resources Information Center

Barrera, Azael

2014-01-01

Historical accounts of trigonometry refer to the works of many Indian and Arab astronomers on the origin of the trigonometric functions as we know them now, in particular Abu al-Wafa (ca. 980 CE), who determined and named all known trigonometric functions from segments constructed on a regular circle and later on a unit circle (Moussa 2011;…

49 CFR 393.134 - What are the rules for securing roll-on/roll-off or hook lift containers?

Code of Federal Regulations, 2011 CFR

2011-10-01

... or hook lift containers? 393.134 Section 393.134 Transportation Other Regulations Relating to... for securing roll-on/roll-off or hook lift containers? (a) Applicability. The rules in this section apply to the transportation of roll-on/roll-off or hook lift containers. (b) Securement of a roll-on...

49 CFR 393.134 - What are the rules for securing roll-on/roll-off or hook lift containers?

Code of Federal Regulations, 2010 CFR

2010-10-01

... or hook lift containers? 393.134 Section 393.134 Transportation Other Regulations Relating to... for securing roll-on/roll-off or hook lift containers? (a) Applicability. The rules in this section apply to the transportation of roll-on/roll-off or hook lift containers. (b) Securement of a roll-on...

Rolling-Element Bearings

NASA Technical Reports Server (NTRS)

Hamrock, B. J.; Anderson, W. J.

1983-01-01

Rolling element bearings are a precision, yet simple, machine element of great utility. A brief history of rolling element bearings is reviewed and the type of rolling element bearings, their geometry and kinematics, as well as the materials they are made from and the manufacturing processes they involve are described. Unloaded and unlubricated rolling element bearings, loaded but unlubricated rolling element bearings and loaded and lubricated rolling element bearings are considered. The recognition and understanding of elastohydrodynamic lubrication covered, represents one of the major development in rolling element bearings.

Computer-aided analysis and design of the shape rolling process for producing turbine engine airfoils

NASA Technical Reports Server (NTRS)

Lahoti, G. D.; Akgerman, N.; Altan, T.

1978-01-01

Mild steel (AISI 1018) was selected as model cold-rolling material and Ti-6Al-4V and INCONEL 718 were selected as typical hot-rolling and cold-rolling alloys, respectively. The flow stress and workability of these alloys were characterized and friction factor at the roll/workpiece interface was determined at their respective working conditions by conducting ring tests. Computer-aided mathematical models for predicting metal flow and stresses, and for simulating the shape-rolling process were developed. These models utilize the upper-bound and the slab methods of analysis, and are capable of predicting the lateral spread, roll-separating force, roll torque and local stresses, strains and strain rates. This computer-aided design (CAD) system is also capable of simulating the actual rolling process and thereby designing roll-pass schedule in rolling of an airfoil or similar shape. The predictions from the CAD system were verified with respect to cold rolling of mild steel plates. The system is being applied to cold and hot isothermal rolling of an airfoil shape, and will be verified with respect to laboratory experiments under controlled conditions.

Optical Coherence Tomography Scan Circle Location and Mean Retinal Nerve Fiber Layer Measurement Variability

PubMed Central

Gabriele, Michelle L.; Ishikawa, Hiroshi; Wollstein, Gadi; Bilonick, Richard A.; Townsend, Kelly A.; Kagemann, Larry; Wojtkowski, Maciej; Srinivasan, Vivek J.; Fujimoto, James G.; Duker, Jay S.; Schuman, Joel S.

2009-01-01

PURPOSE To investigate the effect on optical coherence tomography (OCT) retinal nerve fiber layer (RNFL) thickness measurements of varying the standard 3.4-mm-diameter circle location. METHODS The optic nerve head (ONH) region of 17 eyes of 17 healthy subjects was imaged with high-speed, ultrahigh-resolution OCT (hsUHR-OCT; 501 × 180 axial scans covering a 6 × 6-mm area; scan time, 3.84 seconds) for a comprehensive sampling. This method allows for systematic simulation of the variable circle placement effect. RNFL thickness was measured on this three-dimensional dataset by using a custom-designed software program. RNFL thickness was resampled along a 3.4-mm-diameter circle centered on the ONH, then along 3.4-mm circles shifted horizontally (x-shift), vertically (y-shift) and diagonally up to ±500 µm (at 100-µm intervals). Linear mixed-effects models were used to determine RNFL thickness as a function of the scan circle shift. A model for the distance between the two thickest measurements along the RNFL thickness circular profile (peak distance) was also calculated. RESULTS RNFL thickness tended to decrease with both positive and negative x- and y-shifts. The range of shifts that caused a decrease greater than the variability inherent to the commercial device was greater in both nasal and temporal quadrants than in the superior and inferior ones. The model for peak distance demonstrated that as the scan moves nasally, the RNFL peak distance increases, and as the circle moves temporally, the distance decreases. Vertical shifts had a minimal effect on peak distance. CONCLUSIONS The location of the OCT scan circle affects RNFL thickness measurements. Accurate registration of OCT scans is essential for measurement reproducibility and longitudinal examination (ClinicalTrials.gov number, NCT00286637). PMID:18515577

Synthesis of boron nitride coatings on quartz fibers: Thickness control and mechanism research

NASA Astrophysics Data System (ADS)

Zheng, Yu; Wang, Shubin

2011-10-01

Boron nitride (BN) coatings were successfully synthesized on quartz fibers by dip-coating in boric acid and urea solutions at 700 °C. The SEM micrographs indicated that the quartz fibers were fully covered by coatings with smooth surface. The XRD, FT-IR, XPS spectra and HR-TEM results showed that the composition of the coatings which combined closely with the quartz fibers was polycrystalline h-BN. By changing the dip circles, the coating thickness was well controlled. The thicknesses of samples dipped less than six circles increased linearly with dipping-circles; and the increment of coating thickness would slow down when the fibers were dipped 10 circles. After being dipped for 10 circles, the thickness was about 300 nm. The coating thickness was also established by calculation and the calculated results were consistent with the results measured by micrograph.

Inflationary dynamics with a smooth slow-roll to constant-roll era transition

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

Odintsov, S.D.; Oikonomou, V.K., E-mail: odintsov@ieec.uab.es, E-mail: v.k.oikonomou1979@gmail.com

In this paper we investigate the implications of having a varying second slow-roll index on the canonical scalar field inflationary dynamics. We shall be interested in cases that the second slow-roll can take small values and correspondingly large values, for limiting cases of the function that quantifies the variation of the second slow-roll index. As we demonstrate, this can naturally introduce a smooth transition between slow-roll and constant-roll eras. We discuss the theoretical implications of the mechanism we introduce and we use various illustrative examples in order to better understand the new features that the varying second slow-roll index introduces.more » In the examples we will present, the second slow-roll index has exponential dependence on the scalar field, and in one of these cases, the slow-roll era corresponds to a type of α-attractor inflation. Finally, we briefly discuss how the combination of slow-roll and constant-roll may lead to non-Gaussianities in the primordial perturbations.« less

Computer-aided analysis and design of the shape rolling process for producing turbine engine airfoils

NASA Technical Reports Server (NTRS)

Lahoti, G. D.; Akgerman, N.; Altan, T.

1978-01-01

Mild steel (AISI 1018) was selected as model cold rolling material and Ti-6A1-4V and Inconel 718 were selected as typical hot rolling and cold rolling alloys, respectively. The flow stress and workability of these alloys were characterized and friction factor at the roll/workpiece interface was determined at their respective working conditions by conducting ring tests. Computer-aided mathematical models for predicting metal flow and stresses, and for simulating the shape rolling process were developed. These models utilized the upper bound and the slab methods of analysis, and were capable of predicting the lateral spread, roll separating force, roll torque, and local stresses, strains and strain rates. This computer-aided design system was also capable of simulating the actual rolling process, and thereby designing the roll pass schedule in rolling of an airfoil or a similar shape.

A Historical Note on the Proof of the Area of a Circle

ERIC Educational Resources Information Center

Wilamowsky, Yonah; Epstein, Sheldon; Dickman, Bernard

2011-01-01

Proofs that the area of a circle is nr[superscript 2] can be found in mathematical literature dating as far back as the time of the Greeks. The early proofs, e.g. Archimedes, involved dividing the circle into wedges and then fitting the wedges together in a way to approximate a rectangle. Later more sophisticated proofs relied on arguments…

The Revival of Research Circles: Meeting the Needs of Modern Aging and the Third Age

ERIC Educational Resources Information Center

Ostlund, Britt

2008-01-01

This article provides evidence that it is worthwhile to reconsider the traditional research circle method as a means of involving people in the third age in fulfilling their needs to participate in learning activities and make their voices heard. The findings are based on three cases of research circles consistently driven by the interests of the…

Providing a Full Circle of Support to Teachers in an Inclusive Elementary School

ERIC Educational Resources Information Center

Waldron, Nancy L.; Redd, Lacy

2011-01-01

Providing a full circle of support to teachers in an inclusive elementary school, the Newberry Elementary School (NES) principal and staff have worked for 5 years to ensure the inclusion of students with disabilities in general education classrooms. The authors would like to share their perceptions of how this full circle (the multiple systems) of…

Finding the Maximal Area of Bounded Polygons in a Circle

ERIC Educational Resources Information Center

Rokach, Arie

2005-01-01

The article deals with the area of polygons that are inscribed in a given circle. Naturally, the following question arises: Among all n-polygons that are inscribed in a given circle, which one has the biggest area? Intuitively, it may be guessed that is suitable for secondary students, and without any use id calculus, but only using very…

The Box and the Circle--Two Systems of Life: A Model for Understanding Native-Non-Native Issues.

