Use of Multiple Displacement Amplification as Pre-polymerase Chain Reaction (Pre-PCR) to amplify genomic DNA of siphonapterids preserved for long periods in scientific collections.

PubMed

Avelar, Daniel M; Linardi, Pedro M

2010-09-15

The recently developed Multiple Displacement Amplification technique (MDA) allows for the production of a large quantity of high quality genomic DNA from low amounts of the original DNA. The goal of this study was to evaluate the performance of the MDA technique to amplify genomic DNA of siphonapterids that have been stored for long periods in 70% ethanol at room temperature. We subjected each DNA sample to two different methodologies: (1) amplification of mitochondrial 16S sequences without MDA; (2) amplification of 16S after MDA. All the samples obtained from these procedures were then sequenced. Only 4 samples (15.4%) subjected to method 1 showed amplification. In contrast, the application of MDA (method 2) improved the performance substantially, with 24 samples (92.3%) showing amplification, with significant difference. Interestingly, one of the samples successfully amplified with this method was originally collected in 1909. All of the sequenced samples displayed satisfactory results in quality evaluations (Phred ≥ 20) and good similarities, as identified with the BLASTn tool. Our results demonstrate that the use of MDA may be an effective tool in molecular studies involving specimens of fleas that have traditionally been considered inadequately preserved for such purposes.

Use of Multiple Displacement Amplification as Pre-polymerase Chain Reaction (Pre-PCR) to amplify genomic DNA of siphonapterids preserved for long periods in scientific collections

PubMed Central

2010-01-01

The recently developed Multiple Displacement Amplification technique (MDA) allows for the production of a large quantity of high quality genomic DNA from low amounts of the original DNA. The goal of this study was to evaluate the performance of the MDA technique to amplify genomic DNA of siphonapterids that have been stored for long periods in 70% ethanol at room temperature. We subjected each DNA sample to two different methodologies: (1) amplification of mitochondrial 16S sequences without MDA; (2) amplification of 16S after MDA. All the samples obtained from these procedures were then sequenced. Only 4 samples (15.4%) subjected to method 1 showed amplification. In contrast, the application of MDA (method 2) improved the performance substantially, with 24 samples (92.3%) showing amplification, with significant difference. Interestingly, one of the samples successfully amplified with this method was originally collected in 1909. All of the sequenced samples displayed satisfactory results in quality evaluations (Phred ≥ 20) and good similarities, as identified with the BLASTn tool. Our results demonstrate that the use of MDA may be an effective tool in molecular studies involving specimens of fleas that have traditionally been considered inadequately preserved for such purposes. PMID:20840790

Improved multiple displacement amplification (iMDA) and ultraclean reagents.

PubMed

Motley, S Timothy; Picuri, John M; Crowder, Chris D; Minich, Jeremiah J; Hofstadler, Steven A; Eshoo, Mark W

2014-06-06

Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome. The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.

In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level.

PubMed

Michikawa, Yuichi; Sugahara, Keisuke; Suga, Tomo; Ohtsuka, Yoshimi; Ishikawa, Kenichi; Ishikawa, Atsuko; Shiomi, Naoko; Shiomi, Tadahiro; Iwakawa, Mayumi; Imai, Takashi

2008-12-15

The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5 kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA was carefully released from intact cells into a mildly heated alkaline agarose solution and mixed thoroughly. The solution was then gently aliquoted and allowed to solidify while maintaining the integrity of the diluted DNA. Exogenously provided Phi29 DNA polymerase was used to perform consistent genomic amplification with random hexameric oligonucleotides within the agarose gels. Simple heat melting of the gel allowed recovery of the amplified materials in a solution of the polymerase chain reaction (PCR)-ready form. The haplotypes of seven SNPs spanning 240 kb of the DNA surrounding the human ATM gene region on chromosome 11 were determined for 10 individuals, demonstrating the feasibility of this new method.

Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications.

PubMed

Huang, Lei; Ma, Fei; Chapman, Alec; Lu, Sijia; Xie, Xiaoliang Sunney

2015-01-01

We present a survey of single-cell whole-genome amplification (WGA) methods, including degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). The key parameters to characterize the performance of these methods are defined, including genome coverage, uniformity, reproducibility, unmappable rates, chimera rates, allele dropout rates, false positive rates for calling single-nucleotide variations, and ability to call copy-number variations. Using these parameters, we compare five commercial WGA kits by performing deep sequencing of multiple single cells. We also discuss several major applications of single-cell genomics, including studies of whole-genome de novo mutation rates, the early evolution of cancer genomes, circulating tumor cells (CTCs), meiotic recombination of germ cells, preimplantation genetic diagnosis (PGD), and preimplantation genomic screening (PGS) for in vitro-fertilized embryos.

Development of microbial genome-probing microarrays using digital multiple displacement amplification of uncultivated microbial single cells.

PubMed

Chang, Ho-Won; Sung, Youlboong; Kim, Kyoung-Ho; Nam, Young-Do; Roh, Seong Woon; Kim, Min-Soo; Jeon, Che Ok; Bae, Jin-Woo

2008-08-15

A crucial problem in the use of previously developed genome-probing microarrays (GPM) has been the inability to use uncultivated bacterial genomes to take advantage of the high sensitivity and specificity of GPM in microbial detection and monitoring. We show here a method, digital multiple displacement amplification (MDA), to amplify and analyze various genomes obtained from single uncultivated bacterial cells. We used 15 genomes from key microbes involved in dichloromethane (DCM)-dechlorinating enrichment as microarray probes to uncover the bacterial population dynamics of samples without PCR amplification. Genomic DNA amplified from single cells originating from uncultured bacteria with 80.3-99.4% similarity to 16S rRNA genes of cultivated bacteria. The digital MDA-GPM method successfully monitored the dynamics of DCM-dechlorinating communities from different phases of enrichment status. Without a priori knowledge of microbial diversity, the digital MDA-GPM method could be designed to monitor most microbial populations in a given environmental sample.

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology.

PubMed

Binga, Erik K; Lasken, Roger S; Neufeld, Josh D

2008-03-01

Microbial ecology is a field that applies molecular techniques to analyze genes and communities associated with a plethora of unique environments on this planet. In the past, low biomass and the predominance of a few abundant community members have impeded the application of techniques such as PCR, microarray analysis and metagenomics to complex microbial populations. In the absence of suitable cultivation methods, it was not possible to obtain DNA samples from individual microorganisms. Recently, a method called multiple displacement amplification (MDA) has been used to circumvent these limitations by amplifying DNA from microbial communities in low-biomass environments, individual cells from uncultivated microbial species and active organisms obtained through stable isotope probing incubations. This review describes the development and applications of MDA, discusses its strengths and limitations and highlights the impact of MDA on the field of microbial ecology. Whole genome amplification via MDA has increased access to the genomic DNA of uncultivated microorganisms and low-biomass environments and represents a 'power tool' in the molecular toolbox of microbial ecologists.

Polymerase chain displacement reaction.

PubMed

Harris, Claire L; Sanchez-Vargas, Irma J; Olson, Ken E; Alphey, Luke; Fu, Guoliang

2013-02-01

Quantitative PCR assays are now the standard method for viral diagnostics. These assays must be specific, as well as sensitive, to detect the potentially low starting copy number of viral genomic material. We describe a new technique, polymerase chain displacement reaction (PCDR), which uses multiple nested primers in a rapid, capped, one-tube reaction that increases the sensitivity of normal quantitative PCR (qPCR) assays. Sensitivity was increased by approximately 10-fold in a proof-of-principle test on dengue virus sequence. In PCDR, when extension occurs from the outer primer, it displaces the extension strand produced from the inner primer by utilizing a polymerase that has strand displacement activity. This allows a greater than 2-fold increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR. Increased sensitivity in PCDR would be useful in nucleic acid detection for viral diagnostics.

Multiple displacement amplification on single cell and possible PGD applications.

PubMed

Hellani, Ali; Coskun, Serdar; Benkhalifa, Moncef; Tbakhi, Abelghani; Sakati, Nadia; Al-Odaib, Ali; Ozand, Pinar

2004-11-01

Multiple displacement amplification (MDA) is a technique used in the amplification of very low amounts of DNA and reported to yield large quantities of high-quality DNA. We used MDA to amplify the whole genome directly from a single cell. The most common techniques used in PGD are PCR and fluorescent in-situ hybridization (FISH). There are many limitations to these techniques including, the number of chromosomes diagnosed for FISH or the quality of DNA issued from a single cell PCR. This report shows, for the first time, use of MDA for single cell whole genome amplification. A total of 16 short tandem repeats (STRs) were amplified successfully with a similar pattern to the genomic DNA. Furthermore, allelic drop out (ADO) derived from MDA was assessed in 40 single cells by analysing (i) heterozygosity for a known beta globin mutation (IVSI-5 C-G) and by studying (ii) the heterozygous loci present in the STRs. ADO turned out to be 10.25% for the beta globin gene sequencing and 5% for the fluorescent PCR analysis of STRs. Moreover, the amplification accuracy of MDA permitted the detection of trisomy 21 on a single cell using comparative genome hybridization-array. Altogether, these data suggest that MDA can be used for single cell molecular karyotyping and the diagnosis of any single gene disorder in PGD.

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.

Mechanism of chimera formation during the Multiple Displacement Amplification reaction.

PubMed

Lasken, Roger S; Stockwell, Timothy B

2007-04-12

Multiple Displacement Amplification (MDA) is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them. Here, we characterize the major types of chimeras formed by carrying out an MDA whole genome amplification from a single E. coli cell and sequencing by the 454 Life Sciences method. Analysis of 475 chimeras revealed the predominant reaction mechanisms that create the DNA rearrangements. The highly branched DNA synthesized in MDA can assume many alternative secondary structures. DNA strands extended on an initial template can be displaced becoming available to prime on a second template creating the chimeras. Evidence supports a model in which branch migration can displace 3'-ends freeing them to prime on the new templates. More than 85% of the resulting DNA rearrangements were inverted sequences with intervening deletions that the model predicts. Intramolecular rearrangements were favored, with displaced 3'-ends reannealing to single stranded 5'-strands contained within the same branched DNA molecule. In over 70% of the chimeric junctions, the 3' termini had initiated priming at complimentary sequences of 2-21 nucleotides (nts) in the new templates. Formation of chimeras is an important limitation to the MDA method, particularly for whole genome sequencing. Identification of the mechanism for chimera formation provides new insight into the MDA reaction and suggests methods to reduce chimeras. The 454 sequencing approach used here will provide a rapid method to assess the utility of reaction modifications.

Mechanism of chimera formation during the Multiple Displacement Amplification reaction

PubMed Central

Lasken, Roger S; Stockwell, Timothy B

2007-01-01

Background Multiple Displacement Amplification (MDA) is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them. Results Here, we characterize the major types of chimeras formed by carrying out an MDA whole genome amplification from a single E. coli cell and sequencing by the 454 Life Sciences method. Analysis of 475 chimeras revealed the predominant reaction mechanisms that create the DNA rearrangements. The highly branched DNA synthesized in MDA can assume many alternative secondary structures. DNA strands extended on an initial template can be displaced becoming available to prime on a second template creating the chimeras. Evidence supports a model in which branch migration can displace 3'-ends freeing them to prime on the new templates. More than 85% of the resulting DNA rearrangements were inverted sequences with intervening deletions that the model predicts. Intramolecular rearrangements were favored, with displaced 3'-ends reannealing to single stranded 5'-strands contained within the same branched DNA molecule. In over 70% of the chimeric junctions, the 3' termini had initiated priming at complimentary sequences of 2–21 nucleotides (nts) in the new templates. Conclusion Formation of chimeras is an important limitation to the MDA method, particularly for whole genome sequencing. Identification of the mechanism for chimera formation provides new insight into the MDA reaction and suggests methods to reduce chimeras. The 454 sequencing approach used here will provide a rapid method to assess the utility of reaction modifications. PMID:17430586

Genome-wide single-nucleotide polymorphism arrays demonstrate high fidelity of multiple displacement-based whole-genome amplification.

PubMed

Tzvetkov, Mladen V; Becker, Christian; Kulle, Bettina; Nürnberg, Peter; Brockmöller, Jürgen; Wojnowski, Leszek

2005-02-01

Whole-genome DNA amplification by multiple displacement (MD-WGA) is a promising tool to obtain sufficient DNA amounts from samples of limited quantity. Using Affymetrix' GeneChip Human Mapping 10K Arrays, we investigated the accuracy and allele amplification bias in DNA samples subjected to MD-WGA. We observed an excellent concordance (99.95%) between single-nucleotide polymorphisms (SNPs) called both in the nonamplified and the corresponding amplified DNA. This concordance was only 0.01% lower than the intra-assay reproducibility of the genotyping technique used. However, MD-WGA failed to amplify an estimated 7% of polymorphic loci. Due to the algorithm used to call genotypes, this was detected only for heterozygous loci. We achieved a 4.3-fold reduction of noncalled SNPs by combining the results from two independent MD-WGA reactions. This indicated that inter-reaction variations rather than specific chromosomal loci reduced the efficiency of MD-WGA. Consistently, we detected no regions of reduced amplification, with the exception of several SNPs located near chromosomal ends. Altogether, despite a substantial loss of polymorphic sites, MD-WGA appears to be the current method of choice to amplify genomic DNA for array-based SNP analyses. The number of nonamplified loci can be substantially reduced by amplifying each DNA sample in duplicate.

Isothermal multiple displacement amplification: a methodical approach enhancing molecular routine diagnostics of microcarcinomas and small biopsies.

PubMed

Mairinger, Fabian D; Walter, Robert Fh; Vollbrecht, Claudia; Hager, Thomas; Worm, Karl; Ting, Saskia; Wohlschläger, Jeremias; Zarogoulidis, Paul; Zarogoulidis, Konstantinos; Schmid, Kurt W

2014-01-01

Isothermal multiple displacement amplification (IMDA) can be a powerful tool in molecular routine diagnostics for homogeneous and sequence-independent whole-genome amplification of notably small tumor samples, eg, microcarcinomas and biopsies containing a small amount of tumor. Currently, this method is not well established in pathology laboratories. We designed a study to confirm the feasibility and convenience of this method for routine diagnostics with formalin-fixed, paraffin-embedded samples prepared by laser-capture microdissection. A total of 250 μg DNA (concentration 5 μg/μL) was generated by amplification over a period of 8 hours with a material input of approximately 25 cells, approximately equivalent to 175 pg of genomic DNA. In the generated DNA, a representation of all chromosomes could be shown and the presence of elected genes relevant for diagnosis in clinical samples could be proven. Mutational analysis of clinical samples could be performed without any difficulty and showed concordance with earlier diagnostic findings. We established the feasibility and convenience of IMDA for routine diagnostics. We also showed that small amounts of DNA, which were not analyzable with current molecular methods, could be sufficient for a wide field of applications in molecular routine diagnostics when they are preamplified with IMDA.

Integrating DNA strand displacement circuitry to the nonlinear hybridization chain reaction.

PubMed

Zhang, Zhuo; Fan, Tsz Wing; Hsing, I-Ming

2017-02-23

Programmable and modular attributes of DNA molecules allow one to develop versatile sensing platforms that can be operated isothermally and enzyme-free. In this work, we present an approach to integrate upstream DNA strand displacement circuits that can be turned on by a sequence-specific microRNA analyte with a downstream nonlinear hybridization chain reaction for a cascading hyperbranched nucleic acid assembly. This system provides a two-step amplification strategy for highly sensitive detection of the miRNA analyte, conducive for multiplexed detection. Multiple miRNA analytes were tested with our integrated circuitry using the same downstream signal amplification setting, showing the decoupling of nonlinear self-assembly with the analyte sequence. Compared with the reported methods, our signal amplification approach provides an additional control module for higher-order DNA self-assembly and could be developed into a promising platform for the detection of critical nucleic-acid based biomarkers.

Long-range barcode labeling-sequencing

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

Chen, Feng; Zhang, Tao; Singh, Kanwar K.

Methods for sequencing single large DNA molecules by clonal multiple displacement amplification using barcoded primers. Sequences are binned based on barcode sequences and sequenced using a microdroplet-based method for sequencing large polynucleotide templates to enable assembly of haplotype-resolved complex genomes and metagenomes.

Optimization and evaluation of single-cell whole-genome multiple displacement amplification.

PubMed

Spits, C; Le Caignec, C; De Rycke, M; Van Haute, L; Van Steirteghem, A; Liebaers, I; Sermon, K

2006-05-01

The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minute quantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research. (c) 2006 Wiley-Liss, Inc.

Hotspot Selective Preference of the Chimeric Sequences Formed in Multiple Displacement Amplification.

PubMed

Tu, Jing; Lu, Na; Duan, Mengqin; Huang, Mengting; Chen, Liang; Li, Junji; Guo, Jing; Lu, Zuhong

2017-02-24

Multiple displacement amplification (MDA) is considered to be a conventional approach to comprehensive amplification from low input DNA. The chimeric reads generated in MDA lead to severe disruption in some studies, including those focusing on heterogeneity, structural variation, and genetic recombination. Meanwhile, the generation of by-products gives a new approach to gain insights into the reaction process of φ29 polymerase. Here, we analyzed 36.7 million chimeras and screened 196 billion chimeric hotspots in the human genome, as well as evaluating the hotspot selective preference of chimeras. No significant preference was captured in the distributions of chimeras and hotspots among chromosomes. Hotspots with overlaps for 12-13 nucleotides (nt) were most likely to be selected as templates in chimera generation. Meanwhile, a regularly selective preference was noticed in overlap GC content. The preferences in overlap length and GC content was shown to be pertinent to the sequence denaturation temperature, which pointed out the optimization direction for reducing chimeras. Distance preference between two segments of chimeras was 80-280 nt. The analysis is beneficial for reducing the chimeras in MDA, and the characterization of MDA chimeras is helpful in distinguishing MDA chimeras from chimeric sequences caused by disease.

Hotspot Selective Preference of the Chimeric Sequences Formed in Multiple Displacement Amplification

PubMed Central

Tu, Jing; Lu, Na; Duan, Mengqin; Huang, Mengting; Chen, Liang; Li, Junji; Guo, Jing; Lu, Zuhong

2017-01-01

Multiple displacement amplification (MDA) is considered to be a conventional approach to comprehensive amplification from low input DNA. The chimeric reads generated in MDA lead to severe disruption in some studies, including those focusing on heterogeneity, structural variation, and genetic recombination. Meanwhile, the generation of by-products gives a new approach to gain insights into the reaction process of φ29 polymerase. Here, we analyzed 36.7 million chimeras and screened 196 billion chimeric hotspots in the human genome, as well as evaluating the hotspot selective preference of chimeras. No significant preference was captured in the distributions of chimeras and hotspots among chromosomes. Hotspots with overlaps for 12–13 nucleotides (nt) were most likely to be selected as templates in chimera generation. Meanwhile, a regularly selective preference was noticed in overlap GC content. The preferences in overlap length and GC content was shown to be pertinent to the sequence denaturation temperature, which pointed out the optimization direction for reducing chimeras. Distance preference between two segments of chimeras was 80–280 nt. The analysis is beneficial for reducing the chimeras in MDA, and the characterization of MDA chimeras is helpful in distinguishing MDA chimeras from chimeric sequences caused by disease. PMID:28245591

A cascade autocatalytic strand displacement amplification and hybridization chain reaction event for label-free and ultrasensitive electrochemical nucleic acid biosensing.

PubMed

Chen, Zhiqiang; Liu, Ying; Xin, Chen; Zhao, Jikuan; Liu, Shufeng

2018-08-15

Herein, an autocatalytic strand displacement amplification (ASDA) strategy was proposed for the first time, which was further ingeniously coupled with hybridization chain reaction (HCR) event for the isothermal, label-free and multiple amplification toward nucleic acid detection. During the ASDA module, the target recognition opens the immobilized hairpin probe (IP) and initiates the annealing of the auxiliary DNA strand (AS) with the opened IP for the successive polymerization and nicking reaction in the presence of DNA polymerase and nicking endonuclease. This induces the target recycling and generation of a large amount of intermediate DNA sequences, which can be used as target analogy to execute the autocatalytic strand displacement amplification. Simultaneously, the introduced AS strand can propagate the HCR between two hairpins (H1 and H2) to form a linear DNA concatamer with cytosine (C)-rich loop region, which can facilitate the in-situ synthesis of silver nanoclusters (AgNCs) as electrochemical tags for further amplification toward target responses. With current cascade ASDA and HCR strategy, the detection of target DNA could be achieved with a low detection limit of about 0.16 fM and a good selectivity. The developed biosensor also exhibits the distinct advantages of flexibility and simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus opens a promising avenue for the detection of nucleic acid with low abundance in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier B.V. All rights reserved.

Simplified Real-Time Multiplex Detection of Loop-Mediated Isothermal Amplification Using Novel Mediator Displacement Probes with Universal Reporters.

PubMed

Becherer, Lisa; Bakheit, Mohammed; Frischmann, Sieghard; Stinco, Silvina; Borst, Nadine; Zengerle, Roland; von Stetten, Felix

2018-04-03

A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3-4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, n = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; n = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels.

Development of a multiplex polymerase chain reaction-sequence-specific primer method for NKG2D and NKG2F single-nucleotide polymorphism typing using isothermal multiple displacement amplification products.

PubMed

Kaewmanee, M; Phoksawat, W; Romphruk, A; Romphruk, A V; Jumnainsong, A; Leelayuwat, C

2013-06-01

Natural killer group 2 member D (NKG2D) on immune effector cells recognizes multiple stress-inducible ligands. NKG2D single-nucleotide polymorphism (SNP) haplotypes were related to the levels of cytotoxic activity of peripheral blood mononuclear cells. Indeed, these polymorphisms were also located in NKG2F. Isothermal multiple displacement amplification (IMDA) is used for whole genome amplification (WGA) that can amplify very small genomic DNA templates into microgram with whole genome coverage. This is particularly useful in the cases of limited amount of valuable DNA samples requiring multi-locus genotyping. In this study, we evaluated the quality and applicability of IMDA to genetic studies in terms of sensitivity, efficiency of IMDA re-amplification and stability of IMDA products. The smallest amount of DNA to be effectively amplified by IMDA was 200 pg yielding final DNA of approximately 16 µg within 1.5 h. IMDA could be re-amplified only once (second round of amplification), and could be kept for 5 months at 4°C and more than a year at -20°C without loosing genome coverage. The amplified products were used successfully to setup a multiplex polymerase chain reaction-sequence-specific primer for SNP typing of the NKG2D/F genes. The NKG2D/F multiplex polymerase chain reaction (PCR) contained six PCR mixtures for detecting 10 selected SNPs, including 8 NKG2D/F SNP haplotypes and 2 additional NKG2D coding SNPs. This typing procedure will be applicable in both clinical and research laboratories. Thus, our data provide useful information and limitations for utilization of genome-wide amplification using IMDA and its application for multiplex NKG2D/F typing. © 2013 John Wiley & Sons Ltd.

Invasive reaction assisted strand-displacement signal amplification for sensitive DNA detection.

PubMed

Zou, Bingjie; Song, Qinxin; Wang, Jianping; Liu, Yunlong; Zhou, Guohua

2014-11-18

A novel DNA detection assay was proposed by invasive reaction coupled with molecular beacon assisted strand-displacement signal amplification (IRASA). Target DNAs are firstly hybridized to two probes to initiate invasive reaction to produce amplified flaps. Then these flaps are further amplified by strand-displacement signal amplification. The detection limit was around 0.2 pM.

Single-Cell-Based Platform for Copy Number Variation Profiling through Digital Counting of Amplified Genomic DNA Fragments.

PubMed

Li, Chunmei; Yu, Zhilong; Fu, Yusi; Pang, Yuhong; Huang, Yanyi

2017-04-26

We develop a novel single-cell-based platform through digital counting of amplified genomic DNA fragments, named multifraction amplification (mfA), to detect the copy number variations (CNVs) in a single cell. Amplification is required to acquire genomic information from a single cell, while introducing unavoidable bias. Unlike prevalent methods that directly infer CNV profiles from the pattern of sequencing depth, our mfA platform denatures and separates the DNA molecules from a single cell into multiple fractions of a reaction mix before amplification. By examining the sequencing result of each fraction for a specific fragment and applying a segment-merge maximum likelihood algorithm to the calculation of copy number, we digitize the sequencing-depth-based CNV identification and thus provide a method that is less sensitive to the amplification bias. In this paper, we demonstrate a mfA platform through multiple displacement amplification (MDA) chemistry. When performing the mfA platform, the noise of MDA is reduced; therefore, the resolution of single-cell CNV identification can be improved to 100 kb. We can also determine the genomic region free of allelic drop-out with mfA platform, which is impossible for conventional single-cell amplification methods.

Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

PubMed Central

Ullrich, Thomas; Ermantraut, Eugen; Schulz, Torsten; Steinmetzer, Katrin

2012-01-01

Background State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests. PMID:22539973

Signal-on electrochemical detection of antibiotics at zeptomole level based on target-aptamer binding triggered multiple recycling amplification.

PubMed

Wang, Hongzhi; Wang, Yu; Liu, Su; Yu, Jinghua; Guo, Yuna; Xu, Ying; Huang, Jiadong

2016-06-15

In the work, a signal-on electrochemical DNA sensor based on multiple amplification for ultrasensitive detection of antibiotics has been reported. In the presence of target, the ingeniously designed hairpin probe (HP1) is opened and the polymerase-assisted target recycling amplification is triggered, resulting in autonomous generation of secondary target. It is worth noting that the produced secondary target could not only hybridize with other HP1, but also displace the Helper from the electrode. Consequently, methylene blue labeled HP2 forms a "close" probe structure, and the increase of signal is monitored. The increasing current provides an ultrasensitive electrochemical detection for antibiotics down to 1.3 fM. To our best knowledge, such work is the first report about multiple recycling amplification combing with signal-on sensing strategy, which has been utilized for quantitative determination of antibiotics. It would be further used as a general strategy associated with more analytical techniques toward the detection of a wide spectrum of analytes. Thus, it holds great potential for the development of ultrasensitive biosensing platform for the applications in bioanalysis, disease diagnostics, and clinical biomedicine. Copyright © 2016 Elsevier B.V. All rights reserved.

Preimplantation genetic diagnosis for Duchenne muscular dystrophy by multiple displacement amplification.

PubMed

Ren, Zi; Zeng, Hai-tao; Xu, Yan-wen; Zhuang, Guang-lun; Deng, Jie; Zhang, Cheng; Zhou, Can-quan

2009-02-01

To evaluate the use of multiple displacement amplification (MDA) in preimplantation genetic diagnosis (PGD) for female carriers with Duchenne muscular dystrophy (DMD). MDA was used to amplify a whole genome of single cells. Following the setup on single cells, the test was applied in two clinical cases of PGD. One mutant exon, six short tandem repeats (STR) markers within the dystrophin gene, and amelogenin were incorporated into singleplex polymerase chain reaction (PCR) assays on MDA products of single blastomeres. Center for reproductive medicine in First Affiliated Hospital, Sun Yat-sen University, China. Two female carriers with a duplication of exons 3-11 and a deletion of exons 47-50, respectively. The MDA of single cells and fluorescent PCR assays for PGD. The ability to analyze single blastomeres for DMD using MDA. The protocol setup previously allowed for the accurate diagnosis of each embryo. Two clinical cases resulted in a healthy girl, which was the first successful clinical application of MDA in PGD for DMD. We suggest that this protocol is reliable to increase the accuracy of the PGD for DMD.

Systematic assessment of the performance of whole-genome amplification for SNP/CNV detection and β-thalassemia genotyping.

PubMed

He, Fei; Zhou, Wanjun; Cai, Ren; Yan, Tizhen; Xu, Xiangmin

2018-04-01

In this study, we aimed to assess the performance of two whole-genome amplification methods, multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycle (MALBAC), for β-thalassemia genotyping and single-nucleotide polymorphism (SNP)/copy-number variant (CNV) detection using two DNA sequencing assays. We collected peripheral blood, cell lines, and discarded embryos, and carried out MALBAC and MDA on single-cell and five-cell samples. We detected and statistically analyzed differences in the amplification efficiency, positive predictive value, sensitivity, allele dropout (ADO) rate, SNPs, and CV values between the two methods. Through Sanger sequencing at the single-cell and five-cell levels, we showed that both the amplification rate and ADO rate of MDA were better than those using MALBAC, and the sensitivity and positive predictive value obtained from MDA were higher than those from MALBAC for β-thalassemia genotyping. Using next-generation sequencing (NGS) at the single-cell level, we confirmed that MDA has better properties than MALBAC for SNP detection. However, MALBAC was more stable and homogeneous than MDA using low-depth NGS at the single-cell level for CNV detection. We conclude that MALBAC is the better option for CNV detection, while MDA is better suited for SNV detection.

Calibrating genomic and allelic coverage bias in single-cell sequencing.

PubMed

Zhang, Cheng-Zhong; Adalsteinsson, Viktor A; Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L; Meyerson, Matthew; Love, J Christopher

2015-04-16

Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1-10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (∼0.1 × ) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples.

Calibrating genomic and allelic coverage bias in single-cell sequencing

PubMed Central

Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L.; Meyerson, Matthew; Love, J. Christopher

2016-01-01

Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1–10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (~0.1 ×) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples. PMID:25879913

Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates.

PubMed

Leung, Kaston; Klaus, Anders; Lin, Bill K; Laks, Emma; Biele, Justina; Lai, Daniel; Bashashati, Ali; Huang, Yi-Fei; Aniba, Radhouane; Moksa, Michelle; Steif, Adi; Mes-Masson, Anne-Marie; Hirst, Martin; Shah, Sohrab P; Aparicio, Samuel; Hansen, Carl L

2016-07-26

The genomes of large numbers of single cells must be sequenced to further understanding of the biological significance of genomic heterogeneity in complex systems. Whole genome amplification (WGA) of single cells is generally the first step in such studies, but is prone to nonuniformity that can compromise genomic measurement accuracy. Despite recent advances, robust performance in high-throughput single-cell WGA remains elusive. Here, we introduce droplet multiple displacement amplification (MDA), a method that uses commercially available liquid dispensing to perform high-throughput single-cell MDA in nanoliter volumes. The performance of droplet MDA is characterized using a large dataset of 129 normal diploid cells, and is shown to exceed previously reported single-cell WGA methods in amplification uniformity, genome coverage, and/or robustness. We achieve up to 80% coverage of a single-cell genome at 5× sequencing depth, and demonstrate excellent single-nucleotide variant (SNV) detection using targeted sequencing of droplet MDA product to achieve a median allelic dropout of 15%, and using whole genome sequencing to achieve false and true positive rates of 9.66 × 10(-6) and 68.8%, respectively, in a G1-phase cell. We further show that droplet MDA allows for the detection of copy number variants (CNVs) as small as 30 kb in single cells of an ovarian cancer cell line and as small as 9 Mb in two high-grade serous ovarian cancer samples using only 0.02× depth. Droplet MDA provides an accessible and scalable method for performing robust and accurate CNV and SNV measurements on large numbers of single cells.

Multiple Cross Displacement Amplification Coupled With Nanoparticles-Based Lateral Flow Biosensor for Detection of Staphylococcus aureus and Identification of Methicillin-Resistant S. aureus.

PubMed

Wang, Yi; Yan, Weiqiang; Fu, Shanshan; Hu, Shoukui; Wang, Yan; Xu, Jianguo; Ye, Changyun

2018-01-01

Staphylococcus aureus ( S. aureus ), including methicillin-resistant S. aureus (MRSA), is one of the most important human pathogens, which is responsible for bacteremia, soft-tissue infections, and food poisoning. Hence, multiple cross displacement amplification (MCDA) is employed to detect all S. aureus strains, and differentiates MRSA from methicillin-sensitive S. aureus . Multiplex MCDA (m-MCDA), which targets the nuc gene ( S. aureus -specific gene) and mecA gene (encoding penicillin-binding protein-2'), could detect S. aureus strains and identify MRSA within 85 min. Detection of the m-MCDA products is achieved using disposable lateral flow biosensors. A total of 58 strains, including various species of Gram-positive and Gram-negative strains, are used for evaluating and optimizing m-MCDA assays. The optimal amplification condition is found to be 63°C for 40 min, with detection limits at 100 fg DNA/reaction for nuc and mecA genes in the pure cultures, and 10 CFU/tube for nuc and mecA genes in the blood samples. The analytical specificity of m-MCDA assay is of 100%, and no cross-reactions to non- S. aureus strains are produced according to the specificity testing. Particularly, two additional components, including AUDG enzyme and dUTP, are added into the m-MCDA amplification mixtures, which are used for eliminating the unwanted results arising from carryover contamination. Thus, the m-MCDA technique appears to be a simple, rapid, sensitive, and reliable assay to detect all S. aureus strains, and identify MRSA infection for appropriate antibiotic therapy.

Multiple displacement amplification in combination with high-fidelity PCR improves detection of bacteria from single females or eggs of Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae).

PubMed

Jeyaprakash, Ayyamperumal; Hoy, Marjorie A

2004-07-01

Amplifying microbial DNA by the polymerase chain reaction (PCR) from single phytoseiid mites has been difficult, perhaps due to the low titer of bacteria and to interference by the relatively larger amounts of mite genomic DNA. In this paper we evaluate the efficiency of standard and high-fidelity PCR protocols subsequent to amplification of the whole genome by a multiple displacement amplification (MDA) procedure developed by Dean et al. DNA from the phytoseiid Phytoseiulus persimilis (Athias-Henriot) was tested because it lacks a Cytophaga-like organism (CLO) and we could add known amounts of a plasmid containing a cloned 16S rRNA gene fragment from a CLO from Metaseiulus occidentalis (Nesbitt). P. persimilis genomic DNA was mixed with the serially diluted plasmid and amplified using MDA followed by either standard or high-fidelity PCR. MDA followed by high-fidelity PCR was most efficient and successfully amplified an expected 1.5-kb band from as little as 0.01fg of the plasmid, which is equivalent to about 1 copy. MDA followed by high-fidelity PCR also consistently amplified Wolbachia- or CLO-specific products from naturally infected single females or eggs of M. occidentalis, which will allow detailed studies of infection frequency and transmission of several microorganisms associated with this predatory mite.

A linear concatenation strategy to construct 5'-enriched amplified cDNA libraries using multiple displacement amplification.

PubMed

Gadkar, Vijay J; Filion, Martin

2013-06-01

In various experimental systems, limiting available amounts of RNA may prevent a researcher from performing large-scale analyses of gene transcripts. One way to circumvent this is to 'pre-amplify' the starting RNA/cDNA, so that sufficient amounts are available for any downstream analysis. In the present study, we report the development of a novel protocol for constructing amplified cDNA libraries using the Phi29 DNA polymerase based multiple displacement amplification (MDA) system. Using as little as 200 ng of total RNA, we developed a linear concatenation strategy to make the single-stranded cDNA template amenable for MDA. The concatenation, made possible by the template switching property of the reverse transcriptase enzyme, resulted in the amplified cDNA library with intact 5' ends. MDA generated micrograms of template, allowing large-scale polymerase chain reaction analyses or other large-scale downstream applications. As the amplified cDNA library contains intact 5' ends, it is also compatible with 5' RACE analyses of specific gene transcripts. Empirical validation of this protocol is demonstrated on a highly characterized (tomato) and an uncharacterized (corn gromwell) experimental system.

Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles.

PubMed

Stepanauskas, Ramunas; Fergusson, Elizabeth A; Brown, Joseph; Poulton, Nicole J; Tupper, Ben; Labonté, Jessica M; Becraft, Eric D; Brown, Julia M; Pachiadaki, Maria G; Povilaitis, Tadas; Thompson, Brian P; Mascena, Corianna J; Bellows, Wendy K; Lubys, Arvydas

2017-07-20

Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms. Here we present WGA-X, a method based on multiple displacement amplification of DNA that utilizes a thermostable mutant of the phi29 polymerase. WGA-X enhances genome recovery from individual microbial cells and viral particles while maintaining ease of use and scalability. The greatest improvements are observed when amplifying high G+C content templates, such as those belonging to the predominant bacteria in agricultural soils. By integrating WGA-X with calibrated index-cell sorting and high-throughput genomic sequencing, we are able to analyze genomic sequences and cell sizes of hundreds of individual, uncultured bacteria, archaea, protists, and viral particles, obtained directly from marine and soil samples, in a single experiment. This approach may find diverse applications in microbiology and in biomedical and forensic studies of humans and other multicellular organisms.Single-cell genomics can be used to study uncultured microorganisms. Here, Stepanauskas et al. present a method combining improved multiple displacement amplification and FACS, to obtain genomic sequences and cell size information from uncultivated microbial cells and viral particles in environmental samples.

Preparation of genomic DNA from a single species of uncultured magnetotactic bacterium by multiple-displacement amplification.

PubMed

Arakaki, Atsushi; Shibusawa, Mie; Hosokawa, Masahito; Matsunaga, Tadashi

2010-03-01

Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order to obtain genetic information from uncultured magnetotactic bacteria, a genome preparation method that involves magnetic separation of cells, flow cytometry, and multiple displacement amplification (MDA) using phi29 polymerase was used in this study. The conditions for the MDA reaction using samples containing 1 to 100 cells were evaluated using a pure-culture magnetotactic bacterium, "Magnetospirillum magneticum AMB-1," whose complete genome sequence is available. Uniform gene amplification was confirmed by quantitative PCR (Q-PCR) when 100 cells were used as a template. This method was then applied for genome preparation of uncultured magnetotactic bacteria from complex bacterial communities in an aquatic environment. A sample containing 100 cells of the uncultured magnetotactic coccus was prepared by magnetic cell separation and flow cytometry and used as an MDA template. 16S rRNA sequence analysis of the MDA product from these 100 cells revealed that the amplified genomic DNA was from a single species of magnetotactic bacterium that was phylogenetically affiliated with magnetotactic cocci in the Alphaproteobacteria. The combined use of magnetic separation, flow cytometry, and MDA provides a new strategy to access individual genetic information from magnetotactic bacteria in environmental samples.

Catalytic molecular logic devices by DNAzyme displacement.

PubMed

Brown, Carl W; Lakin, Matthew R; Stefanovic, Darko; Graves, Steven W

2014-05-05

Chemical reactions catalyzed by DNAzymes offer a route to programmable modification of biomolecules for therapeutic purposes. To this end, we have developed a new type of catalytic DNA-based logic gates in which DNAzyme catalysis is controlled via toehold-mediated strand displacement reactions. We refer to these as DNAzyme displacement gates. The use of toeholds to guide input binding provides a favorable pathway for input recognition, and the innate catalytic activity of DNAzymes allows amplification of nanomolar input concentrations. We demonstrate detection of arbitrary input sequences by rational introduction of mismatched bases into inhibitor strands. Furthermore, we illustrate the applicability of DNAzyme displacement to compute logic functions involving multiple logic gates. This work will enable sophisticated logical control of a range of biochemical modifications, with applications in pathogen detection and autonomous theranostics. © 2014 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.

Limits on amplification by Aharonov-Albert-Vaidman weak measurement

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

Koike, Tatsuhiko; Tanaka, Saki

2011-12-15

We analyze the amplification by the Aharonov-Albert-Vaidman weak quantum measurement on a Sagnac interferometer [Dixon et al., Phys. Rev. Lett. 102, 173601 (2009)] up to all orders of the coupling strength between the measured system and the measuring device. The amplifier transforms a small tilt of a mirror into a large transverse displacement of the laser beam. The conventional analysis has shown that the measured value is proportional to the weak value, so that the amplification can be made arbitrarily large in the cost of decreasing output laser intensity. It is shown that the measured displacement and the amplification factormore » are in fact not proportional to the weak value and rather vanish in the limit of infinitesimal output intensity. We derive the optimal overlap of the pre- and postselected states with which the amplification become maximum. We also show that the nonlinear effects begin to arise in the performed experiments so that any improvements in the experiment, typically with an amplification greater than 100, should require the nonlinear theory in translating the observed value to the original displacement.« less

Strand displacement amplification as an in vitro model for rolling-circle replication: deletion formation and evolution during serial transfer.

PubMed Central

Walter, N G; Strunk, G

1994-01-01

Strand displacement amplification is an isothermal DNA amplification reaction based on a restriction endonuclease nicking its recognition site and a polymerase extending the nick at its 3' end, displacing the downstream strand. The reaction resembles rolling-circle replication of single-stranded phages and small plasmids. The displaced sense strand serves as target for an antisense reaction and vice versa, resulting in exponential growth and the autocatalytic nature of this in vitro reaction as long as the template is the limiting agent. We describe the optimization of strand displacement amplification for in vitro evolution experiments under serial transfer conditions. The reaction was followed and controlled by use of the fluorescent dye thiazole orange binding to the amplified DNA. We were able to maintain exponential growth conditions with a doubling time of 3.0 min throughout 100 transfers or approximately 350 molecular generations by using an automatic handling device. Homology of in vitro amplification with rolling-circle replication was mirrored by the occurring evolutionary processes. Deletion events most likely caused by a slipped mispairing mechanism as postulated for in vivo replication took place. Under our conditions, the mutation rate was high and a molecular quasi-species formed with a mutant lacking internal hairpin formation ability and thus outgrowing all other species under dGTP/dCTP deficiency. Images PMID:8058737

Molecular analysis of single oocyst of Eimeria by whole genome amplification (WGA) based nested PCR.

PubMed

Wang, Yunzhou; Tao, Geru; Cui, Yujuan; Lv, Qiyao; Xie, Li; Li, Yuan; Suo, Xun; Qin, Yinghe; Xiao, Lihua; Liu, Xianyong

2014-09-01

PCR-based molecular tools are widely used for the identification and characterization of protozoa. Here we report the molecular analysis of Eimeria species using combined methods of whole genome amplification (WGA) and nested PCR. Single oocyst of Eimeria stiedai or Eimeriamedia was directly used for random amplification of the genomic DNA with either primer extension preamplification (PEP) or multiple displacement amplification (MDA), and then the WGA product was used as template in nested PCR with species-specific primers for ITS-1, 18S rDNA and 23S rDNA of E. stiedai and E. media. WGA-based PCR was successful for the amplification of these genes from single oocyst. For the species identification of single oocyst isolated from mixed E. stiedai or E. media, the results from WGA-based PCR were exactly in accordance with those from morphological identification, suggesting the availability of this method in molecular analysis of eimerian parasites at the single oocyst level. WGA-based PCR method can also be applied for the identification and genetic characterization of other protists. Copyright © 2014 Elsevier Inc. All rights reserved.

Aptamer-based microfluidic beads array sensor for simultaneous detection of multiple analytes employing multienzyme-linked nanoparticle amplification and quantum dots labels.

PubMed

Zhang, He; Hu, Xinjiang; Fu, Xin

2014-07-15

This study reports the development of an aptamer-mediated microfluidic beads-based sensor for multiple analytes detection and quantification using multienzyme-linked nanoparticle amplification and quantum dots labels. Adenosine and cocaine were selected as the model analytes to validate the assay design based on strand displacement induced by target-aptamer complex. Microbeads functionalized with the aptamers and modified electron rich proteins were arrayed within a microfluidic channel and were connected with the horseradish peroxidases (HRP) and capture DNA probe derivative gold nanoparticles (AuNPs) via hybridization. The conformational transition of aptamer induced by target-aptamer complex contributes to the displacement of functionalized AuNPs and decreases the fluorescence signal of microbeads. In this approach, increased binding events of HRP on each nanosphere and enhanced mass transport capability inherent from microfluidics are integrated for enhancing the detection sensitivity of analytes. Based on the dual signal amplification strategy, the developed aptamer-based microfluidic bead array sensor could discriminate as low as 0.1 pM of adenosine and 0.5 pM cocaine, and showed a 500-fold increase in detection limit of adenosine compared to the off-chip test. The results proved the microfluidic-based method was a rapid and efficient system for aptamer-based targets assays (adenosine (0.1 pM) and cocaine (0.5 pM)), requiring only minimal (microliter) reagent use. This work demonstrated the successful application of aptamer-based microfluidic beads array sensor for detection of important molecules in biomedical fields. Copyright © 2014 Elsevier B.V. All rights reserved.

Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification

PubMed Central

Maruyama, Toru; Yamagishi, Keisuke; Mori, Tetsushi; Takeyama, Haruko

2015-01-01

Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 105 droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples. PMID:26389587

Evaluation of the efficacy of constitutional array-based comparative genomic hybridization in the diagnosis of aneuploidy using genomic and amplified DNA.

PubMed

Tan, Niap H; Palmer, Rodger; Wang, Rubin

2010-02-01

Array-based comparative genomic hybridization (array CGH) is a new molecular technique that has the potential to revolutionize cytogenetics. However, use of high resolution array CGH in the clinical setting is plagued by the problem of widespread copy number variations (CNV) in the human genome. Constitutional microarray, containing only clones that interrogate regions of known constitutional syndromes, may circumvent the dilemma of detecting CNV of unknown clinical significance. The present study investigated the efficacy of constitutional microarray in the diagnosis of trisomy. Test samples included genomic DNA from trisomic cell lines, amplification products of 50 ng of genomic DNA and whole genome amplification products of single cells. DNA amplification was achieved by means of multiple displacement amplification (MDA) over 16 h. The trisomic and sex chromosomes copy number imbalances in the genomic DNA were correctly identified by the constitutional microarrays. However, there was a failure to detect the trisomy in the amplification products of 50 ng of genomic DNA and whole genome amplification products of single cells. Using carefully selected clones, Spectral Genomics constitutional microarray was able to detect the chromosomal copy number imbalances in genomic DNA without the confounding effects of CNV. The diagnostic failure in amplified DNA samples could be attributed to the amplification process. The MDA duration of 16 h generated excessive amount of biases and shortening the duration might minimize the problem.

A label-free fluorescent direct detection of live Salmonella typhimurium using cascade triple trigger sequences-regenerated strand displacement amplification and hairpin template-generated-scaffolded silver nanoclusters.

PubMed

Zhang, Peng; Liu, Hui; Li, Xiaocheng; Ma, Suzhen; Men, Shuai; Wei, Heng; Cui, Jingjing; Wang, Hongning

2017-01-15

The harm of Salmonella typhimurium (S. typhimurium) to public health mainly by the consumption of contaminated agricultural products or water stresses an urgent need for rapid detection methods to help control the spread of S. typhimurium. In this work, an intelligently designed sensor system took creative advantage of triple trigger sequences-regenerated strand displacement amplification and self-protective hairpin template-generated-scaffolded silver nanoclusters (AgNCs) for the first time. In the presence of live S. typhimurium, single-stranded trigger sequences were released from aptamer-trigger sequences complex, initiating a branch migration to open the hairpin template I containing complementary scaffolds of AgNCs. Then the first strand displacement amplification was induced to produce numerous scaffolds of AgNCs and reporter strands which initiated a branch migration to open the hairpin template II containing complementary scaffolds of AgNCs. Then the second strand displacement amplification was induced to generate numerous scaffolds of AgNCs and trigger sequences which initiated the third branch migration and strand displacement amplification to produce numerous scaffolds of AgNCs and reporter strands in succession. Cyclically, the reproduction of the trigger sequences and cascade successive production of scaffolds were achieved successfully, forming highly fluorescent AgNCs, thus providing significantly enhanced fluorescent signals to achieve ultrasensitive detection of live S. typhimurium down to 50 CFU/mL with a linear range from 10 2 to 10 7 CFU/mL. It is the first report on a fluorescent biosensor for detecting viable S. typhimurium directly, which can distinguish from heat denatured S. typhimurium. And it develops a new strategy to generate the DNA-scaffolds for forming AgNCs. Copyright © 2016 Elsevier B.V. All rights reserved.

Homogenous assay for protein detection based on proximity DNA hybridization and isothermal circular strand displacement amplification reaction.

PubMed

Zhang, Manjun; Li, Ruimin; Ling, Liansheng

2017-06-01

This work proposed a homogenous fluorescence assay for proteins, based on the target-triggered proximity DNA hybridization in combination with strand displacement amplification (SDA). It benefited from target-triggered proximity DNA hybridization to specifically recognize the target and SDA making recycling signal amplification. The system included a molecular beacon (MB), an extended probe (EP), and an assistant probe (AP), which were not self-assembly in the absence of target proteins, due to the short length of the designed complementary sequence among MB, EP, and AP. Upon addition of the target proteins, EP and AP are bound to the target proteins, which induced the occurrence of proximity hybridization between MB, EP, and AP and followed by strand displacement amplification. Through the primer extension, a tripartite complex of probes and target was displaced and recycled to hybridize with another MB, and the more opened MB enabled the detection signal to amplify. Under optimum conditions, it was used for the detection of streptavidin and thrombin. Fluorescence intensity was proportional to the concentration of streptavidin and thrombin in the range of 0.2-30 and 0.2-35 nmol/L, respectively. Furthermore, this fluorescent method has a good selectivity, in which the fluorescence intensity of thrombin was ~37-fold or even larger than that of the other proteins at the same concentration. It is a new and simple method for SDA-involved target protein detection and possesses a great potential for other protein detection in the future. Graphical abstract A homogenous assay for protein detection is based on proximity DNA hybridization and strand displacement amplification reaction.

[Do Multiplex PCR techniques displace classical cultures in microbiology?].

PubMed

Auckenthaler, Raymond; Risch, Martin

2015-02-01

Multiplex PCR technologies progressively find their way in clinical microbiology. This technique allows the simultaneous amplification of multiple DNA targets in a single test run for the identification of pathogens up to the species level. Various pathogens of infectious diseases can be detected by a symptom-oriented approach clearly and quickly with high reliability. Essentially multiplex PCR panels are available for clarification of gastrointestinal, respiratory, sexually transmitted infections and meningitis. Today's offer from industry, university hospitals and large private laboratories of Switzerland is tabulated and commented.

Highly sensitive chemiluminescent point mutation detection by circular strand-displacement amplification reaction.

PubMed

Shi, Chao; Ge, Yujie; Gu, Hongxi; Ma, Cuiping

2011-08-15

Single nucleotide polymorphism (SNP) genotyping is attracting extensive attentions owing to its direct connections with human diseases including cancers. Here, we have developed a highly sensitive chemiluminescence biosensor based on circular strand-displacement amplification and the separation by magnetic beads reducing the background signal for point mutation detection at room temperature. This method took advantage of both the T4 DNA ligase recognizing single-base mismatch with high selectivity and the strand-displacement reaction of polymerase to perform signal amplification. The detection limit of this method was 1.3 × 10(-16)M, which showed better sensitivity than that of most of those reported detection methods of SNP. Additionally, the magnetic beads as carrier of immobility was not only to reduce the background signal, but also may have potential apply in high through-put screening of SNP detection in human genome. Copyright © 2011 Elsevier B.V. All rights reserved.

Linear nicking endonuclease-mediated strand-displacement DNA amplification.

PubMed

Joneja, Aric; Huang, Xiaohua

2011-07-01

We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. The combined continuous repetitive action of nicking by the endonuclease and strand-displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule. We demonstrate that DNA templates up to 5000 nucleotides can be linearly amplified using a nicking endonuclease with 7-bp recognition sequence and Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of three templates of 500, 1000, and 5000 nucleotides in length is linearly amplified with the original molar ratios of the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic DNA from bacteriophage lambda can be amplified linearly. Copyright © 2011 Elsevier Inc. All rights reserved.

Linear nicking endonuclease-mediated strand displacement DNA amplification

PubMed Central

Joneja, Aric; Huang, Xiaohua

2011-01-01

We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. The combined continuous repetitive action of nicking by the endonuclease and strand displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule. We demonstrate that DNA templates up to five thousand nucleotides can be linearly amplified using a nicking endonuclease with seven base-pair recognition sequence and Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of three templates of 500, 1000, and 5000 nucleotides in length are linearly amplified with the original molar ratios of the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic DNA from bacteriophage lambda can be amplified linearly. PMID:21342654

Amplified electrochemiluminescence detection of DNA based on novel quantum dots signal probe by multiple cycling amplification strategy.

PubMed

Tan, Lu; Ge, Junjun; Jiao, Meng; Jie, Guifen; Niu, Shuyan

2018-06-01

In the present work, we designed a unique enzyme-aided multiple amplification strategy for sensitive electrochemiluminescence (ECL) detection of DNA by using the amplified gold nanoparticles (GNPS)-polyamidoamine (PAMAM)-CdSe quantum dots (QDs) signal probe. Firstly, the novel GNPS-PAMAM dendrimers nanostructure with good biocompatibility and electroconductibility contains many amino groups, which can load a large number of CdSe QDs to develop amplified ECL signal probe. Then, the presence of target DNA activated the enzyme-assisted polymerization strand-displacement cycling reaction, and a large number of the hairpin template was opened. Subsequently, the opened stem further interacted with the capture hairpin (HP) DNA on the electrode, and the GNPS-PAMAM-CdSe signal probe hybridized with the exposed stem of the HP to trigger the second new polymerization reaction. Meanwhile, the first cycle was generating abundant DNA triggers which could directly open the template. As a result of the cascade amplification technique, a large number of CdSe QDs signal probe could be assembled on the electrode, generating much amplified ECL signal for sensitive detection of target DNA. Thus, this novel QDs-based amplified ECL strategy holds great promise for DNA detection and can be further exploited for sensing applications in clinical diagnostics. Copyright © 2018 Elsevier B.V. All rights reserved.

Target-aptamer binding triggered quadratic recycling amplification for highly specific and ultrasensitive detection of antibiotics at the attomole level.

PubMed

Wang, Hongzhi; Wang, Yu; Liu, Su; Yu, Jinghua; Xu, Wei; Guo, Yuna; Huang, Jiadong

2015-05-14

A novel electrochemical aptasensor for ultrasensitive detection of antibiotics by combining polymerase-assisted target recycling amplification with strand displacement amplification with the help of polymerase and nicking endonuclease has been reported. This work is the first time that target-aptamer binding triggered quadratic recycling amplification has been utilized for electrochemical detection of antibiotics.

Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.

PubMed

Hosokawa, Masahito; Nishikawa, Yohei; Kogawa, Masato; Takeyama, Haruko

2017-07-12

Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

Signal amplification of microRNAs with modified strand displacement-based cycling probe technology.

PubMed

Jia, Huning; Bu, Ying; Zou, Bingjie; Wang, Jianping; Kumar, Shalen; Pitman, Janet L; Zhou, Guohua; Song, Qinxin

2016-10-24

Micro ribose nucleic acids (miRNAs) play an important role in biological processes such as cell differentiation, proliferation and apoptosis. Therefore, miRNAs are potentially a powerful marker for monitoring cancer and diagnosis. Here, we present sensitive signal amplification for miRNAs based on modified cycling probe technology with strand displacement amplification. miRNA was captured by the template coupled with beads, and then the first cycle based on SDA was repeatedly extended to the nicking end, which was produced by the extension reaction of miRNA. The products generated by SDA are captured by a molecular beacon (MB), which is designed to initiate the second amplification cycle, with a similar principle to the cycling probe technology (CPT), which is based on repeated digestion of the DNA-RNA hybrid by the RNase H. After one sample enrichment and two steps of signal amplification, 0.1 pM of let-7a can be detected. The miRNA assay exhibits a great dynamic range of over 100 orders of magnitude and high specificity to clearly discriminate a single base difference in miRNA sequences. This isothermal amplification does not require any special temperature control instrument. The assay is also about signal amplification rather than template amplification, therefore minimising contamination issues. In addition, there is no need for the reverse transcription (RT) process. Thus the amplification is suitable for miRNA detection.

[Establishment of a novel HLA genotyping method for preimplantation genetic diagnonis using multiple displacement amplification-polymerase chain reaction-sequencing based technique].

PubMed

Zhang, Yinfeng; Luo, Haining; Zhang, Yunshan

2015-12-01

To establish a novel HLA genotyping method for preimplantation genetic diagnonis (PGD) using multiple displacement amplification-polymerase chain reaction-sequencing based technique (MDA-PCR-SBT). Peripheral blood samples and 76 1PN, 2PN, 3PN discarded embryos from 9 couples were collected. The alleles of HLA-A, B, DR loci were detected from the MDA product with the PCR-SBT method. The HLA genotypes of the parental peripheral blood samples were analyzed with the same protocol. The genotypes of specific HLA region were evaluated for distinguishing the segregation of haplotypes among the family members, and primary HLA matching was performed between the embryos. The 76 embryos were subjected to MDA and 74 (97.4%) were successfully amplified. For the 34 embryos from the single blastomere group, the amplification rate was 94.1%, and for the 40 embryos in the two blastomeres group, the rate was 100%. The dropout rates for DQ allele and DR allele were 1.3% and 0, respectively. The positive rate for MDA in the single blastomere group was 100%, with the dropout rates for DQ allele and DR allele being 1.5% and 0, respectively. The positive rate of MDA for the two blastomere group was 100%, with the dropout rates for both DQ and DR alleles being 0. The recombination rate of fetal HLA was 20.2% (30/148). Due to the improper classification and abnormal fertilized embryos, the proportion of matched embryos HLA was 20.3% (15/74),which was lower than the theoretical value of 25%. PGD with HLA matching can facilitate creation of a HLA-identical donor (saviour child) for umbilical cord blood or bone marrow stem cells for its affected sibling with a genetic disease. Therefore, preimplantation HLA matching may provide a tool for couples desiring to conceive a potential donor progeny for transplantation for its sibling with a life-threatening disorder.

Increasing pumping efficiency in a micro throttle pump by enhancing displacement amplification in an elastomeric substrate

NASA Astrophysics Data System (ADS)

Fujiwara, T.; Johnston, I. D.; Tracey, M. C.; Tan, C. K. L.

2010-06-01

Fluid transport is accomplished in a micro throttle pump (MTP) by alternating deformation of a micro channel cast into a polydimethylsiloxane (PDMS) elastomeric substrate. The active deformation is achieved using a bimorph PZT piezoelectric disc actuator bonded to a glass diaphragm. The bimorph PZT deflects the diaphragm as well as alternately pushing and pulling the elastomer layer providing displacement amplification in the PDMS directly surrounding the micro channel. In order to improve pumping rates we have embedded a polymethylmethacrylate (PMMA) ring into the PMDS substrate which increases the magnitude of the displacement amplification achieved. FEM simulation of the elastomeric substrate deformation predicts that the inclusion of the PMMA ring should increase the channel deformation. We experimentally demonstrate that inclusion of a PMMA ring, having a diameter equal to that of the circular node of the PZT/glass/PDMS composite, increases in the throttle resistance ratio by 40% and the maximum pumping rate by 90% compared to an MTP with no ring.

Systematic evaluation of bias in microbial community profiles induced by whole genome amplification.

PubMed

Direito, Susana O L; Zaura, Egija; Little, Miranda; Ehrenfreund, Pascale; Röling, Wilfred F M

2014-03-01

Whole genome amplification methods facilitate the detection and characterization of microbial communities in low biomass environments. We examined the extent to which the actual community structure is reliably revealed and factors contributing to bias. One widely used [multiple displacement amplification (MDA)] and one new primer-free method [primase-based whole genome amplification (pWGA)] were compared using a polymerase chain reaction (PCR)-based method as control. Pyrosequencing of an environmental sample and principal component analysis revealed that MDA impacted community profiles more strongly than pWGA and indicated that this related to species GC content, although an influence of DNA integrity could not be excluded. Subsequently, biases by species GC content, DNA integrity and fragment size were separately analysed using defined mixtures of DNA from various species. We found significantly less amplification of species with the highest GC content for MDA-based templates and, to a lesser extent, for pWGA. DNA fragmentation also interfered severely: species with more fragmented DNA were less amplified with MDA and pWGA. pWGA was unable to amplify low molecular weight DNA (< 1.5 kb), whereas MDA was inefficient. We conclude that pWGA is the most promising method for characterization of microbial communities in low-biomass environments and for currently planned astrobiological missions to Mars. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Development of small molecule biosensors by coupling the recognition of the bacterial allosteric transcription factor with isothermal strand displacement amplification.

PubMed

Yao, Yongpeng; Li, Shanshan; Cao, Jiaqian; Liu, Weiwei; Fan, Keqiang; Xiang, Wensheng; Yang, Keqian; Kong, Deming; Wang, Weishan

2018-05-08

Here, we demonstrate an easy-to-implement and general biosensing strategy by coupling the small-molecule recognition of the bacterial allosteric transcription factor (aTF) with isothermal strand displacement amplification (SDA) in vitro. Based on this strategy, we developed two biosensors for the detection of an antiseptic, p-hydroxybenzoic acid, and a disease marker, uric acid, using bacterial aTF HosA and HucR, respectively, highlighting the great potential of this strategy for the development of small-molecule biosensors.

Fluorometric determination of nucleic acids based on the use of polydopamine nanotubes and target-induced strand displacement amplification.

PubMed

Ge, Jia; Bai, Dong-Mei; -Geng, Xin; Hu, Ya-Lei; Cai, Qi-Yong; Xing, Ke; Zhang, Lin; Li, Zhao-Hui

2018-01-10

The authors describe a fluorometric method for the quantitation of nucleic acids by combining (a) cycled strand displacement amplification, (b) the unique features of the DNA probe SYBR Green, and (c) polydopamine nanotubes. SYBR Green undergoes strong fluorescence enhancement upon intercalation into double-stranded DNA (dsDNA). The polydopamine nanotubes selectively adsorb single-stranded DNA (ssDNA) and molecular beacons. In the absence of target DNA, the molecular beacon, primer and SYBR Green are adsorbed on the surface of polydopamine nanotubes. This results in quenching of the fluorescence of SYBR Green, typically measured at excitation/emission wavelengths of 488/518 nm. Upon addition of analyte (target DNA) and polymerase, the stem of the molecular beacon is opened so that it can bind to the primer. This triggers target strand displacement polymerization, during which dsDNA is synthesized. The hybridized target is then displaced due to the strand displacement activity of the polymerase. The displaced target hybridizes with another molecular beacon. This triggers the next round of polymerization. Consequently, a large amount of dsDNA is formed which is detected by addition of SYBR Green. Thus, sensitive and selective fluorometric detection is realized. The fluorescent sensing strategy shows very good analytical performances towards DNA detection, such as a wide linear range from 0.05 to 25 nM with a low limit of detection of 20 pM. Graphical abstract Schematic of a fluorometric strategy for highly sensitive and selective determination of nucleic acids by combining strand displacement amplification and the unique features of SYBR Green I (SG) and polydopamine nanotubes.

The spatial and temporal representation of a tone on the guinea pig basilar membrane

NASA Astrophysics Data System (ADS)

Nilsen, K. E.; Russell, I. J.

2000-10-01

School of Biological Sciences, University of Sussex, Falmer Brighton, BN1 9QG, United Kingdom In the mammalian cochlea, the basilar membrane's (BM) mechanical responses are amplified, and frequency tuning is sharpened through active feedback from the electromotile outer hair cells (OHCs). To be effective, OHC feedback must be delivered to the correct region of the BM and introduced at the appropriate time in each cycle of BM displacement. To investigate when OHCs contribute to cochlear amplification, a laser-diode interferometer was used to measure tone-evoked BM displacements in the basal turn of the guinea pig cochlea. Measurements were made at multiple sites acrossthe width of the BM, which are tuned to the same characteristic frequency (CF). In response to CF tones, the largest displacements occur in the OHC region and phase lead those measured beneath the outer pillar cells and adjacent to the spiral ligament by about 90°. Postmortem, responses beneath the OHCs are reduced by up to 65 dB, and all regions across the width of the BM move in unison. We suggest that OHCs amplify BM responses to CF tones when the BM is moving at maximum velocity. In regions of the BM where OHCs contribute to its motion, the responses are compressive and nonlinear. We measured the distribution of nonlinear compressive vibrations along the length of the BM in response to a single frequency tone and estimated that OHC amplification is restricted to a 1.25- to 1.40-mm length of BM centered on the CF place.

Signal amplification of padlock probes by rolling circle replication.

PubMed Central

Banér, J; Nilsson, M; Mendel-Hartvig, M; Landegren, U

1998-01-01

Circularizing oligonucleotide probes (padlock probes) have the potential to detect sets of gene sequences with high specificity and excellent selectivity for sequence variants, but sensitivity of detection has been limiting. By using a rolling circle replication (RCR) mechanism, circularized but not unreacted probes can yield a powerful signal amplification. We demonstrate here that in order for the reaction to proceed efficiently, the probes must be released from the topological link that forms with target molecules upon hybridization and ligation. If the target strand has a nearby free 3' end, then the probe-target hybrids can be displaced by the polymerase used for replication. The displaced probe can then slip off the targetstrand and a rolling circle amplification is initiated. Alternatively, the target sequence itself can prime an RCR after its non-base paired 3' end has been removed by exonucleolytic activity. We found the Phi29 DNA polymerase to be superior to the Klenow fragment in displacing the target DNA strand, and it maintained the polymerization reaction for at least 12 h, yielding an extension product that represents several thousand-fold the length of the padlock probe. PMID:9801302

Ground motion in Anchorage, Alaska, from the 2002 Denali fault earthquake: Site response and Displacement Pulses

USGS Publications Warehouse

Boore, D.M.

2004-01-01

Data from the 2002 Denali fault earthquake recorded at 26 sites in and near Anchorage, Alaska, show a number of systematic features important in studies of site response and in constructing long-period spectra for use in earthquake engineering. The data demonstrate that National Earthquake Hazards Reduction Program (NEHRP) site classes are a useful way of grouping stations according to site amplification. In general, the sites underlain by lower shear-wave velocities have higher amplification. The amplification on NEHRP class D sites exceeds a factor of 2 relative to an average of motions on class C sites. The amplifications are period dependent. They are in rough agreement with those from previous studies, but the new data show that the amplifications extend to at least 10 sec, periods longer than considered in previous studies. At periods longer than about 14 sec, all sites have motion of similar amplitude, and the ground displacements are similar in shape, polarization, and amplitude for all stations. The displacement ground motion is dominated by a series of four pulses, which are associated with the three subevents identified in inversion studies (the first pulse is composed of P waves from the first subevent). Most of the high-frequency ground motion is associated with the S waves from subevent 1. The pulses from subevents 1 and 2, with moment releases corresponding to M 7.1 and 7.0, are similar to the pulse of displacement radiated by the M 7.1 Hector Mine earthquake. The signature from the largest subevent (M 7.6) is more subdued than those from the first two subevents. The two largest pulses produce response spectra with peaks at a period of about 15 sec. The spectral shape at long periods is in good agreement with the recent 2003 NEHRP code spectra but is in poor agreement with the shape obtained from Eurocode 8.

Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.

PubMed

Cheng, Rui; Tao, Mangjuan; Shi, Zhilu; Zhang, Xiafei; Jin, Yan; Li, Baoxin

2015-11-15

Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research. Copyright © 2015 Elsevier B.V. All rights reserved.

A micromechanical analogue mixer with dynamic displacement amplification

NASA Astrophysics Data System (ADS)

Erismis, M. A.

2018-06-01

A new micromechanical device is proposed which is capable of modulation, demodulation and filtering operations. The device uses a patented 3-mass coupled micromechanical resonator which dynamically amplifies the displacement within a frequency range of interest. Modulation can be obtained by exciting different masses of the resonator with the data and the carrier signals. Demodulation can be obtained similarly by exciting the actuator with the input and carrier signals at the same time. With the help of dynamic motion amplification, filtering and signal amplification can be achieved simultaneously. A generic design approach is introduced which can be applied from kHz to MHz regime frequencies of interest. A sample mixer design for an silicon on insulator-based process is provided. A SPICE (Simulation Program with Integrated Circuit Emphasis)-based electro-mechanical co-simulation platform is also developed and the proposed mixer is simulated.

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.

A new photoelectrochemical biosensors based on DNA conformational changes and isothermal circular strand-displacement polymerization reaction.

PubMed

Zhang, Xiaoru; Xu, Yunpeng; Zhao, Yanqing; Song, Weiling

2013-01-15

We report a strategy for the transduction of DNA hybridization into a readily detectable photoelectrochemical signal by means of a conformational change analogous to electrochemical DNA (E-DNA) approach. To demonstrate the effect of distance change for photosensitizer to the surface of electrode on the change of photocurrent, photosensitizer Ru(bpy)(2)(dcbpy)(2+) tagged DNA stem-loop structures were self-assembled onto a nanogold modified ITO electrode. Hybridization induced a large conformational change in DNA structure, which in turn significantly altered the electron-transfer tunneling distance between the electrode and photosensitizer. The resulting change in photocurrent was proportional to the concentration of DNA in the range of 1.0×10(-10)-8.0×10(-9)M. In order to improve the sensitivity of the photoelectrochemical biosensor, an amplified detection method based on isothermal strand displacement polymerization reaction was employed. With multiple rounds of isothermal strand replication, which led to strand displacement and constituted consecutive signal amplification, a detection limit of 9.4×10(-14)M target DNA was achieved. Copyright © 2012 Elsevier B.V. All rights reserved.

Homogeneous real-time detection of single-nucleotide polymorphisms by strand displacement amplification on the BD ProbeTec ET system.

PubMed

Wang, Sha-Sha; Thornton, Keith; Kuhn, Andrew M; Nadeau, James G; Hellyer, Tobin J

2003-10-01

The BD ProbeTec ET System is based on isothermal strand displacement amplification (SDA) of target nucleic acid coupled with homogeneous real-time detection using fluorescent probes. We have developed a novel, rapid method using this platform that incorporates a universal detection format for identification of single-nucleotide polymorphisms (SNPs) and other genotypic variations. The system uses a common pair of fluorescent Detector Probes in conjunction with unlabeled allele-specific Adapter Primers and a universal buffer chemistry to permit analysis of multiple SNP loci under generic assay conditions. We used Detector Probes labeled with different dyes to facilitate differentiation of two alternative alleles in a single reaction with no postamplification manipulation. We analyzed six SNPs within the human beta(2)-adrenergic receptor (beta(2)AR) gene, using whole blood, buccal swabs, and urine samples, and compared results with those obtained by DNA sequencing. Unprocessed whole blood was successfully genotyped with as little as 0.1-1 micro L of sample per reaction. All six beta(2)AR assays were able to accommodate >/==" BORDER="0">20 micro L of unprocessed whole blood. For the 14 individuals tested, genotypes determined with the six beta(2)AR assays agreed with DNA sequencing results. SDA-based allelic differentiation on the BD ProbeTec ET System can detect SNPs rapidly, using whole blood, buccal swabs, or urine.

Optimization of strand displacement amplification-sensitized G-quadruplex DNAzyme-based sensing system and its application in activity detection of uracil-DNA glycosylase.

PubMed

Du, Yi-Chen; Jiang, Hong-Xin; Huo, Yan-Fang; Han, Gui-Mei; Kong, De-Ming

2016-03-15

As an isothermal nucleic acid amplification technique, strand displacement amplification (SDA) reaction has been introduced in G-quadruplex DNAzyme-based sensing system to improve the sensing performance. To further provide useful information for the design of SDA-amplified G-quadruplex DNAzyme-based sensors, the effects of nicking site number in SDA template DNA were investigated. With the increase of the nicking site number from 1 to 2, enrichment of G-quadruplex DNAzyme by SDA is changed from a linear amplification to an exponential amplification, thus greatly increasing the amplification efficiency and subsequently improving the sensing performance of corresponding sensing system. The nicking site number cannot be further increased because more nicking sites might result in high background signals. However, we demonstrated that G-quadruplex DNAzyme enrichment efficiency could be further improved by introducing a cross-triggered SDA strategy, in which two templates each has two nicking sites are used. To validate the proposed cross-triggered SDA strategy, we used it to develop a sensing platform for the detection of uracil-DNA glycosylase (UDG) activity. The sensor enables sensitive detection of UDG activity in the range of 1 × 10(-4)-1 U/mL with a detection limit of 1 × 10(-4)U/mL. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Design of a sensitive aptasensor based on magnetic microbeads-assisted strand displacement amplification and target recycling.

PubMed

Li, Ying; Ji, Xiaoting; Song, Weiling; Guo, Yingshu

2013-04-03

A cross-circular amplification system for sensitive detection of adenosine triphosphate (ATP) in cancer cells was developed based on aptamer-target interaction, magnetic microbeads (MBs)-assisted strand displacement amplification and target recycling. Here we described a new recognition probe possessing two parts, the ATP aptamer and the extension part. The recognition probe was firstly immobilized on the surface of MBs and hybridized with its complementary sequence to form a duplex. When combined with ATP, the probe changed its conformation, revealing the extension part in single-strand form, which further served as a toehold for subsequent target recycling. The released complementary sequence of the probe acted as the catalyst of the MB-assisted strand displacement reaction. Incorporated with target recycling, a large amount of biotin-tagged MB complexes were formed to stimulate the generation of chemiluminescence (CL) signal in the presence of luminol and H2O2 by incorporating with streptavidin-HRP, reaching a detection limit of ATP as low as 6.1×10(-10)M. Moreover, sample assays of ATP in Ramos Burkitt's lymphoma B cells were performed, which confirmed the reliability and practicality of the protocol. Copyright © 2013 Elsevier B.V. All rights reserved.

Electromechanical Displacement Detection With an On-Chip High Electron Mobility Transistor Amplifier

NASA Astrophysics Data System (ADS)

Oda, Yasuhiko; Onomitsu, Koji; Kometani, Reo; Warisawa, Shin-ichi; Ishihara, Sunao; Yamaguchi, Hiroshi

2011-06-01

We developed a highly sensitive displacement detection scheme for a GaAs-based electromechanical resonator using an integrated high electron mobility transistor (HEMT). Piezoelectric voltage generated by the vibration of the resonator is applied to the gate of the HEMT, resulting in the on-chip amplification of the signal voltage. This detection scheme achieves a displacement sensitivity of ˜9 pm·Hz-1/2, which is one of the highest among on-chip purely electrical displacement detection schemes at room temperature.

Sex and PRNP genotype determination in preimplantation caprine embryos.

PubMed

Guignot, F; Perreau, C; Cavarroc, C; Touzé, J-L; Pougnard, J-L; Dupont, F; Beckers, J-F; Rémy, B; Babilliot, J-M; Bed'Hom, B; Lamorinière, J M; Mermillod, P; Baril, G

2011-08-01

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo. © 2010 Blackwell Verlag GmbH.

Label-free thioflavin T/G-quadruplex-based real-time strand displacement amplification for biosensing applications.

PubMed

Du, Yi-Chen; Zhu, Li-Na; Kong, De-Ming

2016-12-15

To promote application of strand-displacement amplification (SDA) techniques in biosensing, a label-free, real-time monitoring strategy for isothermal nucleic acid amplification reactions was designed. G-quadruplex structures were introduced into SDA products using specific recognition of G-quadruplexes by the fluorogenic dye thioflavin T. Performance was good for real-time monitoring of traditional SDA by a linear-amplification mechanism and for exponential cross-triggered SDA amplification. The strategy worked on a commercial real-time PCR instrument, making it suitable for biosensing platforms. As examples, two highly sensitive and specific biosensors were designed for analysis of the activity of uracil-DNA glycosylase (UDG) and the restriction endonuclease EcoRI. Detection limits were 6×10(-5)U/mL for UDG and 0.016U/mL for EcoRI. Detection of corresponding targets in complex matrices such as cell lysates or human serum was also demonstrated. Compared to traditional end-point detection methods, real-time SDA-based approaches have the advantages of simple, fast operation; high sensitivity; low risk of carryover contamination; and very high throughput. The introduction of real-time monitoring strategies may promote application of SDA reactions in biosensor design. Copyright © 2016 Elsevier B.V. All rights reserved.

Strand Displacement Amplification Reaction on Quantum Dot-Encoded Silica Bead for Visual Detection of Multiplex MicroRNAs.

PubMed

Qu, Xiaojun; Jin, Haojun; Liu, Yuqian; Sun, Qingjiang

2018-03-06

The combination of microbead array, isothermal amplification, and molecular signaling enables the continuous development of next-generation molecular diagnostic techniques. Herein we reported the implementation of nicking endonuclease-assisted strand displacement amplification reaction on quantum dots-encoded microbead (Qbead), and demonstrated its feasibility for multiplexed miRNA assay in real sample. The Qbead featured with well-defined core-shell superstructure with dual-colored quantum dots loaded in silica core and shell, respectively, exhibiting remarkably high optical encoding stability. Specially designed stem-loop-structured probes were immobilized onto the Qbead for specific target recognition and amplification. In the presence of low abundance of miRNA target, the target triggered exponential amplification, producing a large quantity of stem-G-quadruplexes, which could be selectively signaled by a fluorescent G-quadruplex intercalator. In one-step operation, the Qbead-based isothermal amplification and signaling generated emissive "core-shell-satellite" superstructure, changing the Qbead emission-color. The target abundance-dependent emission-color changes of the Qbead allowed direct, visual detection of specific miRNA target. This visualization method achieved limit of detection at the subfemtomolar level with a linear dynamic range of 4.5 logs, and point-mutation discrimination capability for precise miRNA analyses. The array of three encoded Qbeads could simultaneously quantify three miRNA biomarkers in ∼500 human hepatoma carcinoma cells. With the advancements in ease of operation, multiplexing, and visualization capabilities, the isothermal amplification-on-Qbead assay could potentially enable the development of point-of-care diagnostics.

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.

Parametric amplification in MoS2 drum resonator.

PubMed

Prasad, Parmeshwar; Arora, Nishta; Naik, A K

2017-11-30

Parametric amplification is widely used in diverse areas from optics to electronic circuits to enhance low level signals by varying relevant system parameters. Parametric amplification has also been performed in several micro-nano resonators including nano-electromechanical system (NEMS) resonators based on a two-dimensional (2D) material. Here, we report the enhancement of mechanical response in a MoS 2 drum resonator using degenerate parametric amplification. We use parametric pumping to modulate the spring constant of the MoS 2 resonator and achieve a 10 dB amplitude gain. We also demonstrate quality factor enhancement in the resonator with parametric amplification. We investigate the effect of cubic nonlinearity on parametric amplification and show that it limits the gain of the mechanical resonator. Amplifying ultra-small displacements at room temperature and understanding the limitations of the amplification in these devices is key for using these devices for practical applications.

UV Decontamination of MDA Reagents for Single Cell Genomics

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

Lee, Janey; Tighe, Damon; Sczyrba, Alexander

2011-03-18

Single cell genomics, the amplification and sequencing of genomes from single cells, can provide a glimpse into the genetic make-up and thus life style of the vast majority of uncultured microbial cells, making it an immensely powerful and increasingly popular tool. This is accomplished by use of multiple displacement amplification (MDA), which can generate billions of copies of a single bacterial genome producing microgram-range DNA required for shotgun sequencing. Here, we address a key challenge inherent to this approach and propose a solution for the improved recovery of single cell genomes. While DNA-free reagents for the amplification of a singlemore » cell genome are a prerequisite for successful single cell sequencing and analysis, DNA contamination has been detected in various reagents, which poses a considerable challenge. Our study demonstrates the effect of UV irradiation in efficient elimination of exogenous contaminant DNA found in MDA reagents, while maintaining Phi29 activity. Consequently, we also find that increased UV exposure to Phi29 does not adversely affect genome coverage of MDA amplified single cells. While additional challenges in single cell genomics remain to be resolved, the proposed methodology is relatively quick and simple and we believe that its application will be of high value for future single cell sequencing projects.« less

Single palindromic molecular beacon-based amplification for genetic analysis of cancers.

PubMed

Li, Feng; Zhao, Hui; Wang, Zheng-Yong; Wu, Zai-Sheng; Yang, Zhe; Li, Cong-Cong; Xu, Huo; Lyu, Jian-Xin; Shen, Zhi-Fa

2017-05-15

The detection of biomarkers is of crucial importance in reducing the morbidity and mortality of complex diseases. Thus, there is a great desire to develop highly efficient and simple sensing methods to fulfill the different diagnostic and therapeutic purposes. Herein, using tumor suppressor p53 gene as model target DNA, we developed a novel palindromic fragment-incorporated molecular beacon (P-MB) that can perform multiple functions, including recognition element, signal reporter, polymerization template and primer. Upon specific hybridization with target DNA, P-MBs can interact with each other and are extended by polymerase without any additional probes. As a result, hybridized targets are peeled off from P-MBs and initiate the next round of reactions, leading to the unique strand displacement amplification (SDA). The newly-proposed enzymatic amplification displays the detection limit as low as 100pM and excellent selectivity in distinguishing single-base mutation with the linear response range from 100pM to 75nM. This is the simplest SDA sensing system so far because of only involving one type of DNA probe. This impressive sensing paradigm is expected to provide new insight into developing new-type of DNA probes that hold tremendous potential with important applications in molecular biology research and clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Label-free and sensitive fluorescence detection of nucleic acid, based on combination of a graphene oxid /SYBR green I dye platform and polymerase assisted signal amplification

NASA Astrophysics Data System (ADS)

Zhu, Xiao; Xing, Da

2012-12-01

A new label-free isothermal fluorescence amplification detection for nucleic acid has been developed. In this paper, we first developed a novel sensitive and specific detection platform with an unmodified hairpin probe (HP) combination of the graphene oxid (GO)/ SYBR green I dye (SG), which was relied on the selective principle of adsorption and the high quenching efficiency of GO. Then for the application of this new strategy, we used Mirco RNA-21 (Mir-21) as the target to evaluate this working principle of our design. When the target was hybridizing with the HP and inducing its conformation of change, an efficient isothermal circular strand-displacement polymerization reaction was activating to assist the first signal amplification. In this format, the formed complex conformation of DNA would interact with its high affinity dye, then detached from the surface of GO after incubating with the platform of GO/intercalating dye. This reaction would accompany with obvious fluorescence recovery, and accomplish farther signal enhancement by a mass of intercalating dye inserting into the minor groove of the long duplex replication product. By taking advantage of the multiple amplification of signal, this method exerted substantial enhancement in sensitivity and could be used for rapid and selective detection of Mir-21 with attomole range. It is expected that this cost-effective GO based sensor might hold considerable potential to apply in bioanalysis studies.

Isothermal amplification detection of nucleic acids by a double-nicked beacon.

PubMed

Shi, Chao; Zhou, Meiling; Pan, Mei; Zhong, Guilin; Ma, Cuiping

2016-03-01

Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use. Copyright © 2015 Elsevier Inc. All rights reserved.

Compact piezoelectric tripod manipulator based on a reverse bridge-type amplification mechanism

NASA Astrophysics Data System (ADS)

Na, Tae-Won; Choi, Jun-Ho; Jung, Jin-Young; Kim, Hyeong-Geon; Han, Jae-Hung; Park, Kwang-Chun; Oh, Il-Kwon

2016-09-01

We report a hierarchical piezoelectric tripod manipulator based on a reverse bridge-type displacement amplifier. The reverse bridge-type amplification mechanism is pre-strained by each piezo-stack actuator up to 60 μm and is cross-stacked in a series arrangement to make a compact and high-stroke manipulator having load-bearing characteristics. The designed manipulator with three degrees of freedom is compact with a height of 56.0 mm, a diameter of 48.6 mm and total weight of 115 g. It achieves a translational stroke of up to 880 μm in heaving motion and a tilting angle of up to 2.0° in rotational motion within the operating voltage and power range of the piezoelectric stack actuator. A key feature of the present design is built-in and pre-strained displacement amplification mechanisms integrated with piezoelectric stacked actuators, resulting in a compact tripod manipulator having exceptionally high stroke and load-bearing capacity.

Precision oncology using a limited number of cells: optimization of whole genome amplification products for sequencing applications.

PubMed

Sho, Shonan; Court, Colin M; Winograd, Paul; Lee, Sangjun; Hou, Shuang; Graeber, Thomas G; Tseng, Hsian-Rong; Tomlinson, James S

2017-07-01

Sequencing analysis of circulating tumor cells (CTCs) enables "liquid biopsy" to guide precision oncology strategies. However, this requires low-template whole genome amplification (WGA) that is prone to errors and biases from uneven amplifications. Currently, quality control (QC) methods for WGA products, as well as the number of CTCs needed for reliable downstream sequencing, remain poorly defined. We sought to define strategies for selecting and generating optimal WGA products from low-template input as it relates to their potential applications in precision oncology strategies. Single pancreatic cancer cells (HPAF-II) were isolated using laser microdissection. WGA was performed using multiple displacement amplification (MDA), multiple annealing and looping based amplification (MALBAC) and PicoPLEX. Quality of amplified DNA products were assessed using a multiplex/RT-qPCR based method that evaluates for 8-cancer related genes and QC-scores were assigned. We utilized this scoring system to assess the impact of de novo modifications to the WGA protocol. WGA products were subjected to Sanger sequencing, array comparative genomic hybridization (aCGH) and next generation sequencing (NGS) to evaluate their performances in respective downstream analyses providing validation of the QC-score. Single-cell WGA products exhibited a significant sample-to-sample variability in amplified DNA quality as assessed by our 8-gene QC assay. Single-cell WGA products that passed the pre-analysis QC had lower amplification bias and improved aCGH/NGS performance metrics when compared to single-cell WGA products that failed the QC. Increasing the number of cellular input resulted in improved QC-scores overall, but a resultant WGA product that consistently passed the QC step required a starting cellular input of at least 20-cells. Our modified-WGA protocol effectively reduced this number, achieving reproducible high-quality WGA products from ≥5-cells as a starting template. A starting cellular input of 5 to 10-cells amplified using the modified-WGA achieved aCGH and NGS results that closely matched that of unamplified, batch genomic DNA. The modified-WGA protocol coupled with the 8-gene QC serve as an effective strategy to enhance the quality of low-template WGA reactions. Furthermore, a threshold number of 5-10 cells are likely needed for a reliable WGA reaction and product with high fidelity to the original starting template.

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.

Systematic Characteristic Exploration of the Chimeras Generated in Multiple Displacement Amplification through Next Generation Sequencing Data Reanalysis

PubMed Central

Gao, Shen; Yao, Bei; Lu, Zuhong

2015-01-01

Background The chimeric sequences produced by phi29 DNA polymerase, which are named as chimeras, influence the performance of the multiple displacement amplification (MDA) and also increase the difficulty of sequence data process. Despite several articles have reported the existence of chimeric sequence, there was only one research focusing on the structure and generation mechanism of chimeras, and it was merely based on hundreds of chimeras found in the sequence data of E. coli genome. Method We finished data mining towards a series of Next Generation Sequencing (NGS) reads which were used for whole genome haplotype assembling in a primary study. We established a bioinformatics pipeline based on subsection alignment strategy to discover all the chimeras inside and achieve their structural visualization. Then, we artificially defined two statistical indexes (the chimeric distance and the overlap length), and their regular abundance distribution helped illustrate of the structural characteristics of the chimeras. Finally we analyzed the relationship between the chimera type and the average insertion size, so that illustrate a method to decrease the proportion of wasted data in the procedure of DNA library construction. Results/Conclusion 131.4 Gb pair-end (PE) sequence data was reanalyzed for the chimeras. Totally, 40,259,438 read pairs (6.19%) with chimerism were discovered among 650,430,811 read pairs. The chimeric sequences are consisted of two or more parts which locate inconsecutively but adjacently on the chromosome. The chimeric distance between the locations of adjacent parts on the chromosome followed an approximate bimodal distribution ranging from 0 to over 5,000 nt, whose peak was at about 250 to 300 nt. The overlap length of adjacent parts followed an approximate Poisson distribution and revealed a peak at 6 nt. Moreover, unmapped chimeras, which were classified as the wasted data, could be reduced by properly increasing the length of the insertion segment size through a linear correlation analysis. Significance This study exhibited the profile of the phi29MDA chimeras by tens of millions of chimeric sequences, and helped understand the amplification mechanism of the phi29 DNA polymerase. Our work also illustrated the importance of NGS data reanalysis, not only for the improvement of data utilization efficiency, but also for more potential genomic information. PMID:26440104

A lateral flow biosensor for detection of single nucleotide polymorphism by circular strand displacement reaction.

PubMed

Xiao, Zhuo; Lie, Puchang; Fang, Zhiyuan; Yu, Luxin; Chen, Junhua; Liu, Jie; Ge, Chenchen; Zhou, Xuemeng; Zeng, Lingwen

2012-09-04

A lateral flow biosensor for detection of single nucleotide polymorphism based on circular strand displacement reaction (CSDPR) has been developed. Taking advantage of high fidelity of T4 DNA ligase, signal amplification by CSDPR, and the optical properties of gold nanoparticles, this assay has reached a detection limit of 0.01 fM.

Microarray-Based Analysis of Subnanogram Quantities of Microbial Community DNAs by Using Whole-Community Genome Amplification†

PubMed Central

Wu, Liyou; Liu, Xueduan; Schadt, Christopher W.; Zhou, Jizhong

2006-01-01

Microarray technology provides the opportunity to identify thousands of microbial genes or populations simultaneously, but low microbial biomass often prevents application of this technology to many natural microbial communities. We developed a whole-community genome amplification-assisted microarray detection approach based on multiple displacement amplification. The representativeness of amplification was evaluated using several types of microarrays and quantitative indexes. Representative detection of individual genes or genomes was obtained with 1 to 100 ng DNA from individual or mixed genomes, in equal or unequal abundance, and with 1 to 500 ng community DNAs from groundwater. Lower concentrations of DNA (as low as 10 fg) could be detected, but the lower template concentrations affected the representativeness of amplification. Robust quantitative detection was also observed by significant linear relationships between signal intensities and initial DNA concentrations ranging from (i) 0.04 to 125 ng (r2 = 0.65 to 0.99) for DNA from pure cultures as detected by whole-genome open reading frame arrays, (ii) 0.1 to 1,000 ng (r2 = 0.91) for genomic DNA using community genome arrays, and (iii) 0.01 to 250 ng (r2 = 0.96 to 0.98) for community DNAs from ethanol-amended groundwater using 50-mer functional gene arrays. This method allowed us to investigate the oligotrophic microbial communities in groundwater contaminated with uranium and other metals. The results indicated that microorganisms containing genes involved in contaminant degradation and immobilization are present in these communities, that their spatial distribution is heterogeneous, and that microbial diversity is greatly reduced in the highly contaminated environment. PMID:16820490

A new class of homogeneous nucleic acid probes based on specific displacement hybridization

PubMed Central

Li, Qingge; Luan, Guoyan; Guo, Qiuping; Liang, Jixuan

2002-01-01

We have developed a new class of probes for homogeneous nucleic acid detection based on the proposed displacement hybridization. Our probes consist of two complementary oligodeoxyribonucleotides of different length labeled with a fluorophore and a quencher in close proximity in the duplex. The probes on their own are quenched, but they become fluorescent upon displacement hybridization with the target. These probes display complete discrimination between a perfectly matched target and single nucleotide mismatch targets. A comparison of double-stranded probes with corresponding linear probes confirms that the presence of the complementary strand significantly enhances their specificity. Using four such probes labeled with different color fluorophores, each designed to recognize a different target, we have demonstrated that multiple targets can be distinguished in the same solution, even if they differ from one another by as little as a single nucleotide. Double-stranded probes were used in real-time nucleic acid amplifications as either probes or as primers. In addition to its extreme specificity and flexibility, the new class of probes is simple to design and synthesize, has low cost and high sensitivity and is accessible to a wide range of labels. This class of probes should find applications in a variety of areas wherever high specificity of nucleic acid hybridization is relevant. PMID:11788731

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.

Toehold strand displacement-driven assembly of G-quadruplex DNA for enzyme-free and non-label sensitive fluorescent detection of thrombin.

PubMed

Xu, Yunying; Zhou, Wenjiao; Zhou, Ming; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

2015-02-15

Based on a new signal amplification strategy by the toehold strand displacement-driven cyclic assembly of G-quadruplex DNA, the development of an enzyme-free and non-label aptamer sensing approach for sensitive fluorescent detection of thrombin is described. The target thrombin associates with the corresponding aptamer of the partial dsDNA probes and liberates single stranded initiation sequences, which trigger the toehold strand displacement assembly of two G-quadruplex containing hairpin DNAs. This toehold strand displacement reaction leads to the cyclic reuse of the initiation sequences and the production of DNA assemblies with numerous G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binds to these G-quadruplex structures and generates significantly amplified fluorescent signals to achieve highly sensitive detection of thrombin down to 5 pM. Besides, this method shows high selectivity towards the target thrombin against other control proteins. The developed thrombin sensing method herein avoids the modification of the probes and the involvement of any enzyme or nanomaterial labels for signal amplification. With the successful demonstration for thrombin detection, our approach can be easily adopted to monitor other target molecules in a simple, low-cost, sensitive and selective way by choosing appropriate aptamer/ligand pairs. Copyright © 2014 Elsevier B.V. All rights reserved.

Signal-on fluorescence biosensor for microRNA-21 detection based on DNA strand displacement reaction and Mg2+-dependent DNAzyme cleavage.

PubMed

Yin, Huan-Shun; Li, Bing-Chen; Zhou, Yun-Lei; Wang, Hai-Yan; Wang, Ming-Hui; Ai, Shi-Yun

2017-10-15

MicroRNAs have been involved into many biological processes and are regarded as disease biomarkers. Simple, rapid, sensitive and selective method for microRNA detection is crucial for early diagnosis and therapy of diseases. In this work, sensitive fluorescence assay was developed for microRNA-21 detection based on DNA polymerase induced strand displacement amplification reaction, Mg 2+ -dependent DNAzyme catalysis reaction, and magnetic separation. In the presence of target microRNA-21, amounts of trigger DNA could be produced with DNA polymerase induced strand displacement amplification reaction, and the trigger DNA could be further hybridized with signal DNA, which was labeled with biotin and AMCA dye. After introduction of Mg 2+ , trigger DNA could form DNAzyme to cleave signal DNA. After magnetic separation, the DNA fragment with AMCA dye could give fluorescence signal, which was related to microRNA-21 concentration. Based on the two efficient signal amplifications, the developed method showed high detection sensitivity with low detection limit of 0.27fM (3σ). In addition, this fluorescence strategy also possessed excellent detection specificity, and could be applied to analyze microRNA-21 expression level in serum of cancer patient. According to the obtained results, the developed fluorescence method might be a promising detection platform for microRNA-21 quantitative analysis in biomedical research and clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a Tandem Repeat-Based Polymerase Chain Displacement Reaction Method for Highly Sensitive Detection of 'Candidatus Liberibacter asiaticus'.

PubMed

Lou, Binghai; Song, Yaqin; RoyChowdhury, Moytri; Deng, Chongling; Niu, Ying; Fan, Qijun; Tang, Yan; Zhou, Changyong

2018-02-01

Huanglongbing (HLB) is one of the most destructive diseases in citrus production worldwide. Early detection of HLB pathogens can facilitate timely removal of infected citrus trees in the field. However, low titer and uneven distribution of HLB pathogens in host plants make reliable detection challenging. Therefore, the development of effective detection methods with high sensitivity is imperative. This study reports the development of a novel method, tandem repeat-based polymerase chain displacement reaction (TR-PCDR), for the detection of 'Candidatus Liberibacter asiaticus', a widely distributed HLB-associated bacterium. A uniquely designed primer set (TR2-PCDR-F/TR2-PCDR-1R) and a thermostable Taq DNA polymerase mutant with strand displacement activity were used for TR-PCDR amplification. Performed in a regular thermal cycler, TR-PCDR could produce more than two amplicons after each amplification cycle. Sensitivity of the developed TR-PCDR was 10 copies of target DNA fragment. The sensitive level was proven to be 100× higher than conventional PCR and similar to real-time PCR. Data from the detection of 'Ca. L. asiaticus' with filed samples using the above three methods also showed similar results. No false-positive TR-PCDR amplification was observed from healthy citrus samples and water controls. These results thereby illustrated that the developed TR-PCDR method can be applied to the reliable, highly sensitive, and cost-effective detection of 'Ca. L. asiaticus'.

Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices.

PubMed

Giuffrida, Maria Chiara; Zanoli, Laura Maria; D'Agata, Roberta; Finotti, Alessia; Gambari, Roberto; Spoto, Giuseppe

2015-02-01

Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells.

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.

Fast implementation of length-adaptive privacy amplification in quantum key distribution

NASA Astrophysics Data System (ADS)

Zhang, Chun-Mei; Li, Mo; Huang, Jing-Zheng; Patcharapong, Treeviriyanupab; Li, Hong-Wei; Li, Fang-Yi; Wang, Chuan; Yin, Zhen-Qiang; Chen, Wei; Keattisak, Sripimanwat; Han, Zhen-Fu

2014-09-01

Post-processing is indispensable in quantum key distribution (QKD), which is aimed at sharing secret keys between two distant parties. It mainly consists of key reconciliation and privacy amplification, which is used for sharing the same keys and for distilling unconditional secret keys. In this paper, we focus on speeding up the privacy amplification process by choosing a simple multiplicative universal class of hash functions. By constructing an optimal multiplication algorithm based on four basic multiplication algorithms, we give a fast software implementation of length-adaptive privacy amplification. “Length-adaptive” indicates that the implementation of privacy amplification automatically adapts to different lengths of input blocks. When the lengths of the input blocks are 1 Mbit and 10 Mbit, the speed of privacy amplification can be as fast as 14.86 Mbps and 10.88 Mbps, respectively. Thus, it is practical for GHz or even higher repetition frequency QKD systems.

Multiple-strand displacement and identification of single nucleotide polymorphisms as markers of genotypic variation of Pasteuria penetrans biotypes infecting root-knot nematodes.

PubMed

Nong, Guang; Chow, Virginia; Schmidt, Liesbeth M; Dickson, Don W; Preston, James F

2007-08-01

Pasteuria species are endospore-forming obligate bacterial parasites of soil-inhabiting nematodes and water-inhabiting cladocerans, e.g. water fleas, and are closely related to Bacillus spp. by 16S rRNA gene sequence. As naturally occurring bacteria, biotypes of Pasteuria penetrans are attractive candidates for the biocontrol of various Meloidogyne spp. (root-knot nematodes). Failure to culture these bacteria outside their hosts has prevented isolation of genomic DNA in quantities sufficient for identification of genes associated with host recognition and virulence. We have applied multiple-strand displacement amplification (MDA) to generate DNA for comparative genomics of biotypes exhibiting different host preferences. Using the genome of Bacillus subtilis as a paradigm, MDA allowed quantitative detection and sequencing of 12 marker genes from 2000 cells. Meloidogyne spp. infected with P. penetrans P20 or B4 contained single nucleotide polymorphisms (SNPs) in the spoIIAB gene that did not change the amino acid sequence, or that substituted amino acids with similar chemical properties. Individual nematodes infected with P. penetrans P20 or B4 contained SNPs in the spoIIAB gene sequenced in MDA-generated products. Detection of SNPs in the spoIIAB gene in a nematode indicates infection by more than one genotype, supporting the need to sequence genomes of Pasteuria spp. derived from single spore isolates.

An Engineered Kinetic Amplification Mechanism for Single Nucleotide Variant Discrimination by DNA Hybridization Probes.

PubMed

Chen, Sherry Xi; Seelig, Georg

2016-04-20

Even a single-nucleotide difference between the sequences of two otherwise identical biological nucleic acids can have dramatic functional consequences. Here, we use model-guided reaction pathway engineering to quantitatively improve the performance of selective hybridization probes in recognizing single nucleotide variants (SNVs). Specifically, we build a detection system that combines discrimination by competition with DNA strand displacement-based catalytic amplification. We show, both mathematically and experimentally, that the single nucleotide selectivity of such a system in binding to single-stranded DNA and RNA is quadratically better than discrimination due to competitive hybridization alone. As an additional benefit the integrated circuit inherits the property of amplification and provides at least 10-fold better sensitivity than standard hybridization probes. Moreover, we demonstrate how the detection mechanism can be tuned such that the detection reaction is agnostic to the position of the SNV within the target sequence. in contrast, prior strand displacement-based probes designed for kinetic discrimination are highly sensitive to position effects. We apply our system to reliably discriminate between different members of the let-7 microRNA family that differ in only a single base position. Our results demonstrate the power of systematic reaction network design to quantitatively improve biotechnology.

Signal-off Electrochemiluminescence Biosensor Based on Phi29 DNA Polymerase Mediated Strand Displacement Amplification for MicroRNA Detection.

PubMed

Chen, Anyi; Gui, Guo-Feng; Zhuo, Ying; Chai, Ya-Qin; Xiang, Yun; Yuan, Ruo

2015-06-16

A target induced cycling strand displacement amplification (SDA) mediated by phi29 DNA polymerase (phi29) was first investigated and applied in a signal-off electrochemiluminescence (ECL) biosensor for microRNA (miRNA) detection. Herein, the target miRNA triggered the phi29-mediated SDA which could produce amounts of single-stranded DNA (assistant probe) with accurate and comprehensive nucleotide sequence. Then, the assistant probe hybridized with the capture probe and the ferrocene-labeled probe (Fc-probe) to form a ternary "Y" structure for ECL signal quenching by ferrocene. Therefore, the ECL intensity would decrease with increasing concentration of the target miRNA, and the sensitivity of biosensor would be promoted on account of the efficient signal amplification of the target induced cycling reaction. Besides, a self-enhanced Ru(II) ECL system was designed to obtain a stable and strong initial signal to further improve the sensitivity. The ECL assay for miRNA-21 detection is developed with excellent sensitivity of a concentration variation from 10 aM to 1.0 pM and limit of detection down to 3.3 aM.

Chemical Amplification with Encapsulated Reagents

NASA Technical Reports Server (NTRS)

Chen, Jian; Koemer, Steffi; Craig, Stephen; Lin, Shirley; Rudkevich, Dmitry M.; Rebek, Julius, Jr.

2002-01-01

Autocatalysis and chemical amplification are characteristic properties of living systems, and they give rise to behaviors such as increased sensitivity, responsiveness, and self-replication. Here we report a synthetic system in which a unique form of compartmentalization leads to nonlinear, autocatalytic behavior. The compartment is a reversibly formed capsule in which a reagent is sequestered. Reaction products displace the reagent from the capsule into solution and the reaction rate is accelerated. The resulting self-regulation is sensitive to the highly selective molecular recognition properties of the capsule.

Targeted sorting of single virus-infected cells of the coccolithophore Emiliania huxleyi.

PubMed

Martínez Martínez, Joaquín; Poulton, Nicole J; Stepanauskas, Ramunas; Sieracki, Michael E; Wilson, William H

2011-01-01

Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing) gave the most unequivocal and earliest separation of cells. Furthermore, we were able to amplify the genomes of single infected cells using Multiple Displacement Amplification. This novel method to reliably discriminate infected from healthy cells in cultures will allow researchers to answer numerous questions regarding the mechanisms and implications of viral infection of E. huxleyi. The method may be transferable to other virus-host systems.

Cascade DNA nanomachine and exponential amplification biosensing.

PubMed

Xu, Jianguo; Wu, Zai-Sheng; Shen, Weiyu; Xu, Huo; Li, Hongling; Jia, Lee

2015-11-15

DNA is a versatile scaffold for the assembly of multifunctional nanostructures, and potential applications of various DNA nanodevices have been recently demonstrated for disease diagnosis and treatment. In the current study, a powerful cascade DNA nanomachine was developed that can execute the exponential amplification of p53 tumor suppressor gene. During the operation of the newly-proposed DNA nanomachine, dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) was ingeniously introduced, where the target trigger is repeatedly used as the fuel molecule and the nicked fragments are dramatically accumulated. Moreover, each displaced nicked fragment is able to activate the another type of cyclical strand-displacement amplification, increasing exponentially the value of fluorescence intensity. Essentially, one target binding event can induce considerable number of subsequent reactions, and the nanodevice was called cascade DNA nanomachine. It can implement several functions, including recognition element, signaling probe, polymerization primer and template. Using the developed autonomous operation of DNA nanomachine, the p53 gene can be quantified in the wide concentration range from 0.05 to 150 nM with the detection limit of 50 pM. If taking into account the final volume of mixture, the detection limit is calculated as lower as 6.2 pM, achieving an desirable assay ability. More strikingly, the mutant gene can be easily distinguished from the wild-type one. The proof-of-concept demonstrations reported herein is expected to promote the development and application of DNA nanomachine, showing great potential value in basic biology and medical diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

A novel regenerative shock absorber with a speed doubling mechanism and its Monte Carlo simulation

NASA Astrophysics Data System (ADS)

Zhang, Ran; Wang, Xu; Liu, Zhenwei

2018-03-01

A novel regenerative shock absorber has been designed and fabricated. The novelty of the presented work is the application of the double speed regenerative shock absorber that utilizes the rack and pinion mechanism to increase the magnet speed with respect to the coils for higher power output. The simulation models with parameters identified from finite element analysis and the experiments are developed. The proposed regenerative shock absorber is compared with the regenerative shock absorber without the rack and pinion mechanism, when they are integrated into the same quarter vehicle suspension system. The sinusoidal wave road profile displacement excitation and the random road profile displacement excitation with peak amplitude of 0.035 m are applied as the inputs in the frequency range of 0-25 Hz. It is found that with the sinusoidal and random road profile displacement input, the proposed innovative design can increase the output power by 4 times comparing to the baseline design. The proposed double speed regenerative shock absorber also presents to be more sensitive to the road profile irregularity than the single speed regenerative shock absorber as suggested by Monte Carlo simulation. Lastly the coil mass and amplification factor are studied for sensitivity analysis and performance optimization, which provides a general design method of the regenerative shock absorbers. It shows that for the system power output, the proposed design becomes more sensitive to either the coil mass or amplification factor depending on the amount of the coil mass. With the specifically selected combination of the coil mass and amplification factor, the optimized energy harvesting performance can be achieved.

Enhanced force sensitivity and noise squeezing in an electromechanical resonator coupled to a nanotransistor

NASA Astrophysics Data System (ADS)

Mahboob, I.; Flurin, E.; Nishiguchi, K.; Fujiwara, A.; Yamaguchi, H.

2010-12-01

A nanofield-effect transistor (nano-FET) is coupled to a massive piezoelectricity based electromechanical resonator integrated with a parametric amplifier. The mechanical parametric amplifier can enhance the resonator's displacement and the resulting electrical signal is further amplified by the nano-FET. This hybrid amplification scheme yields an increase in the mechanical displacement signal by 70 dB resulting in a force sensitivity of 200 aN Hz-1/2 at 3 K. The mechanical parametric amplifier can also squeeze the displacement noise in one oscillation phase by 5 dB enabling a factor of 4 reduction in the thermomechanical noise force level.

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.

A CMOS-MEMS clamped–clamped beam displacement amplifier for resonant switch applications

NASA Astrophysics Data System (ADS)

Liu, Jia-Ren; Lu, Shih-Chuan; Tsai, Chun-Pu; Li, Wei-Chang

2018-06-01

This paper presents a micromechanical clamped–clamped beam (CC-beam) displacement amplifier based on a CMOS-MEMS fabrication process platform. In particular, a 2.0 MHz resonant displacement amplifier composed of two identical CC-beams coupled by a mechanical beam at locations where the two beams have mismatched velocities exhibits a larger displacement, up to 9.96×, on one beam than that of the other. The displacement amplification prevents unwanted input impacting—the structure switches only to the output but not the input—required by resonant switch-based mechanical circuits (Kim et al 2009 22nd IEEE Int. Conf. on Micro Electro Mechanical Systems; Lin et al 2009 15th Int. Conf. on Solid-State Sensors, Actuators, & Microsystems (TRANSDUCERS’09) Li et al 2013 17th Int. Conf. on Solid-State Sensors, Actuators, & Microsystems (TRANSDUCERS’13)). Compared to a single CC-beam displacement amplifier, theory predicts that the displacement amplifying CC-beam array yields a larger overall output displacement for displacement gain beyond 1.13 thanks to the preserved input driving force. A complete analytical model predicts the resultant stiffness and displacement gain of the coupled CC-beam displacement amplifier that match well with finite element analysis (FEA) prediction and measured results.

Levers and linkages: mechanical trade-offs in a power-amplified system.

PubMed

Anderson, Philip S L; Claverie, Thomas; Patek, S N

2014-07-01

Mechanical redundancy within a biomechanical system (e.g., many-to-one mapping) allows morphologically divergent organisms to maintain equivalent mechanical outputs. However, most organisms depend on the integration of more than one biomechanical system. Here, we test whether coupled mechanical systems follow a pattern of amplification (mechanical changes are congruent and evolve toward the same functional extreme) or independence (mechanisms evolve independently). We examined the correlated evolution and evolutionary pathways of the coupled four-bar linkage and lever systems in mantis shrimp (Stomatopoda) ultrafast raptorial appendages. We examined models of character evolution in the framework of two divergent groups of stomatopods-"smashers" (hammer-shaped appendages) and "spearers" (bladed appendages). Smashers tended to evolve toward force amplification, whereas spearers evolved toward displacement amplification. These findings show that coupled biomechanical systems can evolve synergistically, thereby resulting in functional amplification rather than mechanical redundancy. © 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.

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

Jet oscillations caused by vorticity interactions with shock waves

NASA Technical Reports Server (NTRS)

Parthasarathy, S. P.; Harstad, K.; Massier, P. F.

1981-01-01

A linear theory is developed for the amplification of disturbances along a jet containing shock waves. The theory indicates that near grazing angles (i.e., wave angles near 90 deg) horizontal vorticity is greatly amplified after passing through the two shock waves that exist in a shock cell. The cumulative amplification and the mode that is amplified most can be obtained if the changes in shock parameters from cell to cell are known. Rapid rates of growth of disturbances are exhibited by shadowgraphs and rates of angular displacement of about 10 are observed. The linear two-dimensional theory also indicates that such rates of amplification occur, and that the behavior of a two-dimensional jet is qualitatively similar to that of a round jet.

Droplet Microfluidic Device Fabrication and Use for Isothermal Amplification and Detection of MicroRNA.

PubMed

Giuffrida, Maria Chiara; D'Agata, Roberta; Spoto, Giuseppe

2017-01-01

Droplet microfluidics combined with the isothermal circular strand displacement polymerization (ICSDP) represents a powerful new technique to detect both single-stranded DNA and microRNA sequences. The method here described helps in overcoming some drawbacks of the lately introduced droplet polymerase chain reaction (PCR) amplification when implemented in microfluidic devices. The method also allows the detection of nanoliter droplets of nucleic acids sequences solutions, with a particular attention to microRNA sequences that are detected at the picomolar level. The integration of the ICSDP amplification protocol in droplet microfluidic devices reduces the time of analysis and the amount of sample required. In addition, there is also the possibility to design parallel analyses to be integrated in portable devices.

An embeddable optical strain gauge based on a buckled beam.

PubMed

Du, Yang; Chen, Yizheng; Zhu, Chen; Zhuang, Yiyang; Huang, Jie

2017-11-01

We report, for the first time, a low cost, compact, and novel mechanically designed extrinsic Fabry-Perot interferometer (EFPI)-based optical fiber sensor with a strain amplification mechanism for strain measurement. The fundamental design principle includes a buckled beam with a coated gold layer, mounted on two grips. A Fabry-Perot cavity is produced between the buckled beam and the endface of a single mode fiber (SMF). A ceramic ferrule is applied for supporting and orienting the SMF. The principal sensor elements are packaged and protected by two designed metal shells. The midpoint of the buckled beam will experience a deflection vertically when the beam is subjected to a horizontally/axially compressive displacement. It has been found that the vertical deflection of the beam at midpoint can be 6-17 times larger than the horizontal/axial displacement, which forms the basis of a strain amplification mechanism. The user-configurable buckling beam geometry-based strain amplification mechanism enables the strain sensor to achieve a wide range of strain measurement sensitivities. The designed EFPI was used to monitor shrinkage of a square brick of mortar. The strain was measured during the drying/curing stage. We envision that it could be a good strain sensor to be embedded in civil materials/structures under a harsh environment for a prolonged period of time.

An embeddable optical strain gauge based on a buckled beam

NASA Astrophysics Data System (ADS)

Du, Yang; Chen, Yizheng; Zhu, Chen; Zhuang, Yiyang; Huang, Jie

2017-11-01

We report, for the first time, a low cost, compact, and novel mechanically designed extrinsic Fabry-Perot interferometer (EFPI)-based optical fiber sensor with a strain amplification mechanism for strain measurement. The fundamental design principle includes a buckled beam with a coated gold layer, mounted on two grips. A Fabry-Perot cavity is produced between the buckled beam and the endface of a single mode fiber (SMF). A ceramic ferrule is applied for supporting and orienting the SMF. The principal sensor elements are packaged and protected by two designed metal shells. The midpoint of the buckled beam will experience a deflection vertically when the beam is subjected to a horizontally/axially compressive displacement. It has been found that the vertical deflection of the beam at midpoint can be 6-17 times larger than the horizontal/axial displacement, which forms the basis of a strain amplification mechanism. The user-configurable buckling beam geometry-based strain amplification mechanism enables the strain sensor to achieve a wide range of strain measurement sensitivities. The designed EFPI was used to monitor shrinkage of a square brick of mortar. The strain was measured during the drying/curing stage. We envision that it could be a good strain sensor to be embedded in civil materials/structures under a harsh environment for a prolonged period of time.

A novel monolithic piezoelectric actuated flexure-mechanism based wire clamp for microelectronic device packaging.

PubMed

Liang, Cunman; Wang, Fujun; Tian, Yanling; Zhao, Xingyu; Zhang, Hongjie; Cui, Liangyu; Zhang, Dawei; Ferreira, Placid

2015-04-01

A novel monolithic piezoelectric actuated wire clamp is presented in this paper to achieve fast, accurate, and robust microelectronic device packaging. The wire clamp has compact, flexure-based mechanical structure and light weight. To obtain large and robust jaw displacements and ensure parallel jaw grasping, a two-stage amplification composed of a homothetic bridge type mechanism and a parallelogram leverage mechanism was designed. Pseudo-rigid-body model and Lagrange approaches were employed to conduct the kinematic, static, and dynamic modeling of the wire clamp and optimization design was carried out. The displacement amplification ratio, maximum allowable stress, and natural frequency were calculated. Finite element analysis (FEA) was conducted to evaluate the characteristics of the wire clamp and wire electro discharge machining technique was utilized to fabricate the monolithic structure. Experimental tests were carried out to investigate the performance and the experimental results match well with the theoretical calculation and FEA. The amplification ratio of the clamp is 20.96 and the working mode frequency is 895 Hz. Step response test shows that the wire clamp has fast response and high accuracy and the motion resolution is 0.2 μm. High speed precision grasping operations of gold and copper wires were realized using the wire clamper.

Reverse strand-displacement amplification strategy for rapid detection of p53 gene.

PubMed

Wang, Lisha; Han, Ying; Xiao, Shuai; Lv, Sha; Wang, Cong; Zhang, Nan; Wang, Zhengyong; Tang, Yongqiong; Li, Hongbo; Lyu, Jianxin; Xu, Huo; Shen, Zhifa

2018-09-01

The development of rapid approaches to detect prognostic markers is significant in reducing the morbidity and mortality of cancer. In this paper, we describe a rapid and specific biosensing platform for target DNA (p53 gene as a model) detection based on reverse strand displacement amplification (R-SDA). When the p53 gene is added, multifuctional molecular beacon (MMB) is unfolded via the hybridization with p53 gene. With the assist of Klenow fragment (KF) and Nt.BbvCI (the nicking endonuclease), p53 gene recycling could be initiated and considerable amount of complementary sequences for the MMBs (Nicked fragments, NFs) could be formed, generating enhanced fluorescence signal. Using this amplification strategy, the proposed biosensor displays the detection limit of 1 nM and a wide linear range from 1 to 100 nM, even if only one type of probe is involved. Notably, remarkable detection specificity for single-base mismatched target p53 gene is achieved. Moreover, the described biosensor also exhibited the stability in real biological samples (human serum). The rapid detection strategy can be performed less than 30 min without harsh reaction conditions or expensive nanoparticles. This biosensor shows great potential for application in clinic assay, especially, for early cancer diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Optical pulse synthesis using brillouin selective sideband amplification

NASA Technical Reports Server (NTRS)

Yao, X. Steve (Inventor)

2002-01-01

Techniques for producing optical pulses based on Brillouin selective sideband amplification by using a common modulation control signal to modulate both a signal beam to produce multiple sideband signals and a single pump beam to produce multiple pump beams.

The effect of main urine inhibitors on the activity of different DNA polymerases in loop-mediated isothermal amplification.

PubMed

Jevtuševskaja, Jekaterina; Krõlov, Katrin; Tulp, Indrek; Langel, Ülo

2017-04-01

The use of rapid amplification methods to detect pathogens in biological samples is mainly limited by the amount of pathogens present in the sample and the presence of inhibiting substances. Inhibitors can affect the amplification efficiency by either binding to the polymerase, interacting with the DNA, or interacting with the polymerase during primer extension. Amplification is performed using DNA polymerase enzymes and even small changes in their activity can influence the sensitivity and robustness of molecular assays Methods: The main purpose of this research was to examine which compounds present in urine inhibit polymerases with strand displacement activity. To quantify the inhibition, we employed quantitative loop-mediated isothermal amplification Results: The authors found that the presence of BSA, Mg 2+, and urea at physiologically relevant concentrations, as well as acidic or alkaline conditions did not affect the activity of any of the tested polymerases. However, addition of salt significantly affected the activity of the tested polymerases. These findings may aid in the development of more sensitive, robust, cost effective isothermal amplification based molecular assays suitable for both point-of-care testing and on-site screening of pathogens directly from unprocessed urine which avoid the need for long and tedious DNA purification steps prior to amplification.

Target-regulated proximity hybridization with three-way DNA junction for in situ enhanced electronic detection of marine biotoxin based on isothermal cycling signal amplification strategy.