ERIC Educational Resources Information Center

Derrick, Jann

Working as a family systems therapist with Native and non-Native families, the author observed two opposing social systems. Non-native families systems typify "The Box System," whereas native family systems portray "The Circle System." A few characteristics of the Circle System are: (1) a focus on life and peacefulness; (2) females and children…

Understanding Girls' Circle as an Intervention on Perceived Social Support, Body Image, Self-Efficacy, Locus of Control, and Self-Esteem

ERIC Educational Resources Information Center

Steese, Stephanie; Dollette, Maya; Phillips, William; Hossfeld, Elizabeth; Matthews, Gail; Taormina, Giovanna

2006-01-01

The Girls' Circle is a support group for adolescent girls developed by Beth Hossfeld and Giovanna Taormina as a unique program that addresses the needs of girls by focusing on increasing connections, building empathic skills, and developing resiliency. The present study evaluates the effectiveness of the Girls' Circle intervention on improving…

Implementing the patient circle. Call on patients to help improve perceptions of health care quality.

PubMed

Ostasiewski, P; Fugate, D L

1994-01-01

Adapting the quality-circle concept to a health care setting helped one hospital solve a problem and boosted its image among patients. The "patient circle" technique is one step health care providers can take toward delivering "total customer value," a quality perception that can mean the difference between surviving and thriving in the future.

DETAIL VIEW OF PIEDMONT AVENUE TRAFFIC CIRCLE AT INTERSECTION OF ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

DETAIL VIEW OF PIEDMONT AVENUE TRAFFIC CIRCLE AT INTERSECTION OF CHANNING WAY. SEEN FROM EAST SIDE OF CIRCLE LOOKING NORTH AT 2395 PIEDMONT, SIGMA PI HOUSE BY FREDERICK H. REIMERS, 1928. Photograph by Brian Grogan, July 8, 2007 - Piedmont Way & the Berkeley Property Tract, East of College Avenue between Dwight Way & U.C. Memorial Stadium, Berkeley, Alameda County, CA

Evolved Expendable Launch Vehicle (EELV)

DTIC Science & Technology

2015-12-15

FY13+ Phase I Buy Contractor: United Launch Services, LLC Contractor Location: 9501 East Panorama Circle Centennial , CO 80112 Contract Number...Contract Name: FY13+ Phase I Buy Contractor: United Launch Services, LLC Contractor Location: 9501 East Panorama Circle Centennial , CO 80112 Contract...FY12 EELV Launch Services (ELS5) Contractor: United Launch Services, LLC. Contractor Location: 9501 East Panorama Circle Centennial , CO 80112

Geometrical Solutions of Quadratic Equations.

ERIC Educational Resources Information Center

Grewal, A. S.; Godloza, L.

1999-01-01

Demonstrates that the equation of a circle (x-h)2 + (y-k)2 = r2 with center (h; k) and radius r reduces to a quadratic equation x2-2xh + (h2 + k2 -r2) = O at the intersection with the x-axis. Illustrates how to determine the center of a circle as well as a point on a circle. (Author/ASK)

Method for construction of normalized cDNA libraries

DOEpatents

Soares, Marcelo B.; Efstratiadis, Argiris

1998-01-01

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.

Method for construction of normalized cDNA libraries

DOEpatents

Soares, M.B.; Efstratiadis, A.

1998-11-03

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3` noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries. 19 figs.

Sometimes area counts more than number.

PubMed

Hurewitz, Felicia; Gelman, Rochel; Schnitzer, Brian

2006-12-19

Using an interference paradigm, we show across three experiments that adults' order judgments of numbers, sizes, or combined area of dots in pairs of arrays occur spontaneously and automatically, but at different speeds and levels of accuracy. Experiment 1 used circles whose sizes varied between but not within arrays. Variation in circle size interfered with judgments of which array had more circles. Experiment 2 used displays in which circle size varied within and between arrays. Between-array differences in the amount of "circle stuff" (area occupied by circles) interfered with judgments of number. Experiment 3 examined whether variation in number also interferes with judgments of area. Interference between discrete and continuous stimulus dimensions occurred in both directions, although it was stronger from the continuous to the discrete than vice versa. These results bear on interpretations of studies with infants and preschoolers wherein subjects respond on the basis of continuous quantity rather than discrete quantity. In light of our results with adults, these findings do not license the conclusion that young children cannot represent discrete quantity. Absent data on attentional hierarchies and speed of processing, it is premature to conclude that infant and child quantity processes are fundamentally different from that of adults.

Walking straight into circles.

PubMed

Souman, Jan L; Frissen, Ilja; Sreenivasa, Manish N; Ernst, Marc O

2009-09-29

Common belief has it that people who get lost in unfamiliar terrain often end up walking in circles. Although uncorroborated by empirical data, this belief has widely permeated popular culture. Here, we tested the ability of humans to walk on a straight course through unfamiliar terrain in two different environments: a large forest area and the Sahara desert. Walking trajectories of several hours were captured via global positioning system, showing that participants repeatedly walked in circles when they could not see the sun. Conversely, when the sun was visible, participants sometimes veered from a straight course but did not walk in circles. We tested various explanations for this walking behavior by assessing the ability of people to maintain a fixed course while blindfolded. Under these conditions, participants walked in often surprisingly small circles (diameter < 20 m), though rarely in a systematic direction. These results rule out a general explanation in terms of biomechanical asymmetries or other general biases [1-6]. Instead, they suggest that veering from a straight course is the result of accumulating noise in the sensorimotor system, which, without an external directional reference to recalibrate the subjective straight ahead, may cause people to walk in circles.

An unusual case of olecranon tuberculosis.

PubMed

Megas, Panagiotis; Karageorgos, Athanasios; Gliatis, Ioannis; Marangos, Markos

2008-08-01

A 67-year-old woman presented with erythema and swelling over her left elbow. She felt pain during palpation of the olecranon, while the range of elbow motion was slightly reduced (02 in circle-302 in circle-1302 in circle). She had been treated conservatively elsewhere for 3 months with nonsteroidal anti-inflammatory drugs and splint immobilization. Radiographs and computed tomography showed an osteolytic area over the olecranon with cortex disruption. She underwent open biopsy of the olecranon, and the histologic features were compatible with tuberculosis. Erythrocyte sedimentation rate was elevated and she had a positive Mantoux test (14 mm). She mentioned no history of pulmonary disease and had a normal chest radiograph. She received antituberculous treatment (isoniazid, rifambin, and pyrazinamide) for 2 months without obvious improvement. She underwent surgical debridement of the olecranon while she continued antituberculous treatment for 12 months, discontinuing pyrazinamide after the third month. After the completion of antituberculous treatment, the patient was pain-free, without local signs of infection and with improved range of motion (02 in circle-202 in circle-1452 in circle). Early diagnosis and treatment of olecranon tuberculosis is of great value, and the prognosis is good when there is no spread in the elbow joint.

A peptide derived from enzymatic digestion of globulins from amaranth shows strong affinity binding to the replication origin of Tomato yellow leaf curl virus reducing viral replication in Nicotiana benthamiana.

PubMed

Mendoza-Figueroa, J S; Kvarnheden, A; Méndez-Lozano, J; Rodríguez-Negrete, E-A; Arreguín-Espinosa de Los Monteros, R; Soriano-García, M

2018-02-01

Tomato yellow leaf curl virus (TYLCV; genus Begomovirus; family Geminiviridae) infects mainly plants of the family Solanaceae, and the infection induces curling and chlorosis of leaves, dwarfing of the whole plant, and reduced fruit production. Alternatives for direct control of TYLCV and other geminiviruses have been reported, for example, the use of esterified whey proteins, peptide aptamer libraries or artificial zinc finger proteins. The two latter alternatives affect directly the replication of TYLCV as well as of other geminiviruses because the replication structures and sequences are highly conserved within this virus family. Because peptides and proteins offer a potential solution for virus replication control, in this study we show the isolation, biochemical characterization and antiviral activity of a peptide derived from globulins of amaranth seeds (Amaranthus hypochondriacus) that binds to the replication origin sequence (OriRep) of TYLCV and affects viral replication with a consequent reduction of disease symptoms in Nicotiana benthamiana. Aromatic peptides obtained from papain digests of extracted globulins and albumins of amaranth were tested by intrinsic fluorescent titration and localized surface resonance plasmon to analyze their binding affinity to OriRep of TYLCV. The peptide AmPep1 (molecular weight 2.076 KDa) showed the highest affinity value (Kd = 1.8 nM) for OriRep. This peptide shares a high amino acid similarity with a part of an amaranth 11S globulin, and the strong affinity of AmPep1 could be explained by the presence of tryptophan and lysine facilitating interaction with the secondary structure of OriRep. In order to evaluate the effect of the peptide on in vitro DNA synthesis, rolling circle amplification (RCA) was performed using as template DNA from plants infected with TYLCV or another begomovirus, pepper huasteco yellow vein virus (PHYVV), and adding AmPep1 peptide at different concentrations. The results showed a decrease in DNA synthesis of both viruses at increasing concentrations of AmPep1. To further confirm the antiviral activity of the peptide in vivo, AmPep1 was infiltrated into leaves of N. benthamiana plants previously infected with TYLCV. Plants treated with AmPep1 showed a significant decrease in virus titer compared with untreated N. benthamiana plants as well as reduced symptom progression due to the effect of AmPep1 curtailing TYLCV replication in the plant. The peptide also showed antiviral activity in plants infected with PHYVV. This is the first report, in which a peptide is directly used for DNA virus control in plants, supplied as exogenous application and without generation of transgenic lines. Copyright © 2018 Elsevier Inc. All rights reserved.