PubMed

Liu, Bingqian; Chen, Jinfeng; Wei, Qiaohua; Zhang, Bing; Zhang, Lan; Tang, Dianping

2015-07-15

A new signal amplification strategy based on target-regulated DNA proximity hybridization (TRPH) reaction accompanying formation of three-way DNA junction was designed for electronic detection of Microcystin-LR (MC-LR used in this case), coupling with junction-induced isothermal cycling signal amplification. Initially, a sandwiched-type immunoreaction was carried out in a low-cost PCR tube between anti-MC-LR mAb1 antibody-labeled DNA1 (mAb1-DNA1) and anti-MC-LR mAb2-labeled DNA2 (mAb2-DNA2) in the presence of target to form a three-way DNA junction. Then, the junction could undergo an unbiased strand displacement reaction on an h-like DNA nanostructure-modified electrode (labeled with methylene blue redox tag on the short DNA strand), thereby resulting in the dissociation of methylene blue-labeled signal DNA from the electrode. The newly formed double-stranded DNA could be cleaved again by exonuclease III, and the released three-way DNA junction retriggered the strand-displacement reaction with h-like DNA nanostructures for junction recycling. During the strand-displacement reaction, numerous methylene blue-labeled DNA strands were far away from the electrode, thus decreasing the detectable electrochemical signal within the applied potentials. Under optimal conditions, the TRPH-based immunosensing system exhibited good electrochemical responses for detecting target MC-LR at a concentration as low as 1.0ngkg(-1) (1.0ppt). Additionally, the precision, reproducibility, specificity and method accuracy were also investigated with acceptable results. Copyright © 2015 Elsevier B.V. All rights reserved.

Liquefaction-induced lateral spreading in Oceano, California, during the 2003 San Simeon Earthquake

USGS Publications Warehouse

Holzer, Thomas L.; Noce, Thomas E.; Bennett, Michael J.; Di Alessandro, Carola; Boatwright, John; Tinsley, John C.; Sell, Russell W.; Rosenberg, Lewis I.

2004-01-01

The December 22, 2003, San Simeon, California, (M6.5) earthquake caused damage to houses, road surfaces, and underground utilities in Oceano, California. The community of Oceano is approximately 50 miles (80 km) from the earthquake epicenter. Damage at this distance from a M6.5 earthquake is unusual. To understand the causes of this damage, the U.S. Geological Survey conducted extensive subsurface exploration and monitoring of aftershocks in the months after the earthquake. The investigation included 37 seismic cone penetration tests, 5 soil borings, and aftershock monitoring from January 28 to March 7, 2004. The USGS investigation identified two earthquake hazards in Oceano that explain the San Simeon earthquake damage?site amplification and liquefaction. Site amplification is a phenomenon observed in many earthquakes where the strength of the shaking increases abnormally in areas where the seismic-wave velocity of shallow geologic layers is low. As a result, earthquake shaking is felt more strongly than in surrounding areas without similar geologic conditions. Site amplification in Oceano is indicated by the physical properties of the geologic layers beneath Oceano and was confirmed by monitoring aftershocks. Liquefaction, which is also commonly observed during earthquakes, is a phenomenon where saturated sands lose their strength during an earthquake and become fluid-like and mobile. As a result, the ground may undergo large permanent displacements that can damage underground utilities and well-built surface structures. The type of displacement of major concern associated with liquefaction is lateral spreading because it involves displacement of large blocks of ground down gentle slopes or towards stream channels. The USGS investigation indicates that the shallow geologic units beneath Oceano are very susceptible to liquefaction. They include young sand dunes and clean sandy artificial fill that was used to bury and convert marshes into developable lots. Most of the 2003 damage was caused by lateral spreading in two separate areas, one near Norswing Drive and the other near Juanita Avenue. The areas coincided with areas with the highest liquefaction potential found in Oceano. Areas with site amplification conditions similar to those in Oceano are particularly vulnerable to earthquakes. Site amplification may cause shaking from distant earthquakes, which normally would not cause damage, to increase locally to damaging levels. The vulnerability in Oceano is compounded by the widespread distribution of highly liquefiable soils that will reliquefy when ground shaking is amplified as it was during the San Simeon earthquake. The experience in Oceano can be expected to repeat because the region has many active faults capable of generating large earthquakes. In addition, liquefaction and lateral spreading will be more extensive for moderate-size earthquakes that are closer to Oceano than was the 2003 San Simeon earthquake. Site amplification and liquefaction can be mitigated. Shaking is typically mitigated in California by adopting and enforcing up-to-date building codes. Although not a guarantee of safety, application of these codes ensures that the best practice is used in construction. Building codes, however, do not always require the upgrading of older structures to new code requirements. Consequently, many older structures may not be as resistant to earthquake shaking as new ones. For older structures, retrofitting is required to bring them up to code. Seismic provisions in codes also generally do not apply to nonstructural elements such as drywall, heating systems, and shelving. Frequently, nonstructural damage dominates the earthquake loss. Mitigation of potential liquefaction in Oceano presently is voluntary for existing buildings, but required by San Luis Obispo County for new construction. Multiple mitigation procedures are available to individual property owners. These procedures typically involve either

Highly sensitive electrochemical nuclear factor kappa B aptasensor based on target-induced dual-signal ratiometric and polymerase-assisted protein recycling amplification strategy.

PubMed

Peng, Kanfu; Xie, Pan; Yang, Zhe-Han; Yuan, Ruo; Zhang, Keqin

2018-04-15

In this work, an amplified electrochemical ratiometric aptasensor for nuclear factor kappa B (NF-κB) assay based on target binding-triggered ratiometric signal readout and polymerase-assisted protein recycling amplification strategy is described. To demonstrate the effect of "signal-off" and "signal-on" change for the dual-signal electrochemical ratiometric readout, the thiol-hairpin DNA (SH-HD) hybridizes with methylene blue (MB)-modified protection DNA (MB-PD) to form capture probes, which is rationally introduced for the construction of the assay platform. On the interface, the probes can specifically bind to target NF-κB and expose a toehold region which subsequently hybridizes with the ferrocene (Fc)-modified DNA strand to take the Fc group to the electrode surface, accompanied by displacing MB-PD to release the MB group from the electrode surface, leading to the both "signal-on" of Fc (I Fc ) and "signal-off" of MB (I MB ). In order to improve the sensitivity of the electrochemical aptasensor, phi29-assisted target protein recycling amplification strategy was designed to achieve an amplified ratiometric signal. With the above advantages, the prepared aptasensor exhibits a wide linear range of 0.1pgmL -1 to 15ngmL -1 with a low detection limit of 0.03pgmL -1 . This strategy provides a simple and ingenious approach to construct ratiometric electrochemical aptasensor and shows promising potential applications in multiple disease marker detection by changing the recognition probe. Copyright © 2017. Published by Elsevier B.V.

Toehold-mediated strand displacement reaction triggered isothermal DNA amplification for highly sensitive and selective fluorescent detection of single-base mutation.

PubMed

Zhu, Jing; Ding, Yongshun; Liu, Xingti; Wang, Lei; Jiang, Wei

2014-09-15

Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8 pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Proximity-dependent isothermal cycle amplification for small-molecule detection based on surface enhanced Raman scattering.

PubMed

Li, Ying; Zeng, Yan; Mao, Yaning; Lei, Chengcun; Zhang, Shusheng

2014-01-15

A novel proximity-dependent isothermal cycle amplification (PDICA) strategy has been proposed and successfully used for the determination of cocaine coupled with surface enhanced Raman scattering (SERS). For enhancing the SERS signal, Raman dye molecules modified bio-barcode DNA and gold nanoparticles (AuNPs) are used to prepare the Raman probes. Magnetic beads (MBs) are used as the carrier of amplification template and signal output products for circumventing the problem of high background induced by excess bio-barcode DNA. In the presence of target molecules, two label-free proximity probes can hybridize with each other and subsequently opens the hairpin connector-probe to perform the PDICA reaction including the target recycling amplification and strand-displacement amplification. As a result, abundant AuNPs Raman probes can be anchored on the surface of MBs and a low detection limit of 0.1 nM for cocaine is obtained. This assay also exhibits an excellent selectivity and has been successfully performed in human serum, which confirms the reliability and practicality of this protocol. © 2013 Elsevier B.V. All rights reserved.

Ultrasensitive electrochemical biosensor for detection of DNA from Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification.

PubMed

Hu, Yuhua; Xu, Xueqin; Liu, Qionghua; Wang, Ling; Lin, Zhenyu; Chen, Guonan

2014-09-02

A simple, ultrasensitive, and specific electrochemical biosensor was designed to determine the given DNA sequence of Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification. The target DNA (TD, the DNA sequence from the hypervarient region of 16S rDNA of Bacillus subtilis) could be detected by the differential pulse voltammetry (DPV) in a range from 0.1 fM to 20 fM with the detection limit down to 0.08 fM at the 3s(blank) level. This electrochemical biosensor exhibits high distinction ability to single-base mismatch, double-bases mismatch, and noncomplementary DNA sequence, which may be expected to detect single-base mismatch and single nucleotide polymorphisms (SNPs). Moreover, the applicability of the designed biosensor for detecting the given DNA sequence from Bacillus subtilis was investigated. The result obtained by electrochemical method is approximately consistent with that by a real-time quantitative polymerase chain reaction detecting system (QPCR) with SYBR Green.

Hair cell transduction, tuning and synaptic transmission in the mammalian cochlea

PubMed Central

Fettiplace, Robert

2017-01-01

Sound pressure fluctuations striking the ear are conveyed to the cochlea, where they vibrate the basilar membrane on which sit hair cells, the mechanoreceptors of the inner ear. Recordings of hair cell electrical responses have shown that they transduce sound via sub-micrometer deflections of their hair bundles, which are arrays of interconnected stereocilia containing the mechanoelectrical transducer (MET) channels. MET channels are activated by tension in extracellular tip links bridging adjacent stereocilia, and they can respond within microseconds to nanometer displacements of the bundle, facilitated by multiple processes of Ca2+-dependent adaptation. Studies of mouse mutants have produced much detail about the molecular organization of the stereocilia, the tip links and their attachment sites, and the MET channels localized to the lower ends of each tip link. The mammalian cochlea contains two categories of hair cells. Inner hair cells relay acoustic information via multiple ribbon synapses that transmit rapidly without rundown. Outer hair cells are important for amplifying sound-evoked vibrations. The amplification mechanism primarily involves contractions of the outer hair cells, which are driven by changes in membrane potential and mediated by prestin, a motor protein in the outer hair cell lateral membrane. Different sound frequencies are separated along the cochlea, with each hair cell being tuned to a narrow frequency range; amplification sharpens the frequency resolution and augments sensitivity 100-fold around the cell’s characteristic frequency. Genetic mutations and environmental factors such as acoustic overstimulation cause hearing loss through irreversible damage to the hair cells or degeneration of inner hair cell synapses. PMID:28915323

Sensitive SERS detection of DNA methyltransferase by target triggering primer generation-based multiple signal amplification strategy.

PubMed

Li, Ying; Yu, Chuanfeng; Han, Huixia; Zhao, Caisheng; Zhang, Xiaoru

2016-07-15

A novel and sensitive surface-enhanced Raman scattering (SERS) method is proposed for the assay of DNA methyltransferase (MTase) activity and evaluation of inhibitors by developing a target triggering primer generation-based multiple signal amplification strategy. By using of a duplex substrate for Dam MTase, two hairpin templates and a Raman probe, multiple signal amplification mode is achieved. Once recognized by Dam MTase, the duplex substrate can be cleaved by Dpn I endonuclease and two primers are released for triggering the multiple signal amplification reaction. Consequently, a wide dynamic range and remarkably high sensitivity are obtained under isothermal conditions. The detection limit is 2.57×10(-4)UmL(-1). This assay exhibits an excellent selectivity and is successfully applied in the screening of inhibitors for Dam MTase. In addition, this novel sensing system is potentially universal as the recognition element can be conveniently designed for other target analytes by changing the substrate of DNA MTase. Copyright © 2016 Elsevier B.V. All rights reserved.

Topoisomerase expression and amplification in solid tumours: Analysis of 24,262 patients

PubMed Central

Heestand, Gregory M.; Schwaederle, Maria; Gatalica, Zoran; Arguello, David; Kurzrock, Razelle

2017-01-01

Background Topoisomerase I (TOPO1) and topoisomerase IIα (TOP2A) are specific targets of multiple chemotherapy drugs. Increased expression of TOPO1 protein and amplification of the TOP2A gene have been associated with treatment response in colorectal and breast cancers, respectively. TOPO1 and TOP2A may be potential therapeutic targets in other malignancies as well. Summary of methods We analysed TOPO1 protein expression and TOP2A gene amplification in patients (n = 24,262 specimens) with diverse cancers. Since HER2 and TOP2A co-amplification have been investigated for predictive value regarding anthracycline benefit, we analysed specimens for HER2 amplification as well. Results Overexpressed TOPO1 protein was present in 51% of the tumours. Four percent of the tumours had TOP2A amplification, with gallbladder tumours and gastroesophageal/oesophageal tumours having rates over 10%. Overall, 4903 specimens were assessed for both TOP2A and HER2 amplification; 129 (2.6%) had co-amplification. High rates (>40%) of HER2 amplification were seen in patients with TOP2A amplification in breast, ovarian, gastroesophageal/oesophageal and pancreatic cancer. Conclusion Our data indicate that increased TOPO1 expression and TOP2A amplification, as well as HER2 co-alterations, are present in multiple malignancies. The implications of these observations regarding sensitivity to chemotherapy not traditionally administered to these tumour types merits investigation. PMID:28728050

Dual-cyclical nucleic acid strand-displacement polymerization based signal amplification system for highly sensitive determination of p53 gene.

PubMed

Xu, Jianguo; Wu, Zai-Sheng; Li, Hongling; Wang, Zhenmeng; Le, Jingqing; Zheng, Tingting; Jia, Lee

2016-12-15

In the present study, we proposed a novel dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) based signal amplification system for highly sensitive determination of tumor suppressor genes. The system primarily consisted of a signaling hairpin probe (SHP), a label-free hairpin probe (LHP) and an initiating primer (IP). The presence of target DNA was able to induce one CNDP through continuous process of ligation, polymerization and nicking, leading to extensively accumulation of two nicked triggers (NT1 and NT2). Intriguingly, the NT1 could directly hybridize SHP, while the NT2 could act as the target analog to induce another CNDP. The resulting dual-CNDP contributed the striking signal amplification, and only a very weak blank noise existed since the ligation template of target was not involved. In this case, the target could be detected in a wide linear range (5 orders of magnitude), and a low detection limit (78 fM) was obtained, which is superior to most of the existing fluorescent methods. Moreover, the dual-CNDP sensing system provided a high selectivity towards target DNA against mismatched target and was successfully applied to analysis of target gene extracted from cancer cells or in human serum-contained samples, indicating its great potential for practical applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Searching for Helicobacter pylori and Chlamydia pneumoniae in primary endodontic infections.

PubMed

Rôças, Isabela N; Siqueira, José F

2012-04-01

The purpose of this study was to search samples from primary endodontic infections for the presence of two common human bacterial pathogens - Helicobacter pylori and Chlamydia pneumoniae. Genomic DNA isolated from samples taken from 25 root canals of teeth with asymptomatic (chronic) apical periodontitis and 25 aspirates from acute apical abscess was initially amplified by the multiple displacement amplification approach and then used as template in species-specific polymerase chain reaction (PCR) for detection of H. pylori and C. pneumoniae. All clinical samples were positive for the presence of bacterial DNA. However, no clinical sample was positive for either H. pylori or C. pneumoniae. Neither H. pylori nor C. pneumoniae were found in samples from primary endodontic infections. These findings suggest that these species are not candidate endodontic pathogens and that the necrotic root canal does not serve as a reservoir for these human pathogens in healthy patients.

Demonstration of Vibrational Braille Code Display Using Large Displacement Micro-Electro-Mechanical Systems Actuators

NASA Astrophysics Data System (ADS)

Watanabe, Junpei; Ishikawa, Hiroaki; Arouette, Xavier; Matsumoto, Yasuaki; Miki, Norihisa

2012-06-01

In this paper, we present a vibrational Braille code display with large-displacement micro-electro-mechanical systems (MEMS) actuator arrays. Tactile receptors are more sensitive to vibrational stimuli than to static ones. Therefore, when each cell of the Braille code vibrates at optimal frequencies, subjects can recognize the codes more efficiently. We fabricated a vibrational Braille code display that used actuators consisting of piezoelectric actuators and a hydraulic displacement amplification mechanism (HDAM) as cells. The HDAM that encapsulated incompressible liquids in microchambers with two flexible polymer membranes could amplify the displacement of the MEMS actuator. We investigated the voltage required for subjects to recognize Braille codes when each cell, i.e., the large-displacement MEMS actuator, vibrated at various frequencies. Lower voltages were required at vibration frequencies higher than 50 Hz than at vibration frequencies lower than 50 Hz, which verified that the proposed vibrational Braille code display is efficient by successfully exploiting the characteristics of human tactile receptors.

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

PubMed

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

2018-07-30

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

Strong ground motion in Port-au-Prince, Haiti, during the M7.0 12 January 2010 Haiti earthquake

USGS Publications Warehouse

Hough, Susan E; Given, Doug; Taniguchi, Tomoyo; Altidor, J.R.; Anglade, Dieuseul; Mildor, S-L.

2011-01-01

No strong motion records are available for the 12 January 2010 M7.0 Haiti earthquake. We use aftershock recordings as well as detailed considerations of damage to estimate the severity and distribution of mainshock shaking in Port-au-Prince. Relative to ground motions at a hard - rock reference site, peak accelerations are amplified by a factor of approximately 2 at sites on low-lying deposits in central Port-au-Prince and by a factor of 2.5 - 3.5 on a steep foothill ridge in the southern Port-au-Prince metropolitan region. The observed amplification along the ridge cannot be explained by sediment - induced amplification , but is consistent with predicted topographic amplification by a steep, narrow ridge. Although damage was largely a consequence of poor construction , the damage pattern inferred from analysis of remote sensing imagery provides evidence for a correspondence between small-scale (0.1 - 1.0 km) topographic relief and high damage. Mainshock shaking intensity can be estimated crudely from a consideration of macroseismic effects . We further present detailed, quantitative analysis of the marks left on a tile floor by an industrial battery rack displaced during the mainshock, at the location where we observed the highest weak motion amplifications. Results of this analysis indicate that mainshock shaking was significantly higher at this location (~0.5 g , MMI VIII) relative to the shaking in parts of Port-au-Prince that experienced light damage. Our results further illustrate how observations of rigid body horizontal displacement during earthquakes can be used to estimate peak ground accelerations in the absence of instrumental data .

Establishment of a universal and rational gene detection strategy through three-way junction-based remote transduction.

PubMed

Tang, Yidan; Lu, Baiyang; Zhu, Zhentong; Li, Bingling

2018-01-21

The polymerase chain reaction and many isothermal amplifications are able to achieve super gene amplification. Unfortunately, most commonly-used transduction methods, such as dye staining and Taqman-like probing, still suffer from shortcomings including false signals or difficult probe design, or are incompatible with multi-analysis. Here a universal and rational gene detection strategy has been established by translating isothermal amplicons to enzyme-free strand displacement circuits via three-way junction-based remote transduction. An assistant transduction probe was imported to form a partial hybrid with the target single-stranded nucleic acid. After systematic optimization the hybrid could serve as an associative trigger to activate a downstream circuit detector via a strand displacement reaction across the three-way junction. By doing so, the detection selectivity can be double-guaranteed through both amplicon-transducer recognition and the amplicon-circuit reaction. A well-optimized circuit can be immediately applied to a new target detection through simply displacing only 10-12 nt on only one component, according to the target. More importantly, this property for the first time enables multi-analysis and logic-analysis in a single reaction, sharing a single fluorescence reporter. In an applicable model, trace amounts of Cronobacter and Enterobacteria genes have been clearly distinguished from samples with no bacteria or one bacterium, with ultra-high sensitivity and selectivity.

Microbial Ecosystem Analysis in Root Canal Infections Refractory to Endodontic Treatment.

PubMed

Henriques, Luiz Carlos Feitosa; de Brito, Luciana Carla Neves; Tavares, Warley Luciano Faria; Teles, Ricardo Palmier; Vieira, Leda Quércia; Teles, Flávia Rodrigues; Sobrinho, Antônio Paulino Ribeiro

2016-08-01

The purpose of this study was to combine multiple displacement amplification and checkerboard DNA-DNA hybridization to qualitatively and quantitatively evaluate the microbiota present in infections refractory to endodontic treatment. The subjects of this study were 40 patients presenting with periapical lesions refractory to endodontic treatment. Samples were taken by scraping or filing root canal walls with a #10 K-type hand file. Sample DNA was amplified by multiple displacement amplification, and the levels of 107 bacterial taxa were analyzed by checkerboard DNA-DNA hybridization. The taxa were divided into 3 distinct microbial populations depending on their mean proportion in samples (% DNA probe counts ± standard error of the mean) as follows: dominant (≥3.0%), subdominant (>1.6%-3.0%), and residual (≤1.6%) populations. The significance of differences was determined using the Mann-Whitney test. The taxa present with the highest mean proportions (constituting the dominant population) were Corynebacterium diphtheriae (8.03 ± 0.98), Porphyromonas gingivalis (5.42 ± 2.09), Streptococcus sobrinus (5.33 ± 0.69), and Stenotrophomonas maltophilia (4.72 ± 1.73). Among the subdominant population were Eubacterium saphenum (3.85 ± 1.06), Helicobacter pylori (3.16 ± 0.62), Dialister pneumosintes (3.12 ± 1.1), Clostridium difficile (2.74 ± 0.41), Enterobacter agglomerans (2.64 ± 0.54), Salmonella enterica (2.51 ± 0.52), Mobiluncus mulieris (2.44 ± 0.6), and Klebsiella oxytoca (2.32 ± 0.66). In the population of bacteria present at the lowest mean proportions (the residual population), Bacteroides ureolyticus (0.04 ± 0.01), Haemophilus influenzae (0.04 ± 0.02), and Prevotella oris (0.01 ± 0.01) were found at the lowest mean proportions. Enterococcus faecalis was detected in the residual population (0.52 ± 0.26). The microbial climax community in teeth refractory to endodontic treatment not only harbors medically important species but also contains distinct microbial consortia present with different population levels. Copyright © 2016. Published by Elsevier Inc.

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

PubMed

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

2014-03-15

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

A mass spectrometry-based multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification.

PubMed

Park, Jung Hun; Jang, Hyowon; Jung, Yun Kyung; Jung, Ye Lim; Shin, Inkyung; Cho, Dae-Yeon; Park, Hyun Gyu

2017-05-15

We herein describe a new mass spectrometry-based method for multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification (SDA) reaction. In this method, allele-specific ligation is first performed to discriminate base sequence variations at the SNP site within the PCR-amplified target DNA. The primary ligation probe is extended by a universal primer annealing site while the secondary ligation probe has base sequences as an overhang with a nicking enzyme recognition site and complementary mass marker sequence. The ligation probe pairs are ligated by DNA ligase only at specific allele in the target DNA and the resulting ligated product serves as a template to promote the SDA reaction using a universal primer. This process isothermally amplifies short DNA fragments, called mass markers, to be analyzed by mass spectrometry. By varying the sizes of the mass markers, we successfully demonstrated the multiplex SNP genotyping capability of this method by reliably identifying several BRCA mutations in a multiplex manner with mass spectrometry. Copyright © 2016 Elsevier B.V. All rights reserved.

Single-cell transcriptomics using spliced leader PCR: Evidence for multiple losses of photosynthesis in polykrikoid dinoflagellates.

PubMed

Gavelis, Gregory S; White, Richard A; Suttle, Curtis A; Keeling, Patrick J; Leander, Brian S

2015-07-17

Most microbial eukaryotes are uncultivated and thus poorly suited to standard genomic techniques. This is the case for Polykrikos lebouriae, a dinoflagellate with ultrastructurally aberrant plastids. It has been suggested that these plastids stem from a novel symbiosis with either a diatom or haptophyte, but this hypothesis has been difficult to test as P. lebouriae dwells in marine sand rife with potential genetic contaminants. We applied spliced-leader targeted PCR (SLPCR) to obtain dinoflagellate-specific transcriptomes on single-cell isolates of P. lebouriae from marine sediments. Polykrikos lebouriae expressed nuclear-encoded photosynthetic genes that were characteristic of the peridinin-plastids of dinoflagellates, rather than those from a diatom of haptophyte. We confirmed these findings at the genomic level using multiple displacement amplification (MDA) to obtain a partial plastome of P. lebouriae. From these data, we infer that P. lebouriae has retained the peridinin plastids ancestral for dinoflagellates as a whole, while its closest relatives have lost photosynthesis multiple times independently. We discuss these losses with reference to mixotrophy in polykrikoid dinoflagellates. Our findings demonstrate new levels of variation associated with the peridinin plastids of dinoflagellates and the usefulness of SLPCR approaches on single cell isolates. Unlike other transcriptomic methods, SLPCR has taxonomic specificity, and can in principle be adapted to different splice-leader bearing groups.

Single Cell Genome Amplification Accelerates Identification of the Apratoxin Biosynthetic Pathway from a Complex Microbial Assemblage

PubMed Central

Grindberg, Rashel V.; Ishoey, Thomas; Brinza, Dumitru; Esquenazi, Eduardo; Coates, R. Cameron; Liu, Wei-ting; Gerwick, Lena; Dorrestein, Pieter C.; Pevzner, Pavel; Lasken, Roger; Gerwick, William H.

2011-01-01

Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA) and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites. PMID:21533272

First report on an X-linked hypohidrotic ectodermal dysplasia family with X chromosome inversion: Breakpoint mapping reveals the pathogenic mechanism and preimplantation genetics diagnosis achieves an unaffected birth.

PubMed

Wu, Tonghua; Yin, Biao; Zhu, Yuanchang; Li, Guangui; Ye, Lijun; Liang, Desheng; Zeng, Yong

2017-12-01

To investigate the etiology of X-linked hypohidrotic ectodermal dysplasia (XLHED) in a family with an inversion of the X chromosome [inv(X)(p21q13)] and to achieve a healthy birth following preimplantation genetic diagnosis (PGD). Next generation sequencing (NGS) and Sanger sequencing analysis were carried out to define the inversion breakpoint. Multiple displacement amplification, amplification of breakpoint junction fragments, Sanger sequencing of exon 1 of ED1, haplotyping of informative short tandem repeat markers and gender determination were performed for PGD. NGS data of the proband sample revealed that the size of the possible inverted fragment was over 42Mb, spanning from position 26, 814, 206 to position 69, 231, 915 on the X chromosome. The breakpoints were confirmed by Sanger sequencing. A total of 5 blastocyst embryos underwent trophectoderm biopsy. Two embryos were diagnosed as carriers and three were unaffected. Two unaffected blastocysts were transferred and a singleton pregnancy was achieved. Following confirmation by prenatal diagnosis, a healthy baby was delivered. This is the first report of an XLHED family with inv(X). ED1 is disrupted by the X chromosome inversion in this XLHED family and embryos with the X chromosomal abnormality can be accurately identified by means of PGD. Copyright © 2017. Published by Elsevier B.V.

Orbit-based analysis of nonlinear energetic ion dynamics in tokamaks. II. Mechanisms for rapid chirping and convective amplification

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

Bierwage, Andreas; Shinohara, Kouji

2016-04-15

The nonlinear interactions between shear Alfvén modes and tangentially injected beam ions in the 150–400 keV range are studied numerically in realistic geometry for a JT-60U tokamak scenario. In Paper I, which was reported in the companion paper, the recently developed orbit-based resonance analysis method was used to track the resonant frequency of fast ions during their nonlinear evolution subject to large magnetic and electric drifts. Here, that method is applied to map the wave-particle power transfer from the canonical guiding center phase space into the frequency-radius plane, where it can be directly compared with the evolution of the fluctuation spectramore » of fast-ion-driven modes. Using this technique, we study the nonlinear dynamics of strongly driven shear Alfvén modes with low toroidal mode numbers n = 1 and n = 3. In the n = 3 case, both chirping and convective amplification can be attributed to the mode following the resonant frequency of the radially displaced particles, i.e., the usual one-dimensional phase locking process. In the n = 1 case, a new chirping mechanism is found, which involves multiple dimensions, namely, wave-particle trapping in the radial direction and phase mixing across velocity coordinates.« less

Recombinase-based isothermal amplification of nucleic acids with self-avoiding molecular recognition systems (SAMRS).

PubMed

Sharma, Nidhi; Hoshika, Shuichi; Hutter, Daniel; Bradley, Kevin M; Benner, Steven A

2014-10-13

Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single-stranded primers into the duplex DNA product; these are then extended using a strand-displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base-pairs following Watson-Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self-avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS-RPA is expected to be a powerful tool within the range of amplification techniques available to scientists. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

New Downhole Strong-Motion Data Recorded at Tarzana Array

NASA Astrophysics Data System (ADS)

Graizer, V.; Shakal, A.; Haddadi, H.

2001-12-01

Significantly amplified ground accelerations at the Tarzana station were recorded during many, but not all, earthquakes (e.g., Shakal et al., 1988). Peak horizontal ground acceleration at the Tarzana station during the M7.1 Hector Mine earthquake was almost twice as large as the accelerations recorded at nearby stations. After the Northridge earthquake the California Strong Motion Instrumentation Program (CSMIP) significantly increased instrumentation at Tarzana to study the unusual site amplification effect. Current instrumentation at Tarzana consists of an accelerograph at the top of Tarzana hill (Tarzana - Cedar Hill B), a downhole instrument at 60 m depth, and an accelerograph at the foot of the hill (Tarzana - Clubhouse), 180 m from the Cedar Hill B station. The original station, Tarzana - Cedar Hill Nursery A, was lost in 1999 due to construction. Thirteen events, including the Hector Mine earthquake, were simultaneously recorded by these instruments at Tarzana. The downhole instrument (A) was used as a reference site to compare the amplification effects at the top of Tarzana hill (B) and at the foot of the hill (C). Spectral amplification from the bottom of the hole to the top of the hill (B/A) and to the foot of the hill (C/A) is similar along the component parallel to the strike of Tarzana hill. But B/A is almost double C/A along the component transverse to the strike of the hill in period range from 0.04 to 0.8 sec (1.2 to 25 Hz). Comparison of the response spectra demonstrates clear directional site response resonance (perpendicular to the strike of the hill) at Tarzana. In contrast to accelerations recorded during the Mw 7.1 Hector Mine earthquake (high frequency part of seismic signal), displacements (relatively low frequency part of seismic signal) demonstrate almost no site amplification from the bottom of the hole to the surface (B/A) at periods greater than 1.5 sec, in either direction. Ground displacements at other CSMIP downhole arrays which recorded the Hector Mine earthquake also demonstrate almost no near-surface site amplification at long periods. Comparison of empirical and theoretical site amplification effects at Tarzana was performed using SHAKE91 modeling motion separately in the longitudinal and transverse directions. The source of the site amplification that produces large motions at Tarzana is still under investigation. The topography, shear-wave velocity profile and three-dimensional structure of the site apparently all contribute to the higher amplification of ground motion at the Tarzana site. The studies of Tarzana were co-funded by CSMIP and by the National Science Foundation (NSF) through the Resolution of Site Response Issues from the Northridge Earthquake Project (ROSRINE).

Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

PubMed Central

Bhadra, Sanchita; Ellington, Andrew D.

2014-01-01

Nucleic acid circuits are finding increasing real-life applications in diagnostics and synthetic biology. Although DNA has been the main operator in most nucleic acid circuits, transcriptionally produced RNA circuits could provide powerful alternatives for reagent production and their use in cells. Towards these goals, we have implemented a particular nucleic acid circuit, catalytic hairpin assembly, using RNA for both information storage and processing. Our results demonstrated that the design principles developed for DNA circuits could be readily translated to engineering RNA circuits that operated with similar kinetics and sensitivities of detection. Not only could purified RNA hairpins perform amplification reactions but RNA hairpins transcribed in vitro also mediated amplification, even without purification. Moreover, we could read the results of the non-enzymatic amplification reactions using a fluorescent RNA aptamer ‘Spinach’ that was engineered to undergo sequence-specific conformational changes. These advances were applied to the end-point and real-time detection of the isothermal strand displacement amplification reaction that produces single-stranded DNAs as part of its amplification cycle. We were also able to readily engineer gate structures with RNA similar to those that have previously formed the basis of DNA circuit computations. Taken together, these results validate an entirely new chemistry for the implementation of nucleic acid circuits. PMID:24493736

A Framework for Distributed Problem Solving

NASA Astrophysics Data System (ADS)

Leone, Joseph; Shin, Don G.

1989-03-01

This work explores a distributed problem solving (DPS) approach, namely the AM/AG model, to cooperative memory recall. The AM/AG model is a hierarchic social system metaphor for DPS based on the Mintzberg's model of organizations. At the core of the model are information flow mechanisms, named amplification and aggregation. Amplification is a process of expounding a given task, called an agenda, into a set of subtasks with magnified degree of specificity and distributing them to multiple processing units downward in the hierarchy. Aggregation is a process of combining the results reported from multiple processing units into a unified view, called a resolution, and promoting the conclusion upward in the hierarchy. The combination of amplification and aggregation can account for a memory recall process which primarily relies on the ability of making associations between vast amounts of related concepts, sorting out the combined results, and promoting the most plausible ones. The amplification process is discussed in detail. An implementation of the amplification process is presented. The process is illustrated by an example.

NASBA: A detection and amplification system uniquely suited for RNA

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

Sooknanan, R.; Malek, L.T.

1995-06-01

The invention of PCR (polymerase chain reaction) has revolutionized our ability to amplify and manipulate a nucleic acid sequence in vitro. The commercial rewards of this revolution have driven the development of other nuclei acid amplification and detection methodologies. This has created an alphabet soup of technologies that use different amplification methods, including NASBA (nucleic acid sequence-based amplification), LCR (ligase chain reaction), SDA (strand displacement amplification), QBR (Q-beta replicase), CPR (cycling probe reaction), and bDNA (branched DNA). Despite the differences in their processes, these amplification systems can be separated into two broad categories based on how they achieve their goal:more » sequence-based amplification systems, such as PCR, NASBA, and SDA, amplify a target nucleic acid sequence. Signal-based amplification systems, such as LCR, QBR, CPR and bDNA, amplify or alter a signal from a detection reaction that is target-dependent. While the various methods have relative strengths and weaknesses, only NASBA offers the unique ability to homogeneously amplify an RNA analyte in the presence of homologous genomic DNA under isothermal conditions. Since the detection of RNA sequences almost invariably measures biological activity, it is an excellent prognostic indicator of activities as diverse as virus production, gene expression, and cell viability. The isothermal nature of the reaction makes NASBA especially suitable for large-scale manual screening. These features extend NASBA`s application range from research to commercial diagnostic applications. Field test kits are presently under development for human diagnostics as well as the burgeoning fields of food and environmental diagnostic testing. These developments suggest future integration of NASBA into robotic workstations for high-throughput screening as well. 17 refs., 1 tab.« less

A highly sensitive SPRi biosensing strategy for simultaneous detection of multiplex miRNAs based on strand displacement amplification and AuNP signal enhancement.

PubMed

Wei, Xiaotong; Duan, Xiaolei; Zhou, Xiaoyan; Wu, Jiangling; Xu, Hongbing; Min, Xun; Ding, Shijia

2018-06-07

Herein, a dual channel surface plasmon resonance imaging (SPRi) biosensor has been developed for the simultaneous and highly sensitive detection of multiplex miRNAs based on strand displacement amplification (SDA) and DNA-functionalized AuNP signal enhancement. In the presence of target miRNAs (miR-21 or miR-192), the miRNAs could specifically hybridize with the corresponding hairpin probes (H) and initiate the SDA, resulting in massive triggers. Subsequently, the two parts of the released triggers could hybridize with capture probes (CP) and DNA-functionalized AuNPs, assembling DNA sandwiches with great mass on the chip surface. A significantly amplified SPR signal readout was achieved. This established biosensing method was capable of simultaneously detecting multiplex miRNAs with a limit of detection down to 0.15 pM for miR-21 and 0.22 pM for miR-192. This method exhibited good specificity and acceptable reproducibility. Moreover, the developed method was applied to the determination of target miRNAs in a complex matrix. Thus, this developed SPRi biosensing method may present a potential alternative tool for miRNA detection in biomedical research and clinical diagnosis.

Ultrasensitive electrochemical DNA detection based on dual amplification of circular strand-displacement polymerase reaction and hybridization chain reaction.

PubMed

Wang, Cui; Zhou, Hui; Zhu, Wenping; Li, Hongbo; Jiang, Jianhui; Shen, Guoli; Yu, Ruqin

2013-09-15

We developed a novel electrochemical strategy for ultrasensitive DNA detection using a dual amplification strategy based on the circular strand-displacement polymerase reaction (CSDPR) and the hybridization chain reaction (HCR). In this assay, hybridization of hairpin-shaped capture DNA to target DNA resulted in a conformational change of the capture DNA with a concomitant exposure of its stem. The primer was then hybridized with the exposed stem and triggered a polymerization reaction, allowing a cyclic reaction comprising release of target DNA, hybridization of target with remaining capture DNA, polymerization initiated by the primer. Furthermore, the free part of the primer propagated a chain reaction of hybridization events between two DNA hairpin probes with biotin labels, enabling an electrochemical reading using the streptavidin-alkaline phosphatase. The proposed biosensor showed to have very high sensitivity and selectivity with a dynamic response range through 10fM to 1nM, and the detect limit was as low as 8fM. The proposed strategy could have the potential for molecular diagnostics in complex biological systems. Copyright © 2013 Elsevier B.V. All rights reserved.

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

Lee, Hee Yoon; Department of Otolaryngology-Head and Neck Surgery, Stanford University, Stanford, California; Raphael, Patrick D.

Cochlear amplification has been most commonly investigated by measuring the vibrations of the basilar membrane in animal models. Several different techniques have been used for measuring these vibrations such as laser Doppler vibrometry, miniature pressure sensors, low coherence interferometry, and spectral-domain optical coherence tomography (SD-OCT). We have built a swept-source OCT (SS-OCT) system, which is similar to SD-OCT in that it is capable of performing both imaging and vibration measurements within the mouse cochlea in vivo without having to open the bone. In vivo 3D images of a mouse cochlea were obtained, and the basilar membrane, tectorial membrane, Reissner’s membrane,more » tunnel of Corti, and reticular lamina could all be resolved. We measured vibrations of multiple structures within the mouse cochlea to sound stimuli. As well, we measured the radial deflections of the reticular lamina and tectorial membrane to estimate the displacement of the outer hair cell stereocilia. These measurements have the potential to more clearly define the mechanisms underlying the linear and non-linear processes within the mammalian cochlea.« less

Highly multiplexed targeted DNA sequencing from single nuclei.

PubMed

Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

2016-02-01

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

Real-Time Sequence-Validated Loop-Mediated Isothermal Amplification Assays for Detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

PubMed Central

Bhadra, Sanchita; Jiang, Yu Sherry; Kumar, Mia R.; Johnson, Reed F.; Hensley, Lisa E.; Ellington, Andrew D.

2015-01-01

The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens. PMID:25856093

Treatment dilemma in multiple metatarsal fractures: when to operate?

PubMed

Mahan, Susan T; Lierhaus, Anneliese M; Spencer, Samantha A; Kasser, James R

2016-07-01

Fractures of multiple metatarsals in the pediatric population are uncommon; however, indications for surgical treatment have not been delineated. The aim of this study was to review multiple metatarsal fractures to help refine surgical indications. A total of 98 patients had multiple metatarsal fractures; displacement greater than 10% shaft width (displaced) was encountered in 33 (34.0%) patients. Fifteen patients had displacement of more than 75% shaft width of one metatarsal. Patients older than 14 years of age were more likely to have surgery for their injury (52.6%) than those younger than 14 years of age (3.7%) (P<0.0001). Younger patients and those with less than 75% displacement should be considered for nonoperative care. Level IV.

Multisubstrate Isotope Labeling and Metagenomic Analysis of Active Soil Bacterial Communities

PubMed Central

Verastegui, Y.; Cheng, J.; Engel, K.; Kolczynski, D.; Mortimer, S.; Lavigne, J.; Montalibet, J.; Romantsov, T.; Hall, M.; McConkey, B. J.; Rose, D. R.; Tomashek, J. J.; Scott, B. R.

2014-01-01

ABSTRACT Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon (12C) or stable-isotope-labeled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the 13C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. PMID:25028422

The Location of the Cochlear Amplifier: Spatial Representation of a Single Tone on the Guinea Pig Basilar Membrane

NASA Astrophysics Data System (ADS)

Russell, I. J.; Nilsen, K. E.

1997-03-01

Acoustic stimulation vibrates the cochlear basilar membrane, initiating a wave of displacement that travels toward the apex and reaches a peak over a restricted region according to the stimulus frequency. In this characteristic frequency region, a tone at the characteristic frequency maximally excites the sensory hair cells of the organ of Corti, which transduce it into electrical signals to produce maximum activity in the auditory nerve. Saturating, nonlinear, feedback from the motile outer hair cells is thought to provide electromechanical amplification of the travelling wave. However, neither the location nor the extent of the source of amplification, in relation to the characteristic frequency, are known. We have used a laser--diode interferometer to measure in vivo the distribution along the basilar membrane of nonlinear, saturating vibrations to 15 kHz tones. We estimate that the site of amplification for the 15 kHz region is restricted to a 1.25 mm length of basilar membrane centered on the 15 kHz place.

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.

Dynamic protein assembly by programmable DNA strand displacement.

PubMed

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

2018-04-01

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

Dynamic protein assembly by programmable DNA strand displacement

NASA Astrophysics Data System (ADS)

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

2018-03-01

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

Electromechanical displacement of piezoelectric-electrostrictive monolithic bilayer composites

NASA Astrophysics Data System (ADS)

Ngernchuklin, P.; Akdoǧan, E. K.; Safari, A.; Jadidian, B.

2009-02-01

We examine the electromechanical displacement of piezoelectric-electrostrictive monolithic bilayer composites with various piezoelectric volume percentage obtained by cosintering piezoelectric 0.65Pb(Mg1/3Nb2/3)O3-0.35PbTiO3 and electrostrictive 0.9Pb(Mg1/3Nb2/.3)O3-0.1PbTiO3 under unipolar and bipolar electric field excitation up to 10 kV/cm experimentally. It is shown that the effective d33 of the composites is limited by the electrostrictive layer, which acts as a capacitor in series to the piezoelectric layer, causing incomplete poling. We show that by controlling the volume content of the piezoelectric layer and constraining it with an electrostrictor, substantial strain amplification (15 μm for bipolar excitation) can be achieved while inducing asymmetry to the displacement with respect to the polarity of the applied field, which we discuss in the context of symmetry superposition.

Direct RNA detection without nucleic acid purification and PCR: Combining sandwich hybridization with signal amplification based on branched hybridization chain reaction.

PubMed

Xu, Yao; Zheng, Zhi

2016-05-15

We have developed a convenient, robust and low-cost RNA detection system suitable for high-throughput applications. This system uses a highly specific sandwich hybridization to capture target RNA directly onto solid support, followed by on-site signal amplification via 2-dimensional, branched hybridizing chain polymerization through toehold-mediated strand displacement reaction. The assay uses SYBR Green to detect targets at concentrations as low as 1 pM, without involving nucleic acid purification or any enzymatic reaction, using ordinary oligonucleotides without modification or labeling. The system was demonstrated in the detection of malaria RNA in blood and GAPDH gene expression in cell lysate. Copyright © 2015 Elsevier B.V. All rights reserved.

Sensitive electrochemical immunoassay for 2,4,6-trinitrotoluene based on functionalized silica nanoparticle labels

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

Wang, Jun; Liu, Guodong; Wu, Hong

2008-03-03

We present a poly(guanine)-functionalized silica nanoparticle (NP) label-based electrochemical immunoassay for sensitively detecting 2,4,6-trinitrotoluene (TNT). This immunoassay takes advantage of magnetic bead–based platform for competitive displacement immunoreactions and separation, and use electroactive nanoparticles as labels for signal amplification. For this assay, anti-TNT-coated magnetic beads interacted with TNT analog-conjugated poly(guanine)-silica NPs and formed analog-anti-TNT immunocomplexes on magnetic beads. The immunocomplexes coated magnetic beads were exposed to TNT samples, which resulted in displacing the analog conjugated poly(guanine) silica NPs into solution by TNT. In contrast, there are no guanine residues releasing into the solution in the absence of TNT. The reaction solutionmore » was then separated from the magnetic beads and transferred to the electrode surface for electrochemical measurements of guanine oxidation with Ru(bpy)32+ as mediator. The sensitivity of this TNT assay was greatly enhanced through dual signal amplifications: 1) a large amount of guanine residues on silica nanoparticles is introduced into the test solution by displacement immunoreactions and 2) a Ru(bpy)32+-induced guanine catalytic oxidation further enhances the electrochemical signal. Some experimental parameters for the nanoparticle label-based electrochemical immunoassay were studied and the performance of this assay was evaluated. The method is found to be very sensitive and the detection limit of this assay is ~ 0.1 ng mL-1 TNT. The electrochemical immunoassay based on the poly[guanine]-functionalized silica NP label offers a new approach for sensitive detection of explosives.« less

A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans.

PubMed

Mauchline, T H; Mohan, S; Davies, K G; Schaff, J E; Opperman, C H; Kerry, B R; Hirsch, P R

2010-05-01

To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR-based diagnostic tool for P. penetrans. An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria-specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro-organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.

Brillouin Amplification--A Powerful New Scheme for Microwave Photonic Communications

NASA Technical Reports Server (NTRS)

Yao, S.; Maleki, L.

1997-01-01

We introduce the Brillouin selective sideband amplification technique and demonstrate many important applications of this technique in photonic microwave systems, including efficient phase modulation to amplitude modulation conversion, photonic frequency multiplication, photonic signal mixing with gain, and frequency multiplied signal up conversion.

Persistent West Nile Virus Transmission and the Apparent Displacement St. Louis Encephalitis Virus in Southeastern California, 2003−2006

PubMed Central

REISEN, WILLIAM K.; LOTHROP, HUGH D.; WHEELER, SARAH S.; KENNSINGTON, MARC; GUTIERREZ, ARTURO; FANG, YING; GARCIA, SANDRA; LOTHROP, BRANKA

2008-01-01

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) invaded the Colorado Desert biome of southern California during summer 2003 and seemed to displace previously endemic St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV, an antigenically similar Flavivirus in the Japanese encephalitis virus serocomplex). Western equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, WEEV), an antigenically distinct Alphavirus, was detected during 2005 and 2006, indicating that conditions were suitable for encephalitis virus introduction and detection. Cross-protective “avian herd immunity” due to WNV infection possibly may have prevented SLEV reintroduction and/or amplification to detectable levels. During 2003−2006, WNV was consistently active at wetlands and agricultural habitats surrounding the Salton Sea where Culex tarsalis Coquillett served as the primary enzootic maintenance and amplification vector. Based on published laboratory infection studies and the current seroprevalence estimates, house sparrows, house finches, and several Ardeidae may have been important avian amplifying hosts in this region. Transmission efficiency may have been dampened by high infection rates in incompetent avian hosts, including Gamble's quail, mourning doves, common ground doves, and domestic pigeons. Early season WNV amplification and dispersal from North Shore in the southeastern portion of the Coachella Valley resulted in sporadic WNV incursions into the urbanized Upper Valley near Palm Springs, where Culex pipiens quinquefasciatus Say was the primary enzootic and bridge vector. Although relatively few human cases were detected during the 2003−2006 period, all were concentrated in the Upper Valley and were associated with high human population density and WNV infection in peridomestic populations of Cx. p. quinquefasciatus. Intensive early mosquito control during 2006 seemed to interrupt and delay transmission, perhaps setting the stage for the future reintroduction of SLEV. PMID:18533445

Hairpin DNA-Templated Silver Nanoclusters as Novel Beacons in Strand Displacement Amplification for MicroRNA Detection.

PubMed

Zhang, Jingpu; Li, Chao; Zhi, Xiao; Ramón, Gabriel Alfranca; Liu, Yanlei; Zhang, Chunlei; Pan, Fei; Cui, Daxiang

2016-01-19

MicroRNA (miRNA) biomarkers display great potential for cancer diagnosis and prognosis. The development of rapid and specific methods for miRNA detection has become a hotspot. Herein, hairpin DNA-templated silver nanoclusters (AgNCs/HpDNA) were prepared and integrated into strand-displacement amplification (SDA) as a novel beacon for miRNA detection. The light-up platform was established based on guanine (G)-rich fluorescence enhancement that essentially converted the excitation/emission pair of AgNCs/HpDNAs from a shorter wavelength to a longer wavelength, and then achieved fluorescent enhancement at longer wavelength. On the basis of the validation of the method, the single and duplex detection were conducted in two plasma biomarkers (miR-16-5p and miR-19b-3p) for the diagnosis of gastric cancer. The probe (AgNCs/RED 16(7s)C) utilized for miR-16-5p detection adopted a better conformation with high specificity to recognize single-base mismatches by producing dramatically opposite signals (increase or decrease at 580 nm ex/640 nm em) while the probe (AgNCs/GRE 19b(5s)C) for miR-19b-3p generated dual signals (increase at 490 nm ex/570 nm em and decrease at 430 nm ex/530 nm em) with bright fluorescence in one reaction during the amplification, but unexpectedly was partially digested. This is for the first time to allow the generation of enhanced fluorescent AgNCs and the target recognition integrated into a single process, which offers great opportunity for specific miRNA detection in an easy and rapid way.

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

PubMed

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

2015-03-15

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

Observational Evidence for Desert Amplification Using Multiple Satellite Datasets.

PubMed

Wei, Nan; Zhou, Liming; Dai, Yongjiu; Xia, Geng; Hua, Wenjian

2017-05-17

Desert amplification identified in recent studies has large uncertainties due to data paucity over remote deserts. Here we present observational evidence using multiple satellite-derived datasets that desert amplification is a real large-scale pattern of warming mode in near surface and low-tropospheric temperatures. Trend analyses of three long-term temperature products consistently confirm that near-surface warming is generally strongest over the driest climate regions and this spatial pattern of warming maximizes near the surface, gradually decays with height, and disappears in the upper troposphere. Short-term anomaly analyses show a strong spatial and temporal coupling of changes in temperatures, water vapor and downward longwave radiation (DLR), indicating that the large increase in DLR drives primarily near surface warming and is tightly associated with increasing water vapor over deserts. Atmospheric soundings of temperature and water vapor anomalies support the results of the long-term temperature trend analysis and suggest that desert amplification is due to comparable warming and moistening effects of the troposphere. Likely, desert amplification results from the strongest water vapor feedbacks near the surface over the driest deserts, where the air is very sensitive to changes in water vapor and thus efficient in enhancing the longwave greenhouse effect in a warming climate.

Heralded amplification of path entangled quantum states

NASA Astrophysics Data System (ADS)

Monteiro, F.; Verbanis, E.; Caprara Vivoli, V.; Martin, A.; Gisin, N.; Zbinden, H.; Thew, R. T.

2017-06-01

Device-independent quantum key distribution (DI-QKD) represents one of the most fascinating challenges in quantum communication, exploiting concepts of fundamental physics, namely Bell tests of nonlocality, to ensure the security of a communication link. This requires the loophole-free violation of a Bell inequality, which is intrinsically difficult due to losses in fibre optic transmission channels. Heralded photon amplification (HPA) is a teleportation-based protocol that has been proposed as a means to overcome transmission loss for DI-QKD. Here we demonstrate HPA for path entangled states and characterise the entanglement before and after loss by exploiting a recently developed displacement-based detection scheme. We demonstrate that by exploiting HPA we are able to reliably maintain high fidelity entangled states over loss-equivalent distances of more than 50 km.

On dealing with multiple correlation peaks in PIV

NASA Astrophysics Data System (ADS)

Masullo, A.; Theunissen, R.

2018-05-01

A novel algorithm to analyse PIV images in the presence of strong in-plane displacement gradients and reduce sub-grid filtering is proposed in this paper. Interrogation windows subjected to strong in-plane displacement gradients often produce correlation maps presenting multiple peaks. Standard multi-grid procedures discard such ambiguous correlation windows using a signal to noise (SNR) filter. The proposed algorithm improves the standard multi-grid algorithm allowing the detection of splintered peaks in a correlation map through an automatic threshold, producing multiple displacement vectors for each correlation area. Vector locations are chosen by translating images according to the peak displacements and by selecting the areas with the strongest match. The method is assessed on synthetic images of a boundary layer of varying intensity and a sinusoidal displacement field of changing wavelength. An experimental case of a flow exhibiting strong velocity gradients is also provided to show the improvements brought by this technique.

Contribution of gold nanoparticles to the catalytic DNA strand displacement in leakage reduction and signal amplification.

PubMed

Wang, Bei; Zhou, Xiang; Yao, Dongbao; Sun, Xianbao; He, Miao; Wang, Xiaojing; Yin, Xue; Liang, Haojun

2017-10-03

A new model using a gold nanoparticle (AuNP)-DNA system to constrain leakage and improve efficiency of catalytic toehold-mediated strand displacement reactions was outlined. A 10-bp spacer on AuNPs and fourfold amount of fuels were determined for good performance of this model with an optimized toehold strategy. After the reaction at 25 °C for 10 h, a 258 pM target could be identified, which is a remarkable improvement compared with the traditional AuNP-DNA system without fuel. Moreover, this model was also studied to differentiate specific single nucleotide polymorphism on target with superior selection factors. This model may help by introducing a proposition of target detection to guide further investigation.

Experimental demonstration of spatially coherent beam combining using optical parametric amplification.

PubMed

Kurita, Takashi; Sueda, Keiichi; Tsubakimoto, Koji; Miyanaga, Noriaki

2010-07-05

We experimentally demonstrated coherent beam combining using optical parametric amplification with a nonlinear crystal pumped by random-phased multiple-beam array of the second harmonic of a Nd:YAG laser at 10-Hz repetition rate. In the proof-of-principle experiment, the phase jump between two pump beams was precisely controlled by a motorized actuator. For the demonstration of multiple-beam combining a random phase plate was used to create random-phased beamlets as a pump pulse. Far-field patterns of the pump, the signal, and the idler indicated that the spatially coherent signal beams were obtained on both cases. This approach allows scaling of the intensity of optical parametric chirped pulse amplification up to the exa-watt level while maintaining diffraction-limited beam quality.

Partial genome assembly for a candidate division OP11 single cell from an anoxic spring (Zodletone Spring, Oklahoma).

PubMed

Youssef, Noha H; Blainey, Paul C; Quake, Stephen R; Elshahed, Mostafa S

2011-11-01

Members of candidate division OP11 are widely distributed in terrestrial and marine ecosystems, yet little information regarding their metabolic capabilities and ecological role within such habitats is currently available. Here, we report on the microfluidic isolation, multiple-displacement-amplification, pyrosequencing, and genomic analysis of a single cell (ZG1) belonging to candidate division OP11. Genome analysis of the ∼270-kb partial genome assembly obtained showed that it had no particular similarity to a specific phylum. Four hundred twenty-three open reading frames were identified, 46% of which had no function prediction. In-depth analysis revealed a heterotrophic lifestyle, with genes encoding endoglucanase, amylopullulanase, and laccase enzymes, suggesting a capacity for utilization of cellulose, starch, and, potentially, lignin, respectively. Genes encoding several glycolysis enzymes as well as formate utilization were identified, but no evidence for an electron transport chain was found. The presence of genes encoding various components of lipopolysaccharide biosynthesis indicates a Gram-negative bacterial cell wall. The partial genome also provides evidence for antibiotic resistance (β-lactamase, aminoglycoside phosphotransferase), as well as antibiotic production (bacteriocin) and extracellular bactericidal peptidases. Multiple mechanisms for stress response were identified, as were elements of type I and type IV secretion systems. Finally, housekeeping genes identified within the partial genome were used to demonstrate the OP11 affiliation of multiple hitherto unclassified genomic fragments from multiple database-deposited metagenomic data sets. These results provide the first glimpse into the lifestyle of a member of a ubiquitous, yet poorly understood bacterial candidate division.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets.

PubMed

Seok, Youngung; Joung, Hyou-Arm; Byun, Ju-Young; Jeon, Hyo-Sung; Shin, Su Jeong; Kim, Sanghyo; Shin, Young-Beom; Han, Hyung Soo; Kim, Min-Gon

2017-01-01

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 10 2 -10 5 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets

PubMed Central

Seok, Youngung; Joung, Hyou-Arm; Byun, Ju-Young; Jeon, Hyo-Sung; Shin, Su Jeong; Kim, Sanghyo; Shin, Young-Beom; Han, Hyung Soo; Kim, Min-Gon

2017-01-01

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 102-105 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing. PMID:28740546

Sensitive immobilization-free electrochemical DNA sensor based on isothermal circular strand displacement polymerization reaction.

PubMed

Xuan, Feng; Luo, Xiaoteng; Hsing, I-Ming

2012-05-15

A highly sensitive electrochemical DNA sensor that requires no probe immobilization has been developed based on a target recycling mechanism utilizing a DNA polymerase with a strand displacement activity. The electrochemical detection is realized by taking advantage of the difference in diffusivity between a free ferrocene-labeled peptide nucleic acid (Fc-PNA) and a Fc-PNA hybridized with a complementary DNA, while the DNA polymerase-assisted target recycling leads to signal generation and amplification. The hybridization of the target DNA opens up a stem-loop template DNA with the Fc-PNA hybridized to its extruded 5' end and allows a DNA primer to anneal and be extended by the DNA polymerase, which results in sequential displacement of the target DNA and the Fc-PNA from the template DNA. The displaced target DNA will hybridize with another template DNA, triggering another round of primer extension and strand displacement. The released Fc-PNA, due to its neutral backbone, has much higher diffusivity towards a negatively charged electrode, compared to that when it is hybridized with a negatively charged DNA. Therefore, a significantly enhanced signal of Fc can be observed. The outstanding sensitivity and simplicity make this approach a promising candidate for next-generation electrochemical DNA sensing technologies. Copyright © 2012 Elsevier B.V. All rights reserved.

Fluorescence detection of thrombin using autocatalytic strand displacement cycle reaction and a dual-aptamer DNA sandwich assay.

PubMed

Niu, Shuyan; Qu, Lijing; Zhang, Qing; Lin, Jiehua

2012-02-15

A sensitive and specific sandwich assay for the detection of thrombin is described. Two affiliative aptamers were used to increase the assay specificity through sandwich recognition. Recognition DNA loaded on gold nanoparticles (AuNPs) partially hybridized with the initiator DNA, which was displaced by surviving DNA. After the initiator DNA was released into the solution, one hairpin structure was opened, which in turn opened another hairpin structure. The initiator DNA was displaced and released into the solution again by another hairpin structure because of the hybridized reaction. Then the released initiator DNA initiated another autocatalytic strand displacement reaction. A sophisticated network of three such duplex formation cycles was designed to amplify the fluorescence signal. Other proteins, such as bovine serum albumin and lysozyme, did not interfere with the detection of thrombin. This approach enables rapid and specific thrombin detection with reduced costs and minimized material consumption compared with traditional assay processes. The detection limit of thrombin was as low as 4.3 × 10⁻¹³ M based on the AuNP amplification and the autocatalytic strand displacement cycle reaction. This method could be used in biological samples with excellent selectivity. Copyright © 2011 Elsevier Inc. All rights reserved.

Polymerase chain reaction amplification as a diagnostic tool in culture-negative multiple-valve endocarditis.

PubMed

Madershahian, Navid; Strauch, Justus T; Breuer, Martin; Bruhin, Raimund; Straube, Eberhard; Wahlers, Thorsten

2005-03-01

We report a case of culture-negative infectious endocarditis in a 17-year-old boy in which the etiologic diagnosis could only be provided by polymerase chain reaction amplification and sequencing of the bacterial 16S rRNA gene from valve tissue.

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.

Verification of the GIS-based Newmark method through 2D dynamic modelling of slope stability

NASA Astrophysics Data System (ADS)

Torgoev, A.; Havenith, H.-B.

2012-04-01

The goal of this work is to verify the simplified GIS-based Newmark displacement approach through 2D dynamic modelling of slope stability. The research is applied to a landslide-prone area in Central Asia, the Mailuu-Suu Valley, situated in the south of Kyrgyzstan. The comparison is carried out on the basis of 30 different profiles located in the target area, presenting different geological, tectonic and morphological settings. One part of the profiles were selected within landslide zones, the other part was selected in stable areas. Many of the landslides are complex slope failures involving falls, rotational sliding and/or planar sliding and flows. These input data were extracted from a 3D structural geological model built with the GOCAD software. Geophysical and geomechanical parameters were defined on the basis of results obtained by multiple surveys performed in the area over the past 15 years. These include geophysical investigation, seismological experiments and ambient noise measurements. Dynamic modelling of slope stability is performed with the UDEC version 4.01 software that is able to compute deformation of discrete elements. Inside these elements both elasto-plastic and purely elastic materials (similar to rigid blocks) were tested. Various parameter variations were tested to assess their influence on the final outputs. And even though no groundwater flow was included, the numerous simulations are very time-consuming (20 mins per model for 10 secs simulated shaking) - about 500 computation hours have been completed so far (more than 100 models). Preliminary results allow us to compare Newmark displacements computed using different GIS approaches (Jibson et al., 1998; Miles and Ho, 1999, among others) with the displacements computed using the original Newmark method (Newmark, 1965, here simulated seismograms were used) and displacements produced along joints by the corresponding 2D dynamical models. The generation of seismic amplification and its impact on peak-ground-acceleration, Arias Intensity and permanent slope movements (total and slip on joints) is assessed for numerous morphological-lithological settings (curvature, slope angle, surficial geology, various layer dips and orientations) throughout the target area. The final results of our studies should allow us to define the limitations of the simplified GIS-based Newmark displacement modelling; thus, the verified method would make landslide susceptibility and hazard mapping in seismically active regions more reliable.

Distilled single-cell genome sequencing and de novo assembly for sparse microbial communities.

PubMed

Taghavi, Zeinab; Movahedi, Narjes S; Draghici, Sorin; Chitsaz, Hamidreza

2013-10-01

Identification of every single genome present in a microbial sample is an important and challenging task with crucial applications. It is challenging because there are typically millions of cells in a microbial sample, the vast majority of which elude cultivation. The most accurate method to date is exhaustive single-cell sequencing using multiple displacement amplification, which is simply intractable for a large number of cells. However, there is hope for breaking this barrier, as the number of different cell types with distinct genome sequences is usually much smaller than the number of cells. Here, we present a novel divide and conquer method to sequence and de novo assemble all distinct genomes present in a microbial sample with a sequencing cost and computational complexity proportional to the number of genome types, rather than the number of cells. The method is implemented in a tool called Squeezambler. We evaluated Squeezambler on simulated data. The proposed divide and conquer method successfully reduces the cost of sequencing in comparison with the naïve exhaustive approach. Squeezambler and datasets are available at http://compbio.cs.wayne.edu/software/squeezambler/.

Structural health monitoring using DOG multi-scale space: an approach for analyzing damage characteristics

NASA Astrophysics Data System (ADS)

Guo, Tian; Xu, Zili

2018-03-01

Measurement noise is inevitable in practice; thus, it is difficult to identify defects, cracks or damage in a structure while suppressing noise simultaneously. In this work, a novel method is introduced to detect multiple damage in noisy environments. Based on multi-scale space analysis for discrete signals, a method for extracting damage characteristics from the measured displacement mode shape is illustrated. Moreover, the proposed method incorporates a data fusion algorithm to further eliminate measurement noise-based interference. The effectiveness of the method is verified by numerical and experimental methods applied to different structural types. The results demonstrate that there are two advantages to the proposed method. First, damage features are extracted by the difference of the multi-scale representation; this step is taken such that the interference of noise amplification can be avoided. Second, a data fusion technique applied to the proposed method provides a global decision, which retains the damage features while maximally eliminating the uncertainty. Monte Carlo simulations are utilized to validate that the proposed method has a higher accuracy in damage detection.

Sequencing intractable DNA to close microbial genomes.

PubMed

Hurt, Richard A; Brown, Steven D; Podar, Mircea; Palumbo, Anthony V; Elias, Dwayne A

2012-01-01

Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap). The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

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.

Impact of plasma response on plasma displacements in DIII-D during application of external 3D perturbations

DOE PAGES

Wingen, Andreas; Ferraro, Nathaniel M.; Shafer, Morgan W.; ...

2014-05-23

The effects of applied non-axisymmetric resonant magnetic perturbations (RMPs) are predicted without and with self-consistent plasma response by modeling of the magnetic field structure and two-fluid MHD simulations, respectively. A synthetic diagnostic is used to simulate soft X-ray (SXR) emission within the steep gradient region of the pedestal, 0.98 > ψ > 0.94. The entire pedestal and edge region is characterized by large changes in plasma rotation and current density. Those parameters are expected to strongly affect the plasma response to RMPs. The M3D-C1 code takes into account this response self-consistently. The plasma response is investigated in detail and usedmore » in the forward modeling of the simulated local SXR emission, within the framework of the synthetic diagnostic. The resulting synthetic emission is compared to measured SXR data. The latter clearly shows helical m = 11 ± 1 displacements around the 11/3 rational surface of sizes up to 5 cm, which change with the poloidal angle. The synthetic emission with plasma response is used to explain the nature of the measured displacements. Different approaches are tested. One approach is based on the magnetic field structure to simulate local emission, which shows additional structures at the separatrix, that are caused by the lobes. Especially without plasma response, almost only separatrix structures are generated while no significant displacements are found further inside. Another approach to model local emission uses the fluid quantities electron density and temperature, as calculated by M3D-C1. Compared to the previous approach, based on the magnetic field structure, the emission simulated by the fluid approach with plasma response shows better agreement with the measured SXR data. To be specific, it has comparable displacements in the steep gradient region and no lobe structures at all. The helical displacements around the 11/3 surface are identified to be directly related to the kink response, caused by non-resonant amplification of various poloidal RMP modes due to plasma response. Regarding the latter, the role of different plasma parameters is investigated, but it appears that the electron rotation plays a key role in the formation of screening and resonant amplification, while the kinking appears to be sensitive to the edge current density. As a result, it is also hypothesised that the strength of the kink response is also correlated to edge-localized-mode (ELM) stability.« less

39 CFR 777.25 - Additional rules for replacement housing payments.

Code of Federal Regulations, 2011 CFR

2011-07-01

... displacement dwelling; or (2) The difference between (i) the occupant's share of the acquisition cost of the displacement dwelling and (ii) the purchase price of a decent, safe, and sanitary replacement dwelling actually purchased and occupied by the displaced person. (b) Multiple Occupants of One Displacement Dwelling. If two...

39 CFR 777.25 - Additional rules for replacement housing payments.

Code of Federal Regulations, 2010 CFR

2010-07-01

... displacement dwelling; or (2) The difference between (i) the occupant's share of the acquisition cost of the displacement dwelling and (ii) the purchase price of a decent, safe, and sanitary replacement dwelling actually purchased and occupied by the displaced person. (b) Multiple Occupants of One Displacement Dwelling. If two...

39 CFR 777.25 - Additional rules for replacement housing payments.

Code of Federal Regulations, 2013 CFR

2013-07-01

... displacement dwelling; or (2) The difference between (i) the occupant's share of the acquisition cost of the displacement dwelling and (ii) the purchase price of a decent, safe, and sanitary replacement dwelling actually purchased and occupied by the displaced person. (b) Multiple Occupants of One Displacement Dwelling. If two...

39 CFR 777.25 - Additional rules for replacement housing payments.

Code of Federal Regulations, 2012 CFR

2012-07-01

... displacement dwelling; or (2) The difference between (i) the occupant's share of the acquisition cost of the displacement dwelling and (ii) the purchase price of a decent, safe, and sanitary replacement dwelling actually purchased and occupied by the displaced person. (b) Multiple Occupants of One Displacement Dwelling. If two...

39 CFR 777.25 - Additional rules for replacement housing payments.

Code of Federal Regulations, 2014 CFR

2014-07-01

... displacement dwelling; or (2) The difference between (i) the occupant's share of the acquisition cost of the displacement dwelling and (ii) the purchase price of a decent, safe, and sanitary replacement dwelling actually purchased and occupied by the displaced person. (b) Multiple Occupants of One Displacement Dwelling. If two...

[Interest of crizotinib in a lung cancer patient with de novo amplification of MET].

PubMed

Rabeau, A; Rouquette, I; Vantelon, J-M; Taranchon-Clermont, E; Mazières, J

2017-01-01

Targeted therapy in lung cancer changes the prognostic and treatment of patients. MET is an oncogene including exon 14 mutations and gene amplification associated with worse prognosis. We here report the case of a 47-year-old former smoker, woman, with a stage IV lung adenocarcinoma with multiple chemotherapy failure. A MET amplification was identified and the patient consequently received crizotinib. A major response was observed after eight weeks of treatment. MET amplification screening appears to be interesting with some oncogenic-addicted tumor response rate. Those patients should be enrolled in clinical trials dedicated to tumor with MET alteration. Copyright © 2016 SPLF. Published by Elsevier Masson SAS. All rights reserved.

Two passive mechanical conditions modulate power generation by the outer hair cells

PubMed Central

Gracewski, Sheryl M.

2017-01-01

In the mammalian cochlea, small vibrations of the sensory epithelium are amplified due to active electro-mechanical feedback of the outer hair cells. The level of amplification is greater in the base than in the apex of the cochlea. Theoretical studies have used longitudinally varying active feedback properties to reproduce the location-dependent amplification. The active feedback force has been considered to be proportional to the basilar membrane displacement or velocity. An underlying assumption was that organ of Corti mechanics are governed by rigid body kinematics. However, recent progress in vibration measurement techniques reveals that organ of Corti mechanics are too complicated to be fully represented with rigid body kinematics. In this study, two components of the active feedback are considered explicitly—organ of Corti mechanics, and outer hair cell electro-mechanics. Physiological properties for the outer hair cells were incorporated, such as the active force gain, mechano-transduction properties, and membrane RC time constant. Instead of a kinematical model, a fully deformable 3D finite element model was used. We show that the organ of Corti mechanics dictate the longitudinal trend of cochlear amplification. Specifically, our results suggest that two mechanical conditions are responsible for location-dependent cochlear amplification. First, the phase of the outer hair cell’s somatic force with respect to its elongation rate varies along the cochlear length. Second, the local stiffness of the organ of Corti complex felt by individual outer hair cells varies along the cochlear length. We describe how these two mechanical conditions result in greater amplification toward the base of the cochlea. PMID:28880884

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.

Whole genome amplification of DNA extracted from FFPE tissues.

PubMed

Bosso, Mira; Al-Mulla, Fahd

2011-01-01

Whole genome amplification systems were developed to meet the increasing research demands on DNA resources and to avoid DNA shortage. The technology enables amplification of nanogram amounts of DNA into microgram quantities and is increasingly used in the amplification of DNA from multiple origins such as blood, fresh frozen tissue, formalin-fixed paraffin-embedded tissues, saliva, buccal swabs, bacteria, and plant and animal sources. This chapter focuses on the use of GenomePlex(®) tissue Whole Genome Amplification Kit, to amplify DNA directly from archived tissue. In addition, this chapter documents our unique experience with the utilization of GenomePlex(®) amplified DNA using several molecular techniques including metaphase Comparative Genomic Hybridization, array Comparative Genomic Hybridization, and real-time quantitative polymerase chain reaction assays. GenomePlex(®) is a registered trademark of Rubicon Genomics Incorporation.

Embryo Sexing and Sex Chromosomal Chimerism Analysis by Loop-Mediated Isothermal Amplification in Cattle and Water Buffaloes

PubMed Central

HIRAYAMA, Hiroki; KAGEYAMA, Soichi; MORIYASU, Satoru; SAWAI, Ken; MINAMIHASHI, Akira

2013-01-01

Abstract In domestic animals of the family Bovidae, sex preselection of offspring has been demanded for convenience of milk/beef production and animal breeding. Development of the nonsurgical embryo transfer technique and sexing methods of preimplantation embryos made it possible. Sexing based on detection of Y chromosome-specific DNA sequences is considered the most reliable method to date. PCR enables amplification of a target sequence from a small number of blastomeres. However, it requires technical skill and is time consuming. Furthermore, PCR has the risk of false positives because of DNA contamination during handling of the PCR products in duplicate PCR procedures and/or electrophoresis. Therefore, for embryo sexing to become widely used in the cattle embryo transfer industry, a simple, rapid and precise sexing method needs to be developed. Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method, and the reaction is carried out under isothermal conditions (range, 60 to 65 C) using DNA polymerase with strand displacement activity. When the target DNA is amplified by LAMP, a white precipitate derived from magnesium pyrophosphate (a by-product of the LAMP reaction) is observed. It is noteworthy that LAMP does not need special reagents or electrophoresis to detect the amplified DNA. This review describes the development and application of an embryo sexing method using LAMP in cattle and water buffaloes. PMID:23965599

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

PubMed

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

2015-06-30

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

Long-stem shaped multifunctional molecular beacon for highly sensitive nucleic acids determination via intramolecular and intermolecular interactions based strand displacement amplification.

PubMed

Xu, Jianguo; Zheng, Tingting; Le, Jingqing; Jia, Lee

2017-11-20

Occurrence and application of oligonucleotide probes have promoted great progress in the biochemical analysis field due to their unique biological and chemical properties. In this work, a long-stem shaped multifunctional molecular beacon (LS-MMB) that is responsive to a cancer-related gene, p53, is well-prepared. By designing the probe with long-paired bases at its two ends and short-paired bases between the middle region and the 3' end, the LS-MMB is intelligently endowed with the ability to recognize the target analyte, serve as the polymerization primer/template, and signal the hybridization event synchronously, which is distinctly advantageous over the traditional molecular beacons (MBs). Moreover, it is excitingly found that the LS-MMB can be employed to exert intramolecular and intermolecular interactions for strand displacement amplification (SDA) without the involvement of any assistant probes; this therapy results in a really easy and rapid sensing system that provides an extremely low background noise and high target output signal. In this case, an excellent sensitivity and specificity to detect target gene down to picomolar level and resolution to even one nucleotide variation are achieved, respectively. In addition, the application potential for real genomic DNA analysis is realized. We envision that the probe of LS-MMB can act as a universal platform for biosensing and biomedical research.

Palindromic Molecule Beacon-Based Cascade Amplification for Colorimetric Detection of Cancer Genes.

PubMed

Shen, Zhi-Fa; Li, Feng; Jiang, Yi-Fan; Chen, Chang; Xu, Huo; Li, Cong-Cong; Yang, Zhe; Wu, Zai-Sheng

2018-03-06

A highly sensitive and selective colorimetric assay based on a multifunctional molecular beacon with palindromic tail (PMB) was proposed for the detection of target p53 gene. The PMB probe can serve as recognition element, primer, and polymerization template and contains a nicking site and a C-rich region complementary to a DNAzyme. In the presence of target DNA, the hairpin of PMB is opened, and the released palindromic tails intermolecularly hybridize with each other, triggering the autonomous polymerization/nicking/displacement cycles. Although only one type of probe is involved, the system can execute triple and continuous polymerization strand displacement amplifications, generating large amounts of G-quadruplex fragments. These G-rich fragments can bind to hemin and form the DNAzymes that possess the catalytic activity similar to horseradish peroxidase, catalyzing the oxidation of ABTS by H 2 O 2 and producing the colorimetric signal. Utilizing the newly proposed sensing system, target DNA can be detected down to 10 pM with a linear response range from 10 pM to 200 nM, and mutant target DNAs are able to be distinguished even by the naked eye. The desirable detection sensitivity, high specificity, and operation convenience without any separation step and chemical modification demonstrate that the palindromic molecular beacon holds the potential for detecting and monitoring a variety of nucleic acid-related biomarkers.

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.

Sound-Field Amplification to Increase Compliance to Directions in Students with ADHD

ERIC Educational Resources Information Center

Maag, John W.; Anderson, Jean M.

2007-01-01

This study determined the effects of sound-field amplification (SFA) on the speed with which students with attention deficit hyperactivity disorder (ADHD) followed teacher directions. A multiple baseline design was used with 3 elementary-aged students with ADHD to assess the effects of SFA across 4 types of directions: (a) task demand (e.g., get…

Automated high-throughput flow-through real-time diagnostic system

DOEpatents

Regan, John Frederick

2012-10-30

An automated real-time flow-through system capable of processing multiple samples in an asynchronous, simultaneous, and parallel fashion for nucleic acid extraction and purification, followed by assay assembly, genetic amplification, multiplex detection, analysis, and decontamination. The system is able to hold and access an unlimited number of fluorescent reagents that may be used to screen samples for the presence of specific sequences. The apparatus works by associating extracted and purified sample with a series of reagent plugs that have been formed in a flow channel and delivered to a flow-through real-time amplification detector that has a multiplicity of optical windows, to which the sample-reagent plugs are placed in an operative position. The diagnostic apparatus includes sample multi-position valves, a master sample multi-position valve, a master reagent multi-position valve, reagent multi-position valves, and an optical amplification/detection system.

An amplified graphene oxide-based fluorescence aptasensor based on target-triggered aptamer hairpin switch and strand-displacement polymerization recycling for bioassays.

PubMed

Hu, Kun; Liu, Jinwen; Chen, Jia; Huang, Yong; Zhao, Shulin; Tian, Jianniao; Zhang, Guohai

2013-04-15

An amplified graphene oxide (GO) based fluorescence aptasensor based on target-triggered aptamer hairpin switch and strand-displacement polymerization recycling is developed for bioassays. The dye-labeled single-strand DNA (aptamer hairpin) was adsorbed on the surface of GO, which result in the fluorescence quenching of dye, and exhibiting minimal background fluorescence. Upon the target, primer and polymerase, the stem of the aptamer hairpin was opened, and binds with the primer to triggers the circular target strand-displacement polymerization reaction, which produces huge amounts of duplex helixes DNA and lead to strong fluorescence emission due to shielding of nucelobases within its double-helix structure. During the polymerization reaction, the primer was extended, and target was displaced. And the displaced target recognizes and hybridizes with another hairpin probe, triggering the next round of polymerization reaction, and the circle process induces fluorescence signal amplification for the detection of analyte. To test the feasibility of the aptasensor systems, interferon-gamma (IFN-γ) was employed as a model analyte. A detection limit as low as 1.5 fM is obtained based on the GO aptasensor with a linear range of three orders of magnitude. The present method was successfully applied for the detection of IFN-γ in human plasma. Copyright © 2012 Elsevier B.V. All rights reserved.

Multipass laser amplification with near-field far-field optical separation

DOEpatents

Hagen, Wilhelm F.

1979-01-01

This invention discloses two classes of optical configurations for high power laser amplification, one allowing near-field and the other allowing far-field optical separation, for the multiple passage of laser pulses through one or more amplifiers over an open optical path. These configurations may reimage the amplifier or any other part of the cavity on itself so as to suppress laser beam intensity ripples that arise from diffraction and/or non-linear effects. The optical cavities combine the features of multiple passes, spatial filtering and optical reimaging and allow sufficient time for laser gain recovery.

Ultrasensitive aptamer-based protein detection via a dual amplified biocatalytic strategy

PubMed Central

Xiang, Yun; Zhang, Yuyong; Qian, Xiaoqing; Chai, Yaqin; Wang, Joseph; Yuan, Ruo

2010-01-01

We present an ultrasensitive aptasensor for electronic monitoring of proteins through a dual amplified strategy in this paper. The target protein thrombin is sandwiched between an electrode surface confined aptamer and an aptamer-enzyme-carbon nanotube bioconjugate. The analytical signal amplification is achieved by coupling the signal amplification nature of multiple enzymes with the biocatalytic signal enhancement of redox-recycling. Our novel dramatic signal amplification strategy, with a detection limit of 8.3 fM, shows about 4 orders of magnitude improvement in sensitivity for thrombin detection compared to other universal single enzyme-based assay. This makes our approach an attractive alternative to other common PCR-based signal amplification in ultralow level of protein detection. PMID:20452761

High peak-power kilohertz laser system employing single-stage multi-pass amplification

DOEpatents

Shan, Bing; Wang, Chun; Chang, Zenghu

2006-05-23

The present invention describes a technique for achieving high peak power output in a laser employing single-stage, multi-pass amplification. High gain is achieved by employing a very small "seed" beam diameter in gain medium, and maintaining the small beam diameter for multiple high-gain pre-amplification passes through a pumped gain medium, then leading the beam out of the amplifier cavity, changing the beam diameter and sending it back to the amplifier cavity for additional, high-power amplification passes through the gain medium. In these power amplification passes, the beam diameter in gain medium is increased and carefully matched to the pump laser's beam diameter for high efficiency extraction of energy from the pumped gain medium. A method of "grooming" the beam by means of a far-field spatial filter in the process of changing the beam size within the single-stage amplifier is also described.

Dual phase multiplex polymerase chain reaction

DOEpatents

Pemov, Alexander [Charlottesville, VA; Bavykin, Sergei [Darien, IL

2008-10-07

Highly specific and sensitive methods were developed for multiplex amplification of nucleic acids on supports such as microarrays. Based on a specific primer design, methods include five types of amplification that proceed in a reaction chamber simultaneously. These relate to four types of multiplex amplification of a target DNA on a solid support, directed by forward and reverse complex primers immobilized to the support and a fifth type--pseudo-monoplex polymerase chain reaction (PCR) of multiple targets in solution, directed by a single pair of unbound universal primers. The addition of the universal primers in the reaction mixture increases the yield over the traditional "bridge" amplification on a solid support by approximately ten times. Methods that provide multitarget amplification and detection of as little as 0.45-4.5.times.10.sup.-12 g (equivalent to 10.sup.2-10.sup.3 genomes) of a bacterial genomic DNA are disclosed.

A normally-closed piezoelectric micro-valve with flexible stopper

NASA Astrophysics Data System (ADS)

Chen, Song; Lu, Song; Liu, Yong; Wang, Jiantao; Tian, Xiaochao; Liu, Guojun; Yang, Zhigang

2016-04-01

In the field of controlled drug delivery system, there are still many problems on those reported micro-valves, such as the small opening height, unsatisfactory particle tolerance and high cost. To solve the above problems, a novel normally-closed piezoelectric micro-valve is presented in this paper. The micro-valve was driven by circular unimorph piezoelectric vibrator and natural rubber membrane with high elasticity was used as the valve stopper. The small axial displacement of piezoelectric vibrator can be converted into a large stroke of valve stopper based on hydraulic amplification mechanism. The experiment indicates that maximum hydraulic amplification ratio is up to 14, and the cut-off pressure of the micro-valve is 39kPa in the case of no working voltage. The presented micro valve has a large flow control range (ranging from 0 to 8.75mL/min).

Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells

PubMed Central

Gole, Jeff; Gore, Athurva; Richards, Andrew; Chiu, Yu-Jui; Fung, Ho-Lim; Bushman, Diane; Chiang, Hsin-I; Chun, Jerold; Lo, Yu-Hwa; Zhang, Kun

2013-01-01

Genome sequencing of single cells has a variety of applications, including characterizing difficult-to-culture microorganisms and identifying somatic mutations in single cells from mammalian tissues. A major hurdle in this process is the bias in amplifying the genetic material from a single cell, a procedure known as polymerase cloning. Here we describe the microwell displacement amplification system (MIDAS), a massively parallel polymerase cloning method in which single cells are randomly distributed into hundreds to thousands of nanoliter wells and simultaneously amplified for shotgun sequencing. MIDAS reduces amplification bias because polymerase cloning occurs in physically separated nanoliter-scale reactors, facilitating the de novo assembly of near-complete microbial genomes from single E. coli cells. In addition, MIDAS allowed us to detect single-copy number changes in primary human adult neurons at 1–2 Mb resolution. MIDAS will further the characterization of genomic diversity in many heterogeneous cell populations. PMID:24213699

Near-Infrared Ag2S Quantum Dots-Based DNA Logic Gate Platform for miRNA Diagnostics.

PubMed

Miao, Peng; Tang, Yuguo; Wang, Bidou; Meng, Fanyu

2016-08-02

Dysregulation of miRNA expression is correlated with the development and progression of many diseases. These miRNAs are regarded as promising biomarkers. However, it is challenging to measure these low abundant molecules without employing time-consuming radioactive labeling or complex amplification strategies. Here, we present a DNA logic gate platform for miRNA diagnostics with fluorescence outputs from near-infrared (NIR) Ag2S quantum dots (QDs). Carefully designed toehold exchange-mediated strand displacements with different miRNA inputs occur on a solid-state interface, which control QDs release from solid-state interface to solution, responding to multiplex information on initial miRNAs. Excellent fluorescence emission properties of NIR Ag2S QDs certify the great prospect for amplification-free and sensitive miRNA assay. We demonstrate the potential of this platform by achieving femtomolar level miRNA analysis and the versatility of a series of logic circuits computation.

Silver nanoclusters-assisted ion-exchange reaction with CdTe quantum dots for photoelectrochemical detection of adenosine by target-triggering multiple-cycle amplification strategy.

PubMed

Zhao, Yang; Tan, Lu; Gao, Xiaoshan; Jie, Guifen; Huang, Tingyu

2018-07-01

Herein, we successfully devised a novel photoelectrochemical (PEC) platform for ultrasensitive detection of adenosine by target-triggering cascade multiple cycle amplification based on the silver nanoparticles-assisted ion-exchange reaction with CdTe quantum dots (QDs). In the presence of target adenosine, DNA s1 is released from the aptamer and then hybridizes with hairpin DNA (HP1), which could initiate the cycling cleavage process under the reaction of nicking endonuclease. Then the product (DNA b) of cycle I could act as the "DNA trigger" of cycle II to further generate a large number of DNA s1, which again go back to cycle I, thus a cascade multiple DNA cycle amplification was carried out to produce abundant DNA c. These DNA c fragments with the cytosine (C)-rich loop were captured by magnetic beads, and numerous silver nanoclusters (Ag NCs) were synthesized by AgNO 3 and sodium borohydride. The dissolved AgNCs released numerous silver ions which could induce ion exchange reaction with the CdTe QDs, thus resulting in greatly amplified change of photocurrent for target detection. The detection linear range for adenosine was 1.0 fM ~10 nM with the detection limit of 0.5 fM. The present PEC strategy combining cascade multiple DNA cycle amplification and AgNCs-induced ion-exchange reaction with QDs provides new insight into rapid, and ultrasensitive PEC detection of different biomolecules, which showed great potential for detecting trace amounts in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier B.V. All rights reserved.

DNA strand-displacement-induced fluorescence enhancement for highly sensitive and selective assay of multiple microRNA in cancer cells.

PubMed

Wu, Ping; Tu, Yunqiu; Qian, Yingdan; Zhang, Hui; Cai, Chenxin

2014-01-28

We report a new strategy for evaluating multiple miRNA expressions in cancer cells based on DNA strand-displacement-induced fluorescence enhancement. This assay has the ability to discriminate the target from even single-base mismatched sequences or other miRNAs.

RNA signal amplifier circuit with integrated fluorescence output.

PubMed

Akter, Farhima; Yokobayashi, Yohei

2015-05-15

We designed an in vitro signal amplification circuit that takes a short RNA input that catalytically activates the Spinach RNA aptamer to produce a fluorescent output. The circuit consists of three RNA strands: an internally blocked Spinach aptamer, a fuel strand, and an input strand (catalyst), as well as the Spinach aptamer ligand 3,5-difluoro-4-hydroxylbenzylidene imidazolinone (DFHBI). The input strand initially displaces the internal inhibitory strand to activate the fluorescent aptamer while exposing a toehold to which the fuel strand can bind to further displace and recycle the input strand. Under a favorable condition, one input strand was able to activate up to five molecules of the internally blocked Spinach aptamer in 185 min at 30 °C. The simple RNA circuit reported here serves as a model for catalytic activation of arbitrary RNA effectors by chemical triggers.

A programming language for composable DNA circuits

PubMed Central

Phillips, Andrew; Cardelli, Luca

2009-01-01

Recently, a range of information-processing circuits have been implemented in DNA by using strand displacement as their main computational mechanism. Examples include digital logic circuits and catalytic signal amplification circuits that function as efficient molecular detectors. As new paradigms for DNA computation emerge, the development of corresponding languages and tools for these paradigms will help to facilitate the design of DNA circuits and their automatic compilation to nucleotide sequences. We present a programming language for designing and simulating DNA circuits in which strand displacement is the main computational mechanism. The language includes basic elements of sequence domains, toeholds and branch migration, and assumes that strands do not possess any secondary structure. The language is used to model and simulate a variety of circuits, including an entropy-driven catalytic gate, a simple gate motif for synthesizing large-scale circuits and a scheme for implementing an arbitrary system of chemical reactions. The language is a first step towards the design of modelling and simulation tools for DNA strand displacement, which complements the emergence of novel implementation strategies for DNA computing. PMID:19535415

A programming language for composable DNA circuits.

PubMed

Phillips, Andrew; Cardelli, Luca

2009-08-06

Recently, a range of information-processing circuits have been implemented in DNA by using strand displacement as their main computational mechanism. Examples include digital logic circuits and catalytic signal amplification circuits that function as efficient molecular detectors. As new paradigms for DNA computation emerge, the development of corresponding languages and tools for these paradigms will help to facilitate the design of DNA circuits and their automatic compilation to nucleotide sequences. We present a programming language for designing and simulating DNA circuits in which strand displacement is the main computational mechanism. The language includes basic elements of sequence domains, toeholds and branch migration, and assumes that strands do not possess any secondary structure. The language is used to model and simulate a variety of circuits, including an entropy-driven catalytic gate, a simple gate motif for synthesizing large-scale circuits and a scheme for implementing an arbitrary system of chemical reactions. The language is a first step towards the design of modelling and simulation tools for DNA strand displacement, which complements the emergence of novel implementation strategies for DNA computing.

Non-biased and efficient global amplification of a single-cell cDNA library

PubMed Central

Huang, Huan; Goto, Mari; Tsunoda, Hiroyuki; Sun, Lizhou; Taniguchi, Kiyomi; Matsunaga, Hiroko; Kambara, Hideki

2014-01-01

Analysis of single-cell gene expression promises a more precise understanding of molecular mechanisms of a living system. Most techniques only allow studies of the expressions for limited numbers of gene species. When amplification of cDNA was carried out for analysing more genes, amplification biases were frequently reported. A non-biased and efficient global-amplification method, which uses a single-cell cDNA library immobilized on beads, was developed for analysing entire gene expressions for single cells. Every step in this analysis from reverse transcription to cDNA amplification was optimized. By removing degrading excess primers, the bias due to the digestion of cDNA was prevented. Since the residual reagents, which affect the efficiency of each subsequent reaction, could be removed by washing beads, the conditions for uniform and maximized amplification of cDNAs were achieved. The differences in the amplification rates for randomly selected eight genes were within 1.5-folds, which could be negligible for most of the applications of single-cell analysis. The global amplification gives a large amount of amplified cDNA (>100 μg) from a single cell (2-pg mRNA), and that amount is enough for downstream analysis. The proposed global-amplification method was used to analyse transcript ratios of multiple cDNA targets (from several copies to several thousand copies) quantitatively. PMID:24141095

Dependence of displacement-length scaling relations for fractures and deformation bands on the volumetric changes across them

USGS Publications Warehouse

Schultz, R.A.; Soliva, R.; Fossen, H.; Okubo, C.H.; Reeves, D.M.

2008-01-01

Displacement-length data from faults, joints, veins, igneous dikes, shear deformation bands, and compaction bands define two groups. The first group, having a power-law scaling relation with a slope of n = 1 and therefore a linear dependence of maximum displacement and discontinuity length (Dmax = ??L), comprises faults and shear (non-compactional or non-dilational) deformation bands. These shearing-mode structures, having shearing strains that predominate over volumetric strains across them, grow under conditions of constant driving stress, with the magnitude of near-tip stress on the same order as the rock's yield strength in shear. The second group, having a power-law scaling relation with a slope of n = 0.5 and therefore a dependence of maximum displacement on the square root of discontinuity length (Dmax = ??L0.5), comprises joints, veins, igneous dikes, cataclastic deformation bands, and compaction bands. These opening- and closing-mode structures grow under conditions of constant fracture toughness, implying significant amplification of near-tip stress within a zone of small-scale yielding at the discontinuity tip. Volumetric changes accommodated by grain fragmentation, and thus control of propagation by the rock's fracture toughness, are associated with scaling of predominantly dilational and compactional structures with an exponent of n = 0.5. ?? 2008 Elsevier Ltd.

Self-Assembled DNA Tetrahedral Scaffolds for the Construction of Electrochemiluminescence Biosensor with Programmable DNA Cyclic Amplification.

PubMed

Feng, Qiu-Mei; Guo, Yue-Hua; Xu, Jing-Juan; Chen, Hong-Yuan

2017-05-24

A novel DNA tetrahedron-structured electrochemiluminescence (ECL) platform for bioanalysis with programmable DNA cyclic amplification was developed. In this work, glucose oxidase (GOD) was labeled to a DNA sequence (S) as functional conjugation (GOD-S), which could hybridize with other DNA sequences (L and P) to form GOD-S:L:P probe. In the presence of target DNA and a help DNA (A), the programmable DNA cyclic amplification was activated and released GOD-S via toehold-mediated strand displacement. Then, the obtained GOD-S was further immobilized on the DNA tetrahedral scaffolds with a pendant capture DNA and Ru(bpy) 3 2+ -conjugated silica nanoparticles (RuSi NPs) decorated on the electrode surface. Thus, the amount of GOD-S assembled on the electrode surface depended on the concentration of target DNA and GOD could catalyze glucose to generate H 2 O 2 in situ. The ECL signal of Ru(bpy) 3 2+ -TPrA system was quenched by the presence of H 2 O 2 . By integrating the programmable DNA cyclic amplification and in situ generating H 2 O 2 as Ru(bpy) 3 2+ ECL quencher, a sensitive DNA tetrahedron-structured ECL sensing platform was proposed for DNA detection. Under optimized conditions, this biosensor showed a wide linear range from 100 aM to 10 pM with a detection limit of 40 aM, indicating a promising application in DNA analysis. Furthermore, by labeling GOD to different recognition elements, the proposed strategy could be used for the detection of various targets. Thus, this programmable cascade amplification strategy not only retains the high selectivity and good capturing efficiency of tetrahedral-decorated electrode surface but also provides potential applications in the construction of ECL biosensor.

Long-period amplification in deep alluvial basins and consequences for site-specific probabilistic seismic-hazard: the case of Castelleone in the Po Plain (Northern Italy)

NASA Astrophysics Data System (ADS)

Barani, S.; Mascandola, C.; Massa, M.; Spallarossa, D.

2017-12-01

The recent Emilia seismic sequence (Northern Italy) occurred at the end of the first half of 2012 with main shock of Mw6.1 highlighted the importance of studying site effects in the Po Plain, the larger and deeper sedimentary basin in Italy. As has long been known, long-period amplification related to deep sedimentary basins can significantly affect the characteristics of the ground-motion induced by strong earthquakes. It follows that the effects of deep sedimentary deposits on ground shaking require special attention during the definition of the design seismic action. The work presented here analyzes the impact of deep-soil discontinuities on ground-motion amplification, with particular focus on long-period probabilistic seismic-hazard assessment. The study focuses on the site of Castelleone, where a seismic station of the Italian National Seismic Network has been recording since 2009. Our study includes both experimental and numerical site response analyses. Specifically, extensive active and passive geophysical measurements were carried out in order to define a detailed shear-wave velocity (VS) model to be used in the numerical analyses. These latter are needed to assess the site-specific ground-motion hazard. Besides classical seismic refraction profiles and multichannel analysis of surface waves, we analyzed ambient vibration measurements in both single and array configurations. The VS profile was determined via joint inversion of the experimental phase-velocity dispersion curve with the ellipticity curve derived from horizontal-to-vertical spectral ratios. The profile shows two main discontinuities at depths of around 160 and 1350 m, respectively. The probabilistic site-specific hazard was assessed in terms of both spectral acceleration and displacement. A partially non-ergodic approach was adopted. We have found that the spectral acceleration hazard is barely sensitive to long-period (up to 10 s) amplification related to the deeper discontinuity whereas the displacement hazard is strongly affected. Our results show that neglecting the effects of the deeper discontinuity implies an underestimation of the hazard of up to about 49% for a mean return period (MRP) of 475 years and 57% for an MRP of 2475 years, with possible consequences on the design of very tall buildings and large bridges.

Variability of site response in Seattle, Washington

USGS Publications Warehouse

Hartzell, S.; Carver, D.; Cranswick, E.; Frankel, A.

2000-01-01

Ground motion from local earthquakes and the SHIPS (Seismic Hazards Investigation in Puget Sound) experiment is used to estimate site amplification factors in Seattle. Earthquake and SHIPS records are analyzed by two methods: (1) spectral ratios relative to a nearby site on Tertiary sandstone, and (2) a source/site spectral inversion technique. Our results show site amplifications between 3 and 4 below 5 Hz for West Seattle relative to Tertiary rock. These values are approximately 30% lower than amplification in the Duwamish Valley on artificial fill, but significantly higher than the calculated range of 2 to 2.5 below 5 Hz for the till-covered hills east of downtown Seattle. Although spectral amplitudes are only 30% higher in the Duwamish Valley compared to West Seattle, the duration of long-period ground motion is significantly greater on the artificial fill sites. Using a three-dimensional displacement response spectrum measure that includes the effects of ground-motion duration, values in the Duwamish Valley are 2 to 3 times greater than West Seattle. These calculations and estimates of site response as a function of receiver azimuth point out the importance of trapped surface-wave energy within the shallow, low-velocity, sedimentary layers of the Duwamish Valley. One-dimensional velocity models yield spectral amplification factors close to the observations for till sites east of downtown Seattle and the Duwamish Valley, but underpredict amplifications by a factor of 2 in West Seattle. A two-dimensional finite-difference model does equally well for the till sites and the Duwamish Valley and also yields duration estimates consistent with the observations for the Duwamish Valley. The two-dimensional model, however, still underpredicts amplification in West Seattle by up to a factor of 2. This discrepancy is attributed to 3D effects, including basin-edge-induced surface waves and basin-geometry-focusing effects, caused by the proximity of the Seattle thrust fault and the sediment-filled Seattle basin.

Actuatable capacitive transducer for quantitative nanoindentation combined with transmission electron microscopy

DOEpatents

Warren, Oden L.; Asif, S. A. Syed; Cyrankowski, Edward; Kounev, Kalin

2010-09-21

An actuatable capacitive transducer including a transducer body, a first capacitor including a displaceable electrode and electrically configured as an electrostatic actuator, and a second capacitor including a displaceable electrode and electrically configured as a capacitive displacement sensor, wherein the second capacitor comprises a multi-plate capacitor. The actuatable capacitive transducer further includes a coupling shaft configured to mechanically couple the displaceable electrode of the first capacitor to the displaceable electrode of the second capacitor to form a displaceable electrode unit which is displaceable relative to the transducer body, and an electrically-conductive indenter mechanically coupled to the coupling shaft so as to be displaceable in unison with the displaceable electrode unit.-

Actuatable capacitive transducer for quantitative nanoindentation combined with transmission electron microscopy

DOEpatents

Warren, Oden L; Asif, Syed Amanula Syed; Cyrankowski, Edward; Kounev, Kalin

2013-06-04

An actuatable capacitive transducer including a transducer body, a first capacitor including a displaceable electrode and electrically configured as an electrostatic actuator, and a second capacitor including a displaceable electrode and electrically configured as a capacitive displacement sensor, wherein the second capacitor comprises a multi-plate capacitor. The actuatable capacitive transducer further includes a coupling shaft configured to mechanically couple the displaceable electrode of the first capacitor to the displaceable electrode of the second capacitor to form a displaceable electrode unit which is displaceable relative to the transducer body, and an electrically-conductive indenter mechanically coupled to the coupling shaft so as to be displaceable in unison with the displaceable electrode unit.

A label-free colorimetric isothermal cascade amplification for the detection of disease-related nucleic acids based on double-hairpin molecular beacon.

PubMed

Wu, Dong; Xu, Huo; Shi, Haimei; Li, Weihong; Sun, Mengze; Wu, Zai-Sheng

2017-03-08

K-Ras mutations at codon 12 play an important role in an early step of carcinogenesis. Here, a label-free colorimetric isothermal cascade amplification for ultrasensitive and specific detection of K-Ras point mutation is developed based on a double-hairpin molecular beacon (DHMB). The biosensor consists of DHMB probe and a primer-incorporated polymerization template (PPT) designed partly complementary to DHMB. In the presence of polymerase, target DNA is designed to trigger strand displacement amplification (SDA) via promote the hybridization of PPT with DHMB and subsequently initiates cascade amplification process with the help of the nicking endonuclease. During the hybridization and enzymatic reaction, G-quadruplex/hemin DNAzymes are generated, catalyzing the oxidation of ABTS 2- by H 2 O 2 in the presence of hemin. Utilizing the proposed facile colorimetric scheme, the target DNA can be quantified down to 4 pM with the dynamic response range of 5 orders of magnitude, indicating the substantially improved detection capability. Even more strikingly, point mutation in K-ras gene can be readily observed by the naked eye without the need for the labeling or expensive equipment. Given the high-performance for K-Ras analysis, the enhanced signal transduction capability associated with double-hairpin structure of DHMB provides a novel rout to screen biomarkers, and the descripted colorimetric biosensor seems to hold great promise for diagnostic applications of genetic diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparing Newmark

NASA Astrophysics Data System (ADS)

Rodríguez-Peces, M. J.; García-Mayordomo, J.; Azañón-Hernández, J. M.; Jabaloy-Sánchez, A.

2009-04-01

The Lorca Basin (Eastern Betic Cordillera, SE Spain) is one of the most seismically active regions of Spain. In this area there are well known cases of earthquake-induced slope instabilities associated to specific earthquakes (e.g., Bullas 2002, La Paca 2005). Furthermore, this area is characterized by moderate magnitude seismicity which mainly produces rock-falls and avalanches. In this work we present the results of our research at regional and site scales. For the regional scale, we have used a geographic information system (GIS) to develop an implementation of the Newmark's sliding rigid block method. We have particularly proposed a new variation of Newmark's method to consider soil and topographic amplification effects. Subsequently, we produced "Newmark displacement" maps for both probabilistic and deterministic seismic scenarios in the Lorca Basin. Probabilistic seismic scenarios consider three hazard maps in terms of peak ground acceleration (PGA) on rock corresponding to the 475-, 975- and 2475-year return periods (exceedance probability of 10, 5 and 2% in 50 years, respectively) in the Murcia Region. Deterministic seismic scenarios consider the occurrence of the most probable earthquake for a 475-year return period (Mw=5.0) at every location, or either a complete rupture of Lorca-Totana (Mw=6.7) or Puerto Lumbreras-Lorca (Mw=6.8) segments of Alhama de Murcia Fault. The Newmark displacement maps allowed us to identify areas with the highest potential seismic hazard, and also locate areas for future particular studies. We have found that rock-falls produced during the last earthquakes in Lorca Basin (e.g., Bullas 2002, La Paca 2005) match very well with areas with values of Newmark displacement lower than 2 cm in all the seismic scenarios considered. Therefore, it seems that low values of Newmark displacements are very likely associated with rock-falls. To support this hypothesis we have applied the Newmark method at a site scale. To do this, we have selected La Paca rock-fall which was generated during La Paca 2005 earthquake (mbLg=4.7, IEMS=VI-VII). We have used a terrestrial laser scanner in order to obtain a high resolution digital elevation model of La Paca rock-fall area. Moreover, we have performed a back-analysis based on field data to estimate the static safety factor previous to the earthquake and the critical acceleration. Furthermore, we have selected a representative strong ground motion record for La Paca earthquake from international databases. The critical acceleration and the peak ground acceleration values obtained from the strong ground motion record allowed us to estimate the actual soil and topographic amplification effects. Finally, we have calculated analytically the real Newmark displacement at La Paca rock-fall and we have compared this displacement with our GIS estimation in order to improve the calibration of Newmark's method at the regional scale.

Multi-link laser interferometry architecture for interspacecraft displacement metrology

NASA Astrophysics Data System (ADS)

Francis, Samuel P.; Lam, Timothy T.-Y.; McClelland, David E.; Shaddock, Daniel A.

2018-03-01

Targeting a future Gravity Recovery and Climate Experiment (GRACE) mission, we present a new laser interferometry architecture that can be used to recover the displacement between two spacecraft from multiple interspacecraft measurements. We show it is possible to recover the displacement between the spacecraft centers of mass in post-processing by forming linear combinations of multiple, spatially offset, interspacecraft measurements. By canceling measurement error due to angular misalignment of the spacecraft, we remove the need for precise placement or alignment of the interferometer, potentially simplifying spacecraft integration. To realize this multi-link architecture, we propose an all-fiber interferometer, removing the need for any ultrastable optical components such as the GRACE Follow-On mission's triple mirror assembly. Using digitally enhanced heterodyne interferometry, the number of links is readily scalable, adding redundancy to our measurement. We present the concept, an example multi-link implementation and the signal processing required to recover the center of mass displacement from multiple link measurements. Finally, in a simulation, we analyze the limiting noise sources in a 9 link interferometer and ultimately show we can recover the 80 {nm}/√{ {Hz}} displacement sensitivity required by the GRACE Follow-On laser ranging interferometer.

Label-free genotyping of cytochrome P450 2D6*10 using ligation-mediated strand displacement amplification with DNAzyme-based chemiluminescence detection.

PubMed

Wang, Hong-Qi; Wu, Zhan; Zhang, Yan; Tang, Li-Juan; Yu, Ru-Qin; Jiang, Jian-Hui

2012-01-13

Genotyping of cytochrome P450 monooxygenase 2D6*10 (CYP2D6*10) plays an important role in pharmacogenomics, especially in clinical drug therapy of Asian populations. This work reported a novel label-free technique for genotyping of CYP2D6*10 based on ligation-mediated strand displacement amplification (SDA) with DNAzyme-based chemiluminescence detection. Discrimination of single-base mismatch is firstly accomplished using DNA ligase to generate a ligation product. The ligated product then initiates a SDA reaction to produce aptamer sequences against hemin, which can be probed by chemiluminescence detection. The proposed strategy is used for the assay of CYP2D6*10 target and the genomic DNA. The results reveal that the proposed technique displays chemiluminescence responses in linear correlation to the concentrations of DNA target within the range from 1 pM to 1 nM. A detection limit of 0.1 pM and a signal-to-background ratio of 57 are achieved. Besides such high sensitivity, the proposed CYP2D6*10 genotyping strategy also offers superb selectivity, great robustness, low cost and simplified operations due to its label-free, homogeneous, and chemiluminescence-based detection format. These advantages suggest this technique may hold considerable potential for clinical CYP2D6*10 genotyping and association studies. Copyright © 2011 Elsevier B.V. All rights reserved.

Extending interferometric synthetic aperture radar measurements from one to two dimensions

NASA Astrophysics Data System (ADS)

Bechor, Noah

Interferometric synthetic aperture radar (InSAR), a very effective technique for measuring crustal deformation, provides measurements in only one dimension, along the radar line of sight. Imaging radar measurements from satellite-based systems are sensitive to both vertical and across-track displacements, but insensitive to along-track displacement. Multiple observations can resolve the first two components, but the along-track component remains elusive. The best existing method to obtain the along-track displacement involves pixel-level azimuth cross-correlation. The measurements are quite coarse (typically 15 cm precision), and they require large computation times. In contrast, across-track and vertical InSAR measurements can reach centimeter-level precision and are readily derived. We present a new method to extract along-track displacements from InSAR data. The new method, multiple aperture InSAR (MAI), is based on split-beam processing of InSAR data to create forward- and backward-looking interferograms. The phase difference between the two modified interferograms provides the along-track displacement component. Thus, from each conventional InSAR pair we extract two components of the displacement vector: one along the line of sight, the other in the along-track direction. Multiple MAI observations, either at two look angles or from the ascending and descending radar passes, then yield the three-dimensional displacement field. We analyze precision of our method by comparing our solution to GPS and offset-derived along-track displacements from interferograms of the M7.1 1999, Hector Mine earthquake. The RMS error between GPS displacements and our results ranges from 5 to 8.8cm. Our method is consistent with along-track displacements derived by pixel-offsets, themselves limited to 12-15cm precision. The theoretical MAI precision depends on SNR and coherence. For SNR=100 the expected precision is 3, 11cm for coherence of 0.8, 0.4, respectively. Finally, we evaluate how the new measurements improve the determination of the earthquake coseismic slip distribution by comparison of models derived from multiple data types. We find that MAI data help constrain the southern portion of the lip distribution, by adding information where GPS data are sparse and the deformation is below the azimuth pixel-offsets detection threshold.

Small-displacement sensing system based on multiple total internal reflections in heterodyne interferometry.

PubMed

Wang, Shinn-Fwu; Chiu, Ming-Hung; Chen, Wei-Wu; Kao, Fu-Hsi; Chang, Rong-Seng

2009-05-01

A small-displacement sensing system based on multiple total internal reflections in heterodyne interferometry is proposed. In this paper, a small displacement can be obtained only by measuring the variation in phase difference between s- and p-polarization states for the total internal reflection effect. In order to improve the sensitivity, we increase the number of total internal reflections by using a parallelogram prism. The theoretical resolution of the method is better than 0.417 nm. The method has some merits, e.g., high resolution, high sensitivity, and real-time measurement. Also, its feasibility is demonstrated.

Job Displacement and Social Participation over the Lifecourse: Findings for a Cohort of Joiners

PubMed Central

Brand, Jennie E.; Burgard, Sarah A.

2010-01-01

We examine the effects of job displacement, an involuntary event associated with socioeconomic and psychological decline, on social participation. Using more than 45 years of panel data from the Wisconsin Longitudinal Study, we find that job displacement is associated with significant, long-term lower probabilities of subsequent involvement with various forms of social participation for workers displaced during their prime earnings years; displacement is not associated with lower probabilities of involvement for workers displaced in the years approaching retirement. We also find that post-displacement socioeconomic and psychological decline explain very little of the negative effect of job displacement on social participation, and that a single displacement event, rather than a series of multiple displacement events, is most strongly associated with lower probabilities of social involvement. PMID:20827387

Damage identification in beams using speckle shearography and an optimal spatial sampling

NASA Astrophysics Data System (ADS)

Mininni, M.; Gabriele, S.; Lopes, H.; Araújo dos Santos, J. V.

2016-10-01

Over the years, the derivatives of modal displacement and rotation fields have been used to localize damage in beams. Usually, the derivatives are computed by applying finite differences. The finite differences propagate and amplify the errors that exist in real measurements, and thus, it is necessary to minimize this problem in order to get reliable damage localizations. A way to decrease the propagation and amplification of the errors is to select an optimal spatial sampling. This paper presents a technique where an optimal spatial sampling of modal rotation fields is computed and used to obtain the modal curvatures. Experimental measurements of modal rotation fields of a beam with single and multiple damages are obtained with shearography, which is an optical technique allowing the measurement of full-fields. These measurements are used to test the validity of the optimal sampling technique for the improvement of damage localization in real structures. An investigation on the ability of a model updating technique to quantify the damage is also reported. The model updating technique is defined by the variations of measured natural frequencies and measured modal rotations and aims at calibrating the values of the second moment of area in the damaged areas, which were previously localized.

Homogeneous Entropy-Driven Amplified Detection of Biomolecular Interactions.

PubMed

Kim, Donghyuk; Garner, Omai B; Ozcan, Aydogan; Di Carlo, Dino

2016-08-23

While a range of artificial biochemical circuits is likely to play a significant role in biological engineering, one of the challenges in the field is the design of circuits that can transduce between biomolecule classes (e.g., moving beyond nucleic acid only circuits). Herein, we design a transduction mechanism whereby a protein signal is transduced into an amplified nucleic acid output using DNA nanotechnology. In this system, a protein is recognized by nucleic acid bound recognition elements to form a catalytic complex that drives a hybridization/displacement reaction on a multicomponent nucleic acid substrate, releasing multiple target single-stranded oligonucleotides in an amplified fashion. Amplification power and simple one-pot reaction conditions lead us to apply the scheme in an assay format, achieving homogeneous and rapid (∼10 min) analyte detection that is also robust (operable in whole blood and plasma). In addition, we demonstrate the assay in a microfluidic digital assay format leading to improved quantification and sensitivity approaching single-molecule levels. The present scheme we believe will have a significant impact on a range of applications from fundamental molecular interaction studies to design of artificial circuits in vivo to high-throughput, multiplexed assays for screening or point-of-care diagnostics.

Rhombic micro-displacement amplifier for piezoelectric actuator and its linear and hybrid model

NASA Astrophysics Data System (ADS)

Chen, Jinglong; Zhang, Chunlin; Xu, Minglong; Zi, Yanyang; Zhang, Xinong

2015-01-01

This paper proposes rhombic micro-displacement amplifier (RMDA) for piezoelectric actuator (PA). First, the geometric amplification relations are analyzed and linear model is built to analyze the mechanical and electrical properties of this amplifier. Next, the accurate modeling method of amplifier is studied for important application of precise servo control. The classical Preisach model (CPM) is generally implemented using a numerical technique based on the first-order reversal curves (FORCs). The accuracy of CPM mainly depends on the number of FORCs. However, it is generally difficult to achieve enough number of FORCs in practice. So, Support Vector Machine (SVM) is employed in the work to circumvent the deficiency of the CPM. Then the hybrid model, which is based on discrete CPM and SVM is developed to account for hysteresis and dynamic effects. Finally, experimental validation is carried out. The analyzed result shows that this amplifier with the hybrid model is suitable for control application.

Control of helicopter rotorblade aerodynamics

NASA Technical Reports Server (NTRS)

Fabunmi, James A.

1991-01-01

The results of a feasibility study of a method for controlling the aerodynamics of helicopter rotorblades using stacks of piezoelectric ceramic plates are presented. A resonant mechanism is proposed for the amplification of the displacements produced by the stack. This motion is then converted into linear displacement for the actuation of the servoflap of the blades. A design which emulates the actuation of the servoflap on the Kaman SH-2F is used to demonstrate the fact that such a system can be designed to produce the necessary forces and velocities needed to control the aerodynamics of the rotorblades of such a helicopter. Estimates of the electrical power requirements are also presented. A Small Business Innovation Research (SBIR) Phase 2 Program is suggested, whereby a bench-top prototype of the device can be built and tested. A collaborative effort between AEDAR Corporation and Kaman Aerospace Corporation is anticipated for future effort on this project.

Target-triggering multiple-cycle signal amplification strategy for ultrasensitive detection of DNA based on QCM and SPR.

PubMed

Song, Weiling; Yin, Wenshuo; Sun, Wenbo; Guo, Xiaoyan; He, Peng; Yang, Xiaoyan; Zhang, Xiaoru

2018-04-24

Detection of ultralow concentrations of nucleic acid sequences is a central challenge in the early diagnosis of genetic diseases. Herein, we developed a target-triggering cascade multiple cycle amplification for ultrasensitive DNA detection using quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). It was based on the exonuclease Ⅲ (Exo Ⅲ)-assisted signal amplification and the hybridization chain reaction (HCR). The streptavidin-coated Au-NPs (Au-NPs-SA) were assembled on the HCR products as recognition element. Upon sensing of target DNA, the duplex DNA probe triggered the Exo Ⅲ cleavage process, accompanied by generating a new secondary target DNA and releasing target DNA. The released target DNA and the secondary target DNA were recycled. Simultaneously, numerous single strands were liberated and acted as the trigger of HCR to generate further signal amplification, resulting in the immobilization of abundant Au-NPs-SA on the gold substrate. The QCM sensor results were found to be comparable to that achieved using a SPR sensor platform. This method exhibited a high sensitivity toward target DNA with a detection limit of 0.70 fM. The high sensitivity and specificity make this method a great potential for detecting DNA with trace amounts in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier Inc. All rights reserved.

Thermally multiplexed polymerase chain reaction.

PubMed

Phaneuf, Christopher R; Pak, Nikita; Saunders, D Curtis; Holst, Gregory L; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L; Jerris, Robert; Forest, Craig R

2015-07-01

Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously-each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel.

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms.

PubMed

Wong, Y-P; Othman, S; Lau, Y-L; Radu, S; Chee, H-Y

2018-03-01

Loop-mediated isothermal amplification (LAMP) amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands. Following its first discovery by Notomi et al. ( Nucleic Acids Res 28: E63), LAMP was further developed over the years which involved the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification for the detection of infectious diseases caused by micro-organisms in humans, livestock and plants. In this review, available types of LAMP techniques will be discussed together with their applications in detection of various micro-organisms. Up to date, there are varieties of LAMP detection methods available including colorimetric and fluorescent detection, real-time monitoring using turbidity metre and detection using lateral flow device which will also be highlighted in this review. Apart from that, commercialization of LAMP technique had also been reported such as lyophilized form of LAMP reagents kit and LAMP primer sets for detection of pathogenic micro-organisms. On top of that, advantages and limitations of this molecular detection method are also described together with its future potential as a diagnostic method for infectious disease. © 2017 The Society for Applied Microbiology.

Invading stacking primer: A trigger for high-efficiency isothermal amplification reaction with superior selectivity for detecting microRNA variants.