Suppression of cotton leaf curl disease symptoms in Gossypium hirsutum through over expression of host-encoded miRNAs.

PubMed

Akmal, Mohd; Baig, Mirza S; Khan, Jawaid A

2017-12-10

Cotton leaf curl disease (CLCuD), a major factor resulting in the enormous yield losses in cotton crop, is caused by a distinct monopartite begomovirus in association with Cotton leaf curl Multan betasatellite (CLCuMB). Micro(mi)RNAs are known to regulate gene expression in eukaryotes, including antiviral defense in plants. In a previous study, we had computationally identified a set of cotton miRNAs, which were shown to have potential targets in the genomes of Cotton leaf curl Multan virus (CLCuMuV) and CLCuMB at multiple loci. In the current study, effect of Gossypium arboreum-encoded miRNAs on the genome of CLCuMuV and CLCuMB was investigated in planta. Two computationally predicted cotton-encoded miRNAs (miR398 and miR2950) that showed potential to bind multiple Open Reading Frames (ORFs; C1, C4, V1, and non- coding intergenic region) of CLCuMuV, and (βC1) of CLCuMB were selected. Functional validation of miR398 and miR2950 was done by overexpression approach in G. hirsutum var. HS6. A total of ten in vitro cotton plants were generated from independent events and subjected to biological and molecular analyses. Presence of the respective Precursor (pre)-miRNA was confirmed through PCR and Southern blotting, and their expression level was assessed by semi quantitative RT-PCR, Real Time quantitative PCR and northern hybridization in the PCR-positive lines. Southern hybridization revealed 2-4 copy integration of T-DNA in the genome of the transformed lines. Remarkably, expression of pre-miRNAs was shown up to 5.8-fold higher in the transgenic (T 0 ) lines as revealed by Real Time PCR. The virus resistance was monitored following inoculation of the transgenic cotton lines with viruliferous whitefly (Bemisia tabaci) insect vector. After inoculation, four of the transgenic lines remained apparently symptom free. While a very low titre of viral DNA could be detected by Rolling circle amplification, betasatellite responsible for symptom induction could not be detected in any of the healthy looking transgenic lines. In this study for the first time, efficacy of the host (G. arboreum)-encoded miRNAs against CLCuD symptoms was experimentally demonstrated through overexpression of miR398 and miR2950 in G. hirsutum var. HS6 plants. Computational prediction of miRNAs targeting virus genome and their subsequent implication in translational inhibition or cleavage based suppression of viral mRNA via overexpression could help in generating virus resistant plants. Copyright © 2017 Elsevier B.V. All rights reserved.

A model for prediction of profile and flatness of hot and cold rolled flat products in four-high mills

NASA Astrophysics Data System (ADS)

Overhagen, Christian; Mauk, Paul Josef

2018-05-01

For flat rolled products, the thickness profile in the transversal direction is one of the most important product properties. For further processing, a defined crown of the product is necessary. In the rolling process, several mechanical and thermal influences interact with each other to form the strip shape at the roll gap exit. In the present analysis, a process model for rolling of strip and sheet is presented. The core feature of the process model is a two-dimensional stress distribution model based on von Karman's differential equation. Sub models for the mechanical influences of work roll flattening as well as work and backup roll deflection and the thermal influence of work roll expansion have been developed or extended. The two-dimensional stress distribution serves as an input parameter for the roll deformation models. For work roll flattening, a three-dimensional model based on the Boussinesq problem is adopted, while the work and backup roll deflection, including contact flattening is calculated by means of finite beam elements. The thermal work roll crown is calculated with help of an axisymmetric numerical solution of the heat equation for the work roll, considering azimuthal averaging for the boundary conditions at the work roll surface. Results are presented for hot rolling of a strip in a seven-stand finishing train of a hot strip mill, showing the calculated evolution of the strip profile. A variation of the strip profile from the first to the 20th rolled strip is shown. This variation is addressed to the progressive increase of work roll temperature during the first 20 strips. It is shown that a CVC® system can lead to improvements in strip profile and therefore flatness.

Finite-element model to predict roll-separation force and defects during rolling of U-10Mo alloys

NASA Astrophysics Data System (ADS)

Soulami, Ayoub; Burkes, Douglas E.; Joshi, Vineet V.; Lavender, Curt A.; Paxton, Dean

2017-10-01

A major goal of the Convert Program of the U.S. Department of Energy's National Nuclear Security Administration (DOE/NNSA) is to enable high-performance research reactors to operate with low-enriched uranium rather than the high-enriched uranium currently used. To this end, uranium alloyed with 10 wt% molybdenum (U-10Mo) represents an ideal candidate because of its stable gamma phase, low neutron caption cross section, acceptable swelling response, and predictable irradiation behavior. However, because of the complexities of the fuel design and the need for rolled monolithic U-10Mo foils, new developments in processing and fabrication are necessary. This study used a finite-element code, LS-DYNA, as a predictive tool to optimize the rolling process. Simulations of the hot rolling of U-10Mo coupons encapsulated in low-carbon steel were conducted following two different schedules. Model predictions of the roll-separation force and roll pack thicknesses at different stages of the rolling process were compared with experimental measurements. The study reported here discussed various attributes of the rolled coupons revealed by the model (e.g., waviness and thickness non-uniformity like dog-boning). To investigate the influence of the cladding material on these rolling defects, other cases were simulated: hot rolling with alternative can materials, namely, 304 stainless steel and Zircaloy-2, and bare-rolling. Simulation results demonstrated that reducing the mismatch in strength between the coupon and can material improves the quality of the rolled sheet. Bare-rolling simulation results showed a defect-free rolled coupon. The finite-element model developed and presented in this study can be used to conduct parametric studies of several process parameters (e.g., rolling speed, roll diameter, can material, and reduction).

Effect of Rolling Massage on the Vortex Flow in Blood Vessels with Lattice Boltzmann Simulation

NASA Astrophysics Data System (ADS)

Yi, Hou Hui

The rolling massage manipulation is a classic Chinese Medical Massage, which is a nature therapy in eliminating many diseases. Here, the effect of the rolling massage on the cavity flows in blood vessel under the rolling manipulation is studied by the lattice Boltzmann simulation. The simulation results show that the vortex flows are fully disturbed by the rolling massage. The flow behavior depends on the rolling velocity and the rolling depth. Rolling massage has a better effect on the flows in the cavity than that of the flows in a planar blood vessel. The result is helpful to understand the mechanism of the massage and develop the rolling techniques.

Orthonormal vector general polynomials derived from the Cartesian gradient of the orthonormal Zernike-based polynomials.

PubMed

Mafusire, Cosmas; Krüger, Tjaart P J

2018-06-01

The concept of orthonormal vector circle polynomials is revisited by deriving a set from the Cartesian gradient of Zernike polynomials in a unit circle using a matrix-based approach. The heart of this model is a closed-form matrix equation of the gradient of Zernike circle polynomials expressed as a linear combination of lower-order Zernike circle polynomials related through a gradient matrix. This is a sparse matrix whose elements are two-dimensional standard basis transverse Euclidean vectors. Using the outer product form of the Cholesky decomposition, the gradient matrix is used to calculate a new matrix, which we used to express the Cartesian gradient of the Zernike circle polynomials as a linear combination of orthonormal vector circle polynomials. Since this new matrix is singular, the orthonormal vector polynomials are recovered by reducing the matrix to its row echelon form using the Gauss-Jordan elimination method. We extend the model to derive orthonormal vector general polynomials, which are orthonormal in a general pupil by performing a similarity transformation on the gradient matrix to give its equivalent in the general pupil. The outer form of the Gram-Schmidt procedure and the Gauss-Jordan elimination method are then applied to the general pupil to generate the orthonormal vector general polynomials from the gradient of the orthonormal Zernike-based polynomials. The performance of the model is demonstrated with a simulated wavefront in a square pupil inscribed in a unit circle.

75 FR 62566 - Hot-Rolled Flat-Rolled Carbon-Quality Steel Products From Brazil, Japan, and Russia

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-12

...)] Hot-Rolled Flat-Rolled Carbon-Quality Steel Products From Brazil, Japan, and Russia AGENCY: United... antidumping duty investigation on hot-rolled steel from Russia. SUMMARY: The Commission hereby gives notice of... suspended investigation on hot-rolled steel from Russia would be likely to lead to continuation or...