PubMed

Liu, Weipeng; Zhu, Minjun; Liu, Hongxing; Wei, Jitao; Zhou, Xiaoming; Xing, Da

2016-07-15

Searching for a strategy to enhance the efficiency of nucleic acid amplification and achieve exquisite discrimination of nucleic acids at the single-base level for biological detection has become an exciting research direction in recent years. Here, we have developed a simple and universal primer design strategy which produces a fascinating effect on isothermal strand displacement amplification (iSDA). We refer to the resultant primer as "invading stacking primer (IS-Primer)" which is based on contiguous stacking hybridization and toehold-mediated exchange reaction and function by merely changing the hybridization location of the primer. Using the IS-Primer, the sensitivity in detecting the target miR-21 is improved approximately five fold compared with the traditional iSDA reaction. It was further demonstrated that the IS-Primer acts as an invading strand to initiate branch migration which can increase the efficiency of the untwisting of the hairpin probe. This effect is equivalent to reducing the free energy of the stem, and the technique shows superior selectivity for single-base mismatches. By demonstrating the enhanced effect of the IS-Primer in the iSDA reaction, this work may provide a potentially new avenue for developing more sensitive and selective nucleic acids assays. Copyright © 2016 Elsevier B.V. All rights reserved.

Deconstructing stem cell population heterogeneity: Single-cell analysis and modeling approaches

PubMed Central

Wu, Jincheng; Tzanakakis, Emmanuel S.

2014-01-01

Isogenic stem cell populations display cell-to-cell variations in a multitude of attributes including gene or protein expression, epigenetic state, morphology, proliferation and proclivity for differentiation. The origins of the observed heterogeneity and its roles in the maintenance of pluripotency and the lineage specification of stem cells remain unclear. Addressing pertinent questions will require the employment of single-cell analysis methods as traditional cell biochemical and biomolecular assays yield mostly population-average data. In addition to time-lapse microscopy and flow cytometry, recent advances in single-cell genomic, transcriptomic and proteomic profiling are reviewed. The application of multiple displacement amplification, next generation sequencing, mass cytometry and spectrometry to stem cell systems is expected to provide a wealth of information affording unprecedented levels of multiparametric characterization of cell ensembles under defined conditions promoting pluripotency or commitment. Establishing connections between single-cell analysis information and the observed phenotypes will also require suitable mathematical models. Stem cell self-renewal and differentiation are orchestrated by the coordinated regulation of subcellular, intercellular and niche-wide processes spanning multiple time scales. Here, we discuss different modeling approaches and challenges arising from their application to stem cell populations. Integrating single-cell analysis with computational methods will fill gaps in our knowledge about the functions of heterogeneity in stem cell physiology. This combination will also aid the rational design of efficient differentiation and reprogramming strategies as well as bioprocesses for the production of clinically valuable stem cell derivatives. PMID:24035899

Exploring Arabidopsis thaliana Root Endophytes via Single-Cell Genomics

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

Lundberg, Derek; Woyke, Tanja; Tringe, Susannah

2014-03-19

Land plants grow in association with microbial communities both on their surfaces and inside the plant (endophytes). The relationships between microbes and their host can vary from pathogenic to mutualistic. Colonization of the endophyte compartment occurs in the presence of a sophisticated plant immune system, implying finely tuned discrimination of pathogens from mutualists and commensals. Despite the importance of the microbiome to the plant, relatively little is known about the specific interactions between plants and microbes, especially in the case of endophytes. The vast majority of microbes have not been grown in the lab, and thus one of the fewmore » ways of studying them is by examining their DNA. Although metagenomics is a powerful tool for examining microbial communities, its application to endophyte samples is technically difficult due to the presence of large amounts of host plant DNA in the sample. One method to address these difficulties is single-cell genomics where a single microbial cell is isolated from a sample, lysed, and its genome amplified by multiple displacement amplification (MDA) to produce enough DNA for genome sequencing. This produces a single-cell amplified genome (SAG). We have applied this technology to study the endophytic microbes in Arabidopsis thaliana roots. Extensive 16S gene profiling of the microbial communities in the roots of multiple inbred A. thaliana strains has identified 164 OTUs as being significantly enriched in all the root endophyte samples compared to their presence in bulk soil.« less

Four photon parametric amplification. [in unbiased Josephson junction

NASA Technical Reports Server (NTRS)

Parrish, P. T.; Feldman, M. J.; Ohta, H.; Chiao, R. Y.

1974-01-01

An analysis is presented describing four-photon parametric amplification in an unbiased Josephson junction. Central to the theory is the model of the Josephson effect as a nonlinear inductance. Linear, small signal analysis is applied to the two-fluid model of the Josephson junction. The gain, gain-bandwidth product, high frequency limit, and effective noise temperature are calculated for a cavity reflection amplifier. The analysis is extended to multiple (series-connected) junctions and subharmonic pumping.

Fluorescent aptasensor for detection of four tetracycline veterinary drugs in milk based on catalytic hairpin assembly reaction and displacement of G-quadruplex.

PubMed

Zhou, Chen; Zou, Haimin; Sun, Chengjun; Ren, Dongxia; Xiong, Wei; Li, Yongxin

2018-05-01

Based on a novel signal amplification strategy by catalytic hairpin assembly and displacement of G-quadruplex DNA, an enzyme-free, non-label fluorescent aptasensing approach was established for sensitive detection of four tetracycline veterinary drugs in milk. The network consisted of a pair of partially complementary DNA hairpins (HP1 and HP2). The DNA aptamer of four tetracycline veterinary drugs was located at the sticky end of the HP1. The ring region of HP1 rich in G and C could form a stable G-quadruplex structure, which could emit specific fluorescence signal after binding with the fluorescent dye and N-methylmesoporphyrin IX (NMM). When presented in the system, the target analytes would be repeatedly used to trigger a recycling procedure between the hairpins, generating numerous HP1-HP2 duplex complexes and displacing G-quadruplex DNA. Thus, the sensitive detection of target analytes was achieved in a wide linear range (0-1000 μg/L) with the detection limit of 4.6 μg/L. Moreover, this proposed method showed high discrimination efficiency towards target analytes against other common mismatched veterinary drugs, and could be successfully applied to the analysis of milk samples. Graphical abstract Schematic of target analyte detection based on catalytic hairpin assembly reaction and displacement of G-quadruplex.

MEMS tactile display: from fabrication to characterization

NASA Astrophysics Data System (ADS)

Miki, Norihisa; Kosemura, Yumi; Watanabe, Junpei; Ishikawa, Hiroaki

2014-03-01

We report fabrication and characterization of MEMS-based tactile display that can display users various tactile information, such as Braille codes and surface textures. The display consists of 9 micro-actuators that are equipped with hydraulic displacement amplification mechanism (HDAM) to achieve large enough displacement to stimulate the human tactile receptors. HDAM encapsulates incompressible liquids. We developed a liquid encapsulation process, which we termed as Bonding-in-Liquid Technique, where bonding with a UV-curable resin in glycerin is conducted in the liquid, which prevented interfusion of air bubbles and deformation of the membrane during the bonding. HDAM successfully amplified the displacement generated by piezoelectric actuators by a factor of 6. The display could virtually produce "rough" and "smooth" surfaces, by controlling the vibration frequency, displacement, and the actuation periods of an actuator until the adjacent actuator was driven. We introduced a sample comparison method to characterize the surfaces, which involves human tactile sensation. First, we prepared samples whose mechanical properties are known. We displayed a surface texture to the user by controlling the parameters and then, the user selects a sample that has the most similar surface texture. By doing so, we can correlate the parameters with the mechanical properties of the sample as well as find the sets of the parameters that can provide similar tactile information to many users. The preliminary results with respect to roughness and hardness is presented.

Detection of HbsAg and hATIII genetically modified goats (Caprahircus) by loop-mediated isothermal amplification.

PubMed

Tao, Chenyu; Zhang, Qingde; Zhai, Shanli; Liu, Bang

2013-11-01

In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.

Entropy Beacon: A Hairpin-Free DNA Amplification Strategy for Efficient Detection of Nucleic Acids

PubMed Central

2015-01-01

Here, we propose an efficient strategy for enzyme- and hairpin-free nucleic acid detection called an entropy beacon (abbreviated as Ebeacon). Different from previously reported DNA hybridization/displacement-based strategies, Ebeacon is driven forward by increases in the entropy of the system, instead of free energy released from new base-pair formation. Ebeacon shows high sensitivity, with a detection limit of 5 pM target DNA in buffer and 50 pM in cellular homogenate. Ebeacon also benefits from the hairpin-free amplification strategy and zero-background, excellent thermostability from 20 °C to 50 °C, as well as good resistance to complex environments. In particular, based on the huge difference between the breathing rate of a single base pair and two adjacent base pairs, Ebeacon also shows high selectivity toward base mutations, such as substitution, insertion, and deletion and, therefore, is an efficient nucleic acid detection method, comparable to most reported enzyme-free strategies. PMID:26505212

Transmission of vibration across honeycombs and its detection by bee leg receptors

PubMed

Sandeman; Tautz; Lindauer

1996-01-01

Vibration of the rims of open cells in a honeycomb, applied in the plane of the comb face, is transmitted across the comb. Attenuation or amplification of the vibratory signal depends on its frequency and on the type of comb. In general, framed combs, both large and small, strongly attenuate higher frequencies, whereas these are amplified in small open combs. The very poor transmission properties of the large framed combs used in commercial hives may explain the bees' habit of freeing an area of comb from the frame in those areas used for dancing. Extracellular electrical recordings from the leg of a honeybee detect large action potentials from receptors that monitor extension of the tibia on the femur. Measurements of threshold displacement amplitudes show these receptors to be sensitive to low frequencies. The amplification properties of unframed combs extend the range of these receptor systems to include frequencies that are emitted by the bee during its dance, namely the 15 Hz abdomen waggle and 250 Hz thorax vibration.

Design of a Minimum Surface-Effect Tendon-Based Microactuator for Micromanipulation

NASA Technical Reports Server (NTRS)

Goldfarb, Michael; Lipsey, James H.

1997-01-01

A piezoelectric (PZT) stack-based actuator was developed to provide a means of actuation with dynamic characteristics appropriate for small-scale manipulation. In particular, the design incorporates a highly nonlinear, large-ratio transmission that provides approximately two orders of magnitude motion amplification from the PZT stack. In addition to motion amplification, the nonlinear transmission was designed via optimization methods to distort the highly non-uniform properties of a piezoelectric actuator so that the achievable actuation force is nearly constant throughout the actuator workspace. The package also includes sensors that independently measure actuator output force and displacement, so that a manipulator structure need not incorporate sensors nor the associated wires. Specifically, the actuator was designed to output a maximum force of at least one Newton through a stroke of at least one millimeter. For purposes of small-scale precision position and/or force control, the actuator/sensor package was designed to eliminate stick-slip friction and backlash. The overall dimensions of the actuator/sensor package are approximately 40 x 65 x 25 mm.

Signal-on electrochemiluminescence biosensor for microRNA-319a detection based on two-stage isothermal strand-displacement polymerase reaction.

PubMed

Wang, Minghui; Zhou, Yunlei; Yin, Huanshun; Jiang, Wenjing; Wang, Haiyan; Ai, Shiyun

2018-06-01

MicroRNAs play crucial role in regulating gene expression in organism, thus it is very necessary to exploit an efficient method for the sensitive and specific detection of microRNA. Herein, a signal-on electrochemiluminescence biosensor was fabricated for microRNA-319a detection based on two-stage isothermal strand-displacement polymerase reaction (ISDPR). In the presence of target microRNA, amounts of trigger DNA could be generated by the first ISDPR. Then, the trigger DNA and the primer hybridized simultaneously with the hairpin probe to open the stem of the probe, and then the ECL signal will be emitted. In the presence of phi29 DNA polymerase and dNTPs, the trigger DNA could be displaced to initiate a new cycle which was the second ISDPR. Due to the two-stage amplification, this method presented excellent detection sensitivity with a low detection limit of 0.14 fM. Moreover, the applicability of the developed method was demonstrated by detecting the change of microRNA-319a content in the leaves of rice seedlings after the rice seeds were incubated with chemical mutagen of ethyl methanesulfonate. Copyright © 2018 Elsevier B.V. All rights reserved.

Simple Approaches to Minimally-Instrumented, Microfluidic-Based Point-of-Care Nucleic Acid Amplification Tests

PubMed Central

Mauk, Michael G.; Song, Jinzhao; Liu, Changchun; Bau, Haim H.

2018-01-01

Designs and applications of microfluidics-based devices for molecular diagnostics (Nucleic Acid Amplification Tests, NAATs) in infectious disease testing are reviewed, with emphasis on minimally instrumented, point-of-care (POC) tests for resource-limited settings. Microfluidic cartridges (‘chips’) that combine solid-phase nucleic acid extraction; isothermal enzymatic nucleic acid amplification; pre-stored, paraffin-encapsulated lyophilized reagents; and real-time or endpoint optical detection are described. These chips can be used with a companion module for separating plasma from blood through a combined sedimentation-filtration effect. Three reporter types: Fluorescence, colorimetric dyes, and bioluminescence; and a new paradigm for end-point detection based on a diffusion-reaction column are compared. Multiplexing (parallel amplification and detection of multiple targets) is demonstrated. Low-cost detection and added functionality (data analysis, control, communication) can be realized using a cellphone platform with the chip. Some related and similar-purposed approaches by others are surveyed. PMID:29495424

Displacement Theories for In-Flight Deformed Shape Predictions of Aerospace Structures

NASA Technical Reports Server (NTRS)

Ko, William L.; Richards, W. L.; Tran, Van t.

2007-01-01

Displacement theories are developed for a variety of structures with the goal of providing real-time shape predictions for aerospace vehicles during flight. These theories are initially developed for a cantilever beam to predict the deformed shapes of the Helios flying wing. The main structural configuration of the Helios wing is a cantilever wing tubular spar subjected to bending, torsion, and combined bending and torsion loading. The displacement equations that are formulated are expressed in terms of strains measured at multiple sensing stations equally spaced on the surface of the wing spar. Displacement theories for other structures, such as tapered cantilever beams, two-point supported beams, wing boxes, and plates also are developed. The accuracy of the displacement theories is successfully validated by finite-element analysis and classical beam theory using input-strains generated by finite-element analysis. The displacement equations and associated strain-sensing system (such as fiber optic sensors) create a powerful means for in-flight deformation monitoring of aerospace structures. This method serves multiple purposes for structural shape sensing, loads monitoring, and structural health monitoring. Ultimately, the calculated displacement data can be visually displayed to the ground-based pilot or used as input to the control system to actively control the shape of structures during flight.

Strong nonlinear dependence of the spectral amplification factors of deep Vrancea earthquakes magnitude

NASA Astrophysics Data System (ADS)

Marmureanu, Gheorghe; Ortanza Cioflan, Carmen; Marmureanu, Alexandru

2010-05-01

Nonlinear effects in ground motion during large earthquakes have long been a controversial issue between seismologists and geotechnical engineers. Aki wrote in 1993:"Nonlinear amplification at sediments sites appears to be more pervasive than seismologists used to think…Any attempt at seismic zonation must take into account the local site condition and this nonlinear amplification( Local site effects on weak and strong ground motion, Tectonophysics,218,93-111). In other words, the seismological detection of the nonlinear site effects requires a simultaneous understanding of the effects of earthquake source, propagation path and local geological site conditions. The difficulty for seismologists in demonstrating the nonlinear site effects has been due to the effect being overshadowed by the overall patterns of shock generation and path propagation. The researchers from National Institute for Earth Physics ,in order to make quantitative evidence of large nonlinear effects, introduced the spectral amplification factor (SAF) as ratio between maximum spectral absolute acceleration (Sa), relative velocity (Sv) , relative displacement (Sd) from response spectra for a fraction of critical damping at fundamental period and peak values of acceleration(a-max),velocity (v-max) and displacement (d-max),respectively, from processed strong motion record and pointed out that there is a strong nonlinear dependence on earthquake magnitude and site conditions.The spectral amplification factors(SAF) are finally computed for absolute accelerations at 5% fraction of critical damping (β=5%) in five seismic stations: Bucharest-INCERC(soft soils, quaternary layers with a total thickness of 800 m);Bucharest-Magurele (dense sand and loess on 350m); Cernavoda Nuclear Power Plant site (marl, loess, limestone on 270 m) Bacau(gravel and loess on 20m) and Iassy (loess, sand, clay, gravel on 60 m) for last strong and deep Vrancea earthquakes: March 4,1977 (MGR =7.2 and h=95 km);August 30,1986(MGR =7.0 and h=130 km);May 30,1990 (MGR =6.7 and h=90 km) and May 31,1990 (MGR =6.1 and h=87 km). With a view to understand the characteristics of nonlinear soil behavior and the nonlinearity in the seismology and the influence to hazard and risk assessment ,this study examined the ways that nonlinearity would expected to appear on strong motion records made on Romania territory during to last Vrancea earthquake. The effect on nonlinearity is very large. For example, if we maintain the same amplification factor (SAF=5.8942) as for relatively strong earthquake on May 31,1990 with magnitude Ms =6,1 then at Bacau seismic station for earthquake on May 30,1990 (MGR =6.7) the peak acceleration has to be a*max =0.154g( +14.16%) and the actual recorded was only, a max =0.135g. Also, for Vrancea earthquake on August 30,1986, the peak acceleration has to be a*max=0.107g (+45,57%), instead of real value of 0.0736 g recorded at Bacau seismic station. More, the spectral amplification factors(SAF) are function of earthquake magnitude and there is a strong nonlinear dependence of the SAF of earthquake magnitude. The median values of SAF of the last strong Vrancea earthquakes for damping 5% are: 4.16; 3.63 and 3.26 corresponding to May 31,1990 Vrancea earthquake (Ms=6.1),May 30,1990 Vrancea earthquake(Ms=6.7),respectively, August 30,1986 Vrancea one(Ms=7.0). At the same seismic station, for example at Bacau, for 5% damping, SAF for accelerations is 5.22 for May 31,1990 earthquake (Ms =6.1);4.32 for May 30,1990 earthquake (Ms =6.7) and 3,94 for August 30,1986 one (Ms=7.0) etc. Finally, it will be made a comment in connection to U.S. Atomic Energy Commission-Regulatory Guide 1.60 on "Design Response Spectra for seismic design of nuclear power plants " to see spectral amplification factors for deep Vrancea earthquakes are larger and different.

Multiplex Allele-Specific Amplification from Whole Blood for Detecting Multiple Polymorphisms Simultaneously

PubMed Central

Zhu, Jianjie; Chen, Lanxin; Mao, Yong; Zhou, Huan

2013-01-01

Allele-specific amplification on the basis of polymerase chain reaction (PCR) has been widely used for single-nucleotide polymorphism (SNP) genotyping. However, the extraction of PCR-compatible genomic DNA from whole blood is usually required. This process is complicated and tedious, and is prone to cause cross-contamination between samples. To facilitate direct PCR amplification from whole blood without the extraction of genomic DNA, we optimized the pH value of PCR solution and the concentrations of magnesium ions and facilitator glycerol. Then, we developed multiplex allele-specific amplifications from whole blood and applied them to a case–control study. In this study, we successfully established triplex, five-plex, and eight-plex allele-specific amplifications from whole blood for determining the distribution of genotypes and alleles of 14 polymorphisms in 97 gastric cancer patients and 141 healthy controls. Statistical analysis results showed significant association of SNPs rs9344, rs1799931, and rs1800629 with the risk of gastric cancer. This method is accurate, time-saving, cost-effective, and easy-to-do, especially suitable for clinical prediction of disease susceptibility. PMID:23072573

Development of an Improved Mammalian Overexpression Method for Human CD62L

PubMed Central

Brown, Haley A.; Roth, Gwynne; Holzapfel, Genevieve; Shen, Sarek; Rahbari, Kate; Ireland, Joanna; Zou, Zhongcheng; Sun, Peter D.

2014-01-01

We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5 to 30 mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4–6 months to produce. To shorten the construction time, we replaced the muti-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2 months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ~5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines. PMID:25286402

Dual signal amplification for highly sensitive electrochemical detection of uropathogens via enzyme-based catalytic target recycling.

PubMed

Su, Jiao; Zhang, Haijie; Jiang, Bingying; Zheng, Huzhi; Chai, Yaqin; Yuan, Ruo; Xiang, Yun

2011-11-15

We report an ultrasensitive electrochemical approach for the detection of uropathogen sequence-specific DNA target. The sensing strategy involves a dual signal amplification process, which combines the signal enhancement by the enzymatic target recycling technique with the sensitivity improvement by the quantum dot (QD) layer-by-layer (LBL) assembled labels. The enzyme-based catalytic target DNA recycling process results in the use of each target DNA sequence for multiple times and leads to direct amplification of the analytical signal. Moreover, the LBL assembled QD labels can further enhance the sensitivity of the sensing system. The coupling of these two effective signal amplification strategies thus leads to low femtomolar (5fM) detection of the target DNA sequences. The proposed strategy also shows excellent discrimination between the target DNA and the single-base mismatch sequences. The advantageous intrinsic sequence-independent property of exonuclease III over other sequence-dependent enzymes makes our new dual signal amplification system a general sensing platform for monitoring ultralow level of various types of target DNA sequences. Copyright © 2011 Elsevier B.V. All rights reserved.

Detection and signal amplification in zebrafish RNA FISH.

PubMed

Hauptmann, Giselbert; Lauter, Gilbert; Söll, Iris

2016-04-01

In situ hybridization (ISH) has become an invaluable tool for the detection of RNA in cells, tissues and organisms. Due to improvements in target and signal amplification and in probe design remarkable progress has been made concerning sensitivity, specificity and resolution of chromogenic and fluorescent ISH (FISH). These advancements allow for exquisite cellular and sub-cellular resolution and for detecting multiple RNA species at a time by multiplexing. In zebrafish (F)ISH non-enzymatic and enzymatic amplification systems have been employed to obtain enhanced signal intensities and signal-to-noise ratios. These amplification strategies include branched DNA-based RNAscope and in situ hybridization chain reaction (HCR) techniques, as well as alkaline phosphatase (AP)- and horseradish peroxidase (PO)-based immunoassays. For practical application, we provide proven multiplex FISH protocols for AP- and PO-based visualization of mRNAs at high resolution. The protocols take advantage of optimized tyramide signal amplification (TSA) conditions of the PO assay and long-lasting high signal-to-noise ratio of the AP reaction, thereby enabling detection of less abundant transcripts. Copyright © 2016 Elsevier Inc. All rights reserved.

Graphene Ambipolar Nanoelectronics for High Noise Rejection Amplification.

PubMed

Liu, Che-Hung; Chen, Qi; Liu, Chang-Hua; Zhong, Zhaohui

2016-02-10

In a modern wireless communication system, signal amplification is critical for overcoming losses during multiple data transformations/processes and long-distance transmission. Common mode and differential mode are two fundamental amplification mechanisms, and they utilize totally different circuit configurations. In this paper, we report a new type of dual-gate graphene ambipolar device with capability of operating under both common and differential modes to realize signal amplification. The signal goes through two stages of modulation where the phase of signal can be individually modulated to be either in-phase or out-of-phase at two stages by exploiting the ambipolarity of graphene. As a result, both common and differential mode amplifications can be achieved within one single device, which is not possible in the conventional circuit configuration. In addition, a common-mode rejection ratio as high as 80 dB can be achieved, making it possible for low noise circuit application. These results open up new directions of graphene-based ambipolar electronics that greatly simplify the RF circuit complexity and the design of multifunction device operation.

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

PubMed Central

Richards-Kortum, Rebecca

2015-01-01

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest. PMID:25867513

Development of a quantitative recombinase polymerase amplification assay with an internal positive control.

PubMed

Crannell, Zachary A; Rohrman, Brittany; Richards-Kortum, Rebecca

2015-03-30

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

The μ-RWELL: A compact, spark protected, single amplification-stage MPGD

NASA Astrophysics Data System (ADS)

Poli Lener, M.; Bencivenni, G.; de Olivera, R.; Felici, G.; Franchino, S.; Gatta, M.; Maggi, M.; Morello, G.; Sharma, A.

2016-07-01

In this work we present two innovative architectures of resistive MPGDs based on the WELL-amplification concept: - the micro-Resistive WELL (μ-RWELL) is a compact spark-protected single amplification-stage Micro-Pattern Gas Detector (MPGD). The amplification stage, realized with a structure very similar to a GEM foil (called WELL), is embedded through a resistive layer in the readout board. A cathode electrode, defining the gas conversion/drift gap, completes the detector mechanics. The new architecture, showing an excellent space resolution, 50 μm, is a very compact device, robust against discharges and exhibiting a large gain (>104), simple to construct and easy for engineering and then suitable for large area tracking devices as well as digital calorimeters. - the Fast Timing Micro-pattern (FTM): a new device with an architecture based on a stack of several coupled full-resistive layers where drift and multiplication stages (WELL type) alternate in the structure. The signals from each multiplication stage can be read out from any external readout boards through the capacitive couplings, providing a signal with a gain of 104-105. The main advantage of this new device is the improvement of the timing provided by the competition of the ionization processes in the different drift regions, which can be exploited for fast timing at the high luminosity accelerators (e.g. HL-LHC upgrade) as well as for applications like medical imaging.

Host-Guest Recognition-Assisted Electrochemical Release: Its Reusable Sensing Application Based on DNA Cross Configuration-Fueled Target Cycling and Strand Displacement Reaction Amplification.

PubMed

Chang, Yuanyuan; Zhuo, Ying; Chai, Yaqin; Yuan, Ruo

2017-08-15

In this work, an elegantly designed host-guest recognition-assisted electrochemical release was established and applied in a reusable electrochemical biosensor for the detection of microRNA-182-5p (miRNA-182-5p), a prostate cancer biomarker in prostate cancer, based on the DNA cross configuration-fueled target cycling and strand displacement reaction (SDR) amplification. With such a design, the single target miRNA input could be converted to large numbers of single-stranded DNA (S1-Trp and S2-Trp) output, which could be trapped by cucurbit[8]uril methyl viologen (CB-8-MV 2+ ) based on the host-guest recognition, significantly enhancing the sensitivity for miRNA detection. Moreover, the nucleic acids products obtained from the process of cycling amplification could be utilized sufficiently, avoiding the waste and saving the experiment cost. Impressively, by resetting a settled voltage, the proposed biosensor could release S1-Trp and S2-Trp from the electrode surface, attributing that the guest ion methyl viologen (MV 2+ ) was reduced to MV +· under this settled voltage and formed a more-stable CB-8-MV +· -MV +· complex. Once O 2 was introduced in this system, MV +· could be oxidized to MV 2+ , generating the complex of CB-8-MV 2+ for capturing S1-Trp and S2-Trp again in only 5 min. As a result, the simple and fast regeneration of biosensor for target detection was realized on the base of electrochemical redox-driven assembly and release, overcoming the challenges of time-consuming, burdensome operations and expensive experimental cost in traditional reusable biosensors and updating the construction method for a reusable bisensor. Furthermore, the biosensor could be reused for more than 10 times with a regeneration rate of 93.20%-102.24%. After all, the conception of this work provides a novel thought for the construction of effective reusable biosensor to detect miRNA and other biomarkers and has great potential application in the area requiring the release of nucleic acids or proteins.

Computing Fault Displacements from Surface Deformations

NASA Technical Reports Server (NTRS)

Lyzenga, Gregory; Parker, Jay; Donnellan, Andrea; Panero, Wendy

2006-01-01

Simplex is a computer program that calculates locations and displacements of subterranean faults from data on Earth-surface deformations. The calculation involves inversion of a forward model (given a point source representing a fault, a forward model calculates the surface deformations) for displacements, and strains caused by a fault located in isotropic, elastic half-space. The inversion involves the use of nonlinear, multiparameter estimation techniques. The input surface-deformation data can be in multiple formats, with absolute or differential positioning. The input data can be derived from multiple sources, including interferometric synthetic-aperture radar, the Global Positioning System, and strain meters. Parameters can be constrained or free. Estimates can be calculated for single or multiple faults. Estimates of parameters are accompanied by reports of their covariances and uncertainties. Simplex has been tested extensively against forward models and against other means of inverting geodetic data and seismic observations. This work

Establishing reference values for central blood pressure and its amplification in a general healthy population and according to cardiovascular risk factors.

PubMed

Herbert, Annie; Cruickshank, John Kennedy; Laurent, Stéphane; Boutouyrie, Pierre

2014-11-21

Estimated central systolic blood pressure (cSBP) and amplification (Brachial SBP-cSBP) are non-invasive measures potentially prognostic of cardiovascular (CV) disease. No worldwide, multiple-device reference values are available. We aimed to establish reference values for a worldwide general population standardizing between the different available methods of measurement. How these values were significantly altered by cardiovascular risk factors (CVRFs) was then investigated. Existing data from population surveys and clinical trials were combined, whether published or not. Reference values of cSBP and amplification were calculated as percentiles for 'Normal' (no CVRFs) and 'Reference' (any CVRFs) populations. We included 45,436 subjects out of 82,930 that were gathered from 77 studies of 53 centres. Included subjects were apparently healthy, not treated for hypertension or dyslipidaemia, and free from overt CV disease and diabetes. Values of cSBP and amplification were stratified by brachial blood pressure categories and age decade in turn, both being stratified by sex. Amplification decreased with age and more so in males than in females. Sex was the most powerful factor associated with amplification with 6.6 mmHg (5.8-7.4) higher amplification in males than in females. Amplification was marginally but significantly influenced by CVRFs, with smoking and dyslipidaemia decreasing amplification, but increased with increasing levels of blood glucose. Typical values of cSBP and amplification in a healthy population and a population free of traditional CVRFs are now available according to age, sex, and brachial BP, providing values included from different devices with a wide geographical representation. Amplification is significantly influenced by CVRFs, but differently in men and women. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene.

PubMed

Xu, Huo; Jiang, Yifan; Liu, Dengyou; Liu, Kai; Zhang, Yafeng; Yu, Suhong; Shen, Zhifa; Wu, Zai-Sheng

2018-06-29

The sensitive detection of cancer-related genes is of great significance for early diagnosis and treatment of human cancers, and previous isothermal amplification sensing systems were often based on the reuse of target DNA, the amplification of enzymatic products and the accumulation of reporting probes. However, no reporting probes are able to be transformed into target species and in turn initiate the signal of other probes. Herein we reported a simple, isothermal and highly sensitive homogeneous assay system for tumor suppressor p53 gene detection based on a new autonomous DNA machine, where the signaling probe, molecular beacon (MB), was able to execute the function similar to target DNA besides providing the common signal. In the presence of target p53 gene, the operation of DNA machine can be initiated, and cyclical nucleic acid strand-displacement polymerization (CNDP) and nicking/polymerization cyclical amplification (NPCA) occur, during which the MB was opened by target species and cleaved by restriction endonuclease. In turn, the cleaved fragments could activate the next signaling process as target DNA did. According to the functional similarity, the cleaved fragment was called twin target, and the corresponding fashion to amplify the signal was named twin target self-amplification. Utilizing this newly-proposed DNA machine, the target DNA could be detected down to 0.1 pM with a wide dynamic range (6 orders of magnitude) and single-base mismatched targets were discriminated, indicating a very high assay sensitivity and good specificity. In addition, the DNA machine was not only used to screen the p53 gene in complex biological matrix but also was capable of practically detecting genomic DNA p53 extracted from A549 cell line. This indicates that the proposed DNA machine holds the potential application in biomedical research and early clinical diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Sensitive detection of microRNA in complex biological samples by using two stages DSN-assisted target recycling signal amplification method.

PubMed

Zhang, Kai; Wang, Ke; Zhu, Xue; Xu, Fei; Xie, Minhao

2017-01-15

MicroRNA (miRNA) has become an important biomarker candidate for cancer diagnosis, prognosis, and therapy. In this study, we have developed a novel fluorescence method for sensitive and specific miRNA detection via duplex specific nuclease (DSN) signal amplification and demonstrated its practical application in biological samples. Malachite green (MG) was employed as a "label-free" signal transducer since fluorescence of MG could be enhanced by 100-fold when MG were binding to a G-quadruplex structure formed within the d(G 2 T) 13 G sequence. The proposed signal amplification strategy is an integrated "biological circuit" designed to initiate a cascade of enzymatic reactions in order to detect, amplify, and measure a specific miRNA sequence by using the isothermal cleavage property of a DSN. The circuit is composed of two molecular switches operating in series: the amplification reaction activated by a specific miRNA and the strand-displacement polymerization reaction designed to initiate molecular beacon-assisted amplification and signal transduction by using MG/G-quadruplex complex. The hsa-miR-141 (miR141) was chosen as a target miRNA because its level specifically abnormal in a wide range of common human cancers including breast, lung, colon, and prostate cancer. The proposed method allowed quantitative sequence-specific detection of miR141 (with a detection limit of 1.03pM) in a dynamic range from 1pM to 10μM, with an excellent ability to discriminate differences in miRNAs. Moreover, the detection assay was applied to quantify miR141 in cancerous cell lysates. On the basis of these findings, we believe that this proposed sensitive and specific assay has great potential as a miRNA quantification method for use in biomedical research and clinical diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

The uses and limitations of the square‐root‐impedance method for computing site amplification

USGS Publications Warehouse

Boore, David

2013-01-01

The square‐root‐impedance (SRI) method is a fast way of computing approximate site amplification that does not depend on the details from velocity models. The SRI method underestimates the peak response of models with large impedance contrasts near their base, but the amplifications for those models is often close to or equal to the root mean square of the theoretical full resonant (FR) response of the higher modes. On the other hand, for velocity models made up of gradients, with no significant impedance changes across small ranges of depth, the SRI method systematically underestimates the theoretical FR response over a wide frequency range. For commonly used gradient models for generic rock sites, the SRI method underestimates the FR response by about 20%–30%. Notwithstanding the persistent underestimation of amplifications from theoretical FR calculations, however, amplifications from the SRI method may often provide more useful estimates of amplifications than the FR method, because the SRI amplifications are not sensitive to details of the models and will not exhibit the many peaks and valleys characteristic of theoretical full resonant amplifications (jaggedness sometimes not seen in amplifications based on averages of site response from multiple recordings at a given site). The lack of sensitivity to details of the velocity models also makes the SRI method useful in comparing the response of various velocity models, in spite of any systematic underestimation of the response. The quarter‐wavelength average velocity, which is fundamental to the SRI method, is useful by itself in site characterization, and as such, is the fundamental parameter used to characterize the site response in a number of recent ground‐motion prediction equations.

Amplification of light in one-dimensional vibrating metal photonic crystal

NASA Astrophysics Data System (ADS)

Ueta, Tsuyoshi

2012-04-01

Photon-phonon interaction on the analogy of electron-phonon interaction is considered in one-dimensional metal photonic crystal. When lattice vibration is artificially introduced to the photonic crystal, a governing equation of electromagnetic field is derived. A simple model is numerically analyzed, and the following novel phenomena are found out. The lattice vibration generates the light of frequency which added the integral multiple of the vibration frequency to that of the incident wave and also amplifies the incident wave resonantly. On a resonance, the amplification factor increases very rapidly with the number of layers. Resonance frequencies change with the phases of lattice vibration. The amplification phenomenon is analytically discussed for low frequency of the lattice vibration and is confirmed by numerical works.

Antibody-drug conjugate targeting CD46 eliminates multiple myeloma cells.

PubMed

Sherbenou, Daniel W; Aftab, Blake T; Su, Yang; Behrens, Christopher R; Wiita, Arun; Logan, Aaron C; Acosta-Alvear, Diego; Hann, Byron C; Walter, Peter; Shuman, Marc A; Wu, Xiaobo; Atkinson, John P; Wolf, Jeffrey L; Martin, Thomas G; Liu, Bin

2016-12-01

Multiple myeloma is incurable by standard approaches because of inevitable relapse and development of treatment resistance in all patients. In our prior work, we identified a panel of macropinocytosing human monoclonal antibodies against CD46, a negative regulator of the innate immune system, and constructed antibody-drug conjugates (ADCs). In this report, we show that an anti-CD46 ADC (CD46-ADC) potently inhibited proliferation in myeloma cell lines with little effect on normal cells. CD46-ADC also potently eliminated myeloma growth in orthometastatic xenograft models. In primary myeloma cells derived from bone marrow aspirates, CD46-ADC induced apoptosis and cell death, but did not affect the viability of nontumor mononuclear cells. It is of clinical interest that the CD46 gene resides on chromosome 1q, which undergoes genomic amplification in the majority of relapsed myeloma patients. We found that the cell surface expression level of CD46 was markedly higher in patient myeloma cells with 1q gain than in those with normal 1q copy number. Thus, genomic amplification of CD46 may serve as a surrogate for target amplification that could allow patient stratification for tailored CD46-targeted therapy. Overall, these findings indicate that CD46 is a promising target for antibody-based treatment of multiple myeloma, especially in patients with gain of chromosome 1q.

Phase reversal of vibratory signals in honeycomb may assist dancing honeybees to attract their audience.

PubMed

Tautz, J; Casas, J; Sandeman, D

2001-11-01

Forager honeybees dancing on the comb are able to attract dance-followers from distances across the comb that are too remote for tactile or visual signals to play a role. An alternative signal could be the vibrations of the comb at 200-300 Hz generated by dancing bees but which, without amplification, may not be large enough to alert remote dance-followers. We describe here, however, an unexpected property of honeycomb when it is subjected to vibration at around 200 Hz that would represent an effective amplification of the vibratory signals for remote dance-followers. We find that, at a specific distance from the origin of an imposed vibration, the walls across a single comb cell abruptly reverse the phase of their displacement and move in opposite directions to one another. Behavioural measurements show that the distance from which the majority of remote dance-followers are recruited coincides with the location of this phase-reversal phenomenon relative to the signal source. We propose that effective signal amplification by the phase-reversal phenomenon occurs when bees straddle a cell across which the phase reversal is expressed. Such a bee would be subjected to a situation in which the legs were moving towards and away from one another instead of in the same direction. In this manner, remote dance-followers could be alerted to a dancer performing in their vicinity.

Research and design on orthogonal diffraction grating-based 3D nanometer displacement sensor

NASA Astrophysics Data System (ADS)

Liu, Baoshuai; Yuan, Yibao; Yin, Zhehao

2017-10-01

This study concerns an orthogonal diffraction grating-based nanometer displacement sensor. In this study, we performed calculation of displacements in the XYZ directions. In the optical measured path part, we used a two-dimensional orthogonal motion grating and a two-dimensional orthogonal reference grating with the pitch of 0.5um to measure the displacement of XYZ in three directions by detecting ±1st diffraction fringes. The self-collimated structure of the grating greatly extended the Z-axis range. We also simulated the optical path of the sensor with ZEMAX software and verified the feasibility of the scheme. For signal subdivision and processing, we combined large number counting (completed grating line) with small number counting (digital subdivision), realizing high multiples of subdivision of grating interference signals. We used PC to process the interference fringes and greatly improved the processing speed. In the scheme, the theoretical multiples of subdivision could reach 1024 with 10-bit AD conversion, but the actual multiples of subdivision was limited by the quality of the grating interference signals. So we introduced an orthogonal compensation circuit and a filter circuit to improve the signal quality.

Temporomandibular joint disorder in a patient with multiple sclerosis--review of literature with a clinical report.

PubMed

Badel, Tomislav; Carek, Andreja; Podoreski, Dijana; Pavicin, Ivana Savić; Lovko, Sandra Kocijan

2010-09-01

Temporomandibular disorders are a form of musculoskeletal disorders, which reduce the function of stomatognathic system and they are related to some other diseases causing painful conditions and disorders of oral function. The aim of this paper is to describe a one year follow up clinical case of a female patient with comorbid multiple sclerosis and a relatively rare form of articular disc disorder. Primary clinical diagnostics encompassed manual methods of TMJ examination. Definite diagnosis included radiologic examination. Clinical hyperextensive condyle position was palpated bilaterally and subsequently confirmed by a functional panoramic radiograph of TMJ. The anterior displacement of disc with reduction was diagnosed by magnetic resonance and in the right joint there was a disc displacement upon excursive movement. From relevant literature, the relationship of a number of diseases that can be related to functional disorder of the orofacial system, such as multiple sclerosis, has been described from many aspects. Also, apart from the standard classification of one form of anterior displacement of the disc, made primarily by magnetic resonance, cases of disc displacement upon excursive mandibular movement can rarely be found in literature.

MULTIPLE ENZYME RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS FOR HIGH RESOLUTION DISTINCTION OF PSEUDOMONAS (SENSU STRICTO) 16S RRNA GENES

EPA Science Inventory

Pseudomonas specific 16S rDNA PCR amplification and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of Alu I, Hinf I, Rsa I, and Tru 9I distinguished 150 published sequences and reference strains of authentic Pseudomonas...

The positioning logic and copy number control of genes in bacteria under stress

NASA Astrophysics Data System (ADS)

Zhang, Qiucen; Austin, Robert; Vyawahare, Saurabh; Lau, Alexandra

2013-03-01

Escherichia coli (E. coli) cells when challenged with sublethal concentrations of the genotoxic antibiotic ciprofloxacin cease to divide and form long filaments which contain multiple bacterial chromosomes. These filaments are individual mesoscopic environmental niches which provide protection for a community of chromosomes (as opposed to cells) under mutagenic stress and can provide an evolutionary fitness advantage within the niche. We use comparative genomic hybridization to show that the mesoscopic niche evolves within 20 minutes of ciprofloxacin exposure via replication of multiple copies of genes expressing ATP dependent transporters. We show that this rapid genomic amplification is done in a time efficient manner via placement of the genes encoding the pumps near the origin of replication on the bacterial chromosome. The de-amplification of multiple copies back to the wild type number is a function of the duration is a function of the ciprofloxacin exposure duration: the longer the exposure, the slower the removal of the multiple copies. The project described was supported by the National Science Foundation and the National Cancer Institute

Low noise optical position sensor

DOEpatents

Spear, J.D.

1999-03-09

A novel optical position sensor is described that uses two component photodiodes electrically connected in parallel, with opposing polarities. A lens provides optical gain and restricts the acceptance angle of the detector. The response of the device to displacements of an optical spot is similar to that of a conventional bi-cell type position sensitive detector. However, the component photodiode design enables simpler electronic amplification with inherently less electrical noise than the bi-cell. Measurements by the sensor of the pointing noise of a focused helium-neon laser as a function of frequency demonstrate high sensitivity and suitability for optical probe beam deflection experiments. 14 figs.

Low noise optical position sensor

DOEpatents

Spear, Jonathan David

1999-01-01

A novel optical position sensor is described that uses two component photodiodes electrically connected in parallel, with opposing polarities. A lens provides optical gain and restricts the acceptance angle of the detector. The response of the device to displacements of an optical spot is similar to that of a conventional bi-cell type position sensitive detector. However, the component photodiode design enables simpler electronic amplification with inherently less electrical noise than the bi-cell. Measurements by the sensor of the pointing noise of a focused helium-neon laser as a function of frequency demonstrate high sensitivity and suitability for optical probe beam deflection experiments.

Whole genome amplification and real-time PCR in forensic casework

PubMed Central

Giardina, Emiliano; Pietrangeli, Ilenia; Martone, Claudia; Zampatti, Stefania; Marsala, Patrizio; Gabriele, Luciano; Ricci, Omero; Solla, Gianluca; Asili, Paola; Arcudi, Giovanni; Spinella, Aldo; Novelli, Giuseppe

2009-01-01

Background WGA (Whole Genome Amplification) in forensic genetics can eliminate the technical limitations arising from low amounts of genomic DNA (gDNA). However, it has not been used to date because any amplification bias generated may complicate the interpretation of results. Our aim in this paper was to assess the applicability of MDA to forensic SNP genotyping by performing a comparative analysis of genomic and amplified DNA samples. A 26-SNPs TaqMan panel specifically designed for low copy number (LCN) and/or severely degraded genomic DNA was typed on 100 genomic as well as amplified DNA samples. Results Aliquots containing 1, 0.1 and 0.01 ng each of 100 DNA samples were typed for a 26-SNPs panel. Similar aliquots of the same DNA samples underwent multiple displacement amplification (MDA) before being typed for the same panel. Genomic DNA samples showed 0% PCR failure rate for all three dilutions, whilst the PCR failure rate of the amplified DNA samples was 0% for the 1 ng and 0.1 ng dilutions and 0.077% for the 0.01 ng dilution. The genotyping results of both the amplified and genomic DNA samples were also compared with reference genotypes of the same samples obtained by direct sequencing. The genomic DNA samples showed genotype concordance rates of 100% for all three dilutions while the concordance rates of the amplified DNA samples were 100% for the 1 ng and 0.1 ng dilutions and 99.923% for the 0.01 ng dilution. Moreover, ten artificially-degraded DNA samples, which gave no results when analyzed by current forensic methods, were also amplified by MDA and genotyped with 100% concordance. Conclusion We investigated the suitability of MDA material for forensic SNP typing. Comparative analysis of amplified and genomic DNA samples showed that a large number of SNPs could be accurately typed starting from just 0.01 ng of template. We found that the MDA genotyping call and accuracy rates were only slightly lower than those for genomic DNA. Indeed, when 10 pg of input DNA was used in MDA, we obtained 99.923% concordance, indicating a genotyping error rate of 1/1299 (7.7 × 10-4). This is quite similar to the genotyping error rate of STRs used in current forensic analysis. Such efficiency and accuracy of SNP typing of amplified DNA suggest that MDA can also generate large amounts of genome-equivalent DNA from a minimal amount of input DNA. These results show for the first time that MDA material is suitable for SNP-based forensic protocols and in general when samples fail to give interpretable STR results. PMID:19366436

Modeling and performance metrics of MIMO-SDM systems with different amplification schemes in the presence of mode-dependent loss.

PubMed

Antonelli, Cristian; Mecozzi, Antonio; Shtaif, Mark; Winzer, Peter J

2015-02-09

Mode-dependent loss (MDL) is a major factor limiting the achievable information rate in multiple-input multiple-output space-division multiplexed systems. In this paper we show that its impact on system performance, which we quantify in terms of the capacity reduction relative to a reference MDL-free system, may depend strongly on the operation of the inline optical amplifiers. This dependency is particularly strong in low mode-count systems. In addition, we discuss ways in which the signal-to-noise ratio of the MDL-free reference system can be defined and quantify the differences in the predicted capacity loss. Finally, we stress the importance of correctly accounting for the effect of MDL on the accumulation of amplification noise.

Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

PubMed

Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

2015-02-01

In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on paraffin-embedded material to test for other diagnostically, prognostically, or therapeutically relevant genomic mutations in lipomatous tumors.

A Deep Analysis of Center Displacement in An Idealized Tropical Cyclone with Low-wavenumber Asymmetries

NASA Astrophysics Data System (ADS)

Zhao, C.; Song, J.; Leng, H.

2017-12-01

The Tropical Cyclone(TC) center-finding technique plays an important role when diagnostic analyses of TC structure are performed, especially when dealing with low-wavenumber asymmetries. Previous works have already established that structure of TCs can vary greatly depending on the displacement induced by center-finding techniques. As it is difficult to define a true TC center in the real world, this work seeks to explore how low-wavenumber azimuthal Fourier analyses can vary with center displacement using idealized, parametric TC-like vortices with different perturbation structures. It is shown that the errors is sensitive to the location and radial structure of the adding perturbation. In the case of adding azimuthal wavenumber 1 and 3 asymmetries, the increasing radial shear of initial asymmetries will enhance the corresponding spectral energy around radius of maximum wind(RMW) significantly, and they also have a great effect on spectral energy of wavenumber 2. On the contrary, the wavenumber 2 cases show a reduction from 1RMW to outer radius when shear is increasing and has little effect on spectral energy of wavenumber 1 or 2. Pervious findings indicated that the aliasing is dependent on the placement of center relative to the location of the asymmetries, which is also valid in these shearing situations. Moreover, it is found that this aliasing caused by phase displacement is less sensitive with the radial shear in wavenumber 2 and 3 cases, while it shows an significant amplification and deformation when wavenumber 1 asymmetry is added.

Cellular traction force recovery: An optimal filtering approach in two-dimensional Fourier space.

PubMed

Huang, Jianyong; Qin, Lei; Peng, Xiaoling; Zhu, Tao; Xiong, Chunyang; Zhang, Youyi; Fang, Jing

2009-08-21

Quantitative estimation of cellular traction has significant physiological and clinical implications. As an inverse problem, traction force recovery is essentially susceptible to noise in the measured displacement data. For traditional procedure of Fourier transform traction cytometry (FTTC), noise amplification is accompanied in the force reconstruction and small tractions cannot be recovered from the displacement field with low signal-noise ratio (SNR). To improve the FTTC process, we develop an optimal filtering scheme to suppress the noise in the force reconstruction procedure. In the framework of the Wiener filtering theory, four filtering parameters are introduced in two-dimensional Fourier space and their analytical expressions are derived in terms of the minimum-mean-squared-error (MMSE) optimization criterion. The optimal filtering approach is validated with simulations and experimental data associated with the adhesion of single cardiac myocyte to elastic substrate. The results indicate that the proposed method can highly enhance SNR of the recovered forces to reveal tiny tractions in cell-substrate interaction.

Observations of height-dependent pressure-perturbation structure of a strong mesoscale gravity wave

NASA Technical Reports Server (NTRS)

Starr, David O'C.; Korb, C. L.; Schwemmer, Geary K.; Weng, Chi Y.

1992-01-01

Airborne observations using a downward-looking, dual-frequency, near-infrared, differential absorption lidar system provide the first measurements of the height-dependent pressure-perturbation field associated with a strong mesoscale gravity wave. A pressure-perturbation amplitude of 3.5 mb was measured within the lowest 1.6 km of the atmosphere over a 52-km flight line. Corresponding vertical displacements of 250-500 m were inferred from lidar-observed displacement of aerosol layers. Accounting for probable wave orientation, a horizontal wavelength of about 40 km was estimated. Satellite observations reveal wave structure of a comparable scale in concurrent cirrus cloud fields over an extended area. Smaller-scale waves were also observed. Local meteorological soundings are analyzed to confirm the existence of a suitable wave duct. Potential wave-generation mechanisms are examined and discussed. The large pressure-perturbation wave is attributed to rapid amplification or possible wave breaking of a gravity wave as it propagated offshore and interacted with a very stable marine boundary layer capped by a strong shear layer.

A semi-empirical analysis of strong-motion peaks in terms of seismic source, propagation path, and local site conditions

NASA Astrophysics Data System (ADS)

Kamiyama, M.; Orourke, M. J.; Flores-Berrones, R.

1992-09-01

A new type of semi-empirical expression for scaling strong-motion peaks in terms of seismic source, propagation path, and local site conditions is derived. Peak acceleration, peak velocity, and peak displacement are analyzed in a similar fashion because they are interrelated. However, emphasis is placed on the peak velocity which is a key ground motion parameter for lifeline earthquake engineering studies. With the help of seismic source theories, the semi-empirical model is derived using strong motions obtained in Japan. In the derivation, statistical considerations are used in the selection of the model itself and the model parameters. Earthquake magnitude M and hypocentral distance r are selected as independent variables and the dummy variables are introduced to identify the amplification factor due to individual local site conditions. The resulting semi-empirical expressions for the peak acceleration, velocity, and displacement are then compared with strong-motion data observed during three earthquakes in the U.S. and Mexico.

Stability of Markers Used for Real-Time Tumor Tracking After Percutaneous Intrapulmonary Placement

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

Voort van Zyp, Noelle C. van der, E-mail: n.vandervoortvanzyp@erasmusmc.nl; Hoogeman, Mischa S.; Water, Steven van de

2011-11-01

Purpose: To determine the stability of markers used for real-time tumor tracking after percutaneous intrapulmonary placement. Methods and Materials: A total of 42 patients with 44 lesions, 111 markers, and {>=}2 repeat computed tomography (CT) scans were studied. The tumor on the repeat CT scans was registered with the tumor on the planning CT scan. Next, the three-dimensional marker coordinates were determined on the planning CT scan and repeat CT scans. Marker stability was analyzed by the displacement of the markers and the displacement of the center of mass (COM) of the marker configurations. In addition, we assessed the reliabilitymore » of using the intermarker distance as a check for displacements in the COM of the marker configurations. Results: The median marker displacement was 1.3 mm (range, 0.1-53.6). The marker displacement was >5 mm in 12% of the markers and >10 mm in 5% of the markers. The causes of marker displacement >5 mm included marker migration (2 of 13) and target volume changes (5 of 13). Nonsynchronous tumor and marker movement during breathing might have been responsible for the displacements >5 mm in the other 6 of 13 markers. The median displacement in the COM of the marker configurations was 1.0 mm (range, 0.1-23.3). Displacements in the COM of the marker configurations of {>=}2.0 mm were detected by changes in the intermarker distance of >1.5 mm in 96% of the treatment fractions. Conclusion: The median marker displacement was small (1.3 mm). Nevertheless, displacements >5 mm occurred in 12% of the markers. Therefore, we recommend the implantation of multiple markers because multiple markers will enable a quick and reliable check of marker displacement by determining the change in the intermarker distance. A displacement in the COM of the marker configuration of {>=}2.0 mm was almost always detected (96%) by a change in the distance between the markers of >1.5 mm. This enabled the displaced marker to be disabled, such that tumor localization was not compromised.« less

Protein detection using biobarcodes.

PubMed

Müller, Uwe R

2006-10-01

Over the past 50 years the development of assays for the detection of protein analytes has been driven by continuing demands for higher levels of sensitivity and multiplexing. The result has been a progression of sandwich-type immunoassays, starting with simple radioisotopic, colorimetric, or fluorescent labeling systems to include various enzymatic or nanostructure-based signal amplification schemes, with a concomitant sensitivity increase of over 1 million fold. Multiplexing of samples and tests has been enabled by microplate and microarray platforms, respectively, or lately by various molecular barcoding systems. Two different platforms have emerged as the current front-runners by combining a nucleic acid amplification step with the standard two-sided immunoassay. In both, the captured protein analyte is replaced by a multiplicity of oligonucleotides that serve as surrogate targets. One of these platforms employs DNA or RNA polymerases for the amplification step, while detection is by fluorescence. The other is based on gold nanoparticles for both amplification as well as detection. The latter technology, now termed Biobarcode, is completely enzyme-free and offers potentially much higher multiplexing power.

Improved Displacement Transfer Functions for Structure Deformed Shape Predictions Using Discretely Distributed Surface Strains

NASA Technical Reports Server (NTRS)

Ko, William L.; Fleischer, Van Tran

2012-01-01

In the formulations of earlier Displacement Transfer Functions for structure shape predictions, the surface strain distributions, along a strain-sensing line, were represented with piecewise linear functions. To improve the shape-prediction accuracies, Improved Displacement Transfer Functions were formulated using piecewise nonlinear strain representations. Through discretization of an embedded beam (depth-wise cross section of a structure along a strain-sensing line) into multiple small domains, piecewise nonlinear functions were used to describe the surface strain distributions along the discretized embedded beam. Such piecewise approach enabled the piecewise integrations of the embedded beam curvature equations to yield slope and deflection equations in recursive forms. The resulting Improved Displacement Transfer Functions, written in summation forms, were expressed in terms of beam geometrical parameters and surface strains along the strain-sensing line. By feeding the surface strains into the Improved Displacement Transfer Functions, structural deflections could be calculated at multiple points for mapping out the overall structural deformed shapes for visual display. The shape-prediction accuracies of the Improved Displacement Transfer Functions were then examined in view of finite-element-calculated deflections using different tapered cantilever tubular beams. It was found that by using the piecewise nonlinear strain representations, the shape-prediction accuracies could be greatly improved, especially for highly-tapered cantilever tubular beams.

Curved Displacement Transfer Functions for Geometric Nonlinear Large Deformation Structure Shape Predictions

NASA Technical Reports Server (NTRS)

Ko, William L.; Fleischer, Van Tran; Lung, Shun-Fat

2017-01-01

For shape predictions of structures under large geometrically nonlinear deformations, Curved Displacement Transfer Functions were formulated based on a curved displacement, traced by a material point from the undeformed position to deformed position. The embedded beam (depth-wise cross section of a structure along a surface strain-sensing line) was discretized into multiple small domains, with domain junctures matching the strain-sensing stations. Thus, the surface strain distribution could be described with a piecewise linear or a piecewise nonlinear function. The discretization approach enabled piecewise integrations of the embedded-beam curvature equations to yield the Curved Displacement Transfer Functions, expressed in terms of embedded beam geometrical parameters and surface strains. By entering the surface strain data into the Displacement Transfer Functions, deflections along each embedded beam can be calculated at multiple points for mapping the overall structural deformed shapes. Finite-element linear and nonlinear analyses of a tapered cantilever tubular beam were performed to generate linear and nonlinear surface strains and the associated deflections to be used for validation. The shape prediction accuracies were then determined by comparing the theoretical deflections with the finiteelement- generated deflections. The results show that the newly developed Curved Displacement Transfer Functions are very accurate for shape predictions of structures under large geometrically nonlinear deformations.

Measurement of slow-moving along-track displacement from an efficient multiple-aperture SAR interferometry (MAI) stacking

USGS Publications Warehouse

Jo, Min-Jeong; Jung, Hyung-Sup; Won, Joong-Sun; Poland, Michael; Miklius, Asta; Lu, Zhong

2015-01-01

Multiple-aperture SAR interferometry (MAI) has demonstrated outstanding measurement accuracy of along-track displacement when compared to pixel-offset-tracking methods; however, measuring slow-moving (cm/year) surface displacement remains a challenge. Stacking of multi-temporal observations is a potential approach to reducing noise and increasing measurement accuracy, but it is difficult to achieve a significant improvement by applying traditional stacking methods to multi-temporal MAI interferograms. This paper proposes an efficient MAI stacking method, where multi-temporal forward- and backward-looking residual interferograms are individually stacked before the MAI interferogram is generated. We tested the performance of this method using ENVISAT data from Kīlauea Volcano, Hawai‘i, where displacement on the order of several centimeters per year is common. By comparing results from the proposed stacking methods with displacements from GPS data, we documented measurement accuracies of about 1.03 and 1.07 cm/year for the descending and ascending tracks, respectively—an improvement of about a factor of two when compared with that from the conventional stacking approach. Three-dimensional surface-displacement maps can be constructed by combining stacked InSAR and MAI observations, which will contribute to a better understanding of a variety of geological phenomena.

The clinical application of single-sperm-based SNP haplotyping for PGD of osteogenesis imperfecta.