76 FR 34101 - Hot-Rolled Flat-Rolled Carbon-Quality Steel Products From Brazil, Japan, and Russia

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-10

...] Hot-Rolled Flat-Rolled Carbon-Quality Steel Products From Brazil, Japan, and Russia Determinations On...-quality steel products from Russia would be likely to lead to continuation or recurrence of material...) entitled Hot-Rolled Flat-Rolled Carbon-Quality Steel Products from Brazil, Japan, and Russia: Investigation...

75 FR 16504 - Hot-Rolled Flat-Rolled Carbon-Quality Steel Products From Brazil, Japan, and Russia

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-01

...)] Hot-Rolled Flat-Rolled Carbon-Quality Steel Products From Brazil, Japan, and Russia AGENCY: United... investigation on hot-rolled steel from Russia. SUMMARY: The Commission hereby gives notice that it has...-rolled steel from Russia would be likely to lead to continuation or recurrence of material injury...

75 FR 42782 - Hot-Rolled Flat-Rolled Carbon-Quality Steel Products From Brazil, Japan, and Russia

Federal Register 2010, 2011, 2012, 2013, 2014

2010-07-22

...)] Hot-Rolled Flat-Rolled Carbon-Quality Steel Products From Brazil, Japan, and Russia AGENCY: United... Brazil and Japan, and the suspended investigation on hot-rolled steel from Russia. SUMMARY: The... Japan, and the suspended investigation on hot-rolled steel from Russia would be likely to lead to...

On the Influence of Surface Heterogeneities onto Roll Convection

NASA Astrophysics Data System (ADS)

Gryschka, M.; Drüe, C.; Raasch, S.; Etling, D.

2009-04-01

Roll convection is a common phenomenon in atmospheric convective boundary layers (CBL) with background wind. Roll convection is observed both over land and over sea for different synoptic situations. There is still some debate about the different types of roll convection and their causes or rather the necessary conditions for their appearance. The stability parameter ζ = -zi•L (zi: boundary layer height, L: Monin-Obukhov stability length) is widely used as a predictor for roll convection, since numerous studies suggest that convective rolls only appear when 0 < ζ < 20. In other words, roll development becomes unlikely for strong surface heating and weak vertical wind shear. In contrast to those studies the presence of roll convection in almost any polar cold air outbreak (as can be seen in numerous satellite images as cloud streets) reveals that even for large ζ roll convection can develop. Some studies report roll convection in cold air outbreaks for ζ = 250. Our large eddy simulations (LES) on roll convection suggests that the contrasting results concerning the dependency of roll convection on ζ are due to two different types of roll convection: One type which develops purely by self organization if ζ < 20 ("free rolls") and another type which is triggered by heterogeneities in surface temperature and develops also for large ζ ("forced rolls"). We think that most of the cloud streets observed in polar cold air outbreaks over open water are due to rolls of forced type which are tied to upstream located heterogeneities in the sea-ice distribution. The results of this study suggests that the omission of surface inhomogeneities in previous LES is the reason for the absence of rolls in all LES with strong surface heating and weak vertical wind shear so far. In this contribution we will present a large eddy simulation which successfully represents forced rolls under such conditions.

Static roll-tilt over 5 minutes locally distorts the internal estimate of direction of gravity.

PubMed

Tarnutzer, A A; Bockisch, C J; Straumann, D; Marti, S; Bertolini, G

2014-12-01

The subjective visual vertical (SVV) indicates perceived direction of gravity. Even in healthy human subjects, roll angle-dependent misestimations, roll overcompensation (A-effect, head-roll > 60° and <135°) and undercompensation (E-effect, head-roll < 60°), occur. Previously, we demonstrated that, after prolonged roll-tilt, SVV estimates when upright are biased toward the preceding roll position, which indicates that perceived vertical (PV) is shifted by the prior tilt (Tarnutzer AA, Bertolini G, Bockisch CJ, Straumann D, Marti S. PLoS One 8: e78079, 2013). Hypothetically, PV in any roll position could be biased toward the previous roll position. We asked whether such a "global" bias occurs or whether the bias is "local". The SVV of healthy human subjects (N = 9) was measured in nine roll positions (-120° to +120°, steps = 30°) after 5 min of roll-tilt in one of two adaptation positions (±90°) and compared with control trials without adaptation. After adapting, adjustments were shifted significantly (P < 0.05) toward the previous adaptation position for nearby roll-tilted positions (±30°, ±60°) and upright only. We computationally simulated errors based on the sum of a monotonically increasing function (producing roll undercompensation) and a mixture of Gaussian functions (representing roll overcompensation centered around PV). In combination, the pattern of A- and E-effects could be generated. By shifting the function representing local overcompensation toward the adaptation position, the experimental postadaptation data could be fitted successfully. We conclude that prolonged roll-tilt locally distorts PV rather than globally shifting it. Short-term adaptation of roll overcompensation may explain these shifts and could reflect the brain's strategy to optimize SVV estimates around recent roll positions. Thus postural stability can be improved by visually-mediated compensatory responses at any sustained body-roll orientation. Copyright © 2014 the American Physiological Society.

On the impact of forced roll convection on vertical turbulent transport in cold air outbreaks

NASA Astrophysics Data System (ADS)

Gryschka, Micha; Fricke, Jens; Raasch, Siegfried

2014-11-01

We investigated the impact of roll convection on the convective boundary layer and vertical transports in different cold air outbreak (CAO) scenarios using large eddy simulations (LES). The organization of convection into rolls was triggered by upstream heterogeneities in the surface temperature, representing ice and water. By changing the sea ice distribution in our LES, we were able to simulate a roll and a nonroll case for each scenario. Furthermore, the roll wavelength was varied by changing the scale of the heterogeneity. The characteristics of the simulated rolls and cloud streets, such as aspect ratios, orientation of the roll axes, and downstream extensions of single rolls agreed closely with observations in CAO situations. The vertical turbulent fluxes, calculated for each simulation, were decomposed into contributions from rolls and from unorganized turbulence. Even though our results confirmed that rolls triggered by upstream heterogeneities can substantially contribute to vertical turbulent fluxes, the total fluxes were not affected by the rolls.

Coupled thermal-fluid-mechanics analysis of twin roll casting of A7075 aluminum alloy

NASA Astrophysics Data System (ADS)

Lee, Yun-Soo; Kim, Hyoung-Wook; Cho, Jae-Hyung; Chun, Se-Hwan

2017-09-01

Better understanding of temperature distribution and roll separation force during twin roll casting of aluminum alloys is critical to successfully fabricate good quality of aluminum strips. Therefore, the simulation techniques are widely applied to understand the twin roll casting process in a comprehensive way and to reduce the experimental time and cost of trial and error. However, most of the conventional approaches are considered thermally coupled flow, or thermally coupled mechanical behaviors. In this study, a fully coupled thermal-fluid-mechanical analysis of twin roll casting of A7075 aluminum strips was carried out using the finite element method. Temperature profile, liquid fraction and metal flow of aluminum strips with different thickness were predicted. Roll separation force and roll temperatures were experimentally obtained from a pilot-scale twin roll caster, and those results were compared with model predictions. Coupling the fluid of the liquid melt to the thermal and mechanical modeling reasonably predicted roll temperature distribution and roll separation force during twin roll casting.

A Study of Traditional Circle Games of Five-Year-Old United States Public School Kindergarten Children.

ERIC Educational Resources Information Center

Rothlein, Liz; Einspruch, Eric

A total of 203 teachers of 5-year-olds in public school kindergartens representing 35 states responded to a questionnaire about traditional circle games. The teachers were asked to identify three of the favorite traditional circle games used in their classrooms and to indicate how often and how long the games were played. In addition, teachers…

Acquiring Combat Capability through Innovative Uses of Public-Private Partnerships

DTIC Science & Technology

2006-06-01

needed for a vital fiber -optic link near the Arctic Circle. Then, we will explore the history of the Energy Saving Performance Contracts (ESPCs...research examines Hannon Armstrong’s “fee for service contract” solution to funding the vital fiber -optic link near the Arctic Circle. The second...5 A. INNOVATIVE USE OF FEE FOR SERVICE CONTRACT .....................5 1. Introduction to the Arctic Circle Fiber

The Establishment of a Quality Circles Program and Its Effect on Employee Attitudes, Morale, and Performance at an Army Medical Treatment Facility.