PubMed

Chen, Linjun; Diao, Zhenyu; Xu, Zhipeng; Zhou, Jianjun; Yan, Guijun; Sun, Haixiang

2018-05-15

Osteogenesis imperfecta (OI) is a genetically heterogeneous disorder, presenting either autosomal dominant, autosomal recessive or X-linked inheritance patterns. The majority of OI cases are autosomal dominant and are caused by heterozygous mutations in either the COL1A1 or COL1A2 gene. In these dominant disorders, allele dropout (ADO) can lead to misdiagnosis in preimplantation genetic diagnosis (PGD). Polymorphic markers linked to the mutated genes have been used to establish haplotypes for identifying ADO and ensuring the accuracy of PGD. However, the haplotype of male patients cannot be determined without data from affected relatives. Here, we developed a method for single-sperm-based single-nucleotide polymorphism (SNP) haplotyping via next-generation sequencing (NGS) for the PGD of OI. After NGS, 10 informative polymorphic SNP markers located upstream and downstream of the COL1A1 gene and its pathogenic mutation site were linked to individual alleles in a single sperm from an affected male. After haplotyping, a normal blastocyst was transferred to the uterus for a subsequent frozen embryo transfer cycle. The accuracy of PGD was confirmed by amniocentesis at 19 weeks of gestation. A healthy infant weighing 4,250 g was born via vaginal delivery at the 40th week of gestation. Single-sperm-based SNP haplotyping can be applied for PGD of any monogenic disorders or de novo mutations in males in whom the haplotype of paternal mutations cannot be determined due to a lack of affected relatives. ADO: allele dropout; DI: dentinogenesis imperfect; ESHRE: European Society of Human Reproduction and Embryology; FET: frozen embryo transfer; gDNA: genomic DNA; ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization; MDA: multiple displacement amplification; NGS: next-generation sequencing; OI: osteogenesis imperfect; PBS: phosphate buffer saline; PCR: polymerase chain reaction; PGD: preimplantation genetic diagnosis; SNP: single-nucleotide polymorphism; STR: short tandem repeat; TE: trophectoderm; WGA: whole-genome amplification.

Amplified detection of cocaine based on strand-displacement polymerization and fluorescence resonance energy transfer.

PubMed

Huang, Jin; Chen, Yan; Yang, Liu; Zhu, Zhi; Zhu, Guizhi; Yang, Xiaohai; Wang, Kemin; Tan, Weihong

2011-10-15

Cocaine is one of the most abused drugs in the United States and is potentially dangerous when consumed in excess. Its detection is thus important in many areas in the fight against drug trafficking. We have developed an amplified detection method for cocaine based on a strand-displacement polymerization reaction using aptamer recognition. The system mainly consists of a hairpin probe with Cy5 labeled on its 3' end, a primer with FAM labeled on its 5' end, and polymerase. The aptamer sequence is integrated into the 5'-section of the hairpin probe. The primer is designed complementary to the 3' end of the hairpin probe, which is also part of the hairpin stem region. The cocaine induced reaction cycle generates product for detection and thus for signal amplification. The detection limit of this method is 200 nM in about 16 min and the specificity of this approach is excellent. We believe that this strategy will be useful for the development of analytical schemes for a variety of aptamers for small molecules, metal ions, and proteins. This simple scheme employing the strand-displacement polymerization reaction may find wide application in forensic analysis, environmental monitoring, and clinical diagnostics. Copyright © 2011 Elsevier B.V. All rights reserved.

Apical root resorption in orthodontically treated adults.

PubMed

Baumrind, S; Korn, E L; Boyd, R L

1996-09-01

This study analyzed the relationship in orthodontically treated adults between upper central incisor displacement measured on lateral cephalograms and apical root resorption measured on anterior periapical x-ray films. A multiple linear regression examined incisor displacements in four directions (retraction, advancement, intrusion, and extrusion) as independent variables, attempting to account for observed differences in the dependent variable, resorption. Mean apical resorption was 1.36 mm (sd +/- 1.46, n = 73). Mean horizontal displacement of the apex was -0.83 mm (sd +/- 1.74, n = 67); mean vertical displacement was 0.19 mm (sd +/- 1.48, n = 67). The regression coefficients for the intercept and for retraction were highly significant; those for extrusion, intrusion, and advancement were not. At the 95% confidence level, an average of 0.99 mm (se = +/- 0.34) of resorption was implied in the absence of root displacement and an average of 0.49 mm (se = +/- 0.14) of resorption was implied per millimeter of retraction. R2 for all four directional displacement variables (DDVs) taken together was only 0.20, which implied that only a relatively small portion of the observed apical resorption could be accounted for by tooth displacement alone. In a secondary set of univariate analyses, the associations between apical resorption and each of 14 additional treatment-related variables were examined. Only Gender, Elapsed Time, and Total Apical Displacement displayed statistically significant associations with apical resorption. Additional multiple regressions were then performed in which the data for each of these three statistically significant variables were considered separately, with the data for the four directional displacement variables. The addition of information on Elapsed Time or Total Apical Displacement did not explain a significant additional portion of the variability in apical resorption. On the other hand, the addition of information on Gender to the information on the four directional displacement variables yielded an R2 value of 0.35, which indicated that these variables taken together could account for approximately a third of the observed variability in apical resorption in this sample.

'Compromise position' image alignment to accommodate independent motion of multiple clinical target volumes during radiotherapy: A high risk prostate cancer example.

PubMed

Rosewall, Tara; Yan, Jing; Alasti, Hamideh; Cerase, Carla; Bayley, Andrew

2017-04-01

Inclusion of multiple independently moving clinical target volumes (CTVs) in the irradiated volume causes an image guidance conundrum. The purpose of this research was to use high risk prostate cancer as a clinical example to evaluate a 'compromise' image alignment strategy. The daily pre-treatment orthogonal EPI for 14 consecutive patients were included in this analysis. Image matching was performed by aligning to the prostate only, the bony pelvis only and using the 'compromise' strategy. Residual CTV surrogate displacements were quantified for each of the alignment strategies. Analysis of the 388 daily fractions indicated surrogate displacements were well-correlated in all directions (r 2  = 0.95 (LR), 0.67 (AP) and 0.59 (SI). Differences between the surrogates displacements (95% range) were -0.4 to 1.8 mm (LR), -1.2 to 5.2 mm (SI) and -1.2 to 5.2 mm (AP). The distribution of the residual displacements was significantly smaller using the 'compromise' strategy, compared to the other strategies (p 0.005). The 'compromise' strategy ensured the CTV was encompassed by the PTV in all fractions, compared to 47 PTV violations when aligned to prostate only. This study demonstrated the feasibility of a compromise position image guidance strategy to accommodate simultaneous displacements of two independently moving CTVs. Application of this strategy was facilitated by correlation between the CTV displacements and resulted in no geometric excursions of the CTVs beyond standard sized PTVs. This simple image guidance strategy may also be applicable to other disease sites that concurrently irradiate multiple CTVs, such as head and neck, lung and cervix cancer. © 2016 The Royal Australian and New Zealand College of Radiologists.

Multiple pass laser amplifier system

DOEpatents

Brueckner, Keith A.; Jorna, Siebe; Moncur, N. Kent

1977-01-01

A laser amplification method for increasing the energy extraction efficiency from laser amplifiers while reducing the energy flux that passes through a flux limited system which includes apparatus for decomposing a linearly polarized light beam into multiple components, passing the components through an amplifier in delayed time sequence and recombining the amplified components into an in phase linearly polarized beam.

Toehold-mediated strand displacement reaction-dependent fluorescent strategy for sensitive detection of uracil-DNA glycosylase activity.

PubMed

Wu, Yushu; Wang, Lei; Jiang, Wei

2017-03-15

Sensitive detection of uracil-DNA glycosylase (UDG) activity is beneficial for evaluating the repairing process of DNA lesions. Here, toehold-mediated strand displacement reaction (TSDR)-dependent fluorescent strategy was constructed for sensitive detection of UDG activity. A single-stranded DNA (ssDNA) probe with two uracil bases and a trigger sequence were designed. A hairpin probe with toehold domain was designed, and a reporter probe was also designed. Under the action of UDG, two uracil bases were removed from ssDNA probe, generating apurinic/apyrimidinic (AP) sites. Then, the AP sites could inhibit the TSDR between ssDNA probe and hairpin probe, leaving the trigger sequence in ssDNA probe still free. Subsequently, the trigger sequence was annealed with the reporter probe, initiating the polymerization and nicking amplification reaction. As a result, numerous G-quadruplex (G4) structures were formed, which could bind with N-methyl-mesoporphyrin IX (NMM) to generate enhanced fluorescent signal. In the absence of UDG, the ssDNA probe could hybridize with the toehold domain of the hairpin probe to initiate TSDR, blocking the trigger sequence, and then the subsequent amplification reaction would not occur. The proposed strategy was successfully implemented for detecting UDG activity with a detection limit of 2.7×10 -5 U/mL. Moreover, the strategy could distinguish UDG well from other interference enzymes. Furthermore, the strategy was also applied for detecting UDG activity in HeLa cells lysate with low effect of cellular components. These results indicated that the proposed strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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

2015-08-18

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

Engineering a Cell-surface Aptamer Circuit for Targeted and Amplified Photodynamic Cancer Therapy

PubMed Central

Han, Da; Zhu, Guizhi; Wu, Cuichen; Zhu, Zhi; Chen, Tao; Zhang, Xiaobing

2013-01-01

Photodynamic therapy (PDT) is one of the most promising and noninvasive methods for clinical treatment of different malignant diseases. Here, we present a novel strategy of designing an aptamer-based DNA nanocircuit capable of the selective recognition of cancer cells, controllable activation of photosensitizer and amplification of photodynamic therapeutic effect. The aptamers can selectively recognize target cancer cells and bind to the specific proteins on cell membranes. Then the overhanging catalyst sequence on aptamer can trigger a toehold-mediated catalytic strand displacement to activate photosensitizer and achieve amplified therapeutic effect. The specific binding-induced activation allows the DNA circuit to distinguish diseased cells from healthy cells, reducing damage to nearby healthy cells. Moreover, the catalytic amplification reaction will only take place close to the target cancer cells, resulting in a high local concentration of singlet oxygen to selectively kill the target cells. The principle employed in this study demonstrated the feasibility of assembling a DNA circuit on cell membranes and could further broaden the utility of DNA circuits for applications in biology, biotechnology, and biomedicine. PMID:23397942

Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification

PubMed Central

Wu, Lucia R.; Chen, Sherry X.; Wu, Yalei; Patel, Abhijit A.; Zhang, David Yu

2018-01-01

Rare DNA-sequence variants hold important clinical and biological information, but existing detection techniques are expensive, complex, allele-specific, or don’t allow for significant multiplexing. Here, we report a temperature-robust polymerase-chain-reaction method, which we term blocker displacement amplification (BDA), that selectively amplifies all sequence variants, including single-nucleotide variants (SNVs), within a roughly 20-nucleotide window by 1,000-fold over wild-type sequences. This allows for easy detection and quantitation of hundreds of potential variants originally at ≤0.1% in allele frequency. BDA is compatible with inexpensive thermocycler instrumentation and employs a rationally designed competitive hybridization reaction to achieve comparable enrichment performance across annealing temperatures ranging from 56 °C to 64 °C. To show the sequence generality of BDA, we demonstrate enrichment of 156 SNVs and the reliable detection of single-digit copies. We also show that the BDA detection of rare driver mutations in cell-free DNA samples extracted from the blood plasma of lung-cancer patients is highly consistent with deep sequencing using molecular lineage tags, with a receiver operator characteristic accuracy of 95%. PMID:29805844

Model experiment of magnetic field amplification in laser-produced plasmas via the Richtmyer-Meshkov instability

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

Kuramitsu, Y., E-mail: yasu@ncu.edu.tw; Moritaka, T.; Ohnishi, N.

2016-03-15

A model experiment of magnetic field amplification (MFA) via the Richtmyer-Meshkov instability (RMI) in supernova remnants (SNRs) was performed using a high-power laser. In order to account for very-fast acceleration of cosmic rays observed in SNRs, it is considered that the magnetic field has to be amplified by orders of magnitude from its background level. A possible mechanism for the MFA in SNRs is stretching and mixing of the magnetic field via the RMI when shock waves pass through dense molecular clouds in interstellar media. In order to model the astrophysical phenomenon in laboratories, there are three necessary factors formore » the RMI to be operative: a shock wave, an external magnetic field, and density inhomogeneity. By irradiating a double-foil target with several laser beams with focal spot displacement under influence of an external magnetic field, shock waves were excited and passed through the density inhomogeneity. Radiative hydrodynamic simulations show that the RMI evolves as the density inhomogeneity is shocked, resulting in higher MFA.« less

Integrated control and health monitoring capacitive displacement sensor development task. Orbit transfer rocket engine technology program

NASA Technical Reports Server (NTRS)

Collamore, Frank N.

1989-01-01

The development of a miniature multifunction turbomachinery shaft displacement sensor using state-of-the-art non-contract capacitive sensing technology is described. Axial displacement, radial displacement, and speed are sensed using a single probe within the envelope normally required for a single function. A survey of displacement sensing technology is summarized including inductive, capacitive, optical and ultrasonic techniques. The design and operation of an experimental triple function sensor is described. Test results are included showing calibration tests and simultaneous dynamic testing of multiple functions. Recommendations for design changes are made to improve low temperature performance, reliability, and for design of a flight type signal conditioning unit.

Nucleic acid detection system and method for detecting influenza

DOEpatents

Cai, Hong; Song, Jian

2015-03-17

The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.

Analysis of multiple cell upset sensitivity in bulk CMOS SRAM after neutron irradiation

NASA Astrophysics Data System (ADS)

Pan, Xiaoyu; Guo, Hongxia; Luo, Yinhong; Zhang, Fengqi; Ding, Lili

2018-03-01

In our previous studies, we have proved that neutron irradiation can decrease the single event latch-up (SEL) sensitivity of CMOS SRAM. And one of the key contributions to the multiple cell upset (MCU) is the parasitic bipolar amplification, it bring us to study the impact of neutron irradiation on the SRAM’s MCU sensitivity. After the neutron experiment, we test the devices’ function and electrical parameters. Then, we use the heavy ion fluence to examine the changes on the devices’ MCU sensitivity pre- and post-neutron-irradiation. Unfortunately, neutron irradiation makes the MCU phenomenon worse. Finally, we use the electric static discharge (ESD) testing technology to deduce the experimental results and find that the changes on the WPM region take the lead rather than the changes on the parasitic bipolar amplification for the 90 nm process.

Multiple signal amplification strategies for ultrasensitive label-free electrochemical immunoassay for carbohydrate antigen 24-2 based on redox hydrogel.

PubMed

Tang, Zhongxue; Fu, Yuanyuan; Ma, Zhanfang

2017-05-15

In this work, multiple signal amplification strategies for ultrasensitive label-free electrochemical immunoassay for carbohydrate antigen 24-2 (CA242) were developed using redox sodium alginate-Pb 2+ -graphene oxide (SA-Pb 2+ -GO) hydrogel. The SA-Pb 2+ -GO hydrogel was synthesised by simply mixing SA, GO, and Pb 2+ and then implemented as a novel redox species with a strong current signal at -0.46V (vs. Ag/AgCl). After the three-dimensional and porous SA-Pb 2+ -GO hydrogel was in situ generated on a glassy carbon electrode (GCE), chitosan was adsorbed on the obtained electrode to further enrich Pb 2+ . When chitosan-Pb 2+ /SA-Pb 2+ -GO/GCE was incubated with anti-CA242 using glutaraldehyde and blocked by bovine serum albumin, the immunoassay platform for CA242 was obtained. Owing to the addition of GO, the obtained conductive SA-GO/GCE was beneficial for signal amplification. After incubating SA-GO/GCE with excessive amounts of Pb 2+ , the resistance of SA-Pb 2+ -GO/GCE further decreased and a strong redox signal was obtained. The chitosan fixed by electrostatic adsorption resulted in further adsorption of Pb 2+ , behaving as further amplifying the signal and improving conductivity. In this case, multiple signal amplification strategies were involved in the proposed immunosensor for the ultrasensitive detection of CA242. Under the optimal conditions, the proposed immunosensor exhibited a wide linear range from 0.005UmL -1 to 500UmL -1 with an ultralow detection limit of 0.067mUmL -1 . In comparison to previous works, the sensitivity of this method was 32.98μA (log 10 C CA242 ) -1 , which was a five-fold increase from the previous works. Copyright © 2016 Elsevier B.V. All rights reserved.

A Noncontact FMCW Radar Sensor for Displacement Measurement in Structural Health Monitoring

PubMed Central

Li, Cunlong; Chen, Weimin; Liu, Gang; Yan, Rong; Xu, Hengyi; Qi, Yi

2015-01-01

This paper investigates the Frequency Modulation Continuous Wave (FMCW) radar sensor for multi-target displacement measurement in Structural Health Monitoring (SHM). The principle of three-dimensional (3-D) displacement measurement of civil infrastructures is analyzed. The requirements of high-accuracy displacement and multi-target identification for the measuring sensors are discussed. The fundamental measuring principle of FMCW radar is presented with rigorous mathematical formulas, and further the multiple-target displacement measurement is analyzed and simulated. In addition, a FMCW radar prototype is designed and fabricated based on an off-the-shelf radar frontend and data acquisition (DAQ) card, and the displacement error induced by phase asynchronism is analyzed. The conducted outdoor experiments verify the feasibility of this sensing method applied to multi-target displacement measurement, and experimental results show that three targets located at different distances can be distinguished simultaneously with millimeter level accuracy. PMID:25822139

A noncontact FMCW radar sensor for displacement measurement in structural health monitoring.

PubMed

Li, Cunlong; Chen, Weimin; Liu, Gang; Yan, Rong; Xu, Hengyi; Qi, Yi

2015-03-26

This paper investigates the Frequency Modulation Continuous Wave (FMCW) radar sensor for multi-target displacement measurement in Structural Health Monitoring (SHM). The principle of three-dimensional (3-D) displacement measurement of civil infrastructures is analyzed. The requirements of high-accuracy displacement and multi-target identification for the measuring sensors are discussed. The fundamental measuring principle of FMCW radar is presented with rigorous mathematical formulas, and further the multiple-target displacement measurement is analyzed and simulated. In addition, a FMCW radar prototype is designed and fabricated based on an off-the-shelf radar frontend and data acquisition (DAQ) card, and the displacement error induced by phase asynchronism is analyzed. The conducted outdoor experiments verify the feasibility of this sensing method applied to multi-target displacement measurement, and experimental results show that three targets located at different distances can be distinguished simultaneously with millimeter level accuracy.

Uniform amplification of phage display libraries in monodisperse emulsions.

PubMed

Matochko, Wadim L; Ng, Simon; Jafari, Mohammad R; Romaniuk, Joseph; Tang, Sindy K Y; Derda, Ratmir

2012-09-01

In this paper, we describe a complete experimental setup for the uniform amplification of libraries of phage. Uniform amplification, which multiplies every phage clone by the same amount irrespective of the growth rate of the clone is essential for phage-display screening. Amplification of phage libraries in a common solution is often non-uniform: it favors fast-growing clones and eliminates those that grow slower. This competition leads to elimination of many useful binding clones, and it is a major barrier to identification of ligands for targets with multiple binding sites such as cells, tissues, or mixtures of proteins. Uniform amplification is achieved by encapsulating individual phage clones into isolated compartments (droplets) of identical volume. Each droplet contains culture medium and an excess of host (Escherichia coli). Here, we describe microfluidics devices that generate mono-disperse droplet-based compartments, and optimal conditions for amplification of libraries of different size. We also describe the detailed synthesis of a perfluoro surfactant, which gives droplets exceptional stability. Droplets stabilized by this compound do not coalesce after many hours in shaking culture. We identified a commercially available compound (Krytox), which destabilizes these droplets to recover the amplified libraries. Overall, uniform amplification is a sequence of three simple steps: (1) encapsulation of mixture of phage and bacteria in droplets using microfluidics; (2) incubation of droplets in a shaking culture; (3) destabilization of droplets to harvest the amplified phage. We anticipate that this procedure can be easily adapted in any academic or industrial laboratory that uses phage display. Copyright © 2012 Elsevier Inc. All rights reserved.

Raman accumulator as a fusion laser driver

DOEpatents

George, E. Victor; Swingle, James C.

1985-01-01

Apparatus for simultaneous laser pulse amplification and compression, using multiple pass Raman scattering in one Raman cell and pulse switchout from the optical cavity through use of a dichroic device associated with the Raman cell.

Raman accumulator as a fusion laser driver

DOEpatents

George, E.V.; Swingle, J.C.

1982-03-31

Apparatus for simultaneous laser pulse amplification and compression, using multiple pass Raman scattering in one Raman cell and pulse switchout from the optical cavity through use of a dichroic device associated with the Raman cell.

Simulation of tsunamis from great earthquakes on the cascadia subduction zone.

PubMed

Ng, M K; Leblond, P H; Murty, T S

1990-11-30

Large earthquakes occur episodically in the Cascadia subduction zone. A numerical model has been used to simulate and assess the hazards of a tsunami generated by a hypothetical earthquake of magnitude 8.5 associated with rupture of the northern sections of the subduction zone. Wave amplitudes on the outer coast are closely related to the magnitude of sea-bottom displacement (5.0 meters). Some amplification, up to a factor of 3, may occur in some coastal embayments. Wave amplitudes in the protected waters of Puget Sound and the Strait of Georgia are predicted to be only about one fifth of those estmated on the outer coast.

Broadband pulsed flow using piezoelectric microjets

NASA Astrophysics Data System (ADS)

Hogue, Joshua; Solomon, John; Hays, Michael; Alvi, Farrukh; Oates, William

2010-04-01

A piezohydraulic microjet design and experimental results are presented to demonstrate broadband active flow control for applications on various aircraft structures including impinging jets, rotor blades, cavity bays, etc. The microjet actuator includes a piezoelectric stack actuator and hydraulic circuit that is used to throttle a 400 μm diameter microjet using hydraulic amplification of the piezoelectric stack actuator. This system is shown to provide broadband pulsed flow actuation up to 800 Hz. Unsteady pressure measurements of the microjet's exit flow are coupled with high-speed phase imagery using micro-Schlieren techniques to quantify the flow field. These results are compared with in situ stack actuator displacements using strain gauge measurements.

Rolling Circle Amplification of Complete Nematode Mitochondrial Genomes

PubMed Central

Tang, Sha; Hyman, Bradley C.

2005-01-01

To enable investigation of nematode mitochondrial DNA evolution, methodology has been developed to amplify intact nematode mitochondrial genomes in preparative yields using a rolling circle replication strategy. Successful reactions were generated from whole cell template DNA prepared by alkaline lysis of the rhabditid nematode Caenorhabditis elegans and a mermithid nematode, Thaumamermis cosgrovei. These taxa, representing the two major nematode classes Chromodorea and Enoplea, maintain mitochondrial genomes of 13.8 kb and 20.0 kb, respectively. Efficient amplifications were conducted on template DNA isolated from individual or pooled nematodes that were alive or stored at -80°C. Unexpectedly, these experiments revealed that multiple T. cosgrovei mitochondrial DNA haplotypes are maintained in our local population. Rolling circle amplification products can be used as templates for standard PCR reactions with specific primers that target mitochondrial genes or for direct DNA sequencing. PMID:19262866

Signal persistence and amplification in cancer development and possible, related opportunities for novel therapies.

PubMed

Ford, Shea A; Blanck, George

2015-01-01

Research in cancer biology has been largely driven by experimental approaches whereby discreet inputs are used to assess discreet outputs, for example, gene-knockouts to assess cancer occurrence. However, cancer hallmarks are only rarely, if ever, exclusively dependent on discreet regulatory processes. Rather, cancer-related regulatory factors affect multiple cancer hallmarks. Thus, novel approaches and paradigms are needed for further advances. Signal pathway persistence and amplification, rather than signal pathway activation resulting from an on/off switch, represent emerging paradigms for cancer research, closely related to developmental regulatory paradigms. In this review, we address both mechanisms and effects of signal pathway persistence and amplification in cancer settings; and address the possibility that hyper-activation of pro-proliferative signal pathways in certain cancer settings could be exploited for therapy. Copyright © 2014. Published by Elsevier B.V.

Jumping translocations of 1q12 in multiple myeloma: a novel mechanism for deletion of 17p in cytogenetically defined high-risk disease.

PubMed

Sawyer, Jeffrey R; Tian, Erming; Heuck, Christoph J; Epstein, Joshua; Johann, Donald J; Swanson, Charles M; Lukacs, Janet L; Johnson, Marian; Binz, Regina; Boast, Angela; Sammartino, Gael; Usmani, Saad; Zangari, Maurizio; Waheed, Sarah; van Rhee, Frits; Barlogie, Bart

2014-04-17

Multiple myeloma (MM) is a B-cell malignancy driven in part by increasing copy number alterations (CNAs) during disease progression. Prognostically significant CNAs accumulate during clonal evolution and include gains of 1q21 and deletions of 17p, among others. Unfortunately, the mechanisms underlying the accumulation of CNAs and resulting subclonal heterogeneity in high-risk MM are poorly understood. To investigate the impact of jumping translocations of 1q12 (JT1q12) on receptor chromosomes (RCs) and subsequent clonal evolution, we analyzed specimens from 86 patients selected for unbalanced 1q12 aberrations by G-banding. Utilizing spectral karyotyping and locus-specific fluorescence in situ hybridization, we identified 10 patients with unexpected focal amplifications of an RC that subsequently translocated as part of a sequential JT1q12 to one or more additional RCs. Four patients exhibited amplification and translocation of 8q24 (MYC), 3 showed amplification of 16q11, and 1 each displayed amplification of 18q21.3 (BCL2), 18q23, or 4p16 (FGFR3). Unexpectedly, in 6 of 14 patients with the combination of the t(4;14) and deletion of 17p, we identified the loss of 17p as resulting from a JT1q12. Here, we provide evidence that the JT1q12 is a mechanism for the simultaneous gain of 1q21 and deletion of 17p in cytogenetically defined high-risk disease.

Two approximations for the geometric model of signal amplification in an electron-multiplying charge-coupled device detector

PubMed Central

Chao, Jerry; Ram, Sripad; Ward, E. Sally; Ober, Raimund J.

2014-01-01

The extraction of information from images acquired under low light conditions represents a common task in diverse disciplines. In single molecule microscopy, for example, techniques for superresolution image reconstruction depend on the accurate estimation of the locations of individual particles from generally low light images. In order to estimate a quantity of interest with high accuracy, however, an appropriate model for the image data is needed. To this end, we previously introduced a data model for an image that is acquired using the electron-multiplying charge-coupled device (EMCCD) detector, a technology of choice for low light imaging due to its ability to amplify weak signals significantly above its readout noise floor. Specifically, we proposed the use of a geometrically multiplied branching process to model the EMCCD detector’s stochastic signal amplification. Geometric multiplication, however, can be computationally expensive and challenging to work with analytically. We therefore describe here two approximations for geometric multiplication that can be used instead. The high gain approximation is appropriate when a high level of signal amplification is used, a scenario which corresponds to the typical usage of an EMCCD detector. It is an accurate approximation that is computationally more efficient, and can be used to perform maximum likelihood estimation on EMCCD image data. In contrast, the Gaussian approximation is applicable at all levels of signal amplification, but is only accurate when the initial signal to be amplified is relatively large. As we demonstrate, it can importantly facilitate the analysis of an information-theoretic quantity called the noise coefficient. PMID:25075263

Continuous in vitro evolution of a ribozyme ligase: a model experiment for the evolution of a biomolecule.

PubMed

Ledbetter, Michael P; Hwang, Tony W; Stovall, Gwendolyn M; Ellington, Andrew D

2013-01-01

Evolution is a defining criterion of life and is central to understanding biological systems. However, the timescale of evolutionary shifts in phenotype limits most classroom evolution experiments to simple probability simulations. In vitro directed evolution (IVDE) frequently serves as a model system for the study of Darwinian evolution but produces noticeable phenotypic shifts in a matter of hours. An IVDE demonstration lab would serve to both directly demonstrate how Darwinian selection can act on a pool of variants and introduce students to an essential method of modern molecular biology. To produce an IVDE demonstration lab, continuous IVDE of a T500 ribozyme ligase population has been paired with a fluorescent strand displacement reporter system to visualize the selection of improved catalytic function. A ribozyme population is taken through rounds of isothermal amplification dependent on the self-ligation of a T7 promoter. As the population is selectively enriched with better ligase activity, the strand displacement system allows for the monitoring of the population's ligation rate. The strand displacement reporter system permits the detection of ligated ribozyme. Once ligated with the T7 promoter, the 5' end of the ribozyme displaces paired fluorophore-quencher oligonucleotides, in turn, generating visible signal upon UV light excitation. As the ligation rate of the population increases, due to the selection for faster ligating species, the fluorescent signal develops more rapidly. The pairing of the continuous isothermal system with the fluorescent reporting scheme allows any user, provided with minimal materials, to model the continuous directed evolution of a biomolecule. Copyright © 2013 Wiley-Liss, Inc.

Ankle morphology amplifies calcaneus movement relative to triceps surae muscle shortening.

PubMed

Csapo, R; Hodgson, J; Kinugasa, R; Edgerton, V R; Sinha, S

2013-08-15

The present study investigated the mechanical role of the dorsoventral curvature of the Achilles tendon in the conversion of the shortening of the plantarflexor muscles into ankle joint rotation. Dynamic, sagittal-plane magnetic resonance spin-tagged images of the ankle joint were acquired in six healthy subjects during both passive and active plantarflexion movements driven by a magnetic resonance compatible servomotor-controlled foot-pedal device. Several points on these images were tracked to determine the 1) path and deformation of the Achilles tendon, 2) ankle's center of rotation, and 3) tendon moment arms. The degree of mechanical amplification of joint movement was calculated as the ratio of the displacements of the calcaneus and myotendinous junction. In plantarflexion, significant deflection of the Achilles tendon was evident in both the passive (165.7 ± 7.4°; 180° representing a straight tendon) and active trials (166.9 ± 8.8°). This bend in the dorsoventral direction acts to move the Achilles tendon closer to the ankle's center of rotation, resulting in an ∼5% reduction of moment arm length. Over the entire range of movement, the overall displacement of the calcaneus exceeded the displacement of the myotendinous junction by ∼37%, with the mechanical gains being smaller in dorsi- and larger in plantarflexed joint positions. This is the first study to assess noninvasively and in vivo using MRI the curvature of the Achilles tendon during both passive and active plantarflexion movements. The dorsoventral tendon curvature amplifies the shortening of the plantarflexor muscles, resulting in a greater displacement of the tendon's insertion into the calcaneus compared with its origin.

Prospective identification of parasitic sequences in phage display screens

PubMed Central

Matochko, Wadim L.; Cory Li, S.; Tang, Sindy K.Y.; Derda, Ratmir

2014-01-01

Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.01% of the available diversity. Specifically, a library of 109 random 7-mer peptides (Ph.D.-7) includes a few thousand sequences that grow quickly (the ‘parasites’), which are the sequences that are typically identified in phage display screens published to date. A similar collapse was observed in other libraries. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of Ph.D.-7 library, we identified a focused population of 770 ‘parasites’. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g. target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: (i) target-binding selection, and (ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ∼106 diverse clones prevents the biased selection of parasitic clones. PMID:24217917

Optimization of multiplexed PCR on an integrated microfluidic forensic platform for rapid DNA analysis.

PubMed

Estes, Matthew D; Yang, Jianing; Duane, Brett; Smith, Stan; Brooks, Carla; Nordquist, Alan; Zenhausern, Frederic

2012-12-07

This study reports the design, prototyping, and assay development of multiplexed polymerase chain reaction (PCR) on a plastic microfluidic device. Amplification of 17 DNA loci is carried out directly on-chip as part of a system for continuous workflow processing from sample preparation (SP) to capillary electrophoresis (CE). For enhanced performance of on-chip PCR amplification, improved control systems have been developed making use of customized Peltier assemblies, valve actuators, software, and amplification chemistry protocols. Multiple enhancements to the microfluidic chip design have been enacted to improve the reliability of sample delivery through the various on-chip modules. This work has been enabled by the encapsulation of PCR reagents into a solid phase material through an optimized Solid Phase Encapsulating Assay Mix (SPEAM) bead-based hydrogel fabrication process. SPEAM bead technology is reliably coupled with precise microfluidic metering and dispensing for efficient amplification and subsequent DNA short tandem repeat (STR) fragment analysis. This provides a means of on-chip reagent storage suitable for microfluidic automation, with the long shelf-life necessary for point-of-care (POC) or field deployable applications. This paper reports the first high quality 17-plex forensic STR amplification from a reference sample in a microfluidic chip with preloaded solid phase reagents, that is designed for integration with up and downstream processing.

Quinone Methide Signal Amplification: Covalent Reporter Labeling of Cancer Epitopes using Alkaline Phosphatase Substrates.

PubMed

Polaske, Nathan W; Kelly, Brian D; Ashworth-Sharpe, Julia; Bieniarz, Christopher

2016-03-16

Diagnostic assays with the sensitivity required to improve cancer therapeutics depend on the development of new signal amplification technologies. Herein, we report the development and application of a novel amplification system which utilizes latent quinone methides (QMs) activated by alkaline phosphatase (AP) for signal amplification in solid-phase immunohistochemical (IHC) assays. Phosphate-protected QM precursor substrates were prepared and conjugated to either biotin or a fluorophore through an amine-functionalized linker group. Upon reaction with AP, the phosphate group is cleaved, followed by elimination of the leaving group and formation of the highly reactive and short-lived QM. The QMs either react with tissue nucleophiles in close proximity to their site of generation, or are quenched by nucleophiles in the reaction media. The reporter molecules that covalently bind to the tissue were then detected visually by fluorescence microscopy in the case of fluorophore reporters, or brightfield microscopy using diaminobenzidine (DAB) in the case of biotin reporters. With multiple reporters deposited per enzyme, significant signal amplification was observed utilizing QM precursor substrates containing either benzyl difluoro or benzyl monofluoro leaving group functionalities. However, the benzyl monofluoro leaving group gave superior results with respect to both signal intensity and discretion, the latter of which was found to be imperative for use in diagnostic IHC assays.

Formation of template-switching artifacts by linear amplification.

PubMed

Chakravarti, Dhrubajyoti; Mailander, Paula C

2008-07-01

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

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.

ILF2 Is a Regulator of RNA Splicing and DNA Damage Response in 1q21-Amplified Multiple Myeloma.

PubMed

Marchesini, Matteo; Ogoti, Yamini; Fiorini, Elena; Aktas Samur, Anil; Nezi, Luigi; D'Anca, Marianna; Storti, Paola; Samur, Mehmet Kemal; Ganan-Gomez, Irene; Fulciniti, Maria Teresa; Mistry, Nipun; Jiang, Shan; Bao, Naran; Marchica, Valentina; Neri, Antonino; Bueso-Ramos, Carlos; Wu, Chang-Jiun; Zhang, Li; Liang, Han; Peng, Xinxin; Giuliani, Nicola; Draetta, Giulio; Clise-Dwyer, Karen; Kantarjian, Hagop; Munshi, Nikhil; Orlowski, Robert; Garcia-Manero, Guillermo; DePinho, Ronald A; Colla, Simona

2017-07-10

Amplification of 1q21 occurs in approximately 30% of de novo and 70% of relapsed multiple myeloma (MM) and is correlated with disease progression and drug resistance. Here, we provide evidence that the 1q21 amplification-driven overexpression of ILF2 in MM promotes tolerance of genomic instability and drives resistance to DNA-damaging agents. Mechanistically, elevated ILF2 expression exerts resistance to genotoxic agents by modulating YB-1 nuclear localization and interaction with the splicing factor U2AF65, which promotes mRNA processing and the stabilization of transcripts involved in homologous recombination in response to DNA damage. The intimate link between 1q21-amplified ILF2 and the regulation of RNA splicing of DNA repair genes may be exploited to optimize the use of DNA-damaging agents in patients with high-risk MM. Copyright © 2017 Elsevier Inc. All rights reserved.

Plug-and-play, infrared, laser-mediated PCR in a microfluidic chip.

PubMed

Pak, Nikita; Saunders, D Curtis; Phaneuf, Christopher R; Forest, Craig R

2012-04-01

Microfluidic polymerase chain reaction (PCR) systems have set milestones for small volume (100 nL-5 μL), amplification speed (100-400 s), and on-chip integration of upstream and downstream sample handling including purification and electrophoretic separation functionality. In practice, the microfluidic chips in these systems require either insertion of thermocouples or calibration prior to every amplification. These factors can offset the speed advantages of microfluidic PCR and have likely hindered commercialization. We present an infrared, laser-mediated, PCR system that features a single calibration, accurate and repeatable precision alignment, and systematic thermal modeling and management for reproducible, open-loop control of PCR in 1 μL chambers of a polymer microfluidic chip. Total cycle time is less than 12 min: 1 min to fill and seal, 10 min to amplify, and 1 min to recover the sample. We describe the design, basis for its operation, and the precision engineering in the system and microfluidic chip. From a single calibration, we demonstrate PCR amplification of a 500 bp amplicon from λ-phage DNA in multiple consecutive trials on the same instrument as well as multiple identical instruments. This simple, relatively low-cost plug-and-play design is thus accessible to persons who may not be skilled in assembly and engineering.

Variable-Displacement Hydraulic Drive Unit

NASA Technical Reports Server (NTRS)

Lang, D. J.; Linton, D. J.; Markunas, A.

1986-01-01

Hydraulic power controlled through multiple feedback loops. In hydraulic drive unit, power closely matched to demand, thereby saving energy. Hydraulic flow to and from motor adjusted by motor-control valve connected to wobbler. Wobbler angle determines motor-control-valve position, which in turn determines motor displacement. Concept applicable to machine tools, aircraft controls, and marine controls.

Shape and 3D acoustically induced vibrations of the human eardrum characterized by digital holography

NASA Astrophysics Data System (ADS)

Khaleghi, Morteza; Furlong, Cosme; Cheng, Jeffrey Tao; Rosowski, John J.

2014-07-01

The eardrum or Tympanic Membrane (TM) transfers acoustic energy from the ear canal (at the external ear) into mechanical motions of the ossicles (at the middle ear). The acousto-mechanical-transformer behavior of the TM is determined by its shape and mechanical properties. For a better understanding of hearing mysteries, full-field-of-view techniques are required to quantify shape, nanometer-scale sound-induced displacement, and mechanical properties of the TM in 3D. In this paper, full-field-of-view, three-dimensional shape and sound-induced displacement of the surface of the TM are obtained by the methods of multiple wavelengths and multiple sensitivity vectors with lensless digital holography. Using our developed digital holographic systems, unique 3D information such as, shape (with micrometer resolution), 3D acoustically-induced displacement (with nanometer resolution), full strain tensor (with nano-strain resolution), 3D phase of motion, and 3D directional cosines of the displacement vectors can be obtained in full-field-ofview with a spatial resolution of about 3 million points on the surface of the TM and a temporal resolution of 15 Hz.

Amplification of a Gene Related to Mammalian mdr Genes in Drug-Resistant Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Wilson, Craig M.; Serrano, Adelfa E.; Wasley, Annemarie; Bogenschutz, Michael P.; Shankar, Anuraj H.; Wirth, Dyann F.

1989-06-01

The malaria parasite Plasmodium falciparum contains at least two genes related to the mammalian multiple drug resistance genes, and at least one of the P. falciparum genes is expressed at a higher level and is present in higher copy number in a strain that is resistant to multiple drugs than in a strain that is sensitive to the drugs.

Study of the Pan-ERBB Inhibitor Neratinib Given in Combination With Everolimus, Palbociclib or Trametinib in Advanced Cancer Subjects With EGFR Mutation/Amplification, HER2 Mutation/Amplification or HER3/4 Mutation

ClinicalTrials.gov

2018-03-26

Malignant Neoplasm of Breast; Malignant Neoplasms of Digestive Organs; Malignant Neoplasms of Female Genital Organs; Malignant Neoplasms of Ill-defined Secondary and Unspecified Sites; Malignant Neoplasms of Independent (Primary) Multiple Sites; Malignant Neoplasms of Lip Oral Cavity and Pharynx; Malignant Neoplasms of Mesothelial and Soft Tissue; Malignant Neoplasms of Respiratory and Intrathoracic Organs; Malignant Neoplasms of Thyroid and Other Endocrine Glands; Malignant Neoplasms of Urinary Tract; Neoplasms of Uncertain or Unknown Behavior

Surmounting a PCR challenge using a Contradictory matrix from the Theory of Inventive Problem Solving (TRIZ).

PubMed

Drábek, Jiří

2016-01-01

In this paper I tested whether Contradictory Matrix with 40 Inventive Principles, the simplest instrument from the Theory of Inventive Problem Solving (TRIZ), is a useful approach to a real-life PCR scenario. The PCR challenge consisted of standardization of fluorescence melting curve measurements in Competitive Amplification of Differentially Melting Amplicons (CADMA) PCR for multiple targets. Here I describe my way of using the TRIZ Matrix to generate seven alternative solutions from which I can choose the successful solution, consisting of repeated cycles of amplification and melting in a single PCR run.

Isothermal amplification of environmental DNA (eDNA) for direct field-based monitoring and laboratory confirmation of Dreissena sp.

PubMed

Williams, Maggie R; Stedtfeld, Robert D; Engle, Cathrine; Salach, Paul; Fakher, Umama; Stedtfeld, Tiffany; Dreelin, Erin; Stevenson, R Jan; Latimore, Jo; Hashsham, Syed A

2017-01-01

Loop-mediated isothermal amplification (LAMP) of aquatic invasive species environmental DNA (AIS eDNA) was used for rapid, sensitive, and specific detection of Dreissena sp. relevant to the Great Lakes (USA) basin. The method was validated for two uses including i) direct amplification of eDNA using a hand filtration system and ii) confirmation of the results after DNA extraction using a conventional thermal cycler run at isothermal temperatures. Direct amplification eliminated the need for DNA extraction and purification and allowed detection of target invasive species in grab or concentrated surface water samples, containing both free DNA as well as larger cells and particulates, such as veligers, eggs, or seeds. The direct amplification method validation was conducted using Dreissena polymorpha and Dreissena bugensis and uses up to 1 L grab water samples for high target abundance (e.g., greater than 10 veligers (larval mussels) per L for Dreissena sp.) or 20 L samples concentrated through 35 μm nylon screens for low target abundance, at less than 10 veligers per liter water. Surface water concentrate samples were collected over a period of three years, mostly from inland lakes in Michigan with the help of a network of volunteers. Field samples collected from 318 surface water locations included i) filtered concentrate for direct amplification validation and ii) 1 L grab water sample for eDNA extraction and confirmation. Though the extraction-based protocol was more sensitive (resulting in more positive detections than direct amplification), direct amplification could be used for rapid screening, allowing for quicker action times. For samples collected between May and August, results of eDNA direct amplification were consistent with known presence/absence of selected invasive species. A cross-platform smartphone application was also developed to disseminate the analyzed results to volunteers. Field tests of the direct amplification protocol using a portable device (Gene-Z) showed the method could be used in the field to obtain results within one hr (from sample to result). Overall, the direct amplification has the potential to simplify the eDNA-based monitoring of multiple aquatic invasive species. Additional studies are warranted to establish quantitative correlation between eDNA copy number, veliger, biomass or organismal abundance in the field.

Isothermal amplification of environmental DNA (eDNA) for direct field-based monitoring and laboratory confirmation of Dreissena sp.

PubMed Central

Stedtfeld, Robert D.; Engle, Cathrine; Salach, Paul; Fakher, Umama; Stedtfeld, Tiffany; Dreelin, Erin; Stevenson, R. Jan; Latimore, Jo; Hashsham, Syed A.

2017-01-01

Loop-mediated isothermal amplification (LAMP) of aquatic invasive species environmental DNA (AIS eDNA) was used for rapid, sensitive, and specific detection of Dreissena sp. relevant to the Great Lakes (USA) basin. The method was validated for two uses including i) direct amplification of eDNA using a hand filtration system and ii) confirmation of the results after DNA extraction using a conventional thermal cycler run at isothermal temperatures. Direct amplification eliminated the need for DNA extraction and purification and allowed detection of target invasive species in grab or concentrated surface water samples, containing both free DNA as well as larger cells and particulates, such as veligers, eggs, or seeds. The direct amplification method validation was conducted using Dreissena polymorpha and Dreissena bugensis and uses up to 1 L grab water samples for high target abundance (e.g., greater than 10 veligers (larval mussels) per L for Dreissena sp.) or 20 L samples concentrated through 35 μm nylon screens for low target abundance, at less than 10 veligers per liter water. Surface water concentrate samples were collected over a period of three years, mostly from inland lakes in Michigan with the help of a network of volunteers. Field samples collected from 318 surface water locations included i) filtered concentrate for direct amplification validation and ii) 1 L grab water sample for eDNA extraction and confirmation. Though the extraction-based protocol was more sensitive (resulting in more positive detections than direct amplification), direct amplification could be used for rapid screening, allowing for quicker action times. For samples collected between May and August, results of eDNA direct amplification were consistent with known presence/absence of selected invasive species. A cross-platform smartphone application was also developed to disseminate the analyzed results to volunteers. Field tests of the direct amplification protocol using a portable device (Gene-Z) showed the method could be used in the field to obtain results within one hr (from sample to result). Overall, the direct amplification has the potential to simplify the eDNA-based monitoring of multiple aquatic invasive species. Additional studies are warranted to establish quantitative correlation between eDNA copy number, veliger, biomass or organismal abundance in the field. PMID:29036210

Measuring relative-story displacement and local inclination angle using multiple position-sensitive detectors.

PubMed

Matsuya, Iwao; Katamura, Ryuta; Sato, Maya; Iba, Miroku; Kondo, Hideaki; Kanekawa, Kiyoshi; Takahashi, Motoichi; Hatada, Tomohiko; Nitta, Yoshihiro; Tanii, Takashi; Shoji, Shuichi; Nishitani, Akira; Ohdomari, Iwao

2010-01-01

We propose a novel sensor system for monitoring the structural health of a building. The system optically measures the relative-story displacement during earthquakes for detecting any deformations of building elements. The sensor unit is composed of three position sensitive detectors (PSDs) and lenses capable of measuring the relative-story displacement precisely, even if the PSD unit was inclined in response to the seismic vibration. For verification, laboratory tests were carried out using an Xθ-stage and a shaking table. The static experiment verified that the sensor could measure the local inclination angle as well as the lateral displacement. The dynamic experiment revealed that the accuracy of the sensor was 150 μm in the relative-displacement measurement and 100 μrad in the inclination angle measurement. These results indicate that the proposed sensor system has sufficient accuracy for the measurement of relative-story displacement in response to the seismic vibration.

Static non-reciprocity in mechanical metamaterials.

PubMed

Coulais, Corentin; Sounas, Dimitrios; Alù, Andrea

2017-02-23

Reciprocity is a general, fundamental principle governing various physical systems, which ensures that the transfer function-the transmission of a physical quantity, say light intensity-between any two points in space is identical, regardless of geometrical or material asymmetries. Breaking this transmission symmetry offers enhanced control over signal transport, isolation and source protection. So far, devices that break reciprocity (and therefore show non-reciprocity) have been mostly considered in dynamic systems involving electromagnetic, acoustic and mechanical wave propagation associated with fields varying in space and time. Here we show that it is possible to break reciprocity in static systems, realizing mechanical metamaterials that exhibit vastly different output displacements under excitation from different sides, as well as one-way displacement amplification. This is achieved by combining large nonlinearities with suitable geometrical asymmetries and/or topological features. In addition to extending non-reciprocity and isolation to statics, our work sheds light on energy propagation in nonlinear materials with asymmetric crystalline structures and topological properties. We anticipate that breaking reciprocity will open avenues for energy absorption, conversion and harvesting, soft robotics, prosthetics and optomechanics.

New type of eddy current sensor for large-displacement test

NASA Astrophysics Data System (ADS)

Pan, Haifeng; Zhu, Huizhong; Fu, Zhibin; Xu, Yuzheng; Feng, Guanping

2001-09-01

In this paper a new type of large displacement eddy current sensor is developed to overcome the three main disadvantages of the traditional eddy current senor. For a traditional sensor, the measurement range is limited and less than one half of the diameter of the measuring coil. The output is high sensitivity to the changes of the target material and the cable length connected between the probe and the preamplifier. When the material or the cable length changed, it is necessary to readjust the preamplifier. The probe of the new eddy current sensor has three coaxial measuring coils, one is an exciting coil and the other two are receiving coils. The diameter of measuring coils is (Phi) 11 mm. The measurement range of this sensor is 40mm and almost four times of the diameter of the coil. The form of differential input and feedback amplification is used in signal amplifier. Thus the effect of the common modules, such as the changes of the target material and the cable length, can be counteracted well.

A micropower miniature piezoelectric actuator for implantable middle ear hearing device.

PubMed

Wang, Zhigang; Mills, Robert; Luo, Hongyan; Zheng, Xiaolin; Hou, Wensheng; Wang, Lijun; Brown, Stuart I; Cuschieri, Alfred

2011-02-01

This paper describes the design and development of a small actuator using a miniature piezoelectric stack and a flextensional mechanical amplification structure for an implantable middle ear hearing device (IMEHD). A finite-element method was used in the actuator design. Actuator vibration displacement was measured using a laser vibrometer. Preliminary evaluation of the actuator for an IMEHD was conducted using a temporal bone model. Initial results from one temporal bone study indicated that the actuator was small enough to be implanted within the middle ear cavity, and sufficient stapes displacement can be generated for patients with mild to moderate hearing losses, especially at higher frequency range, by the actuator suspended onto the stapes. There was an insignificant mass-loading effect on normal sound transmission (<3 dB) when the actuator was attached to the stapes and switched off. Improved vibration performance is predicted by more firm attachment. The actuator power consumption and its generated equivalent sound pressure level are also discussed. In conclusion, the actuator has advantages of small size, lightweight, and micropower consumption for potential use as IMHEDs.

War, forced displacement and growth in Laotian adults.

PubMed

Clarkin, Patrick F

2012-01-01

Evidence from several populations suggests that war negatively impacts civilian nutrition, physical growth and overall health. This effect is often enduring or permanent, particularly if experienced early in life. To assess whether the number of lifetime displacement experiences and being displaced in infancy were associated with adult height, sitting height, leg length and the sitting height ratio. Retrospective questionnaires on displacement and resettlement experiences and anthropometric data were collected from a sample of Laotian adult refugees (ethnic Hmong and Lao; n = 365). All were born in Laos or Thailand and had resettled in French Guiana or the US. Many had been displaced several times by military conflict in Laos. In bivariate analyses, being displaced in infancy and the number of lifetime displacement experiences one had were negatively associated with final adult height and leg length in both sexes. The association was stronger in females, particularly Hmong females. There was no significant association between total displacement experiences and the sitting height ratio. In multiple regression analyses, linear growth in males was negatively associated with being displaced in infancy; in females, the number of lifetime displacement experiences was a significant predictor. Forced displacement from war appears to have a lasting effect on final adult height, sitting height and leg length, although not necessarily on the sitting height ratio in this sample.

Screw-actuated displacement micropumps for thermoplastic microfluidics.

PubMed

Han, J Y; Rahmanian, O D; Kendall, E L; Fleming, N; DeVoe, D L

2016-10-05

The fabrication of on-chip displacement pumps integrated into thermoplastic chips is explored as a simple and low cost method for achieving precise and programmable flow control for disposable microfluidic systems. The displacement pumps consist of stainless steel screws inserted into threaded ports machined into a thermoplastic substrate which also serve as on-chip reagent storage reservoirs. Three different methods for pump sealing are investigated to enable high pressure flows without leakage, and software-defined control of multiple pumps is demonstrated in a self-contained platform using a compact and self-contained microcontroller for operation. Using this system, flow rates ranging from 0.5-40 μl min -1 are demonstrated. The pumps are combined with on-chip burst valves to fully seal multiple reagents into fabricated chips while providing on-demand fluid distribution in a downstream microfluidic network, and demonstrated for the generation of size-tunable water-in-oil emulsions.

Investigating acoustic-induced deformations in a foam using multiple light scattering.

PubMed

Erpelding, M; Guillermic, R M; Dollet, B; Saint-Jalmes, A; Crassous, J

2010-08-01

We have studied the effect of an external acoustic wave on bubble displacements inside an aqueous foam. The signature of the acoustic-induced bubble displacements is found using a multiple light scattering technique, and occurs as a modulation on the photon correlation curve. Measurements for various sound frequencies and amplitudes are compared to analytical predictions and numerical simulations. These comparisons finally allow us to elucidate the nontrivial acoustic displacement profile inside the foam; in particular, we find that the acoustic wave creates a localized shear in the vicinity of the solid walls holding the foam, as a consequence of inertial contributions. This study of how bubbles "dance" inside a foam as a response to sound turns out to provide new insights on foam acoustics and sound transmission into a foam, foam deformation at high frequencies, and analysis of light scattering data in samples undergoing nonhomogeneous deformations.

Displaceable Spur Gear Torque Controlled Driver and Method

NASA Technical Reports Server (NTRS)

Cook, Joseph S., Jr. (Inventor)

1996-01-01

Methods and apparatus are provided for a torque driver including a laterally displaceable gear support member to carry an output spur gear. A biasing assembly biases the output spur gear into engagement with a pinion to which is applied an input torque greater than a desired output torque limit for a threaded fastener such as a nut or screw. A coiled output linkage connects the output spur gear with a fastener adaptor which may be a socket for a nut. A gear tooth profile provides a separation force that overcomes the bias to limit torque at the desired torque limit. Multiple fasteners may be rotated simultaneously to a desired torque limit if additional output spur gears are provided. A gauged selector mechanism is provided to laterally displace multiple driven members for fasteners arranged in differing configurations. The torque limit is selectably adjustable and may be different for fasteners within the same fastener configuration.

Displaceable spur gear torque controlled driver and method

NASA Technical Reports Server (NTRS)

Cook, Joseph S., Jr. (Inventor)

1994-01-01

Methods and apparatus are provided for a torque driver including a laterally displaceable gear support member to carry an output spur gear. A biasing assembly biases the output spur gear into engagement with a pinion to which is applied an input torque greater than a desired output torque limit for a threaded fastener such as a nut or screw. A coiled output linkage connects the output spur gear with a fastener adaptor which may be a socket for a nut. A gear tooth profile provides a separation force that overcomes the bias to limit torque at the desired torque limit. Multiple fasteners may be rotated simultaneously to a desired torque limit if additional output spur gears are provided. A gauged selector mechanism is provided to laterally displace multiple driver members for fasteners arranged in differing configurations. The torque limit is selectably adjustable and may be different for fasteners within the same fastener configuration.

Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells

PubMed Central

Han, Lin; Wu, Hua-Jun; Zhu, Haiying; Kim, Kun-Yong; Marjani, Sadie L.; Riester, Markus; Euskirchen, Ghia; Zi, Xiaoyuan; Yang, Jennifer; Han, Jasper; Snyder, Michael; Park, In-Hyun; Irizarry, Rafael; Weissman, Sherman M.

2017-01-01

Abstract Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population. PMID:28126923

Chronic low back pain and the transdiagnostic process: How do cognitive and emotional dysregulations contribute to the intensity of risk factors and pain?

PubMed

Le Borgne, Margaux; Boudoukha, Abdel Halim; Petit, Audrey; Roquelaure, Yves

2017-10-01

Based on a transdiagnostic approach, this study assesses the impact of cognitive and emotional processes (difficulties in emotional regulation, impulsiveness, rumination and somatosensory amplification) on the psychological risk factors of chronic low-back pain. The study was carried out with 256 patients with chronic low-back pain. All the variables were assessed through a booklet of 10 validated questionnaires. Multiple regression analysis and moderation analysis were performed. Predictors included in multiple regression models explain 3%-42% (adjusted R 2 ) of the variance in psychological risk factors. Moreover, analyses reveal a significant moderator effect of somatosensory amplification on the link between fear-avoidance beliefs linked to work and pain intensity (F (3;250) =12.33; p=.00), of somatosensory amplification and brooding on the link between depression and functional repercussions (FR) on everyday life (F (3;252) =13.36; p=.000; F (1;252) =12.42; p=.00), of the reflection dimension of rumination on the link between the helplessness dimension of catastrophizing and FRs on sociability (F (3;252) =37.02; p=.00). There is also a moderation analysis with a significant trend concerning the lack of emotional awareness and the difficulties in controlling impulsive behaviours. Our results indicate an important role of some dimensions of difficulties in emotional regulation, somatosensory amplification and rumination in the increase in negative affects and dysfunctional beliefs, and in the links between those psychological risk factors and pain/disability. This study identifies some cognitive and emotional dysregulations substantially involved in work-related chronic pain. This contribute to put in place psychotherapeutic protocols to tackle these deficits and dysregulations in a relevant way. Copyright © 2017 Scandinavian Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Determination of EGFR mutations in single cells microdissected from enriched lung tumor cells in peripheral blood.

PubMed

Ran, Ran; Li, Longyun; Wang, Mengzhao; Wang, Shulan; Zheng, Zhi; Lin, Peter Ping

2013-09-01

A minimally invasive and repeatable approach for real-time epidermal growth factor receptor (EGFR) mutation surveillance would be highly beneficial for individualized therapy of late stage lung cancer patients whose surgical specimens are often not available. We aim to develop a viable method to detect EGFR mutations in single circulating tumor cells (CTCs). Using a model CTC system of spiked tumor cells in whole blood, we evaluated EGFR mutation determination in single tumor cells enriched from blood. We used magnetic beads labeled with antibody against leukocyte surface antigens to deplete leukocytes and enrich native CTCs independent of epithelial marker expression level. We then used laser cell microdissection (LCM) to isolate individual CTCs, followed by whole-genome amplification of the DNA for exon 19 microdeletion, L858R and T790M mutation detection by PCR sequencing. EGFR mutations were successfully measured in individual spiked tumor cells enriched from 7.5 ml whole blood. Whole-genome amplification provided sufficient DNA for mutation determination at multiple sites. Ninety-five percent of the single CTCs microdissected by LCM (19/20) yielded PCR amplicons for at least one of the three mutation sites. The amplification success rates were 55 % (11/20) for exon 19 deletion, 45 % (9/20) for T790M, and 85 % (17/20) for L858R. Sequencing of the amplicons showed allele dropout in the amplification reactions, but mutations were correctly identified in 80 % of the amplicons. EGFR mutation determination from single captured tumor cells from blood is feasible with the approach described here. However, to overcome allele dropout and to obtain reliable information about the tumor's EGFR status, multiple individual tumor cells should be assayed.

Examination of the site amplification factor of OBS and their application to magnitude estimation and ground-motion prediction for EEW

NASA Astrophysics Data System (ADS)

Hayashimoto, N.; Hoshiba, M.