DTIC Science & Technology

1984-08-01

more times. Dr. Kaoru Ishikawa , a professor at the University of Tokyo and a board member of JUSE, used the efforts of Dr. Juran as the background for...his later work with quality circles. Dr. Ishikawa and the JUSE are generally credited with formalizing the modern Japanese quality circle technique in

Fostering Positive Peer Relations in the Primary Classroom through Circle Time and Co-Operative Games

ERIC Educational Resources Information Center

Mary, Latisha

2014-01-01

The aim of this study was to investigate the role of co-operative games and circle time activities in fostering positive peer relations in two French Primary classrooms (N = 40). It presents French teachers' and pupils' perceptions of a set of co-operative games and circle time activities implemented within a year long study on personal, social…

Media Literacy as Mindful Practice for Democratic Education. A Response to "Transaction Circles with Digital Texts as a Foundation for Democratic Practices"

ERIC Educational Resources Information Center

Redmond, Theresa

2016-01-01

This essay is a response to Brown's (2015) article describing her strategy of transaction circles as a student-centered, culturally responsive, and democratic literacy practice. In my response, I provide further evidence from the field of media literacy education (MLE) that serves to enhance Brown's argument for using transaction circles in order…

Air Vehicle Path Planning

DTIC Science & Technology

2001-12-13

6-18 6.13. Apollonius Circle for the Case of Two Unequal Power Radars . . . 6-20 6.14. Solution Triangle...Voronoi edge is an Apollonius circle [32, 19]. In this section, we are concerned with the optimality of the Voronoi path for the two radar exposure...Comparison of Cost vs. Path Length for Constrained Trajectories Around and Between Two Radars 6-18 from the two radars is an Apollonius circle

Short-Term Impacts of Student Listening Circles on Student Perceptions of School Climate and of Their Own Competencies. REL 2017-210

ERIC Educational Resources Information Center

Hanson, Thomas; Polik, Jeff; Cerna, Rebeca

2017-01-01

An activity for eliciting student involvement in collaborative decision-making and problem-solving with adults--the student listening circle workshop--is examined for the first time through an experimental study of its effects on participating students. A student listening circle is a facilitated focus group in which students articulate to adults…

Study Circles at the Pharmacy--A New Model for Diabetes Education in Groups.

ERIC Educational Resources Information Center

Sarkadi, Anna; Rosenqvist, Urban

1999-01-01

Tests the feasibility of a one-year group education model for patients with type 2 diabetes in Sweden. Within study circles led by pharmacists, participants learned to self-monitor glucose, to interpret the results and to act upon them. Results show that study circles held at pharmacies are a feasible way of education persons with type 2 diabetes.…

Talking Circles for Adolescent Girls in an Urban High School: A Restorative Practices Program for Building Friendships and Developing Emotional Literacy Skills

ERIC Educational Resources Information Center

Schumacher, Martha Ann

2012-01-01

Restorative Practices and Restorative Justice programs in schools are a new and emerging field. Meeting in Circles to build community, resolve conflict, and learn interactively are core components of these programs. This ethnographic study, which took place February 2010 to December 2011, evaluates 12 small, out-of-classroom Talking Circles for 60…

CIRCLE Enhancement After Myopic SMILE.

PubMed

Siedlecki, Jakob; Luft, Nikolaus; Mayer, Wolfgang J; Siedlecki, Martin; Kook, Daniel; Meyer, Bertram; Bechmann, Martin; Wiltfang, Rainer; Priglinger, Siegfried G; Dirisamer, Martin

2018-05-01

To report the outcomes of enhancement after small incision lenticule extraction (SMILE) using the VisuMax CIRCLE option (Carl Zeiss Meditec AG, Jena, Germany), which converts the SMILE cap into a femtosecond LASIK flap for secondary excimer laser application. Of 2,065 SMILE procedures, 22 eyes (1.1%) re-treated with CIRCLE with a follow-up of 3 months were included in the analysis. SMILE was performed in the usual manner. For re-treatment, the CIRCLE procedure was performed with pattern D flap creation on the VisuMax system and subsequent excimer laser ablation with a Zeiss MEL 90 laser (Carl Zeiss Meditec) with plano target in all cases. Spherical equivalent was -5.56 ± 2.22 diopters (D) before SMILE and -0.51 ± 1.08 D before CIRCLE. CIRCLE enhancement was performed after a mean of 10.0 ± 7.9 months, allowed for safe flap lifting in all eyes, and resulted in a final manifest refraction spherical equivalent of 0.18 ± 0.31 D at 3 months (P < .008). The number of eyes within 0.50 and 1.00 D from target refraction increased from 31.8% to 90.9% and from 77.3% to 100%, respectively. Mean uncorrected distance visual acuity (UDVA) had already improved from 0.37 ± 0.16 to 0.08 ± 0.16 logMAR at 1 week (P < .0001), resulting in 0.03 ± 0.07 logMAR at 3 months (P < .0001). All eyes gained at least one line of UDVA. Corrected distance visual acuity (CDVA) remained unchanged at all time points (before vs after CIRCLE, P = .40). Two eyes (9.1 %) lost one line of CDVA; no eye lost two or more lines. The safety and efficacy indices were 1.03 and 0.97 at 3 months. The CIRCLE procedure represents an effective re-treatment option after SMILE. Compared to surface ablation re-treatment after SMILE, CIRCLE seems to offer advantages in respect to speed of visual recovery, safety, and predictability, but at the price of flap creation. [J Refract Surg. 2018;34(5):304-309.]. Copyright 2018, SLACK Incorporated.

75 FR 75455 - Certain Hot-Rolled Flat-Rolled Carbon-Quality Steel Products From Brazil: Final Results of Full...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-03

...-Rolled Carbon-Quality Steel Products From Brazil: Final Results of Full Sunset Review of Countervailing... of the countervailing duty (CVD) order on certain hot-rolled flat-rolled carbon-quality steel products (hot-rolled steel) from Brazil, pursuant to section 751(c) of the Tariff Act of 1930, as amended...

21 CFR 136.180 - Whole wheat bread, rolls, and buns.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 2 2010-04-01 2010-04-01 false Whole wheat bread, rolls, and buns. 136.180... § 136.180 Whole wheat bread, rolls, and buns. (a) Each of the foods whole wheat bread, graham bread, entire wheat bread, whole wheat rolls, graham rolls, entire wheat rolls, whole wheat buns, graham buns...

Noncanonical self-assembly of multifunctional DNA nanoflowers for biomedical applications.

PubMed

Zhu, Guizhi; Hu, Rong; Zhao, Zilong; Chen, Zhuo; Zhang, Xiaobing; Tan, Weihong

2013-11-06

DNA nanotechnology has been extensively explored to assemble various functional nanostructures for versatile applications. Mediated by Watson-Crick base-pairing, these DNA nanostructures have been conventionally assembled through hybridization of many short DNA building blocks. Here we report the noncanonical self-assembly of multifunctional DNA nanostructures, termed as nanoflowers (NFs), and the versatile biomedical applications. These NFs were assembled from long DNA building blocks generated via rolling circle replication (RCR) of a designer template. NF assembly was driven by liquid crystallization and dense packaging of building blocks, without relying on Watson-Crick base-pairing between DNA strands, thereby avoiding the otherwise conventional complicated DNA sequence design. NF sizes were readily tunable in a wide range, by simply adjusting such parameters as assembly time and template sequences. NFs were exceptionally resistant to nuclease degradation, denaturation, or dissociation at extremely low concentration, presumably resulting from the dense DNA packaging in NFs. The exceptional biostability is critical for biomedical applications. By rational design, NFs can be readily incorporated with myriad functional moieties. All these properties make NFs promising for versatile applications. As a proof-of-principle demonstration, in this study, NFs were integrated with aptamers, bioimaging agents, and drug loading sites, and the resultant multifunctional NFs were demonstrated for selective cancer cell recognition, bioimaging, and targeted anticancer drug delivery.

Self-assembled Multifunctional DNA Nanoflowers for the Circumvention of Multidrug Resistance in Targeted Anticancer Drug Delivery.

PubMed

Mei, Lei; Zhu, Guizhi; Qiu, Liping; Wu, Cuichen; Chen, Huapei; Liang, Hao; Cansiz, Sena; Lv, Yifan; Zhang, Xiaobing; Tan, Weihong

2015-11-01

Cancer chemotherapy has been impeded by side effects and multidrug resistance (MDR) partially caused by drug efflux from cancer cells, which call for targeted drug delivery systems additionally able to circumvent MDR. Here we report multifunctional DNA nanoflowers (NFs) for targeted drug delivery to both chemosensitive and MDR cancer cells and circumvent MDR in both leukemia and breast cancer cell models. NFs are self-assembled via liquid crystallization of DNA generated by Rolling Circle Replication, during which NFs are incorporated with aptamers for specific cancer cell recognition, fluorophores for bioimaging, and Doxorubicin (Dox)-binding DNA for drug delivery. NF sizes are tunable (down to ~200 nm in diameter), and the densely packed drug-binding motifs and porous intrastructures endow NFs with high drug loading capacity (71.4%, wt/wt). The Dox-loaded NFs (NF-Dox) are stable at physiological pH, yet drug release is facilitated in acidic or basic conditions. NFs deliver Dox into target chemosensitive and MDR cancer cells, preventing drug efflux and enhancing drug retention in MDR cells. Consequently, NF-Dox induces potent cytotoxicity in both target chemosensitive cells and MDR cells, but not nontarget cells, thus concurrently circumventing MDR and reducing side effects. Overall, these NFs are promising to circumvent MDR in targeted cancer therapy.

Noncanonical self-assembly of multifunctional DNA nanoflowers for biomedical applications

PubMed Central

Zhu, Guizhi; Hu, Rong; Zhao, Zilong; Chen, Zhuo; Zhang, Xiaobing; Tan, Weihong

2013-01-01

DNA nanotechnology has been extensively explored to assemble various functional nanostructures for versatile applications. Mediated by Watson-Crick base-pairing, these DNA nanostructures have been conventionally assembled through hybridization of many short DNA building blocks. Here we report the noncanonical self-assembly of multifunctional DNA nanostructures, termed as nanoflowers (NFs), and the versatile biomedical applications. These NFs were assembled from long DNA building blocks generated via Rolling Circle Replication (RCR) of a designer template. NF assembly was driven by liquid crystallization and dense packaging of building blocks, without relying on Watson-Crick base-pairing between DNA strands, thereby avoiding the otherwise conventional complicated DNA sequence design. NF sizes were readily tunable in a wide range, by simply adjusting such parameters as assembly time and template sequences. NFs were exceptionally resistant to nuclease degradation, denaturation, or dissociation at extremely low concentration, presumably resulting from the dense DNA packaging in NFs. The exceptional biostability is critical for biomedical applications. By rational design, NFs can be readily incorporated with myriad functional moieties. All these properties make NFs promising for versatile applications. As a proof-of-principle demonstration, in this study, NFs were integrated with aptamers, bioimaging agents, and drug loading sites, and the resultant multifunctional NFs were demonstrated for selective cancer cell recognition, bioimaging, and targeted anticancer drug delivery. PMID:24164620

Subcellular Localization and Rolling Circle Replication of Peach Latent Mosaic Viroid: Hallmarks of Group A Viroids

PubMed Central

Bussière, F.; Lehoux, J.; Thompson, D. A.; Skrzeczkowski, L. J.; Perreault, J.-P.

1999-01-01

We characterized the peach latent mosaic viroid (PLMVd) replication intermediates that accumulate in infected peach leaves and determined the tissue and subcellular localization of the RNA species. Using in situ hybridization, we showed that PLMVd strands of both plus and minus polarities concentrate in the cells forming the palisade parenchyma. At the cellular level, PLMVd was found to accumulate predominantly in chloroplasts. Northern blot analyses demonstrated that PLMVd replicates via a symmetric mode involving the accumulation of both circular and linear monomeric strands of both polarities. No multimeric conformer was detected, indicating that both strands self-cleave efficiently via their hammerhead sequences. Dot blot hybridizations revealed that PLMVd strands of both polarities accumulate equally but that the relative concentrations vary by more than 50-fold between peach cultivars. Taken together these results establish two hallmarks for the classification of viroids. Group A viroids (e.g., PLMVd), which possess hammerhead structures, replicate in the chloroplasts via the symmetric mode. By contrast, group B viroids, which share a conserved central region, replicate in the nucleus via an asymmetric mechanism. This is an important difference between self-cleaving and non-self-cleaving viroids, and the implications for the evolutionary origin and replication are discussed. PMID:10400727

DNA packaging by the Bacillus subtilis defective bacteriophage PBSX.

PubMed Central

Anderson, L M; Bott, K F

1985-01-01

Defective bacteriophage PBSX, a resident of all Bacillus subtilis 168 chromosomes, packages fragments of DNA from all portions of the host chromosome when induced by mitomycin C. In this study, the physical process for DNA packaging of both chromosomal and plasmid DNAs was examined. Discrete 13-kilobase (kb) lengths of DNA were packaged by wild-type phage, and the process was DNase I resistant and probably occurred by a head-filling mechanism. Genetically engineered isogenic host strains having a chloramphenicol resistance determinant integrated as a genetic flag at two different regions of the chromosome were used to monitor the packaging of specific chromosomal regions. No dramatic selectivity for these regions could be documented. If the wild-type strain 168 contains autonomously replicating plasmids, especially pC194, the mitomycin C induces an increase in size of resident plasmid DNA, which is then packaged as 13-kb pieces into phage heads. In strain RB1144, which lacks substantial portions of the PBSX resident phage region, mitomycin C treatment did not affect the structure of resident plasmids. Induction of PBSX started rolling circle replication on plasmids, which then became packaged as 13-kb fragments. This alteration or cannibalization of plasmid replication resulting from mitomycin C treatment requires for its function some DNA within the prophage deletion of strain RB1144. Images PMID:3923209

Characterization of the replication region of the Bacillus subtilis plasmid pLS20: a novel type of replicon.

PubMed Central

Meijer, W J; de Boer, A J; van Tongeren, S; Venema, G; Bron, S

1995-01-01

A 3.1 kb fragment of the large (approximately 55 kb) Bacillus subtilis plasmid pLS20 containing all the information for autonomous replication was cloned and sequenced. In contrast to the parental plasmid, derived minireplicons were unstably maintained. Using deletion analysis the fragment essential and sufficient for replication was delineated to 1.1 kb. This 1.1 kb fragment is located between two divergently transcribed genes, denoted orfA and orfB, neither of which is required for replication. orfA shows homology to the B.subtilis chromosomal genes rapA (spoOL, gsiA) and rapB (spoOP). The 1.1 kb fragment, which is characterized by the presence of several regions of dyad symmetry, contains no open reading frames of more than 85 codons and shows no similarity with other known plasmid replicons. The structural organization of the pLS20 minimal replicon is entirely different from that of typical rolling circle plasmids from Gram-positive bacteria. The pLS20 minireplicons replicate in polA5 and recA4 B.subtilis strains. Taken together, these results strongly suggest that pLS20 belongs to a new class of theta replicons. PMID:7667098

The Work Softening by Deformation-Induced Disordering and Cold Rolling of 6.5 wt pct Si Steel Thin Sheets

NASA Astrophysics Data System (ADS)

Wang, Xianglong; Li, Haoze; Zhang, Weina; Liu, Zhenyu; Wang, Guodong; Luo, Zhonghan; Zhang, Fengquan

2016-09-01

As-cast strip of 6.5 wt pct Si steel was fabricated by twin-roll strip casting. After hot rolling at 1323 K (1050 °C), thin sheets with the thickness of 0.35 mm were produced by warm rolling at 373 K (100 °C) with rolling reductions of 15, 25, 35, 45, 55, and 65 pct. Influence of warm rolling reduction on ductility was investigated by room temperature bending test. The measurement of macro-hardness showed that "work softening" could begin when the warm rolling reduction exceeded 35 pct. The room temperature ductility of the thin sheets gradually increased with the increase of warm rolling reductions, and the plastic deformation during bending began to form when the warm rolling reduction was greater than 45 pct, the 65 pct rolled thin sheet exhibited the maximum plastic deformation of about 0.6 pct during bending at room temperature, with a few small dimples having been observed on the fracture surfaces. B2-ordered domains were formed in the 15, 25, 35, 45, and 55 pct rolled specimens, and their average size decreased with the increase of warm rolling reductions. By contrast, no B2-ordered domain could be found in the 65 pct rolled specimen. It had been observed that large-ordered domains could be split into several small parts by the slip of partial super-dislocations during warm rolling, which led to significant decrease of the order degree to cause the phenomenon of deformation-induced disordering. On the basis of these results, cold rolling schedule was developed to successfully fabricate 0.25-mm-thick sheets with good surface qualities and magnetic properties from warm rolled sheets.

Texture and mechanical properties of Al-0.5Mg-1.0Si-0.5Cu alloy sheets manufactured via a cross rolling method

NASA Astrophysics Data System (ADS)

Jeon, Jae-Yeol; Son, Hyeon-Taek; Woo, Kee-Do; Lee, Kwang-Jin

2012-04-01

The relationship between the texture and mechanical properties of 6xxx aluminum alloy sheets processed via cross rolling was investigated. The microstructures of the conventional rolled and cross rolled sheets after annealing were analyzed using optical micrographs (OM). The texture distribution across the thickness in the Al-Mg-Si-Cu alloy, conventional rolled sheets, and cross rolled sheets both before and after annealing was investigated via X-ray texture measurements. The texture was analyzed in three layers from the surface to the center of the sheet. The β-fiber texture of the conventional rolled sheet was typical of the texture obtained using aluminumoll ring. After annealing, the typical β-fiber orientations were changed to recrystallization textures: cube{001}<100> and normal direction (ND)-rotated cubes. However, the texture of the cross rolled sheet was composed of an asymmetrical, rolling direction (RD)-rotated cubes. After annealing, the asymmetrical orientations in the cross rolled sheet were changed to a randomized texture. The average R-value of the annealed cross rolled sheets was higher than that of the conventional rolled sheets. The limit dome height (LDH) test results demonstrated that cross rolling is effective in improving the formability of the Al-Mg-Si-Cu alloy sheets.

40 CFR 467.20 - Applicability; description of the rolling with emulsions subcategory.

Code of Federal Regulations, 2010 CFR

2010-07-01

... rolling with emulsions subcategory. 467.20 Section 467.20 Protection of Environment ENVIRONMENTAL... Rolling With Emulsions Subcategory § 467.20 Applicability; description of the rolling with emulsions... the rolling with emulsions subcategory. ...