2013-12-01

1. Introduction Ocean bottom seismograph (OBS) is useful for making Earthquake Early Warning (EEW) earlier. However, careful handling of these data is required because the installation environment of OBSs may be different from that of land stations. Site amplification factor is an important factor to estimate the magnitudes, and to predict ground motions (e.g. seismic intensity) in EEW. In this presentation, we discuss the site amplification factor of OBS in the Tonankai area of Japan from these two points of view. 2. Examination of magnitude correction of OBS In the EEW of JMA, the magnitude is estimated from the maximum amplitude of the displacement in real time. To provide the fast magnitude estimation, the magnitude-estimation algorithm switches from the P to S formula (Meew(P) to Meew(S)) depending on the expected S-phase arrival (Kamigaichi,2004). To estimate the magnitude correction for OBS, we determine Meew(P) and Meew(S) at OBSs and compare them with JMA magnitude (Mjma). We find Meew(S) at OBS is generally larger than Mjma by approximately 0.6. The slight differences of spatial distribution of Meew(S) amplification are also found among other OBSs. From the numerical simulations, Nakamura et al. (MGR,submitted) pointed out that the oceanic layer and the low-velocity sediment layers causes the large amplifications in low frequency range (0.1-0.2Hz) at OBSs. We conclude that the site effect of OBS characterized by such a low velocity sediment layers causes those amplification of Magnitude. 3. The frequency-dependent site factor of OBS estimated from Fourier spectrum ratio and their application for prediction of seismic intensity of land station We compare Fourier spectra of S-wave portion on OBSs with those on adjacent land stations. Station pair whose distance is smaller than 50 km is analyzed, and we obtain that spectral ratio of land station (MIEH05 of the KiK-net/NIED) to OBS (KMA01 of the DONET/JAMSTEC) is 5-20 for frequencies 10-20Hz for both horizontal and vertical components, whereas it is approximately 0.2 at less than 2Hz for the horizontal component, which corresponds to the relative site amplification factors in the frequency domain. In addition, we compare the accuracies of expected seismic intensity of land stations using the average of seismic intensity difference with those using the spectral ratio as the empirical amplification factor. In an example of station pair mentioned above, the RMS of the difference between measured and predicted seismic intensity is improved by about 38% by using a spectral ratio as the amplification factor. These results indicate that the frequency-dependent site factor is crucial factor to predict seismic intensity from OBS data, and also show that OBS can be used as front stations in the method for prediction of ground motion based on the real-time monitoring (Hoshiba, 2013). Acknowledgement: Waveform data were obtained from the JMA network, DONET of the JAMSTEC, K-net and KiK-net of the NIED.

Experimental demonstration of beaconless beam displacement tracking for an orbital angular momentum multiplexed free-space optical link.

PubMed

Li, Long; Zhang, Runzhou; Xie, Guodong; Ren, Yongxiong; Zhao, Zhe; Wang, Zhe; Liu, Cong; Song, Haoqian; Pang, Kai; Bock, Robert; Tur, Moshe; Willner, Alan E

2018-05-15

In this Letter, we experimentally demonstrate beaconless beam displacement tracking for free-space optical communication link multiplexing multiple orbital angular momentum (OAM) beams, where the data-carrying OAM beams are used for position detection. 400 Gbit/s data transmission is demonstrated under emulated lateral displacement of up to ±10  mm with power penalties of less than 3 dB for all channels. Channel crosstalk is reduced by the beam tracking system to below -18  dB. Moreover, we investigate using a Gaussian beacon for beam displacement tracking, and achieve similar channel crosstalk and power penalties, compared with using the beaconless beam tracking.

Development of a laser Doppler displacement encoder system with ultra-low-noise-level for linear displacement measurement with subnanometer resolution - Final CRADA Report

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

Shu, Deming

An U.S. DOE Cooperative Research and Development Agreement (CRADA) between ANL and Optodyne, Inc. has been established to develop a prototype laser Doppler displacement encoder system with ultra-low noise level for linear measurements to sub-nanometer resolution for synchrotron radiation applications. We have improved the heterodyne efficiency and reduced the detector shot noises by proper shielding and adding a low-pass filter. The laser Doppler displacement encoder system prototype demonstrated a ~ 1 nm system output noise floor with single reflection optics. With multiple-pass optical arrangement, 0.1 nm scale closed-loop feedback control is achieved.

Competing disturbance amplification mechanisms in two-fluid boundary layers

NASA Astrophysics Data System (ADS)

Saha, Sandeep; Page, Jacob; Zaki, Tamer

2015-11-01

The linear stability of boundary layers above a thin wall film of lower viscosity is analyzed. Appropriate choice of the film thickness and viscosity excludes the possibility of interfacial instabilities. Transient amplification of disturbances is therefore the relevant destabilizing influence, and can take place via three different mechanisms in the two-fluid configuration. Each is examined in detail by solving an initial value problem whose initial condition comprises a pair of appropriately chosen eigenmodes from the discrete, continuous and interface modes. Two regimes are driven by the lift-up mechanism: (i) The response to a streamwise vortex and (ii) the normal vorticity generated by a stable Tollmien-Schlichting wave. Both are damped due to the film. The third regime is associated with the wall-normal vorticity that is generated by the interface displacement. It can lead to appreciable streamwise velocity disturbances in the near-wall region at relatively low viscosity ratios. The results demonstrate that a wall film can stabilize the early linear stages of boundary-layer transition, and explain the observations from the recent nonlinear direct numerical simulations of this configuration by Jung & Zaki (J. Fluid Mech., vol 772, 2015, 330-360).

Dynamic interaction of twin vertically overlapping lined tunnels in an elastic half space subjected to incident plane waves

NASA Astrophysics Data System (ADS)

Liu, Zhongxian; Wang, Yirui; Liang, Jianwen

2016-06-01

The scattering of plane harmonic P and SV waves by a pair of vertically overlapping lined tunnels buried in an elastic half space is solved using a semi-analytic indirect boundary integration equation method. Then the effect of the distance between the two tunnels, the stiffness and density of the lining material, and the incident frequency on the seismic response of the tunnels is investigated. Numerical results demonstrate that the dynamic interaction between the twin tunnels cannot be ignored and the lower tunnel has a significant shielding effect on the upper tunnel for high-frequency incident waves, resulting in great decrease of the dynamic hoop stress in the upper tunnel; for the low-frequency incident waves, in contrast, the lower tunnel can lead to amplification effect on the upper tunnel. It also reveals that the frequency-spectrum characteristics of dynamic stress of the lower tunnel are significantly different from those of the upper tunnel. In addition, for incident P waves in low-frequency region, the soft lining tunnels have significant amplification effect on the surface displacement amplitude, which is slightly larger than that of the corresponding single tunnel.

Heterogeneous patterns of amplification of the NUP214-ABL1 fusion gene in T-cell acute lymphoblastic leukemia.

PubMed

Graux, C; Stevens-Kroef, M; Lafage, M; Dastugue, N; Harrison, C J; Mugneret, F; Bahloula, K; Struski, S; Grégoire, M J; Nadal, N; Lippert, E; Taviaux, S; Simons, A; Kuiper, R P; Moorman, A V; Barber, K; Bosly, A; Michaux, L; Vandenberghe, P; Lahortiga, I; De Keersmaecker, K; Wlodarska, I; Cools, J; Hagemeijer, A; Poirel, H A

2009-01-01

Episomes with the NUP214-ABL1 fusion gene have been observed in 6% of T-ALL. In this multicentric study we collected 27 cases of NUP214-ABL1-positive T-ALL. Median age was 15 years with male predominance. Outcome was poor in 12 patients. An associated abnormality involving TLX1 or TLX3 was found in all investigated cases. Fluorescent in situ hybridization revealed a heterogeneous pattern of NUP214-ABL1 amplification. Multiple episomes carrying the fusion were detected in 24 patients. Episomes were observed in a significant number of nuclei in 18 cases, but in only 1-5% of nuclei in 6. In addition, intrachromosomal amplification (small hsr) was identified either as the only change or in association with episomes in four cases and two T-ALL cell lines (PEER and ALL-SIL). One case showed insertion of apparently non-amplified NUP214-ABL1 sequences at 14q12. The amplified sequences were analyzed using array-based CGH.These findings confirm that the NUP214-ABL1 gene requires amplification for oncogenicity; it is part of a multistep process of leukemogenesis; and it can be a late event present only in subpopulations. Data also provide in vivo evidence for a model of episome formation, amplification and optional reintegration into the genome. Implications for the use of kinase inhibitors are discussed.

A novel signal amplification technology for ELISA based on catalyzed reporter deposition. Demonstration of its applicability for measuring aflatoxin B(1).

PubMed

Bhattacharya, D; Bhattacharya, R; Dhar, T K

1999-11-19

In an earlier communication we have described a novel signal amplification technology termed Super-CARD, which is able to significantly improve antigen detection sensitivity in conventional Dot-ELISA by approximately 10(5)-fold. The method utilizes hitherto unreported synthesized electron rich proteins containing multiple phenolic groups which, when immobilized over a solid phase as blocking agent, markedly increases the signal amplification capability of the existing CARD method (Bhattacharya, R., Bhattacharya, D., Dhar, T.K., 1999. A novel signal amplification technology based on catalyzed reporter deposition and its application in a Dot-ELISA with ultra high sensitivity. J. Immunol. Methods 227, 31.). In this paper we describe the utilization of this Super-CARD amplification technique in ELISA and its applicability for the rapid determination of aflatoxin B(1) (AFB(1)) in infected seeds. Using this method under identical conditions, the increase in absorbance over the CARD method was approximately 400%. The limit of detection of AFB(1) by this method was 0.1 pg/well, the sensitivity enhancement being 5-fold over the optimized CARD ELISA. Furthermore, the total incubation time was reduced to 16 min compared to 50 min for the CARD method. Assay specificity was not adversely affected and the amount of AFB(1) measured in seed extracts correlated well with the values obtained by conventional ELISA.

Smart Cup: A Minimally-Instrumented, Smartphone-Based Point-of-Care Molecular Diagnostic Device.

PubMed

Liao, Shih-Chuan; Peng, Jing; Mauk, Michael G; Awasthi, Sita; Song, Jinzhao; Friedman, Harvey; Bau, Haim H; Liu, Changchun

2016-06-28

Nucleic acid amplification-based diagnostics offer rapid, sensitive, and specific means for detecting and monitoring the progression of infectious diseases. However, this method typically requires extensive sample preparation, expensive instruments, and trained personnel. All of which hinder its use in resource-limited settings, where many infectious diseases are endemic. Here, we report on a simple, inexpensive, minimally-instrumented, smart cup platform for rapid, quantitative molecular diagnostics of pathogens at the point of care. Our smart cup takes advantage of water-triggered, exothermic chemical reaction to supply heat for the nucleic acid-based, isothermal amplification. The amplification temperature is regulated with a phase-change material (PCM). The PCM maintains the amplification reactor at a constant temperature, typically, 60-65°C, when ambient temperatures range from 12 to 35°C. To eliminate the need for an optical detector and minimize cost, we use the smartphone's flashlight to excite the fluorescent dye and the phone camera to record real-time fluorescence emission during the amplification process. The smartphone can concurrently monitor multiple amplification reactors and analyze the recorded data. Our smart cup's utility was demonstrated by amplifying and quantifying herpes simplex virus type 2 (HSV-2) with LAMP assay in our custom-made microfluidic diagnostic chip. We have consistently detected as few as 100 copies of HSV-2 viral DNA per sample. Our system does not require any lab facilities and is suitable for use at home, in the field, and in the clinic, as well as in resource-poor settings, where access to sophisticated laboratories is impractical, unaffordable, or nonexistent.

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.

Highly-sensitive microRNA detection based on bio-bar-code assay and catalytic hairpin assembly two-stage amplification.

PubMed

Tang, Songsong; Gu, Yuan; Lu, Huiting; Dong, Haifeng; Zhang, Kai; Dai, Wenhao; Meng, Xiangdan; Yang, Fan; Zhang, Xueji

2018-04-03

Herein, a highly-sensitive microRNA (miRNA) detection strategy was developed by combining bio-bar-code assay (BBA) with catalytic hairpin assembly (CHA). In the proposed system, two nanoprobes of magnetic nanoparticles functionalized with DNA probes (MNPs-DNA) and gold nanoparticles with numerous barcode DNA (AuNPs-DNA) were designed. In the presence of target miRNA, the MNP-DNA and AuNP-DNA hybridized with target miRNA to form a "sandwich" structure. After "sandwich" structures were separated from the solution by the magnetic field and dehybridized by high temperature, the barcode DNA sequences were released by dissolving AuNPs. The released barcode DNA sequences triggered the toehold strand displacement assembly of two hairpin probes, leading to recycle of barcode DNA sequences and producing numerous fluorescent CHA products for miRNA detection. Under the optimal experimental conditions, the proposed two-stage amplification system could sensitively detect target miRNA ranging from 10 pM to 10 aM with a limit of detection (LOD) down to 97.9 zM. It displayed good capability to discriminate single base and three bases mismatch due to the unique sandwich structure. Notably, it presented good feasibility for selective multiplexed detection of various combinations of synthetic miRNA sequences and miRNAs extracted from different cell lysates, which were in agreement with the traditional polymerase chain reaction analysis. The two-stage amplification strategy may be significant implication in the biological detection and clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Elbow joint angle and elbow movement velocity estimation using NARX-multiple layer perceptron neural network model with surface EMG time domain parameters.

PubMed

Raj, Retheep; Sivanandan, K S

2017-01-01

Estimation of elbow dynamics has been the object of numerous investigations. In this work a solution is proposed for estimating elbow movement velocity and elbow joint angle from Surface Electromyography (SEMG) signals. Here the Surface Electromyography signals are acquired from the biceps brachii muscle of human hand. Two time-domain parameters, Integrated EMG (IEMG) and Zero Crossing (ZC), are extracted from the Surface Electromyography signal. The relationship between the time domain parameters, IEMG and ZC with elbow angular displacement and elbow angular velocity during extension and flexion of the elbow are studied. A multiple input-multiple output model is derived for identifying the kinematics of elbow. A Nonlinear Auto Regressive with eXogenous inputs (NARX) structure based multiple layer perceptron neural network (MLPNN) model is proposed for the estimation of elbow joint angle and elbow angular velocity. The proposed NARX MLPNN model is trained using Levenberg-marquardt based algorithm. The proposed model is estimating the elbow joint angle and elbow movement angular velocity with appreciable accuracy. The model is validated using regression coefficient value (R). The average regression coefficient value (R) obtained for elbow angular displacement prediction is 0.9641 and for the elbow anglular velocity prediction is 0.9347. The Nonlinear Auto Regressive with eXogenous inputs (NARX) structure based multiple layer perceptron neural networks (MLPNN) model can be used for the estimation of angular displacement and movement angular velocity of the elbow with good accuracy.

Pulse transmission receiver with higher-order time derivative pulse generator

DOEpatents

Dress, Jr., William B.; Smith, Stephen F.

2003-08-12

Systems and methods for pulse-transmission low-power communication modes are disclosed. A pulse transmission receiver includes: a front-end amplification/processing circuit; a synchronization circuit coupled to the front-end amplification/processing circuit; a clock coupled to the synchronization circuit; a trigger signal generator coupled to the clock; and at least one higher-order time derivative pulse generator coupled to the trigger signal generator. The systems and methods significantly reduce lower-frequency emissions from pulse transmission spread-spectrum communication modes, which reduces potentially harmful interference to existing radio frequency services and users and also simultaneously permit transmission of multiple data bits by utilizing specific pulse shapes.

Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

PubMed

Fu, Glenn K; Wilhelmy, Julie; Stern, David; Fan, H Christina; Fodor, Stephen P A

2014-03-18

We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification.

Whole genome amplification of Chelex-extracted DNA from a single mite: a method for studying genetics of the predatory mite Phytoseiulus persimilis.

PubMed

Konakandla, Bhanu; Park, Yoonseong; Margolies, David

2006-01-01

We developed and optimized a method using Chelex DNA extraction followed by whole genome amplification (WGA) to overcome problems conducting molecular genetic studies due to the limited amount of DNA obtainable from individual small organisms such as predatory mites. The DNA from a single mite, Phytoseiulus persimilis Athias-Henrot (Acari: Phytoseiidae), isolated in Chelex suspension was subjected to WGA. More than 1000-fold amplification of the DNA was achieved using as little as 0.03 ng genomic DNA template. The DNA obtained by the WGA was used for polymerase chain reaction followed by direct sequencing. From WGA DNA, nuclear DNA intergenic spacers ITS1 and ITS2 and a mitochondrial DNA 12S marker were tested in three different geographical populations of the predatory mite: California, the Netherlands, and Sicily. We found a total of four different alleles of the 12S in the Sicilian population, but no polymorphism was identified in the ITS marker. The combination of Chelex DNA extraction and WGA is thus shown to be a simple and robust technique for examining molecular markers for multiple loci by using individual mites. We conclude that the methods, Chelex extraction of DNA followed by WGA, provide a large quantity of DNA template that can be used for multiple PCR reactions useful for genetic studies requiring the genotypes of individual mites.

Goos-Hänchen-like shift in biased silicene

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

Zhu, Bang-Shan; Wang, Yu, E-mail: ywang@semi.ac.cn; Lou, Yi-Yi

2016-04-28

We have theoretically studied the Goos-Hänchen-like shift of spinor-unpolarized beams tunneling through various gate-biased silicene nanostructures. Following the stationary-phase method, lateral displacement in single-, dual-, and multiple-gated silicene systems has been systematically demonstrated. It is shown for simple single-gated silicene that lateral displacement can be generally enhanced by Fabry-Perot interference, and near the transition point turning on the evanescent mode a very large lateral shift could be observed. For the dual-gated structure, we have also shown the crucial role of localized modes like quantum well states in enhancing the beam lateral displacement, while for the multiple gate-biased systems the resultingmore » superlattice subbands are also favorable for lateral displacement enhancement. Importantly, including the degeneracy-broken mechanisms such as gate-field and magnetic modulations, a fully spinor-resolved beam can be distinguished from the rest counterparts by aligning the incident beam with a proper spinor-resolved transition point, localized state, and subband, all of which can be flexibly modulated via electric means, offering the very desirable strategies to achieve the fully spinor-polarized beam for functional electronic applications.« less

Defense Mechanisms: Discussions and Bibliographies; General or Multiple, and Specific.

ERIC Educational Resources Information Center

Pedrini, D. T.; Pedrini, Bonnie C.

This publication considers some Freudian ego mechanisms. The first discussion and bibliography concerns defense mechanisms, in general or in multiple; after which, the discussions and bibliographies concern specific defense mechanisms: denial; displacement, substitution, sublimation; fixation; identification, introjection, incorporation,…

Ultrasensitive and Multiple Disease-Related MicroRNA Detection Based on Tetrahedral DNA Nanostructures and Duplex-Specific Nuclease-Assisted Signal Amplification.

PubMed

Xu, Fang; Dong, Haifeng; Cao, Yu; Lu, Huiting; Meng, Xiangdan; Dai, Wenhao; Zhang, Xueji; Al-Ghanim, Khalid Abdullah; Mahboob, Shahid

2016-12-14

A highly sensitive and multiple microRNA (miRNA) detection method by combining three-dimensional (3D) DNA tetrahedron-structured probes (TSPs) to increase the probe reactivity and accessibility with duplex-specific nuclease (DSN) for signal amplification for sensitive miRNA detection was proposed. Briefly, 3D DNA TSPs labeled with different fluorescent dyes for specific target miRNA recognition were modified on a gold nanoparticle (GNP) surface to increase the reactivity and accessibility. Upon hybridization with a specific target, the TSPs immobilized on the GNP surface hybridized with the corresponding target miRNA to form DNA-RNA heteroduplexes, and the DSN can recognize the formed DNA-RNA heteroduplexes to hydrolyze the DNA in the heteroduplexes to produce a specific fluorescent signal corresponding to a specific miRNA, while the released target miRNA strands can initiate another cycle, resulting in a significant signal amplification for sensitive miRNA detection. Different targets can produce different fluorescent signals, leading to the development of a sensitive detection for multiple miRNAs in a homogeneous solution. Under optimized conditions, the proposed assay can simultaneously detect three different miRNAs in a homogeneous solution with a logarithmic linear range spanning 5 magnitudes (10 -12 -10 -16 ) and achieving a limit of detection down to attomolar concentrations. Meanwhile, the proposed miRNA assay exhibited the capability of discriminating single bases (three bases mismatched miRNAs) and showed good eligibility in the analysis of miRNAs extracted from cell lysates and miRNAs in cell incubation media, which indicates its potential use in biomedical research and clinical analysis.

Development and Evolution of Character Displacement

PubMed Central

Pfennig, David W.; Pfennig, Karin S.

2012-01-01

Character displacement occurs when competition for either resources or successful reproduction imposes divergent selection on interacting species, causing divergence in traits associated with resource use or reproduction. Here, we describe how character displacement can be mediated either by genetically canalized changes (i.e., changes that reflect allelic or genotype frequency changes) or by phenotypic plasticity. We also discuss how these two mechanisms influence the tempo of character displacement. Specifically, we suggest that, under some conditions, character displacement mediated by phenotypic plasticity might occur more rapidly than that mediated by genetically canalized changes. Finally, we describe how these two mechanisms may act together and determine character displacement’s mode, such that it proceeds through an initial phase in which trait divergence is environmentally induced to a later phase in which divergence becomes genetically canalized. This plasticity-first hypothesis predicts that character displacement should be generally mediated by ancestral plasticity and that it will arise similarly in multiple, independently evolving populations. We conclude by highlighting future directions for research that would test these predictions. PMID:22257002

Multiple-degree-of-freedom vehicle

DOEpatents

Borenstein, Johann

1995-01-01

A multi-degree-of-freedom vehicle employs a compliant linkage to accommodate the need for a variation in the distance between drive wheels or drive systems which are independently steerable and drivable. The subject vehicle is provided with rotary encodes to provide signals representative of the orientation of the steering pivot associated with each such drive wheel or system, and a linear encoder which issues a signal representative of the fluctuations in the distance between the drive elements. The wheels of the vehicle are steered and driven in response to the linear encoder signal, there being provided a controller system for minimizing the fluctuations in the distance. The controller system is a software implementation of a plurality of controllers, operating at the chassis level and at the vehicle level. A trajectory interpolator receives x-displacement, y-displacement, and .theta.-displacement signals and produces to the vehicle level controller trajectory signals corresponding to interpolated control signals. The x-displacement, y-displacement, and .theta.-displacement signals are received from a human operator, via a manipulable joy stick.

Methods for In-Flight Wing Shape Predictions of Highly Flexible Unmanned Aerial Vehicles: Formulation of Ko Displacement Theory

NASA Technical Reports Server (NTRS)

Ko, William L.; Fleischer, Van Tran

2010-01-01

The Ko displacement theory is formulated for a cantilever tubular wing spar under bending, torsion, and combined bending and torsion loading. The Ko displacement equations are expressed in terms of strains measured at multiple sensing stations equally spaced on the surface of the wing spar. The bending and distortion strain data can then be input to the displacement equations to calculate slopes, deflections, and cross-sectional twist angles of the wing spar at the strain-sensing stations for generating the deformed shapes of flexible aircraft wing spars. The displacement equations have been successfully validated for accuracy by finite-element analysis. The Ko displacement theory that has been formulated could also be applied to calculate the deformed shape of simple and tapered beams, plates, and tapered cantilever wing boxes. The Ko displacement theory and associated strain-sensing system (such as fiber optic sensors) form a powerful tool for in-flight deformation monitoring of flexible wings and tails, such as those often employed on unmanned aerial vehicles. Ultimately, the calculated displacement data can be visually displayed in real time to the ground-based pilot for monitoring the deformed shape of unmanned aerial vehicles during flight.

Ecological multiplex interactions determine the role of species for parasite spread amplification

PubMed Central

Stella, Massimo; Selakovic, Sanja; Antonioni, Alberto

2018-01-01

Despite their potential interplay, multiple routes of many disease transmissions are often investigated separately. As a unifying framework for understanding parasite spread through interdependent transmission paths, we present the ‘ecomultiplex’ model, where the multiple transmission paths among a diverse community of interacting hosts are represented as a spatially explicit multiplex network. We adopt this framework for designing and testing potential control strategies for Trypanosoma cruzi spread in two empirical host communities. We show that the ecomultiplex model is an efficient and low data-demanding method to identify which species enhances parasite spread and should thus be a target for control strategies. We also find that the interplay between predator-prey and host-parasite interactions leads to a phenomenon of parasite amplification, in which top predators facilitate T. cruzi spread, offering a mechanistic interpretation of previous empirical findings. Our approach can provide novel insights in understanding and controlling parasite spreading in real-world complex systems. PMID:29683427

Thrust Stand for Electric Propulsion Performance Evaluation

NASA Technical Reports Server (NTRS)

Polzin, Kurt A.; Markusic, Thomas E.; Stanojev, Boris J.; Dehoyos, Amado; Spaun, Benjamin

2006-01-01

An electric propulsion thrust stand capable of supporting testing of thrusters having a total mass of up to 125 kg and producing thrust levels between 100 microN to 1 N has been developed and tested. The design features a conventional hanging pendulum arm attached to a balance mechanism that converts horizontal deflections produced by the operating thruster into amplified vertical motion of a secondary arm. The level of amplification is changed through adjustment of the location of one of the pivot points linking the system. Response of the system depends on the relative magnitudes of the restoring moments applied by the displaced thruster mass and the twisting torsional pivots connecting the members of the balance mechanism. Displacement is measured using a non-contact, optical linear gap displacement transducer and balance oscillatory motion is attenuated using a passive, eddy-current damper. The thrust stand employs an automated leveling and thermal control system. Pools of liquid gallium are used to deliver power to the thruster without using solid wire connections, which can exert undesirable time-varying forces on the balance. These systems serve to eliminate sources of zero-drift that can occur as the stand thermally or mechanically shifts during the course of an experiment. An in-situ calibration rig allows for steady-state calibration before, during and after thruster operation. Thrust measurements were carried out on a cylindrical Hall thruster that produces mN-level thrust. The measurements were very repeatable, producing results that compare favorably with previously published performance data, but with considerably smaller uncertainty.

Amplification of flood frequencies with local sea level rise and emerging flood regimes

NASA Astrophysics Data System (ADS)

Buchanan, Maya K.; Oppenheimer, Michael; Kopp, Robert E.

2017-06-01

The amplification of flood frequencies by sea level rise (SLR) is expected to become one of the most economically damaging impacts of climate change for many coastal locations. Understanding the magnitude and pattern by which the frequency of current flood levels increase is important for developing more resilient coastal settlements, particularly since flood risk management (e.g. infrastructure, insurance, communications) is often tied to estimates of flood return periods. The Intergovernmental Panel on Climate Change’s Fifth Assessment Report characterized the multiplication factor by which the frequency of flooding of a given height increases (referred to here as an amplification factor; AF). However, this characterization neither rigorously considered uncertainty in SLR nor distinguished between the amplification of different flooding levels (such as the 10% versus 0.2% annual chance floods); therefore, it may be seriously misleading. Because both historical flood frequency and projected SLR are uncertain, we combine joint probability distributions of the two to calculate AFs and their uncertainties over time. Under probabilistic relative sea level projections, while maintaining storm frequency fixed, we estimate a median 40-fold increase (ranging from 1- to 1314-fold) in the expected annual number of local 100-year floods for tide-gauge locations along the contiguous US coastline by 2050. While some places can expect disproportionate amplification of higher frequency events and thus primarily a greater number of historically precedented floods, others face amplification of lower frequency events and thus a particularly fast growing risk of historically unprecedented flooding. For example, with 50 cm of SLR, the 10%, 1%, and 0.2% annual chance floods are expected respectively to recur 108, 335, and 814 times as often in Seattle, but 148, 16, and 4 times as often in Charleston, SC.

Parvovirus B19 is a bystander in adult myocarditis.

PubMed

Koepsell, Scott A; Anderson, Daniel R; Radio, Stanley J

2012-01-01

The genomic DNA of parvovirus B19, a small single-stranded DNA virus of the genus Erythrovirus, has been shown to persist in solid tissues of constitutionally healthy, immunocompetent individuals. Despite these data, many case reports and series have linked the presence of parvovirus B19 genomic DNA, detected through nucleic acid amplification testing, with myocarditis and cardiomyopathy. Herein, we use multiple tools to better assess the relationship between parvovirus B19 and myocarditis and cardiomyopathy. Nucleic acid amplification testing, immunohistochemistry, in situ hybridization, and electron microscopy were used to assess the location and activity of parvovirus B19 in cases of myocarditis and in cases with no significant cardiac disease. Nucleic acid amplification testing for parvovirus B19 genomic DNA was positive in 73% of patients with myocarditis/cardiomyopathy and in 26% of patients with no significant disease. In situ hybridization and immunohistochemistry showed that, in cases with amplifiable parvovirus B19 DNA, parvovirus B19 genomic DNA and viral protein production were present in rare mononuclear cells. In a majority of cases of myocarditis and a significant number of otherwise normal hearts, nucleic acid amplification testing detected persistent parvovirus B19 genomic DNA that did not play a significant pathogenic role. The source of parvovirus B19 DNA appeared to be interstitial mononuclear inflammatory cells and not myocardial or endothelial cells. Therefore, nucleic acid amplification testing alone is not diagnostically helpful for determining the etiology of adult myocarditis. Copyright © 2012 Elsevier Inc. All rights reserved.

Coexpression network analysis identifies transcriptional modules associated with genomic alterations in neuroblastoma.

PubMed

Yang, Liulin; Li, Yun; Wei, Zhi; Chang, Xiao

2018-06-01

Neuroblastoma is a highly complex and heterogeneous cancer in children. Acquired genomic alterations including MYCN amplification, 1p deletion and 11q deletion are important risk factors and biomarkers in neuroblastoma. Here, we performed a co-expression-based gene network analysis to study the intrinsic association between specific genomic changes and transcriptome organization. We identified multiple gene coexpression modules which are recurrent in two independent datasets and associated with functional pathways including nervous system development, cell cycle, immune system process and extracellular matrix/space. Our results also indicated that modules involved in nervous system development and cell cycle are highly associated with MYCN amplification and 1p deletion, while modules responding to immune system process are associated with MYCN amplification only. In summary, this integrated analysis provides novel insights into molecular heterogeneity and pathogenesis of neuroblastoma. This article is part of a Special Issue entitled: Accelerating Precision Medicine through Genetic and Genomic Big Data Analysis edited by Yudong Cai & Tao Huang. Copyright © 2017. Published by Elsevier B.V.

SSRPrimer and SSR Taxonomy Tree: Biome SSR discovery

PubMed Central

Jewell, Erica; Robinson, Andrew; Savage, David; Erwin, Tim; Love, Christopher G.; Lim, Geraldine A. C.; Li, Xi; Batley, Jacqueline; Spangenberg, German C.; Edwards, David

2006-01-01

Simple sequence repeat (SSR) molecular genetic markers have become important tools for a broad range of applications such as genome mapping and genetic diversity studies. SSRs are readily identified within DNA sequence data and PCR primers can be designed for their amplification. These PCR primers frequently cross amplify within related species. We report a web-based tool, SSR Primer, that integrates SPUTNIK, an SSR repeat finder, with Primer3, a primer design program, within one pipeline. On submission of multiple FASTA formatted sequences, the script screens each sequence for SSRs using SPUTNIK. Results are then parsed to Primer3 for locus specific primer design. We have applied this tool for the discovery of SSRs within the complete GenBank database, and have designed PCR amplification primers for over 13 million SSRs. The SSR Taxonomy Tree server provides web-based searching and browsing of species and taxa for the visualisation and download of these SSR amplification primers. These tools are available at . PMID:16845092

SSRPrimer and SSR Taxonomy Tree: Biome SSR discovery.

PubMed

Jewell, Erica; Robinson, Andrew; Savage, David; Erwin, Tim; Love, Christopher G; Lim, Geraldine A C; Li, Xi; Batley, Jacqueline; Spangenberg, German C; Edwards, David

2006-07-01

Simple sequence repeat (SSR) molecular genetic markers have become important tools for a broad range of applications such as genome mapping and genetic diversity studies. SSRs are readily identified within DNA sequence data and PCR primers can be designed for their amplification. These PCR primers frequently cross amplify within related species. We report a web-based tool, SSR Primer, that integrates SPUTNIK, an SSR repeat finder, with Primer3, a primer design program, within one pipeline. On submission of multiple FASTA formatted sequences, the script screens each sequence for SSRs using SPUTNIK. Results are then parsed to Primer3 for locus specific primer design. We have applied this tool for the discovery of SSRs within the complete GenBank database, and have designed PCR amplification primers for over 13 million SSRs. The SSR Taxonomy Tree server provides web-based searching and browsing of species and taxa for the visualisation and download of these SSR amplification primers. These tools are available at http://bioinformatics.pbcbasc.latrobe.edu.au/ssrdiscovery.html.

Earthquake response of storey building in Jakarta using accelerographs data analysis

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

Julius, Admiral Musa, E-mail: admiralmusajulius@yahoo.com; Jakarta Geophysics Observatory, Indonesia Agency of Meteorology Climatology and Geophysics; Sunardi, Bambang, E-mail: b.sunardi@gmail.com

As seismotectonic, the Jakarta city will be greatly affected by the earthquake which originated from the subduction zone of the Sunda Strait and south of Java. Some occurrences of earthquakes in these location are often perceived by the occupants in the upper floors of multi-storey buildings in Jakarta but was not perceived by the occupants on the ground floor. The case shows the difference in ground-motion parameters on each floor height. The analysis of the earthquake data recorded by accelerographs on different floors need to be done to know the differences in ground-motion parameters. Data used in this research ismore » accelerograph data installed on several floors in the main building of Meteorology Climatology and Geophysics Agency with a case study of Kebumen earthquake on January 25{sup th} 2014. Parameters analyzed include the Peak Ground Acceleration (PGA), Peak Ground Displacement (PGD), Peak Spectral Acceleration (PSA), Amplification (Ag), and the Effective Duration of earthquake (t{sub e}). Research stages include accelerographs data acquisition in three (3) different floors, conversion and data partition for each component, conversion to units of acceleration, determination of PGA, PGD, PSA, Ag and t{sub e} as well as data analysis. The study shows the value of PGA on the ground floor, 7{sup th} floor and 15{sup th} floors, respectively are 0.016 g, 0.053 g and 0.116 g. PGD on the ground floor, 7{sup th} floor and 15{sup th} floor respectively are 2.15 cm, 2.98 cm and 4.92 cm. PSA on the ground floor, 7{sup th} floor and 15{sup th} floor respectively are 0.067 g, 0.308 g and 0.836 g. Amplification of the peak acceleration value on the ground floor, 7{sup th} floor and 15{sup th} floor to the surface rock are 4.37, 6.07 and 7.30. Effective duration of the earthquake on the ground floor, 7{sup th} floor and 15{sup th} floor respectively are 222.28 s, 202.28 s and 91.58 s. In general, with increasing floor of the building, the value of the peak ground acceleration, peak ground displacement, peak spectral acceleration and amplification growing, contrary to the value of the effective duration of earthquake decreases. The difference in this parameter is strongly influenced by local soil conditions and building construction.« less

Droplet microfluidics for amplification-free genetic detection of single cells.

PubMed

Rane, Tushar D; Zec, Helena C; Puleo, Chris; Lee, Abraham P; Wang, Tza-Huei

2012-09-21

In this article we present a novel droplet microfluidic chip enabling amplification-free detection of single pathogenic cells. The device streamlines multiple functionalities to carry out sample digitization, cell lysis, probe-target hybridization for subsequent fluorescent detection. A peptide nucleic acid fluorescence resonance energy transfer probe (PNA beacon) is used to detect 16S rRNA present in pathogenic cells. Initially the sensitivity and quantification abilities of the platform are tested using a synthetic target mimicking the actual expression level of 16S rRNA in single cells. The capability of the device to perform "sample-to-answer" pathogen detection of single cells is demonstrated using E. coli as a model pathogen.

Methods and devices based on brillouin selective sideband amplification

NASA Technical Reports Server (NTRS)

Yao, X. Steve (Inventor)

2003-01-01

Opto-electronic devices and techniques using Brillouin scattering to select a sideband in a modulated optical carrier signal for amplification. Two lasers respectively provide a carrier signal beam and a Brillouin pump beam which are fed into an Brillouin optical medium in opposite directions. The relative frequency separation between the lasers is adjusted to align the frequency of the backscattered Brillouin signal with a desired sideband in the carrier signal to effect a Brillouin gain on the sideband. This effect can be used to implement photonic RF signal mixing and conversion with gain, conversion from phase modulation to amplitude modulation, photonic RF frequency multiplication, optical and RF pulse generation and manipulation, and frequency-locking of lasers.

A nanoscale piezoelectric transformer for low-voltage transistors.

PubMed

Agarwal, Sapan; Yablonovitch, Eli

2014-11-12

A novel piezoelectric voltage transformer for low-voltage transistors is proposed. Placing a piezoelectric transformer on the gate of a field-effect transistor results in the piezoelectric transformer field-effect transistor that can switch at significantly lower voltages than a conventional transistor. The piezoelectric transformer operates by using one piezoelectric to squeeze another piezoelectric to generate a higher output voltage than the input voltage. Multiple piezoelectrics can be used to squeeze a single piezoelectric layer to generate an even higher voltage amplification. Coupled electrical and mechanical modeling in COMSOL predicts a 12.5× voltage amplification for a six-layer piezoelectric transformer. This would lead to more than a 150× reduction in the power needed for communications.

High-frequency performance for a spiral-shaped piezoelectric bimorph

NASA Astrophysics Data System (ADS)

Huang, Fang Sheng; Feng, Zhi Hua; Ma, Yu Ting; Pan, Qiao Sheng; Zhang, Lian Sheng; Liu, Yong Bin; He, Liang Guo

2018-04-01

Piezoelectric cantilever is suitable as an actuator for micro-flapping-wing aircraft. Higher resonant frequency brings about stronger flight energy, and the flight amplitude can be compensated by displacement-amplification mechanism, such as lever. To obtain a higher resonant frequency, straight piezoelectric bimorph was rolled into spiral-shaped piezoelectric bimorph with identical effective length in this study, which is verified in COMSOL simulations. Simulation results show that compared with the straight piezoelectric bimorph, the spiral-shaped piezoelectric bimorph with two turns has higher inherent frequencies (from 204.79 Hz to 504.84 Hz in terms of axial oscillation mode, and from 319.77 Hz to 704.48 Hz in terms of tangential torsional mode). The spiral-shaped piezoelectric bimorph is fabricated by a precise laser cutting process and consists of two turns with effective length of 60 mm, width of 2.5 mm, and thickness of 1.6 mm, respectively. With the excitation voltage of 100 Vpp applying an electric field across the thickness of the bimorph, the tip displacement of the actuator in the axial oscillation and tangential torsional modes are 85 μm and 15 μm, respectively.

The Study on the Durability of Submerged Structure Displacement due to Concrete Failure

NASA Astrophysics Data System (ADS)

Mohd, M.; Zainon, O.; Rasib, A. W.; Majid, Z.

2016-09-01

Concrete structures that exposed to marine environments are subjected to multiple deterioration mechanisms. An overview of the existing technology for submerged concrete, pressure resistant, concrete structures which related such as cracks, debonds, and delamination are discussed. Basic knowledge related to drowning durability such as submerged concrete structures in the maritime environment are the durability of a concrete and the ability to resist to weathering, chemical attack, abrasion or other deterioration processes. The measuring techniques and instrumentation for geometrical monitoring of submerged structural displacements have traditionally been categorized into two groups according to the two main groups, namely as geodetic surveying and geotechnical structural measurements of local displacements. This paper aims to study the durability of submerged concrete displacement and harmful effects of submerged concrete structures.

Computer-assisted time-averaged holograms of the motion of the surface of the mammalian tympanic membrane with sound stimuli of 0.4 to 25 kHz

PubMed Central

Rosowski, John J.; Cheng, Jeffrey Tao; Ravicz, Michael E.; Hulli, Nesim; Hernandez-Montes, Maria; Harrington, Ellery; Furlong, Cosme

2009-01-01

Time-averaged holograms describing the sound-induced motion of the tympanic membrane (TM) in cadaveric preparations from three mammalian species and one live ear were measured using opto-electronic holography. This technique allows rapid measurements of the magnitude of motion of the tympanic membrane surface at frequencies as high as 25 kHz. The holograms measured in response to low and middle-frequency sound stimuli are similar to previously reported time-averaged holograms. However, at higher frequencies (f > 4 kHz), our holograms reveal unique TM surface displacement patterns that consist of highly-ordered arrangements of multiple local displacement magnitude maxima, each of which is surrounded by nodal areas of low displacement magnitude. These patterns are similar to modal patterns (two-dimensional standing waves) produced by either the interaction of surface waves traveling in multiple directions or the uniform stimulation of modes of motion that are determined by the structural properties and boundary conditions of the TM. From the ratio of the displacement magnitude peaks to nodal valleys in these apparent surface waves, we estimate a Standing Wave Ratio of at least 4 that is consistent with energy reflection coefficients at the TM boundaries of at least 0.35. It is also consistent with small losses within the uniformly stimulated modal surface waves. We also estimate possible TM surface wave speeds that vary with frequency and species from 20 to 65 m/s, consistent with other estimates in the literature. The presence of standing wave or modal phenomena has previously been intuited from measurements of TM function, but is ignored in some models of tympanic membrane function. Whether these standing waves result either from the interactions of multiple surface waves that travel along the membrane, or by uniformly excited modal displacement patterns of the entire TM surface is still to be determined. PMID:19328841

Computer-assisted time-averaged holograms of the motion of the surface of the mammalian tympanic membrane with sound stimuli of 0.4-25 kHz.

PubMed

Rosowski, John J; Cheng, Jeffrey Tao; Ravicz, Michael E; Hulli, Nesim; Hernandez-Montes, Maria; Harrington, Ellery; Furlong, Cosme

2009-07-01

Time-averaged holograms describing the sound-induced motion of the tympanic membrane (TM) in cadaveric preparations from three mammalian species and one live ear were measured using opto-electronic holography. This technique allows rapid measurements of the magnitude of motion of the tympanic membrane surface at frequencies as high as 25 kHz. The holograms measured in response to low and middle-frequency sound stimuli are similar to previously reported time-averaged holograms. However, at higher frequencies (f>4 kHz), our holograms reveal unique TM surface displacement patterns that consist of highly-ordered arrangements of multiple local displacement magnitude maxima, each of which is surrounded by nodal areas of low displacement magnitude. These patterns are similar to modal patterns (two-dimensional standing waves) produced by either the interaction of surface waves traveling in multiple directions or the uniform stimulation of modes of motion that are determined by the structural properties and boundary conditions of the TM. From the ratio of the displacement magnitude peaks to nodal valleys in these apparent surface waves, we estimate a Standing Wave Ratio of at least 4 that is consistent with energy reflection coefficients at the TM boundaries of at least 0.35. It is also consistent with small losses within the uniformly stimulated modal surface waves. We also estimate possible TM surface wave speeds that vary with frequency and species from 20 to 65 m/s, consistent with other estimates in the literature. The presence of standing wave or modal phenomena has previously been intuited from measurements of TM function, but is ignored in some models of tympanic membrane function. Whether these standing waves result either from the interactions of multiple surface waves that travel along the membrane, or by uniformly excited modal displacement patterns of the entire TM surface is still to be determined.

Next-Generation in Situ Hybridization Chain Reaction: Higher Gain, Lower Cost, Greater Durability

PubMed Central

2014-01-01

Hybridization chain reaction (HCR) provides multiplexed, isothermal, enzyme-free, molecular signal amplification in diverse settings. Within intact vertebrate embryos, where signal-to-background is at a premium, HCR in situ amplification enables simultaneous mapping of multiple target mRNAs, addressing a longstanding challenge in the biological sciences. With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The properties of HCR lead to straightforward multiplexing, deep sample penetration, high signal-to-background, and sharp subcellular signal localization within fixed whole-mount zebrafish embryos, a standard model system for the study of vertebrate development. However, RNA reagents are expensive and vulnerable to enzymatic degradation. Moreover, the stringent hybridization conditions used to destabilize nonspecific hairpin binding also reduce the energetic driving force for HCR polymerization, creating a trade-off between minimization of background and maximization of signal. Here, we eliminate this trade-off by demonstrating that low background levels can be achieved using permissive in situ amplification conditions (0% formamide, room temperature) and engineer next-generation DNA HCR amplifiers that maximize the free energy benefit per polymerization step while preserving the kinetic trapping property that underlies conditional polymerization, dramatically increasing signal gain, reducing reagent cost, and improving reagent durability. PMID:24712299

A multivariate test of disease risk reveals conditions leading to disease amplification.

PubMed

Halliday, Fletcher W; Heckman, Robert W; Wilfahrt, Peter A; Mitchell, Charles E

2017-10-25

Theory predicts that increasing biodiversity will dilute the risk of infectious diseases under certain conditions and will amplify disease risk under others. Yet, few empirical studies demonstrate amplification. This contrast may occur because few studies have considered the multivariate nature of disease risk, which includes richness and abundance of parasites with different transmission modes. By combining a multivariate statistical model developed for biodiversity-ecosystem-multifunctionality with an extensive field manipulation of host (plant) richness, composition and resource supply to hosts, we reveal that (i) host richness alone could not explain most changes in disease risk, and (ii) shifting host composition allowed disease amplification, depending on parasite transmission mode. Specifically, as predicted from theory, the effect of host diversity on parasite abundance differed for microbes (more density-dependent transmission) and insects (more frequency-dependent transmission). Host diversity did not influence microbial parasite abundance, but nearly doubled insect parasite abundance, and this amplification effect was attributable to variation in host composition. Parasite richness was reduced by resource addition, but only in species-rich host communities. Overall, this study demonstrates that multiple drivers, related to both host community and parasite characteristics, can influence disease risk. Furthermore, it provides a framework for evaluating multivariate disease risk in other systems. © 2017 The Author(s).

Outbreak or illusion: consequences of 'improved' diagnostics for gonorrhoea.

PubMed

Bennett, Amy; Jeffery, Katie; O'Neill, Eunan; Sherrard, Jackie

2017-06-01

The sexual health service in Oxford introduced gonorrhoea nucleic amplification acid testing using the BD Viper XTR™ System. For practical reasons, a confirmatory nucleic amplification acid testing using a different platform was not used initially. Following the introduction of nucleic amplification acid testing, the rates of gonorrhoea increased threefold. Concerns were raised that this increase represented an outbreak. A retrospective review of cases over six months suggested that there may have been a number of false-positive results. A prospective study was then undertaken over six months, where all gonorrhoea positive samples were sent for confirmatory testing. This evaluation of all gonorrhoea cases in an English county found that the overall presumptive false-positive rates for gonorrhoea nucleic amplification acid testing using BD Viper XTR™ in our population are significant at 27% of female samples, 13.2% of heterosexual male samples, 3.5% of anogenital multiple site men who have sex with men samples and 62.8% of pharyngeal only men who have sex with men samples. The data demonstrate the need for confirmatory testing using a second nucleic acid target, as per BASHH/Public Health England guidelines, especially in low-prevalence settings and extragenital sites, due to cross-reactivity with commensal Neisseria species and low positive predictive values.

Interactive fluorophore and quencher pairs for labeling fluorescent nucleic acid hybridization probes.

PubMed

Marras, Salvatore A E

2008-03-01

The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.

A lab-on-a-chip system with integrated sample preparation and loop-mediated isothermal amplification for rapid and quantitative detection of Salmonella spp. in food samples.

PubMed

Sun, Yi; Quyen, Than Linh; Hung, Tran Quang; Chin, Wai Hoe; Wolff, Anders; Bang, Dang Duong

2015-04-21

Foodborne disease is a major public health threat worldwide. Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture or molecular-based methods are time consuming and usually take a few hours to days to complete. In response to the demand for rapid on line or on site detection of pathogens, in this study, we describe for the first time an eight-chamber lab-on-a-chip (LOC) system with integrated magnetic bead-based sample preparation and loop-mediated isothermal amplification (LAMP) for rapid and quantitative detection of Salmonella spp. in food samples. The whole diagnostic procedures including DNA isolation, isothermal amplification, and real-time detection were accomplished in a single chamber. Up to eight samples could be handled simultaneously and the system was capable to detect Salmonella at concentration of 50 cells per test within 40 min. The simple design, together with high level of integration, isothermal amplification, and quantitative analysis of multiple samples in short time, will greatly enhance the practical applicability of the LOC system for rapid on-site screening of Salmonella for applications in food safety control, environmental surveillance, and clinical diagnostics.

Binding-induced DNA walker for signal amplification in highly selective electrochemical detection of protein.

PubMed

Ji, Yuhang; Zhang, Lei; Zhu, Longyi; Lei, Jianping; Wu, Jie; Ju, Huangxian

2017-10-15

A binding-induced DNA walker-assisted signal amplification was developed for highly selective electrochemical detection of protein. Firstly, the track of DNA walker was constructed by self-assembly of the high density ferrocene (Fc)-labeled anchor DNA and aptamer 1 on the gold electrode surface. Sequentially, a long swing-arm chain containing aptamer 2 and walking strand DNA was introduced onto gold electrode through aptamers-target specific recognition, and thus initiated walker strand sequences to hybridize with anchor DNA. Then, the DNA walker was activated by the stepwise cleavage of the hybridized anchor DNA by nicking endonuclease to release multiple Fc molecules for signal amplification. Taking thrombin as the model target, the Fc-generated electrochemical signal decreased linearly with logarithm value of thrombin concentration ranging from 10pM to 100nM with a detection limit of 2.5pM under the optimal conditions. By integrating the specific recognition of aptamers to target with the enzymatic cleavage of nicking endonuclease, the aptasensor showed the high selectivity. The binding-induced DNA walker provides a promising strategy for signal amplification in electrochemical biosensor, and has the extensive applications in sensitive and selective detection of the various targets. Copyright © 2017 Elsevier B.V. All rights reserved.

Household displacement and health risk behaviors among AIDS-affected children in rural China

PubMed Central

Zhao, Qun; Zhao, Junfeng; Li, Xiaoming; Fang, Xiaoyi; Zhao, Guoxiang; Lin, Xiuyun; Zhang, Liying

2011-01-01

When parents die of or are infected with HIV, children might have to leave their own household and be displaced to other living arrangement and some may even be displaced multiple times. The objective of this study is to examine the association between household displacement and health risk behaviors among AIDS orphans (children who have lost one or both of their parents to HIV/AIDS) and vulnerable children (children living with HIV-infected parents) in rural China. The sample consisted of 1015 children (549 AIDS orphans, 466 vulnerable children) in family-based care. The children were assigned to three displacement groups according to the number of household displacement (i.e., none, once, at least twice) after their parents became ill or died of AIDS. Cigarette smoking, alcohol use, violence, public property destruction, suicidal ideation, and suicide attempt were used to assess the health risk behaviors of these children. Both bivariate and multivariate tests were used to assess the differences in health risk behaviors among displacement groups. The findings indicated that children who were displaced at least twice were more likely to report a higher frequency of public property destruction and suicide ideation than those who were never displaced or displaced once. Multivariate analysis revealed that public property destruction, suicide ideation and suicide attempt were significantly associated with the household displacement among these children, controlling for gender, age, child status (AIDS orphans vs. vulnerable children) and the duration of household displacement. Results in the current study suggest that a stable living environment was important for both AID orphans and vulnerable children in communities of high prevalence of HIV/AIDS. The government, community and other agencies need to make efforts to avoid frequent household displacement among these children after the HIV-related infection or death of their parents. PMID:21400311

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.

Development of a wireless displacement measurement system using acceleration responses.

PubMed

Park, Jong-Woong; Sim, Sung-Han; Jung, Hyung-Jo; Spencer, Billie F

2013-07-01

Displacement measurements are useful information for various engineering applications such as structural health monitoring (SHM), earthquake engineering and system identification. Most existing displacement measurement methods are costly, labor-intensive, and have difficulties particularly when applying to full-scale civil structures because the methods require stationary reference points. Indirect estimation methods converting acceleration to displacement can be a good alternative as acceleration transducers are generally cost-effective, easy to install, and have low noise. However, the application of acceleration-based methods to full-scale civil structures such as long span bridges is challenging due to the need to install cables to connect the sensors to a base station. This article proposes a low-cost wireless displacement measurement system using acceleration. Developed with smart sensors that are low-cost, wireless, and capable of on-board computation, the wireless displacement measurement system has significant potential to impact many applications that need displacement information at multiple locations of a structure. The system implements an FIR-filter type displacement estimation algorithm that can remove low frequency drifts typically caused by numerical integration of discrete acceleration signals. To verify the accuracy and feasibility of the proposed system, laboratory tests are carried out using a shaking table and on a three storey shear building model, experimentally confirming the effectiveness of the proposed system.

Development of a Wireless Displacement Measurement System Using Acceleration Responses

PubMed Central

Park, Jong-Woong; Sim, Sung-Han; Jung, Hyung-Jo; Spencer, Billie F.

2013-01-01

Displacement measurements are useful information for various engineering applications such as structural health monitoring (SHM), earthquake engineering and system identification. Most existing displacement measurement methods are costly, labor-intensive, and have difficulties particularly when applying to full-scale civil structures because the methods require stationary reference points. Indirect estimation methods converting acceleration to displacement can be a good alternative as acceleration transducers are generally cost-effective, easy to install, and have low noise. However, the application of acceleration-based methods to full-scale civil structures such as long span bridges is challenging due to the need to install cables to connect the sensors to a base station. This article proposes a low-cost wireless displacement measurement system using acceleration. Developed with smart sensors that are low-cost, wireless, and capable of on-board computation, the wireless displacement measurement system has significant potential to impact many applications that need displacement information at multiple locations of a structure. The system implements an FIR-filter type displacement estimation algorithm that can remove low frequency drifts typically caused by numerical integration of discrete acceleration signals. To verify the accuracy and feasibility of the proposed system, laboratory tests are carried out using a shaking table and on a three storey shear building model, experimentally confirming the effectiveness of the proposed system. PMID:23881123

A magnetostrictive acceleration sensor for registration of chest wall displacements

NASA Astrophysics Data System (ADS)

Kaniusas, E.; Mehnen, L.; Krell, C.; Pfützner, H.

2000-06-01

The present study concerns a novel acceleration sensor built up using a bimetallic strip with one freely oscillating end. The strip consists of a magnetostrictive amorphous ribbon glued on a nonmagnetic metal ribbon. The sensor's effectiveness is demonstrated for the case of chest wall displacements as resulting from cardiorespiratory activity. The cheap and easy-to-handle novel sensor offers multiple information on the so-called sleep apnea syndrome.

Simultaneous full-field 3-D vibrometry of the human eardrum using spatial-bandwidth multiplexed holography

NASA Astrophysics Data System (ADS)

Khaleghi, Morteza; Guignard, Jérémie; Furlong, Cosme; Rosowski, John J.

2015-11-01

Holographic interferometric methods typically require the use of three sensitivity vectors in order to obtain three-dimensional (3-D) information. Methods based on multiple directions of illumination have limited applications when studying biological tissues that have temporally varying responses such as the tympanic membrane (TM). Therefore, to measure 3-D displacements in such applications, the measurements along all the sensitivity vectors have to be done simultaneously. We propose a multiple-illumination directions approach to measure 3-D displacements from a single-shot hologram that contains displacement information from three sensitivity vectors. The hologram of an object of interest is simultaneously recorded with three incoherently superimposed pairs of reference and object beams. The incident off-axis angles of the reference beams are adjusted such that the frequency components of the multiplexed hologram are completely separate. Because of the differences in the directions and wavelengths of the reference beams, the positions of each reconstructed image corresponding to each sensitivity vector are different. We implemented a registration algorithm to accurately translate individual components of the hologram into a single global coordinate system to calculate 3-D displacements. The results include magnitudes and phases of 3-D sound-induced motions of a human cadaveric TM at several excitation frequencies showing modal and traveling wave motions on its surface.

How does tooth eruption relate to vertical mandibular growth displacement?

PubMed

Liu, Sean Shih-Yao; Buschang, Peter H

2011-06-01

Our objectives were to investigate the eruptive patterns of the mandibular teeth and assess their associations with mandibular growth displacements. Cephalograms for a mixed-longitudinal sample of 124 French-Canadian girls were evaluated between 10 and 15 years of age. Vertical mandibular displacement and mandibular eruption were evaluated by using cranial and mandibular superimpositions, respectively. Multilevel modeling procedures were used to estimate each subject's growth change over time. Stepwise multiple regressions were used to determine the amount and relative magnitudes of variations in mandibular eruption explained by mandibular growth displacement, controlling for vertical maxillary tooth movements. Cubic polynomial models explained between 91% and 98% of the variations in eruption and vertical growth displacement. All curves showed acceleration of eruption until approximately 12 years of age, after which eruption decelerated. The eruption of the mandibular teeth demonstrated greater relative variability than did vertical mandibular growth displacements. Independent of the overall movements of the maxillary molars, inferior mandibular growth displacement explained approximately 54% of the variation in mandibular molar eruption between 10.5 and 14.5 years of age. Inferior mandibular growth displacement and dental eruption followed similar patterns of change during adolescence. Based on their associations and the differences in variability identified, mandibular eruption appears to compensate for or adapt to growth displacements. Copyright © 2011 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

A comparison of single-cycle versus multiple-cycle proof testing strategies

NASA Technical Reports Server (NTRS)

Hudak, S. J., Jr.; Mcclung, R. C.; Bartlett, M. L.; Fitzgerald, J. H.; Russell, D. A.

1990-01-01

An evaluation of single-cycle and multiple-cycle proof testing (MCPT) strategies for SSME components is described. Data for initial sizes and shapes of actual SSME hardware defects are analyzed statistically. Closed-form estimates of the J-integral for surface flaws are derived with a modified reference stress method. The results of load- and displacement-controlled stable crack growth tests on thin IN-718 plates with deep surface flaws are summarized. A J-resistance curve for the surface-cracked configuration is developed and compared with data from thick compact tension specimens. The potential for further crack growth during large unload/reload cycles is discussed, highlighting conflicting data in the literature. A simple model for ductile crack growth during MCPT based on the J-resistance curve is used to study the potential effects of key variables. The projected changes in the crack size distribution during MCPT depend on the interactions between several key parameters, including the number of proof cycles, the nature of the resistance curve, the initial crack size distribution, the component boundary conditions (load vs. displacement control), and the magnitude of the applied load or displacement. The relative advantages of single-cycle and multiple-cycle proof testing appear to be specific, therefore, to individual component geometry, material, and loading.

Steady Boundary Layer Disturbances Created By Two-Dimensional Surface Ripples

NASA Astrophysics Data System (ADS)

Kuester, Matthew

2017-11-01

Multiple experiments have shown that surface roughness can enhance the growth of Tollmien-Schlichting (T-S) waves in a laminar boundary layer. One of the common observations from these studies is a ``wall displacement'' effect, where the boundary layer profile shape remains relatively unchanged, but the origin of the profile pushes away from the wall. The objective of this work is to calculate the steady velocity field (including this wall displacement) of a laminar boundary layer over a surface with small, 2D surface ripples. The velocity field is a combination of a Blasius boundary layer and multiple disturbance modes, calculated using the linearized Navier-Stokes equations. The method of multiple scales is used to include non-parallel boundary layer effects of O (Rδ- 1) ; the non-parallel terms are necessary, because a wall displacement is mathematically inconsistent with a parallel boundary layer assumption. This technique is used to calculate the steady velocity field over ripples of varying height and wavelength, including cases where a separation bubble forms on the leeward side of the ripple. In future work, the steady velocity field will be the input for stability calculations, which will quantify the growth of T-S waves over rough surfaces. The author would like to acknowledge the support of the Kevin T. Crofton Aerospace & Ocean Engineering Department at Virginia Tech.