40 CFR 467.20 - Applicability; description of the rolling with emulsions subcategory.

Code of Federal Regulations, 2011 CFR

2011-07-01

... rolling with emulsions subcategory. 467.20 Section 467.20 Protection of Environment ENVIRONMENTAL... Rolling With Emulsions Subcategory § 467.20 Applicability; description of the rolling with emulsions... the rolling with emulsions subcategory. ...

CLASSICAL AREAS OF PHENOMENOLOGY: Lattice Boltzmann simulation of behaviour of particles moving in blood vessels under the rolling massage

NASA Astrophysics Data System (ADS)

Yi, Hou-Hui; Yang, Xiao-Feng; Wang, Cai-Feng; Li, Hua-Bing

2009-07-01

The rolling massage is one of the most important manipulations in Chinese massage, which is expected to eliminate many diseases. Here, the effect of the rolling massage on a pair of particles moving in blood vessels under rolling massage manipulation is studied by the lattice Boltzmann simulation. The simulated results show that the motion of each particle is considerably modified by the rolling massage, and it depends on the relative rolling velocity, the rolling depth, and the distance between particle position and rolling position. Both particles' translational average velocities increase almost linearly as the rolling velocity increases, and obey the same law. The increment of the average relative angular velocity for the leading particle is smaller than that of the trailing one. The result is helpful for understanding the mechanism of the massage and to further develop the rolling techniques.

Primer-optimized results and trends for circular phasing and other circle-to-circle impulsive coplanar rendezvous

NASA Astrophysics Data System (ADS)

Sandrik, Suzannah

Optimal solutions to the impulsive circular phasing problem, a special class of orbital maneuver in which impulsive thrusts shift a vehicle's orbital position by a specified angle, are found using primer vector theory. The complexities of optimal circular phasing are identified and illustrated using specifically designed Matlab software tools. Information from these new visualizations is applied to explain discrepancies in locally optimal solutions found by previous researchers. Two non-phasing circle-to-circle impulsive rendezvous problems are also examined to show the applicability of the tools developed here to a broader class of problems and to show how optimizing these rendezvous problems differs from the circular phasing case.

Rolling into spatial disorientation: simulator demonstration of the post-roll (Gillingham) illusion.

PubMed

Nooij, Suzanne A E; Groen, Eric L

2011-05-01

Spatial disorientation (SD) is still a contributing factor in many aviation accidents, stressing the need for adequate SD training scenarios. In this article we focused on the post-roll effect (the sensation of rolling back after a roll maneuver, such as an entry of a coordinated turn) and investigated the effect of roll stimuli on the pilot's ability to stabilize their roll attitude. This resulted in a ground-based demonstration scenario for pilots. The experiments took place in the advanced 6-DOF Desdemona motion simulator, with the subject in a supine position. Roll motions were either fully automated with the subjects blindfolded (BLIND), automated with the subject viewing the cockpit interior (COCKPIT), or self-controlled (LEAD). After the roll stimulus subjects had to cancel all perceived simulator motion without any visual feedback. Both the roll velocity and duration were varied. In 68% of all trials subjects corrected for the perceived motion of rolling back by initiating a roll motion in the same direction as the preceeding roll. The effect was dependent on both rate and duration, in a manner consistent with semicircular canal dynamics. The effect was smallest in the BLIND scenario, but differences between simulation scenarios were non-significant. The results show that the effects of the post-roll illusion on aircraft control can be demonstrated adequately in a flight simulator using an attitude control task. The effect is present even after short roll movements, occurring frequently in flight. Therefore this demonstration is relevant for spatial disorientation training programs for pilots.

Defect Analysis of Roll-to-Roll SAIL Manufactured Flexible Display Backplanes

DTIC Science & Technology

2011-01-01

tenting defect through the SAIL process Figure 5: Flexible backplane electrical tester Figure 6: R2R optical inspection system Figure 7: TEM of TFT ...Analysis of Roll-to-Roll SAIL Manufactured Flexible Display...Marcia Almanza-Workman, Robert A. Garcia, HanJun Kim, Ohseung Kwon, Frank Jeffrey HP Laboratories HPL-2011-35 SAIL, flexible displays, roll-to-roll HP

Wind-tunnel investigation at supersonic speeds of a remote-controlled canard missile with a free-rolling-tail brake torque system

NASA Technical Reports Server (NTRS)

Blair, A. B., Jr.

1985-01-01

Wind tunnel tests were conducted at Mach numbers 1.70, 2.16, and 2.86 to determine the static aerodynamic characteristics of a cruciform canard-controlled missile with fixed or free rolling tailfin afterbodies. Mechanical coupling effects of the free-rolling-tail afterbody were investigated by using an electronic electromagnetic brake system providing arbitrary tail-fin brake torques with continuous measurements of tail-to-mainframe torque and tail roll rate. Remote-controlled canards were deflected to provide pitch, yaw, and roll control. Results indicate that the induced rolling moment coefficients due to canard yaw control are reduced and linearized for the free-rolling-tail (free-tail) configuration. The canards of the latter provide conventional roll control for the entire angle-of-attack test range. For the free-tail configuration, the induced rolling moment coefficient due to canard yaw control increased and the canard roll control decreased with increases in brake torque, which simulated bearing friction torque. It appears that a compromise in regard to bearing friction, for example, low-cost bearings with some friction, may allow satisfactory free-tail aerodynamic characteristics that include reductions in adverse rolling-moment coefficients and lower tail roll rates.

Open ended tubing cutters

NASA Technical Reports Server (NTRS)

Girala, A. S. (Inventor)

1981-01-01

A self clamping cutting tool which includes a handle attached to a C-shaped housing is described. Rotatably mounted within the housing is a C-shaped tool body carrying a set of clamping rolls, two support rolls, and an edged cutting roll (64). The support rolls are disposed to one side of the axis of a pipe and the cutting roll is disposed to the other side of a pipe axis so that these rolls contact a pipe at three circumferential points. Cutter advancing apparatus advance the cutting roll toward the support rollers. The support rolls and cutting roll are rotatable independently of the C-shaped housing. A one way ratchet mechanism disposed between the C-shaped housing and the C-shaped tool body permits operation by movement in one rotational direction about the pipe axis.

Literature Circles in a Fifth Grade Classroom: A Qualitative Study Examining How the Teacher and Students Used Literature Circles and the Impact They Have on Student Learning

ERIC Educational Resources Information Center

Pambianchi, Laura Claypool

2017-01-01

State and national standards, including the Common Core State Standards, state that students should be expected to learn to discuss and analyze texts, comprehend ideas in increasingly complex texts, and justify their thinking. Literature circles are an instructional practice suggested by many educational writers as an instructional practice that…

Change in Ragweed Pollen Season, 1995-2015

EPA Pesticide Factsheets

This figure shows how the length of ragweed pollen season changed at 11 locations in the central United States and Canada between 1995 and 2015. Data were provided by Dr. Lewis Ziska of the U.S. Department of Agriculture, Agricultural Research Service. Red circles represent a longer pollen season; the blue circle represents a shorter season. Larger circles indicate larger changes. For more information: www.epa.gov/climatechange/science/indicators

Discovering Social Circles in Ego Networks (Author’s Manuscript)

DTIC Science & Technology

2013-01-10

ego-network. We expect that circles are formed by densely-connected sets of alters ( Newman , 2006). However, different circles overlap heavily, i.e...umbrella of community detection (Lancichinetti and Fortunato, 2009a; Schaeffer, 2007; Leskovec et al., 2010; Porter et al., 2009; Newman , 2004). While...MCMC) sampler ( Newman and Barkema, 1999) which efficiently updates node-community memberships by ‘collapsing’ nodes that have common features and

Sets that Contain Their Circle Centers

ERIC Educational Resources Information Center

Martin, Greg

2008-01-01

Say that a subset S of the plane is a "circle-center set" if S is not a subset of a line, and whenever we choose three non-collinear points from S, the center of the circle through those three points is also an element of S. A problem appearing on the Macalester College Problem of the Week website stated that a finite set of points in the plane,…

Generic Environmental Impact Statement. Air Force Low Altitude Flying Operations. Preliminary Draft. Volume 3

DTIC Science & Technology

1990-01-01

landing strip at Circle. Lodging is available primarily at Circle Hot Springs, approximately 30 miles southwest of Circle. In general, hiking, snowmobiling... timberline ), coniferous forest, and several widespread deciduous species. Coniferous and deciduous forest, alpine and deciduous scrub, shrub tundra, and...white and paper birch, quaking aspen, and balsam poplar. Common shrub species (above and below timberline ) are alder, willow, glandular birch

Promoting retention, enabling success: Discovering the potential of student support circles.

PubMed

Bass, Janice; Walters, Caroline; Toohill, Jocelyn; Sidebotham, Mary

2016-09-01

Retention of students is critical to education programs and future workforce. A mixed methods study evaluated student engagement within a Bachelor of Midwifery program and connection with career choice through participation in student support circles. Centred on the Five Senses of Success Framework (sense of capability, purpose, identity, resourcefulness and connectedness) and including four stages of engagement (creating space, preparing self, sharing stories, focused conversations), the circles support and develop student and professional identity. Of 80 students 43 (54%) provided responses to a two item survey assessed against a five point Likert scale to determine utility. Using a nominal group technique, student's voices gave rich insight into the personal and professional growth that participation in the student support circles provided. Evaluated as helpful to first year students in orientating to university study and early socialisation into the profession, the circles appear to influence the development of a strong sense of professional identity and personal midwifery philosophy based on the relational nature of the midwife being with woman rather than doing midwifery. This suggests that student support circles positively influence perceptions and expectations, contributing to a shared sense of purpose and discipline connection, for enhancing student retention and future workforce participation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of stern hull shape on turning circle of ships

NASA Astrophysics Data System (ADS)

Jaswar, Maimun, A.; Wahid, M. A.; Priyanto, A.; Zamani, Pauzi, Saman

2012-06-01

Many factors such as: stern hull shape, length, draught, trim, propulsion system and external forces affecting the drift angle influence rate of turn and size of turning circle of ships. This paper discusses turning circle characteristics of U and V stern hull shape of Very Large Crude Oil Carrier (VLCC) ships. The ships have same principal dimension such as length, beam, and draught. The turning circle characteristics of the VLCC ships are simulated at 35 degree of rudder angle. In the analysis, firstly, turning circle performance of U-type VLCC ship is simulated. In the simulation, initial ship speed is determined using given power and rpm. Hydrodynamic derivatives coefficients are determined by including effect of fullness of aft run. Using the obtained, speed and hydrodynamic coefficients, force and moment acting on hull, force and moment induced by propeller, force and moment induced by rudder are determined. Finally, ship trajectory, ratio of speed, yaw angle and drift angle are determined. Results of simulation results of the VLCC ship are compared with the experimental one as validation. Using the same method, V-type VLCC is simulated and the simulation results are compared with U-type VLCC ship. Results shows the turning circle of U-type is larger than V-type due to effect stern hul results of simulation are.

The Effect of the Type and Colour of Placebo Stimuli on Placebo Effects Induced by Observational Learning.

PubMed

Świder, Karolina; Bąbel, Przemysław

2016-01-01

Research shows that placebo analgesia and nocebo hyperalgesia can be induced through observational learning. Our aim was to replicate and extend these results by studying the influence of the type and colour of stimuli used as placebos on the placebo effects induced by observational learning. Three experimental and two control groups were tested. All participants received pain stimuli of the same intensity preceded by colour lights (green and red) or geometric shapes (circles and squares). Before receiving pain stimuli, participants in the experimental groups, but not in the control groups, observed a model who rated pain stimuli that were preceded by either green lights (green placebo group), red lights (red placebo group), or circles (circle placebo group) as being less painful than those preceded by either red lights (green placebo group), green lights (red placebo group), or squares (circle placebo group). As a result participants in the experimental groups rated pain stimuli preceded by either green lights (green placebo group), red lights (red placebo group), or circles (circle placebo group) as being less painful than the participants in the control groups did, indicating that placebo effect was induced. No statistically significant differences were found in the magnitudes of the placebo effects between the three experimental groups (green placebo, red placebo, and circle placebo groups), indicating that neither the type nor the colour of placebo stimuli affected the placebo effects induced by observational learning. The placebo effects induced by observational learning were found to be unrelated to the individual differences in pain anxiety, fear of pain, and empathy.

The Effect of the Type and Colour of Placebo Stimuli on Placebo Effects Induced by Observational Learning

PubMed Central

Świder, Karolina; Bąbel, Przemysław

2016-01-01

Research shows that placebo analgesia and nocebo hyperalgesia can be induced through observational learning. Our aim was to replicate and extend these results by studying the influence of the type and colour of stimuli used as placebos on the placebo effects induced by observational learning. Three experimental and two control groups were tested. All participants received pain stimuli of the same intensity preceded by colour lights (green and red) or geometric shapes (circles and squares). Before receiving pain stimuli, participants in the experimental groups, but not in the control groups, observed a model who rated pain stimuli that were preceded by either green lights (green placebo group), red lights (red placebo group), or circles (circle placebo group) as being less painful than those preceded by either red lights (green placebo group), green lights (red placebo group), or squares (circle placebo group). As a result participants in the experimental groups rated pain stimuli preceded by either green lights (green placebo group), red lights (red placebo group), or circles (circle placebo group) as being less painful than the participants in the control groups did, indicating that placebo effect was induced. No statistically significant differences were found in the magnitudes of the placebo effects between the three experimental groups (green placebo, red placebo, and circle placebo groups), indicating that neither the type nor the colour of placebo stimuli affected the placebo effects induced by observational learning. The placebo effects induced by observational learning were found to be unrelated to the individual differences in pain anxiety, fear of pain, and empathy. PMID:27362552

Long Terminal Repeat Circular DNA as Markers of Active Viral Replication of Human T Lymphotropic Virus-1 in Vivo.

PubMed

Fox, James M; Hilburn, Silva; Demontis, Maria-Antonietta; Brighty, David W; Rios Grassi, Maria Fernanda; Galvão-Castro, Bernardo; Taylor, Graham P; Martin, Fabiola

2016-03-14

Clonal expansion of human T-lymphotropic virus type-1 (HTLV-1) infected cells in vivo is well documented. Unlike human immunodeficiency virus type 1 (HIV-1), HTLV-1 plasma RNA is sparse. The contribution of the "mitotic" spread of HTLV-1 compared with infectious spread of the virus to HTLV-1 viral burden in established infection is uncertain. Since extrachromosomal long terminal repeat (LTR) DNA circles are indicators of viral replication in HIV-1 carriers with undetectable plasma HIV RNA, we hypothesised that HTLV-1 LTR circles could indicate reverse transcriptase (RT) usage and infectious activity. 1LTR and 2LTR DNA circles were measured in HTLV-1 cell lines and peripheral blood mononuclear cells (PBMC) of asymptomatic carriers (ACs) and patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukaemia/lymphoma (ATLL). 1LTR DNA circles were detected in 14/20 patients at a mean of 1.38/100 PBMC but did not differentiate disease status nor correlate with HTLV-1 DNA copies. 2LTR DNA circles were detected in 30/31 patients and at higher concentrations in patients with HTLV-1-associated diseases, independent of HTLV-1 DNA load. In an incident case the 2LTR DNA circle concentration increased 2.1 fold at the onset of HAM/TSP compared to baseline. Detectable and fluctuating levels of HTLV-1 DNA circles in patients indicate viral RT usage and virus replication. Our results indicate HTLV-1 viral replication capacity is maintained in chronic infection and may be associated with disease onset.

Evaluation of roll designs on a roll-crusher/ crusher/splitter biomass harvester: test bench results

Treesearch

Colin Ashmore; Donald L. Sirois; Bryce J. Stokes

1987-01-01

Four different roll designs were evaluated on a test bench roll crusher/splitter to determine feeding and crushing efficiencies. For each design, different gap settings for the primary and secondary rolls were tested at two hydraulic cylinder pressures on the primary crush roll to determine their ability to crush and/or feed tree bolts. Seven different diameter classes...

Distinct molecular and cellular contributions to stabilizing selectin-mediated rolling under flow

PubMed Central

Yago, Tadayuki; Leppänen, Anne; Qiu, Haiying; Marcus, Warren D.; Nollert, Matthias U.; Zhu, Cheng; Cummings, Richard D.; McEver, Rodger P.

2002-01-01

Leukocytes roll on selectins at nearly constant velocities over a wide range of wall shear stresses. Ligand-coupled microspheres roll faster on selectins and detach quickly as wall shear stress is increased. To examine whether the superior performance of leukocytes reflects molecular features of native ligands or cellular properties that favor selectin-mediated rolling, we coupled structurally defined selectin ligands to microspheres or K562 cells and compared their rolling on P-selectin. Microspheres bearing soluble P-selectin glycoprotein ligand (sPSGL)-1 or 2-glycosulfopeptide (GSP)-6, a GSP modeled after the NH2-terminal P-selectin–binding region of PSGL-1, rolled equivalently but unstably on P-selectin. K562 cells displaying randomly coupled 2-GSP-6 also rolled unstably. In contrast, K562 cells bearing randomly coupled sPSGL-1 or 2-GSP-6 targeted to a membrane-distal region of the presumed glycocalyx rolled more like leukocytes: rolling steps were more uniform and shear resistant, and rolling velocities tended to plateau as wall shear stress was increased. K562 cells treated with paraformaldehyde or methyl-β-cyclodextrin before ligand coupling were less deformable and rolled unstably like microspheres. Cells treated with cytochalasin D were more deformable, further resisted detachment, and rolled slowly despite increases in wall shear stress. Thus, stable, shear-resistant rolling requires cellular properties that optimize selectin–ligand interactions. PMID:12177042

Hot rolling of thick uranium molybdenum alloys

DOEpatents

DeMint, Amy L.; Gooch, Jack G.

2015-11-17

Disclosed herein are processes for hot rolling billets of uranium that have been alloyed with about ten weight percent molybdenum to produce cold-rollable sheets that are about one hundred mils thick. In certain embodiments, the billets have a thickness of about 7/8 inch or greater. Disclosed processes typically involve a rolling schedule that includes a light rolling pass and at least one medium rolling pass. Processes may also include reheating the rolling stock and using one or more heavy rolling passes, and may include an annealing step.