Ultrasensitive Electrochemical Detection of Glycoprotein Based on Boronate Affinity Sandwich Assay and Signal Amplification with Functionalized SiO2@Au Nanocomposites.

PubMed

You, Min; Yang, Shuai; Tang, Wanxin; Zhang, Fan; He, Pin-Gang

2017-04-26

Herein we propose a multiple signal amplification strategy designed for ultrasensitive electrochemical detection of glycoproteins. This approach introduces a new type of boronate-affinity sandwich assay (BASA), which was fabricated by using gold nanoparticles combined with reduced graphene oxide (AuNPs-GO) to modify sensing surface for accelerating electron transfer, the composite of molecularly imprinted polymer (MIP) including 4-vinylphenylboronic acid (VPBA) for specific capturing glycoproteins, and SiO 2 nanoparticles carried gold nanoparticles (SiO 2 @Au) labeled with 6-ferrocenylhexanethiol (FcHT) and 4-mercaptophenylboronic acid (MPBA) (SiO 2 @Au/FcHT/MPBA) as tracing tag for binding glycoprotein and generating electrochemical signal. As a sandwich-type sensing, the SiO 2 @Au/FcHT/MPBA was captured by glycoprotein on the surface of imprinting film for further electrochemical detection in 0.1 M PBS (pH 7.4). Using horseradish peroxidase (HRP) as a model glycoprotein, the proposed approach exhibited a wide linear range from 1 pg/mL to 100 ng/mL, with a low detection limit of 0.57 pg/mL. To the best of our knowledge, this is first report of a multiple signal amplification approach based on boronate-affinity molecularly imprinted polymer and SiO 2 @Au/FcHT/MPBA, exhibiting greatly enhanced sensitivity for glycoprotein detection. Furthermore, the newly constructed BASA based glycoprotein sensor demonstrated HRP detection in real sample, such as human serum, suggesting its promising prospects in clinical diagnostics.

Loop-mediated isothermal amplification (LAMP) assays for rapid detection and differentiation of Nosema apis and N. ceranae in honeybees.

PubMed

Ptaszyńska, Aneta A; Borsuk, Grzegorz; Woźniakowski, Grzegorz; Gnat, Sebastian; Małek, Wanda

2014-08-01

Nosemosis is a contagious disease of honeybees (Apis mellifera) manifested by increased winter mortality, poor spring build-up and even the total extinction of infected bee colonies. In this paper, loop-mediated isothermal amplifications (LAMP) were used for the first time to identify and differentiate N. apis and N. ceranae, the causative agents of nosemosis. LAMP assays were performed at a constant temperature of 60 °C using two sets of six species-specific primers, recognising eight distinct fragments of 16S rDNA gene and GspSSD polymerase with strand displacement activity. The optimal time for LAMP and its Nosema species sensitivity and specificity were assessed. LAMP only required 30 min for robust identification of the amplicons. Ten-fold serial dilutions of total DNA isolated from bees infected with microsporidia were used to determine the detection limit of N. apis and N. ceranae DNAs by LAMP and standard PCR assays. LAMP appeared to be 10(3) -fold more sensitive than a standard PCR in detecting N. apis and N. ceranae. LAMP methods developed by us are highly Nosema species specific and allow to identify and differentiate N. apis and N. ceranae. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Kinematic amplification strategies in plants and engineering

NASA Astrophysics Data System (ADS)

Charpentier, Victor; Hannequart, Philippe; Adriaenssens, Sigrid; Baverel, Olivier; Viglino, Emmanuel; Eisenman, Sasha

2017-06-01

While plants are primarily sessile at the organismal level, they do exhibit a vast array of movements at the organ or sub-organ level. These movements can occur for reasons as diverse as seed dispersal, nutrition, protection or pollination. Their advanced mechanisms generate a myriad of movement typologies, many of which are not fully understood. In recent years, there has been a renewal of interest in understanding the mechanical behavior of plants from an engineering perspective, with an interest in developing novel applications by up-sizing these mechanisms from the micro- to the macro-scale. This literature review identifies the main strategies used by plants to create and amplify movements and anatomize the most recent mechanical understanding of compliant engineering mechanics. The paper ultimately demonstrates that plant movements, rooted in compliance and multi-functionality, can effectively inspire better kinematic/adaptive structures and materials. In plants, the actuators and the deployment structures are fused into a single system. The understanding of those natural movements therefore starts with an exploration of mechanisms at the origins of movements. Plant movements, whether slow or fast, active or passive, reversible or irreversible, are presented and detailed for their mechanical significance. With a focus on displacement amplification, the most recent promising strategies for actuation and adaptive systems are examined with respect to the mechanical principles of shape morphing plant tissues.

Cochlear partition tuning within the 2nd apical turn of the intact gerbil cochlea

NASA Astrophysics Data System (ADS)

Dong, Wei; Xia, Anping; Puria, Sunil; Applegate, Brian E.; Oghalai, John S.

2018-05-01

Our understanding of cochlear amplification has been mainly informed by observations of the high-frequency basal region. Results from the few existing in vivo studies from the low-frequency apical region have suggested that cochlear amplification may be more broadly tuned and not as nonlinear as in the base. The current study explored micromechanics via sound evoked vibrations of the cochlear partition, including the organ of Corti (OoC), the basilar membrane (BM) and the tectorial membrane (TM), within the 2nd apical turn, corresponding to the 2.5 kHz best frequency region, of the gerbil cochlea using volumetric optical coherence tomography vibrometry (VOCTV), imaged non-invasively through the otic capsule bone in vivo. Sound induced radial displacements below 4 kHz showed similarities and differences to the well-established BM transverse responses at high-frequency basal region. Responses showed broader tuning, but similar gains at the best frequency and similar phase accumulation. The distinct difference found was the presence of compressive nonlinear growth with SPL below the best frequency down to 0.1 kHz observed at the outer hair cell (OHC) and reticular laminar (RL) locations but not in the BM or inner hair cell regions. These relative motions provide further insight on how OHC somatic forces are distributed within the cochlear partition.

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

Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes.

PubMed

Treder, Krzysztof; Chołuj, Joanna; Zacharzewska, Bogumiła; Babujee, Lavanya; Mielczarek, Mateusz; Burzyński, Adam; Rakotondrafara, Aurélie M

2018-02-01

Potato virus Y (PVY) infection has been a global challenge for potato production and the leading cause of downgrading and rejection of seed crops for certification. Accurate and timely diagnosis is a key for effective disease control. Here, we have optimized a reverse transcription loop-mediated amplification (RT-LAMP) assay to differentiate the PVY O and N serotypes. The RT-LAMP assay is based on isothermal autocyclic strand displacement during DNA synthesis. The high specificity of this method relies heavily on the primer sets designed for the amplification of the targeted regions. We designed specific primer sets targeting a region within the coat protein gene that contains nucleotide signatures typical for O and N coat protein types, and these primers differ in their annealing temperature. Combining this assay with total RNA extraction by magnetic capture, we have established a highly sensitive, simplified and shortened RT-LAMP procedure as an alternative to conventional nucleic acid assays for diagnosis. This optimized procedure for virus detection may be used as a preliminary test for identifying the viral serotype prior to investing time and effort in multiplex RT-PCR tests when a specific strain is needed.

Rogue waves in shallow water

NASA Astrophysics Data System (ADS)

Soomere, T.

2010-07-01

Most of the processes resulting in the formation of unexpectedly high surface waves in deep water (such as dispersive and geometrical focusing, interactions with currents and internal waves, reflection from caustic areas, etc.) are active also in shallow areas. Only the mechanism of modulational instability is not active in finite depth conditions. Instead, wave amplification along certain coastal profiles and the drastic dependence of the run-up height on the incident wave shape may substantially contribute to the formation of rogue waves in the nearshore. A unique source of long-living rogue waves (that has no analogues in the deep ocean) is the nonlinear interaction of obliquely propagating solitary shallow-water waves and an equivalent mechanism of Mach reflection of waves from the coast. The characteristic features of these processes are (i) extreme amplification of the steepness of the wave fronts, (ii) change in the orientation of the largest wave crests compared with that of the counterparts and (iii) rapid displacement of the location of the extreme wave humps along the crests of the interacting waves. The presence of coasts raises a number of related questions such as the possibility of conversion of rogue waves into sneaker waves with extremely high run-up. Also, the reaction of bottom sediments and the entire coastal zone to the rogue waves may be drastic.

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

PubMed

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

2012-01-01

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

Scaling and characterisation of a 2-DoF velocity amplified electromagnetic vibration energy harvester

NASA Astrophysics Data System (ADS)

O’Donoghue, D.; Frizzell, R.; Punch, J.

2018-07-01

Vibration energy harvesters (VEHs) offer an alternative to batteries for the autonomous operation of low-power electronics. Understanding the influence of scaling on VEHs is of great importance in the design of reduced scale harvesters. The nonlinear harvesters investigated here employ velocity amplification, a technique used to increase velocity through impacts, to improve the power output of multiple-degree-of-freedom VEHs, compared to linear resonators. Such harvesters, employing electromagnetic induction, are referred to as velocity amplified electromagnetic generators (VAEGs), with gains in power achieved by increasing the relative velocity between the magnet and coil in the transducer. The influence of scaling on a nonlinear 2-DoF VAEG is presented. Due to the increased complexity of VAEGs, compared to linear systems, linear scaling theory cannot be directly applied to VAEGs. Therefore, a detailed nonlinear scaling method is utilised. Experimental and numerical methods are employed. This nonlinear scaling method can be used for analysing the scaling behaviour of all nonlinear electromagnetic VEHs. It is demonstrated that the electromagnetic coupling coefficient degrades more rapidly with scale for systems with larger displacement amplitudes, meaning that systems operating at low frequencies will scale poorly compared to those operating at higher frequencies. The load power of the 2-DoF VAEG is predicted to scale as {P}L\\propto {s}5.51 (s = volume1/3), suggesting that achieving high power densities in a VAEG with low device volume is extremely challenging.

Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells.

PubMed

Han, Lin; Wu, Hua-Jun; Zhu, Haiying; Kim, Kun-Yong; Marjani, Sadie L; Riester, Markus; Euskirchen, Ghia; Zi, Xiaoyuan; Yang, Jennifer; Han, Jasper; Snyder, Michael; Park, In-Hyun; Irizarry, Rafael; Weissman, Sherman M; Michor, Franziska; Fan, Rong; Pan, Xinghua

2017-06-02

Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells

PubMed Central

Berry, David; Mader, Esther; Lee, Tae Kwon; Woebken, Dagmar; Wang, Yun; Zhu, Di; Palatinszky, Marton; Schintlmeister, Arno; Schmid, Markus C.; Hanson, Buck T.; Shterzer, Naama; Mizrahi, Itzhak; Rauch, Isabella; Decker, Thomas; Bocklitz, Thomas; Popp, Jürgen; Gibson, Christopher M.; Fowler, Patrick W.; Huang, Wei E.; Wagner, Michael

2015-01-01

Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy. Incorporation of D2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Raman-based cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/or glucosamine were identified, demonstrating the potential of the nondestructive D2O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics. PMID:25550518

Ultrasensitive electrochemical detection of tumor cells based on multiple layer CdS quantum dots-functionalized polystyrene microspheres and graphene oxide - polyaniline composite.

PubMed

Wang, Jidong; Wang, Xiaoyu; Tang, Hengshan; Gao, Zehua; He, Shengquan; Li, Jian; Han, Shumin

2018-02-15

In this work, a novel ultrasensitive electrochemical biosensor was developed for the detection of K562 cell by a signal amplification strategy based on multiple layer CdS QDs functionalized polystyrene microspheres(PS) as bioprobe and graphene oxide(GO) -polyaniline(PANI) composite as modified materials of capture electrode. Due to electrostatic force of different charge, CdS QDs were decorated on the surface of PS by PDDA (poly(diallyldimethyl-ammonium chloride)) through a layer-by-layer(LBL) assemble technology, in which the structure of multiple layer CdS QDs increased the detection signal intensity. Moreover, GO-PANI composite not only enhanced the electron transfer rate, but also increased tumor cells load ratio. The resulting electrochemical biosensor was used to detect K562 cells with a lower detection limit of 3 cellsmL -1 (S/N = 3) and a wider linear range from 10 to 1.0 × 10 7 cellsmL -1 . This sensor was also used for mannosyl groups on HeLa cells and Hct116 cells, which showed high specificity and sensitivity. This signal amplification strategy would provide a novel approach for detection, diagnosis and treatment for tumor cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Ko Displacement Theory for Structural Shape Predictions

NASA Technical Reports Server (NTRS)

Ko, William L.

2010-01-01

The development of the Ko displacement theory for predictions of structure deformed shapes was motivated in 2003 by the Helios flying wing, which had a 247-ft (75-m) wing span with wingtip deflections reaching 40 ft (12 m). The Helios flying wing failed in midair in June 2003, creating the need to develop new technology to predict in-flight deformed shapes of unmanned aircraft wings for visual display before the ground-based pilots. Any types of strain sensors installed on a structure can only sense the surface strains, but are incapable to sense the overall deformed shapes of structures. After the invention of the Ko displacement theory, predictions of structure deformed shapes could be achieved by feeding the measured surface strains into the Ko displacement transfer functions for the calculations of out-of-plane deflections and cross sectional rotations at multiple locations for mapping out overall deformed shapes of the structures. The new Ko displacement theory combined with a strain-sensing system thus created a revolutionary new structure- shape-sensing technology.

Variability of site response in the Los Angeles urban area

USGS Publications Warehouse

Hartzell, S.; Cranswick, E.; Frankel, A.; Carver, D.; Meremonte, M.

1997-01-01

This article addresses the variability of site response in the Los Angeles area and possible structural causes for the observations. Aftershock records from 231 sites in the San Fernando and Los Angeles basins and the surrounding mountains are used in this study. Spectral ratios, taken with respect to a low-amplitude reference site, are used to document the variation in site amplification in the frequency range 2 to 6 Hz, both spatially and with backazimuth to the source. At higher frequencies (6 to 10 Hz), spectral ratios are shown to have greater spatial variability. Interstation spectral ratios are used to measure the standard deviation among sources as a function of station separation. An increase in the variation in ground motion is shown to take place at a station separation of 1 km. Relative site-response estimates between nearby stations are used to demonstrate that preferred directions of motion can exist even in areas with no surface topographic effects. Significant variations in site response exist over short baselines (up to a factor of 2 over 200 m) that are not explained by differences in surficial geology or shallow shear-wave velocity. A variety of investigative approaches is used, including spectral ratios, arrival-time variations, 1D and 2D waveform modeling, and comparison with seismic reflection lines, to determine the most likely causes of these observations. A correlation is demonstrated between late arrival times of P and S waves and larger site amplification in Sherman Oaks and Northridge. This observation, in conjunction with waveform modeling and seismic reflection profiles, is used to infer that sedimentary structures in the upper 1 to 2 km and topography on the sediment-basement interface play an important role in determining site amplification. These structures, in the form of folds and buried basins, focus energy in spatially restricted areas at the surface. Comparison of displacement waveforms at nearby stations having disparate site amplifications, complemented by known shallow shear-wave velocities at selected sites, is used to support the argument that these structures, in some cases, can be the dominant factor in the modification of local ground motions.

Lattice Boltzmann simulations of immiscible displacement process with large viscosity ratios

NASA Astrophysics Data System (ADS)

Rao, Parthib; Schaefer, Laura

2017-11-01

Immiscible displacement is a key physical mechanism involved in enhanced oil recovery and carbon sequestration processes. This multiphase flow phenomenon involves a complex interplay of viscous, capillary, inertial and wettability effects. The lattice Boltzmann (LB) method is an accurate and efficient technique for modeling and simulating multiphase/multicomponent flows especially in complex flow configurations and media. In this presentation we present numerical simulation results of displacement process in thin long channels. The results are based on a new psuedo-potential multicomponent LB model with multiple relaxation time collision (MRT) model and explicit forcing scheme. We demonstrate that the proposed model is capable of accurately simulating the displacement process involving fluids with a wider range of viscosity ratios (>100) and which also leads to viscosity-independent interfacial tension and reduction of some important numerical artifacts.

Identification of Geomorphic Conditions Favoring Preservation of Multiple Individual Displacements Across Transform Faults

NASA Astrophysics Data System (ADS)

Williams, P. L.; Phillips, D. A.; Bowles-Martinez, E.; Masana, E.; Stepancikova, P.

2010-12-01

Terrestrial and airborne LiDAR data, and low altitude aerial photography have been utilized in conjunction with field work to identify and map single and multiple-event stream-offsets along all strands of the San Andreas fault in the Coachella Valley. Goals of the work are characterizing the range of displacements associated with the fault’s prehistoric surface ruptures, evaluating patterns of along-fault displacement, and disclosing processes associated with the prominent Banning-Mission Creek fault junction. Preservation offsets is associated with landscape conditions including: (1) well-confined and widely spaced source streams up-slope of the fault; (2) persistent geomorphic surfaces below the fault; (3) slope directions oriented approximately perpendicular to the fault. Notably, a pair of multiple-event offset sites have been recognized in coarse fan deposits below the Mission Creek fault near 1000 Palms oasis. Each of these sites is associated with a single source drainage oriented approximately perpendicular to the fault, and preserves a record of individual fault displacements affecting the southern portion of the Mission Creek branch of the San Andreas fault. The two sites individually record long (>10 event) slip-per-event histories. Documentation of the sites indicates a prevalence of moderate displacements and a small number of large offsets. This is consistent with evidence developed in systematic mapping of individual and multiple event stream offsets in the area extending 70 km south to Durmid Hill. Challenges to site interpretation include the presence of closely spaced en echelon fault branches and indications of stream avulsion in the area of the modern fault crossing. Conversely, strong bar and swale topography produce high quality offset indicators that can be identified across en echelon branches in most cases. To accomplish the detailed mapping needed to fully recover the complex yet well-preserved geomorphic features under investigation, a program of terrestrial laser scanning (TLS) was conducted at the 1000 Palms oasis stream offset sites. Data products and map interpretations will be presented along with initial applications of the study to characterizing San Andreas fault rupture hazard. Continuing work will seek to more fully populate the dataset of larger offsets, evaluate means to objectively date the larger offsets, and, as completely as possible, to characterize magnitudes of past surface ruptures of the San Andreas fault in the Coachella Valley.

Protein Analysis Using Real-Time PCR Instrumentation: Incorporation in an Integrated, Inquiry-Based Project

ERIC Educational Resources Information Center

Southard, Jonathan N.

2014-01-01

Instrumentation for real-time PCR is used primarily for amplification and quantitation of nucleic acids. The capability to measure fluorescence while controlling temperature in multiple samples can also be applied to the analysis of proteins. Conformational stability and changes in stability due to ligand binding are easily assessed. Protein…

The increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections.

PubMed

Gray, J; Coupland, L J

2014-01-01

On 14 January 2013, the US Food and Drug Administration (FDA) announced permission for a multiplex nucleic acid test, the xTAG® Gastrointestinal Pathogen Panel (GPP) (Luminex Corporation, USA), which simultaneously detects 11 common viral, bacterial and parasitic causes of infectious gastroenteritis, to be marketed in the USA. This announcement reflects the current move towards the development and commercialization of detection technologies based on nucleic acid amplification techniques for diagnosis of syndromic infections. We discuss the limitations and advantages of nucleic acid amplification techniques and the recent advances in Conformité Européene - in-vitro diagnostic (CE-IVD)-approved multiplex real-time PCR kits for the simultaneous detection of multiple targets within the clinical diagnostics market.

Light-effect transistor (LET) with multiple independent gating controls for optical logic gates and optical amplification

NASA Astrophysics Data System (ADS)

Marmon, Jason; Rai, Satish; Wang, Kai; Zhou, Weilie; Zhang, Yong

2016-03-01

Modern electronics are developing electronic-optical integrated circuits, while their electronic backbone, e.g. field-effect transistors (FETs), remains the same. However, further FET down scaling is facing physical and technical challenges. A light-effect transistor (LET) offers electronic-optical hybridization at the component level, which can continue Moore’s law to quantum region without requiring a FET’s fabrication complexity, e.g. physical gate and doping, by employing optical gating and photoconductivity. Multiple independent gates are therefore readily realized to achieve unique functionalities without increasing chip space. Here we report LET device characteristics and novel digital and analog applications, such as optical logic gates and optical amplification. Prototype CdSe-nanowire-based LETs show output and transfer characteristics resembling advanced FETs, e.g. on/off ratios up to ~1.0x106 with a source-drain voltage of ~1.43 V, gate-power of ~260 nW, and subthreshold swing of ~0.3 nW/decade (excluding losses). Our work offers new electronic-optical integration strategies and electronic and optical computing approaches.

Highly Sensitive Detection of Low-Abundance White Spot Syndrome Virus by a Pre-Amplification PCR Method.

PubMed

Pan, Xiaoming; Zhang, Yanfang; Sha, Xuejiao; Wang, Jing; Li, Jing; Dong, Ping; Liang, Xingguo

2017-03-28

White spot syndrome virus (WSSV) is a major threat to the shrimp farming industry and so far there is no effective therapy for it, and thus early diagnostic of WSSV is of great importance. However, at the early stage of infection, the extremely low-abundance of WSSV DNA challenges the detection sensitivity and accuracy of PCR. To effectively detect low-abundance WSSV, here we developed a pre-amplification PCR (pre-amp PCR) method to amplify trace amounts of WSSV DNA from massive background genomic DNA. Combining with normal specific PCR, 10 copies of target WSSV genes were detected from ~10 10 magnitude of backgrounds. In particular, multiple target genes were able to be balanced amplified with similar efficiency due to the usage of the universal primer. The efficiency of the pre-amp PCR was validated by nested-PCR and quantitative PCR, and pre-amp PCR showed higher efficiency than nested-PCR when multiple targets were detected. The developed method is particularly suitable for the super early diagnosis of WSSV, and has potential to be applied in other low-abundance sample detection cases.

A label-free ultrasensitive fluorescence detection of viable Salmonella enteritidis using enzyme-induced cascade two-stage toehold strand-displacement-driven assembly of G-quadruplex DNA.

PubMed

Zhang, Peng; Liu, Hui; Ma, Suzhen; Men, Shuai; Li, Qingzhou; Yang, Xin; Wang, Hongning; Zhang, Anyun

2016-06-15

The harm of Salmonella enteritidis (S. enteritidis ) to public health mainly by contaminating fresh food and water emphasizes the urgent need for rapid detection techniques to help control the spread of the pathogen. In this assay, an newly designed capture probe complex that contained specific S. enteritidis-aptamer and hybridized signal target sequence was used for viable S. enteritidis recognition directly. In the presence of the target S. enteritidis, single-stranded target sequences were liberated and initiated the replication-cleavage reaction, producing numerous G-quadruplex structures with a linker on the 3'-end. And then, the sensing system took innovative advantage of quadratic linker-induced strand-displacement for the first time to release target sequence in succession, leading to the cyclic reuse of the target sequences and cascade signal amplification, thereby achieving the successive production of G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binded to these G-quadruplex structures and generated significantly enhanced fluorescent signals to achieve highly sensitive detection of S. enteritidis down to 60 CFU/mL with a linear range from 10(2) to 10(7)CFU/mL. By coupling the cascade two-stage target sequences-recyclable toehold strand-displacement with aptamer-based target recognition successfully, it is the first report on a novel non-label, modification-free and DNA extraction-free ultrasensitive fluorescence biosensor for detecting viable S. enteritidis directly, which can discriminate from dead S. enteritidis. Copyright © 2016 Elsevier B.V. All rights reserved.

Emotional, physical, and social needs among 0-5-year-old children displaced by the 2010 Chilean earthquake: associated characteristics and exposures.

PubMed

Arbour, MaryCatherine; Murray, Kara A; Yoshikawa, Hirokazu; Arriet, Felipe; Moraga, Cecilia; Vega, Miguel Angel Cordero

2017-04-01

An 8.8-magnitude earthquake occurred off the coast of Chile on 27 February 2010, displacing nearly 2,000 children aged less than five years to emergency housing camps. Nine months later, this study assessed the needs of 140 displaced 0-5-year-old children in six domains: caregiver stability and protection; health; housing; nutrition; psychosocial situation; and stimulation. Multivariate regression was applied to examine the degree to which emotional, physical, and social needs were associated with baseline characteristics and exposure to the earthquake, to stressful events, and to ongoing risks in the proximal post-earthquake context. In each domain, 20 per cent or fewer children had unmet needs. Of all children in the sample, 20 per cent had unmet needs in multiple domains. Children's emotional, physical, and social needs were associated with ongoing exposures amenable to intervention, more than with baseline characteristics or epicentre proximity. Relief efforts should address multiple interrelated domains of child well-being and ongoing risks in post-disaster settings. © 2017 The Author(s). Disasters © Overseas Development Institute, 2017.

Designing for Multiple Stakeholder Interests within the Humanitarian Market: The Case of Off-Grid Energy Devices

ERIC Educational Resources Information Center

Nielsen, Brita Fladvad; Rodrigues Santos, Ana Laura

2013-01-01

A "humanitarian market" for off-grid renewable energy technologies for displaced populations in remote areas has emerged. Within this market, there are multiple stakeholder agendas. End-user needs and sustainable development goals are currently not considered through the customer-enterprise relationship and the applied product and…

Strong Effects of Vs30 Heterogeneity on Physics-Based Scenario Ground-Shaking Computations

NASA Astrophysics Data System (ADS)

Louie, J. N.; Pullammanappallil, S. K.

2014-12-01

Hazard mapping and building codes worldwide use the vertically time-averaged shear-wave velocity between the surface and 30 meters depth, Vs30, as one predictor of earthquake ground shaking. Intensive field campaigns a decade ago in Reno, Los Angeles, and Las Vegas measured urban Vs30 transects with 0.3-km spacing. The Clark County, Nevada, Parcel Map includes urban Las Vegas and comprises over 10,000 site measurements over 1500 km2, completed in 2010. All of these data demonstrate fractal spatial statistics, with a fractal dimension of 1.5-1.8 at scale lengths from 0.5 km to 50 km. Vs measurements in boreholes up to 400 m deep show very similar statistics at 1 m to 200 m lengths. When included in physics-based earthquake-scenario ground-shaking computations, the highly heterogeneous Vs30 maps exhibit unexpectedly strong influence. In sensitivity tests (image below), low-frequency computations at 0.1 Hz display amplifications (as well as de-amplifications) of 20% due solely to Vs30. In 0.5-1.0 Hz computations, the amplifications are a factor of two or more. At 0.5 Hz and higher frequencies the amplifications can be larger than what the 1-d Building Code equations would predict from the Vs30 variations. Vs30 heterogeneities at one location have strong influence on amplifications at other locations, stretching out in the predominant direction of wave propagation for that scenario. The sensitivity tests show that shaking and amplifications are highly scenario-dependent. Animations of computed ground motions and how they evolve with time suggest that the fractal Vs30 variance acts to trap wave energy and increases the duration of shaking. Validations of the computations against recorded ground motions, possible in Las Vegas Valley due to the measurements of the Clark County Parcel Map, show that ground motion levels and amplifications match, while recorded shaking has longer duration than computed shaking. Several mechanisms may explain the amplification and increased duration of shaking in the presence of heterogeneous spatial distributions of Vs: conservation of wave energy across velocity changes; geometric focusing of waves by low-velocity lenses; vertical resonance and trapping; horizontal resonance and trapping; and multiple conversion of P- to S-wave energy.

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

PubMed Central

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

2013-01-01

Dynamic DNA nanotechnology often uses toehold-mediated strand displacement for controlling reaction kinetics. Although the dependence of strand displacement kinetics on toehold length has been experimentally characterized and phenomenologically modeled, detailed biophysical understanding has remained elusive. Here, we study strand displacement at multiple levels of detail, using an intuitive model of a random walk on a 1D energy landscape, a secondary structure kinetics model with single base-pair steps and a coarse-grained molecular model that incorporates 3D geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Two factors explain the dependence of strand displacement kinetics on toehold length: (i) the physical process by which a single step of branch migration occurs is significantly slower than the fraying of a single base pair and (ii) initiating branch migration incurs a thermodynamic penalty, not captured by state-of-the-art nearest neighbor models of DNA, due to the additional overhang it engenders at the junction. Our findings are consistent with previously measured or inferred rates for hybridization, fraying and branch migration, and they provide a biophysical explanation of strand displacement kinetics. Our work paves the way for accurate modeling of strand displacement cascades, which would facilitate the simulation and construction of more complex molecular systems. PMID:24019238

Fluid volume displacement at the oval and round windows with air and bone conduction stimulation.

PubMed

Stenfelt, Stefan; Hato, Naohito; Goode, Richard L

2004-02-01

The fluids in the cochlea are normally considered incompressible, and the fluid volume displacement of the oval window (OW) and the round window (RW) should be equal and of opposite phase. However, other channels, such as the cochlear and vestibular aqueducts, may affect the fluid flow. To test if the OW and RW fluid flows are equal and of opposite phase, the volume displacement was assessed by multiple point measurement at the windows with a laser Doppler vibrometer. This was done during air conduction (AC) stimulation in seven fresh human temporal bones, and with bone conduction (BC) stimulation in eight temporal bones and one human cadaver head. With AC stimulation, the average volume displacement of the two windows is within 3 dB, and the phase difference is close to 180 degrees for the frequency range 0.1 to 10 kHz. With BC stimulation, the average volume displacement difference between the two windows is greater: below 2 kHz, the volume displacement at the RW is 5 to 15 dB greater than at the OW and above 2 kHz more fluid is displaced at the OW. With BC stimulation, lesions at the OW caused only minor changes of the fluid flow at the RW.

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

PubMed

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

2013-12-01

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

A wireless laser displacement sensor node for structural health monitoring.

PubMed

Park, Hyo Seon; Kim, Jong Moon; Choi, Se Woon; Kim, Yousok

2013-09-30

This study describes a wireless laser displacement sensor node that measures displacement as a representative damage index for structural health monitoring (SHM). The proposed measurement system consists of a laser displacement sensor (LDS) and a customized wireless sensor node. Wireless communication is enabled by a sensor node that consists of a sensor module, a code division multiple access (CDMA) communication module, a processor, and a power module. An LDS with a long measurement distance is chosen to increase field applicability. For a wireless sensor node driven by a battery, we use a power control module with a low-power processor, which facilitates switching between the sleep and active modes, thus maximizing the power consumption efficiency during non-measurement and non-transfer periods. The CDMA mode is also used to overcome the limitation of communication distance, which is a challenge for wireless sensor networks and wireless communication. To evaluate the reliability and field applicability of the proposed wireless displacement measurement system, the system is tested onsite to obtain the required vertical displacement measurements during the construction of mega-trusses and an edge truss, which are the primary structural members in a large-scale irregular building currently under construction. The measurement values confirm the validity of the proposed wireless displacement measurement system and its potential for use in safety evaluations of structural elements.

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

PubMed

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

2014-06-17

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

Detection of Stimulus Displacements Across Saccades is Capacity-Limited and Biased in Favor of the Saccade Target

PubMed Central

Irwin, David E.; Robinson, Maria M.

2015-01-01

Retinal image displacements caused by saccadic eye movements are generally unnoticed. Recent theories have proposed that perceptual stability across saccades depends on a local evaluation process centered on the saccade target object rather than on remapping and evaluating the positions of all objects in a display. In three experiments, we examined whether objects other than the saccade target also influence perceptual stability by measuring displacement detection thresholds across saccades for saccade targets and a variable number of non-saccade objects. We found that the positions of multiple objects are maintained across saccades, but with variable precision, with the saccade target object having priority in the perception of displacement, most likely because it is the focus of attention before the saccade and resides near the fovea after the saccade. The perception of displacement of objects that are not the saccade target is affected by acuity limitations, attentional limitations, and limitations on memory capacity. Unlike previous studies that have found that a postsaccadic blank improves the detection of displacement direction across saccades, we found that postsaccadic blanking hurt the detection of displacement per se by increasing false alarms. Overall, our results are consistent with the hypothesis that visual working memory underlies the perception of stability across saccades. PMID:26640430

Displacement, county social cohesion, and depression after a large-scale traumatic event.

PubMed

Lê, Félice; Tracy, Melissa; Norris, Fran H; Galea, Sandro

2013-11-01

Depression is a common and potentially debilitating consequence of traumatic events. Mass traumatic events cause wide-ranging disruptions to community characteristics, influencing the population risk of depression. In the aftermath of such events, population displacement is common. Stressors associated with displacement may increase risk of depression directly. Indirectly, persons who are displaced may experience erosion in social cohesion, further exacerbating their risk for depression. Using data from a population-based cross-sectional survey of adults living in the 23 southernmost counties of Mississippi (N = 708), we modeled the independent and joint relations of displacement and county-level social cohesion with depression 18-24 months after Hurricane Katrina. After adjustment for individual- and county-level socio-demographic characteristics and county-level hurricane exposure, joint exposure to both displacement and low social cohesion was associated with substantially higher log-odds of depression (b = 1.34 [0.86-1.83]). Associations were much weaker for exposure only to low social cohesion (b = 0.28 [-0.35-0.90]) or only to displacement (b = 0.04 [-0.80-0.88]). The associations were robust to additional adjustment for individually perceived social cohesion and social support. Addressing the multiple, simultaneous disruptions that are a hallmark of mass traumatic events is important to identify vulnerable populations and understand the psychological ramifications of these events.

Fluid volume displacement at the oval and round windows with air and bone conduction stimulation

NASA Astrophysics Data System (ADS)

Stenfelt, Stefan; Hato, Naohito; Goode, Richard L.

2004-02-01

The fluids in the cochlea are normally considered incompressible, and the fluid volume displacement of the oval window (OW) and the round window (RW) should be equal and of opposite phase. However, other channels, such as the cochlear and vestibular aqueducts, may affect the fluid flow. To test if the OW and RW fluid flows are equal and of opposite phase, the volume displacement was assessed by multiple point measurement at the windows with a laser Doppler vibrometer. This was done during air conduction (AC) stimulation in seven fresh human temporal bones, and with bone conduction (BC) stimulation in eight temporal bones and one human cadaver head. With AC stimulation, the average volume displacement of the two windows is within 3 dB, and the phase difference is close to 180° for the frequency range 0.1 to 10 kHz. With BC stimulation, the average volume displacement difference between the two windows is greater: below 2 kHz, the volume displacement at the RW is 5 to 15 dB greater than at the OW and above 2 kHz more fluid is displaced at the OW. With BC stimulation, lesions at the OW caused only minor changes of the fluid flow at the RW.

Improved amplification results following episodes of failure to amplify at the Amelogenin Locus using PowerPlex® ESI 16 Fast System.

PubMed

Berlyne, Sigal; Oz, Carla; Einot, Naftaly; Avraham, Shlomit; Ram, Tanya; Goldberg, Miri D; Gafny, Ron

2017-07-01

In 2012 the Israel Police DNA Casework laboratory adopted the 16 STR PowerPlex ® ESI kit for routine use. The Promega Company updated this kit and developed the PowerPlex ® ESI 16 Fast System in which all autosomal primer pairs remained identical to the original set, except at the amelogenin site. The master mix was improved and optimized which allowed for direct, faster and more robust amplification. Prior to implementing the PowerPlex ® ESI 16 Fast System in our lab, we conducted a preliminary assay where 213 casework samples were amplified using the new kit. These samples had previously been extracted by one of two extraction kits employed by our lab. (the PrepFiler ExpressTM and PrepFiler BTATM Forensic DNA Extraction Kits). The amplification results from these samples were compared to samples amplified using the original PowerPlex ® ESI 16 kit. Multiple incidents of failure to amplify at the amelogenin locus were noted using the new system with the recommended protocol at a rate of 13% (28 of 213 samples). Experiments were performed to understand whether these amplification failures could be a result of primer binding site mutations, extraction method reagents and/or inhibitors. The conclusions reached following these experiments, in conjunction with consultation with the manufacturer, led to the trial of a modified amplification protocol where the suggested annealing temperature was reduced by 2 degrees. To evaluate the efficiency of this altered protocol, a comparison study was undertaken where 88 additional casework samples were chosen and amplified using both the modified 58°C and the recommended 60°C annealing temperatures. We concluded that the most effective method in our laboratory for achieving a consistent and balanced amplification at the amelogenin locus was to reduce the annealing temperature from the manufacturer's recommended 60°C to 58°C. This modification resulted in a reduction of the failure to amplify at the amelogenin locus from 13% (28/213) to 1.1% (1/88) without any observed changes to the autosomal STR amplification results. Copyright © 2017 Elsevier B.V. All rights reserved.

Capillary electrophoresis with electrochemiluminescent detection for highly sensitive assay of genetically modified organisms.

PubMed

Guo, Longhua; Yang, Huanghao; Qiu, Bin; Xiao, Xueyang; Xue, Linlin; Kim, Donghwan; Chen, Guonan

2009-12-01

A capillary electrophoresis coupled with electrochemiluminescent detection system (CE-ECL) was developed for the detection of polymerase chain reaction (PCR) amplicons. The ECL luminophore, tris(1,10-phenanthroline) ruthenium(II) (Ru(phen)(3)(2+)), was labeled to the PCR primers before amplification. Ru(phen)(3)(2+) was then introduced to PCR amplicons by PCR amplification. Eventually, the PCR amplicons were separated and detected by the homemade CE-ECL system. The detection of a typical genetically modified organism (GMO), Roundup Ready Soy (RRS), was shown as an example to demonstrate the reliability of the proposed approach. Four pairs of primers were amplified by multiple PCR (MPCR) simultaneously, three of which were targeted on the specific sequence of exogenous genes of RRS, and another was targeted on the endogenous reference gene of soybean. Both the conditions for PCR amplification and CE-ECL separation and detection were investigated in detail. Results showed that, under the optimal conditions, the proposed method can accurately identifying RRS. The corresponding limit of detection (LOD) was below 0.01% with 35 PCR cycles.

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.

Direct duplex real-time loop mediated isothermal amplification assay for the simultaneous detection of cow and goat species origin of milk and yogurt products for field use.

PubMed

Kim, Mi-Ju; Kim, Hae-Yeong

2018-04-25

A multiple loop-mediated isothermal amplification (LAMP) method was developed to detect cow and goat milk in the field using a portable fluorescence device. For rapid on-site detection, this duplex LAMP assay was used in combination with direct amplification, without DNA extraction. The cow- and goat-specific LAMP primer sets were designed based on the mitochondrial cytochrome b gene, and showed specificity against 13 other animal species in the reactions. The sensitivity of the duplex LAMP assay for cow and goat was 0.1 and 1 pg, respectively. The detection limit for both target species in milk mixtures was 2%. This assay successfully amplified and identified the two target species in 24 samples of commercial milk and yogurt products, with 30 min sampling-to-result analysis time. Therefore, this direct duplex real-time LAMP assay is useful for on-site simultaneous detection of cow and goat milk in commercial products, a capability needed to confirm accurate labeling. Copyright © 2017 Elsevier Ltd. All rights reserved.

Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip

PubMed Central

Yeh, Erh-Chia; Fu, Chi-Cheng; Hu, Lucy; Thakur, Rohan; Feng, Jeffrey; Lee, Luke P.

2017-01-01

Portable, low-cost, and quantitative nucleic acid detection is desirable for point-of-care diagnostics; however, current polymerase chain reaction testing often requires time-consuming multiple steps and costly equipment. We report an integrated microfluidic diagnostic device capable of on-site quantitative nucleic acid detection directly from the blood without separate sample preparation steps. First, we prepatterned the amplification initiator [magnesium acetate (MgOAc)] on the chip to enable digital nucleic acid amplification. Second, a simplified sample preparation step is demonstrated, where the plasma is separated autonomously into 224 microwells (100 nl per well) without any hemolysis. Furthermore, self-powered microfluidic pumping without any external pumps, controllers, or power sources is accomplished by an integrated vacuum battery on the chip. This simple chip allows rapid quantitative digital nucleic acid detection directly from human blood samples (10 to 105 copies of methicillin-resistant Staphylococcus aureus DNA per microliter, ~30 min, via isothermal recombinase polymerase amplification). These autonomous, portable, lab-on-chip technologies provide promising foundations for future low-cost molecular diagnostic assays. PMID:28345028

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

PubMed Central

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

2012-01-01

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

Direct identification of non-polio enteroviruses in residual paralysis cases by analysis of VP1 sequences.

PubMed

Rahimi, Pooneh; Tabatabaie, H; Gouya, Mohammad M; Mahmudi, M; Musavi, T; Rad, K Samimi; Azad, T Mokhtari; Nategh, R

2009-06-01

The 66 serotypes of human enteroviruses (EVs) are classified into four species A-D, based on phylogenetic relationships in multiple genome regions. Partial VP(1) amplification and sequence analysis are reliable methods for identifying non-polio enterovirus serotypes, especially in negative cell culture specimens from patients with residual paralysis. In Iran during the years 2000-2002, there were 29 residual paralysis cases with negative cell (RD, HEp(2) and L(20)B) culture results. The genomic RNA was extracted from stool specimens from cases of residual paralysis and detected by amplification of the 5'-nontranslated region using RT-PCR with Pan-EV primers. Partial VP(1) amplification by semi-nested RT-PCR (snRT-PCR) and sequence analysis were done. Specimens from the 29 culture-negative cases contained echoviruses of six different serotypes. The global eradication of wild polioviruses is near and study of non-polio enteroviruses, which can cause poliomyelitis, is increasingly important to understand their pathogenesis. The VP(1) sequences, derived from the snRT-PCR products, allowed rapid molecular analysis of these non-polio strains.

Cross-subtype Detection of HIV-1 Using Reverse Transcription and Recombinase Polymerase Amplification

PubMed Central

Lillis, Lorraine; Lehman, Dara A.; Siverson, Joshua B.; Weis, Julie; Cantera, Jason; Parker, Mathew; Piepenburg, Olaf; Overbaugh, Julie; Boyle, David S.

2016-01-01

A low complexity diagnostic test that rapidly and reliably detects HIV infection in infants at the point of care could facilitate early treatment, improving outcomes. However, many infant HIV diagnostics can only be performed in laboratory settings. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that can rapidly amplify proviral DNA from multiple subtypes of HIV-1 in under twenty minutes without complex equipment. In this study we added reverse transcription (RT) to RPA to allow detection of both HIV-1 RNA and DNA. We show that this RT-RPA HIV-1 assay has a limit of detection of 10 to 30 copies of an exact sequence matched DNA or RNA, respectively. In addition, at 100 copies of RNA or DNA, the assay detected 171 of 175 (97.7 %) sequence variants that represent all the major subtypes and recombinant forms of HIV-1 Groups M and O. This data suggests that the application of RT-RPA for the combined detection of HIV-1 viral RNA and proviral DNA may prove a highly sensitive tool for rapid and accurate diagnosis of infant HIV. PMID:26821087

Displacement-type quartz crystal microbalance immunosensing platform for ultrasensitive monitoring of small molecular toxins.

PubMed

Tang, Dianping; Zhang, Bing; Tang, Juan; Hou, Li; Chen, Guonan

2013-07-16

A novel displacement-type quartz crystal microbalance (QCM) immunosensing strategy, based on glucose and its analogue dextran for concanavalin A (ConA) binding sites, was designed for ultrasensitive monitoring of small molecular biotoxins (brevetoxin B, PbTx-2, used as a model) with signal amplification on a graphene-functionalized sensing interface. To construct such a QCM immunosensing platform, phenoxy-functionalized dextran (DexP) was initially assembled onto the surface of graphene-coated QCM probe via the π-stacking interaction, and ConA-labeled monoclonal mouse anti-PbTx-2 capture antibody was then immobilized on the DexP-modified probe by dextran-ConA binding. Gold nanoparticle heavily functionalized with glucoamylase and bovine serum albumin-PbTx-2 (PbTx-2-BSA) conjugate was employed as the trace tag. A competitive-type immunoassay format was adopted for the online monitoring of PbTx-2 between anti-PbTx-2 antibody immobilized on the QCM probe and PbTx-2-BSA labeled on the gold nanoparticle. Accompanying the gold nanoparticle, the carried glucoamylase could hydrolyze amylopectin in glucose. The produced glucose competed with dextran for ConA and displaced the ConA-streptavidin-anti-PbTx-2 complex from the QCM probe, resulting in the frequency change. Under optimal conditions, the frequency of the QCM immunosensor was indirectly proportional to the concentration of target PbTx-2 in the sample and exhibited a dynamic range from 1.0 pg·mL(-1) to 10 ng·mL(-1) with a detection limit (LOD) of 0.6 pg·mL(-1) at the 3Sblank level. Intra- and interassay coefficients of variation were below 7.5% and 9.5%, respectively. In addition, the methodology was evaluated for analysis of PbTx-2 in 15 spiked seafood samples and showed good accordance between results obtained by the displacement-type QCM immunosensor and a commercialized enzyme-linked immunosorbent assay (ELISA) method.

Arctic Amplification and Potential Mid-Latitude Weather Linkages

NASA Astrophysics Data System (ADS)

Overland, J. E.

2014-12-01

Increasing temperatures and other changes continued in the Arctic over the last decade, even though the rate of global warming has decreased in part due to a cool Pacific Ocean. Thus Arctic temperatures have increased at least 3 times the rate of mid-latitude temperatures. Credibility for persistent Arctic change comes from multiple indicators which are now available for multiple decades. Further, the spatial pattern of Arctic Amplification differs from patterns of natural variability. The role of the Arctic in the global climate system is based on multiple interacting feedbacks represented by these indicators as a causal basis for Arctic Amplification driven by modest global change. Many of these processes act on a regional basis and their non-linear interactions are not well captured by climate models. For example, future loss of sea ice due to increases in CO2 are demonstrated by these models but the rates of loss appear slow. It is reasonable to suspect that Arctic change which can produce the largest temperature anomalies on the planet and demonstrate recent extremes in the polar vortex could be linked to mid-latitude weather, especially as Arctic change will continue over the next decades. The meteorological community remains skeptical, however, in the sense of "not proven." Natural variability in chaotic atmospheric flow remains the main dynamic process, and it is difficult to determine whether Arctic forcing of a north-south linkage is emerging from the most recent period of Arctic change since 2007. Nonetheless, such a hypothesis is worthy of investigation, given the need to further understand Arctic dynamic atmospheric processes, and the potential for improving mid-latitude seasonal forecasts base on high-latitude forcing. Several AGU sessions and other forums over the next year (WWRP, IASC,CliC) address this issue, but the topic is not ready for a firm answer. The very level of controversy indicates the state of the science.

Simultaneous full-field 3-D vibrometry of the human eardrum using spatial-bandwidth multiplexed holography

PubMed Central

Khaleghi, Morteza; Guignard, Jérémie; Furlong, Cosme; Rosowski, John J.

2015-01-01

Abstract. Holographic interferometric methods typically require the use of three sensitivity vectors in order to obtain three-dimensional (3-D) information. Methods based on multiple directions of illumination have limited applications when studying biological tissues that have temporally varying responses such as the tympanic membrane (TM). Therefore, to measure 3-D displacements in such applications, the measurements along all the sensitivity vectors have to be done simultaneously. We propose a multiple-illumination directions approach to measure 3-D displacements from a single-shot hologram that contains displacement information from three sensitivity vectors. The hologram of an object of interest is simultaneously recorded with three incoherently superimposed pairs of reference and object beams. The incident off-axis angles of the reference beams are adjusted such that the frequency components of the multiplexed hologram are completely separate. Because of the differences in the directions and wavelengths of the reference beams, the positions of each reconstructed image corresponding to each sensitivity vector are different. We implemented a registration algorithm to accurately translate individual components of the hologram into a single global coordinate system to calculate 3-D displacements. The results include magnitudes and phases of 3-D sound-induced motions of a human cadaveric TM at several excitation frequencies showing modal and traveling wave motions on its surface. PMID:25984986

Selection of fluorophore and quencher pairs for fluorescent nucleic acid hybridization probes.

PubMed

Marras, Salvatore A E

2006-01-01

With the introduction of simple and relatively inexpensive methods for labeling nucleic acids with nonradioactive labels, doors have been opened that enable nucleic acid hybridization probes to be used for research and development, as well as for clinical diagnostic applications. The use of fluorescent hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. The use of hybridization probes that bind to the amplification products in real-time markedly improves the ability to obtain quantitative results. Furthermore, real-time nucleic acid amplification assays can be carried out in sealed tubes, eliminating carryover contamination. Because fluorescent hybridization probes are available in a wide range of colors, multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. It is therefore important to carefully select the labels of hybridization probes, based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This chapter outlines different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers.

A SPR biosensor based on signal amplification using antibody-QD conjugates for quantitative determination of multiple tumor markers

PubMed Central

Wang, Huan; Wang, Xiaomei; Wang, Jue; Fu, Weiling; Yao, Chunyan

2016-01-01

The detection of tumor markers is very important in early cancer diagnosis; however, tumor markers are usually present at very low concentrations, especially in the early stages of tumor development. Surface plasmon resonance (SPR) is widely used to detect biomolecular interactions; it has inherent advantages of being high-throughput, real-time, and label-free technique. However, its sensitivity needs essential improvement for practical applications. In this study, we developed a signal amplification strategy using antibody-quantum dot (QD) conjugates for the sensitive and quantitative detection of α-fetoprotein (AFP), carcinoembryonic antigen (CEA) and cytokeratin fragment 21-1 (CYFRA 21-1) in clinical samples. The use of a dual signal amplification strategy using AuNP-antibody and antibody-QD conjugates increased the signal amplification by 50-folds. The constructed SPR biosensor showed a detection limit as low as 0.1 ng/mL for AFP, CEA, and CYFRA 21-1. Moreover, the results obtained using this SPR biosensor were consistent with those obtained using the electrochemiluminescence method. Thus, the constructed SPR biosensor provides a highly sensitive and specific approach for the detection of tumor markers. This SPR biosensor can be expected to be readily applied for the detection of other tumor markers and can offer a potentially powerful solution for tumor screening. PMID:27615417

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

Guta, Madalin; Matsumoto, Keiji; Quantum Computation and Information Project, JST, Hongo 5-28-3, Bunkyo-ku, Tokyo 113-0033

We construct the optimal one to two cloning transformation for the family of displaced thermal equilibrium states of a harmonic oscillator, with a fixed and known temperature. The transformation is Gaussian and it is optimal with respect to the figure of merit based on the joint output state and norm distance. The proof of the result is based on the equivalence between the optimal cloning problem and that of optimal amplification of Gaussian states which is then reduced to an optimization problem for diagonal states of a quantum oscillator. A key concept in finding the optimum is that of stochasticmore » ordering which plays a similar role in the purely classical problem of Gaussian cloning. The result is then extended to the case of n to m cloning of mixed Gaussian states.« less

Outer Hair Cell Electromotility in vivo

NASA Astrophysics Data System (ADS)

Ramamoorthy, Sripriya; Nuttall, Alfred L.

2011-11-01

The effectiveness of outer hair cell (OHC) electro-motility in vivo has been challenged by the expected low-pass filtering of the transmembrane potential due to the cell's own capacitance. The OHC electromotility is characterized here by an electromechanical ratio defined as the ratio of the OHC contraction to the transmembrane potential. This ratio has been measured in isolated cells to be approximately 26 nm/mV. We estimate the OHC electromechanical ratio in vivo from the recently measured displacements of the reticular lamina and the basilar membrane near the 19 kHz characteristic frequency in the basal region of guinea pig cochlea. Our analysis strongly suggests OHC electromotility process is effective for cochlear amplification in vivo at least around the characteristic frequency of the basal location in spite of the low-pass filtering.

[Displaced people's healthcare use in Bucaramanga, Colombia].

PubMed

Ruiz-Rodríguez, Myriam; López-Moreno, Sergio; Avila-Burgos, Leticia; Acosta-Ramírez, Naydú

2006-01-01

Analysing the factors and barriers associated with the population displaced by armed conflict using medical services in Bucaramanga, Colombia. Data from the "Diagnosing the health of both displaced and non-displaced populations in Bucaramanga and its metropolitan area" study, designed and executed by the Industrial University of Santander and financed by the Pan-American Health Organisation, analysed medical service use in individuals aged over 15 years. This agreed with Andersen and Newman's model and evaluated the effect of associated factors by means of multiple logistical regression. One out of each five people who became ill during the fifteen days prior to the survey had used the medical services. This percentage was smaller than that found in the Colombian population as a whole. The type of social security regime (contributory health insurance), previous service use and being from the region were associated with greater service use. Displaced people having subsidised health insurance did not present differences in service use compared to those who had no access to such insurance. The distance from dwelling to institution was another barrier against using health services (RM = 0.64; IC 0.42-0.97). There was differential medical service use amongst the displaced population, determined by their health insurance coverage and economic position.

Calculation of Stress Intensity Factors for Interfacial Cracks in Fiber Metal Laminates

NASA Technical Reports Server (NTRS)

Wang, John T.

2009-01-01

Stress intensity factors for interfacial cracks in Fiber Metal Laminates (FML) are computed by using the displacement ratio method recently developed by Sun and Qian (1997, Int. J. Solids. Struct. 34, 2595-2609). Various FML configurations with single and multiple delaminations subjected to different loading conditions are investigated. The displacement ratio method requires the total energy release rate, bimaterial parameters, and relative crack surface displacements as input. Details of generating the energy release rates, defining bimaterial parameters with anisotropic elasticity, and selecting proper crack surface locations for obtaining relative crack surface displacements are discussed in the paper. Even though the individual energy release rates are nonconvergent, mesh-size-independent stress intensity factors can be obtained. This study also finds that the selection of reference length can affect the magnitudes and the mode mixity angles of the stress intensity factors; thus, it is important to report the reference length used with the calculated stress intensity factors.

Household displacement and health risk behaviors among HIV/AIDS-affected children in rural China.

PubMed

Zhao, Qun; Zhao, Junfeng; Li, Xiaoming; Fang, Xiaoyi; Zhao, Guoxiang; Lin, Xiuyun; Zhang, Liying

2011-07-01

When parents die of or are infected with HIV, children might have to leave their own household and be displaced to other living arrangements and some may even be displaced multiple times. The objective of this study is to examine the association between household displacement and health risk behaviors among AIDS orphans (children who have lost one or both of their parents to HIV/AIDS) and vulnerable children (children living with HIV-infected parents) in rural China. The sample consisted of 1015 children (549 AIDS orphans, 466 vulnerable children) in family-based care. The children were assigned to three displacement groups according to the number of household displacement (i.e., none, once, at least twice) after their parents became ill or died of HIV/AIDS. Cigarette smoking, alcohol use, violence, public property destruction, suicidal ideation, and suicide attempt were used to assess the health risk behaviors of these children. Both bivariate and multivariate tests were used to assess the differences in health risk behaviors among displacement groups. The findings indicated that children who were displaced at least twice were more likely to report a higher frequency of public property destruction and suicide ideation than those who were never displaced or displaced once. Multivariate analysis revealed that public property destruction, suicide ideation and suicide attempt were significantly associated with the household displacement among these children, controlling for gender, age, child status (AIDS orphans vs. vulnerable children), and the duration of household displacement. Results in the current study suggest that a stable living environment was important for both AIDS orphans and vulnerable children in communities with a high prevalence of HIV/AIDS. The government, community, and other agencies need to make efforts to avoid frequent household displacement among these children after the HIV-related infection or death of their parents.

Finite Element Modelling of the Indo-Gangetic Basin to Study Site Amplification

NASA Astrophysics Data System (ADS)

Sivasubramonian, J.; Jaya, D.; Raghukanth, S. T. G.; Mai, P. M.

2017-12-01

We have developed a finite-element model of the 3D velocity structure of the Indo-Gangetic basin (IG basin) to quantify site amplifications due to seismic waves emanated from regional earthquakes. Estimating seismic wave amplifications is difficult in case of limited instrumentation, thus motivating us to propose a new simulation-based approach. The input required for the finite-element model include the spatial coordinates and the material properties (density, P-wave and S-wave velocities, Q factor) of the basin. Recent studies in the basin demarcate sediment layers of varying thickness, reaching down to a depth of 6 km and S-wave velocities ranging from 0.4-2.4 km/s (Srinivas et al., 2013). In the present study, our regional model has dimensions 900 x 900 x 80 km in x, y and z directions, discretized into 320 x 320 x 53 hexahedral elements. The top 6 km of the IG basin is divided into 8 different sediment layers with varying material properties. We use kinematic rupture models for the earthquake sources to simulate past as well as hypothetical future events. Two past earthquakes (Mw4.9, Delhi; Mw5.2, Chamoli) and two hypothetical earthquakes (Mw7.1; Mw8.5) are considered in our study. The rupture plane dimensions (L and W) and the slip distribution are estimated using the method of Mai and Beroza (2002). Based on focal-mechanism solutions and the depths of seismicity, we define the strike (580, 3090), the dip (650, 210), the rake (160, 770), and the depth of top edge of fault (5 km, 19 km) for the two large hypothetical earthquakes. Based on these parameters, the Centroid Moment Tensor (CMT) solution of the source is obtained. Ground motions are then simulated by solving the three-dimensional wave equation using the spectral element method (Komatitsch and Tromp, 1999). The key observations from our results are: 1) basin amplification factors for Peak Ground Velocity (PGV) are twice as high as Peak Ground Displacement (PGD) 2) PGV amplifications are as high as a factor of 6 for earthquakes occurring inside the basin, and a factor of 4 for Himalayan earthquakes (to the north of the study region) 3) The simulated shake maps of PGV and PGD show directivity. Based on the above observations, we conclude that it is important to include our model into low-frequency ground-motion estimation for seismic hazard analysis.

Comparative Genomic Analysis Reveals Multiple Long Terminal Repeats, Lineage-Specific Amplification, and Frequent Interelement Recombination for Cassandra Retrotransposon in Pear (Pyrus bretschneideri Rehd.)

PubMed Central

Yin, Hao; Du, Jianchang; Li, Leiting; Jin, Cong; Fan, Lian; Li, Meng; Wu, Jun; Zhang, Shaoling

2014-01-01

Cassandra transposable elements belong to a specific group of terminal-repeat retrotransposons in miniature (TRIM). Although Cassandra TRIM elements have been found in almost all vascular plants, detailed investigations on the nature, abundance, amplification timeframe, and evolution have not been performed in an individual genome. We therefore conducted a comprehensive analysis of Cassandra retrotransposons using the newly sequenced pear genome along with four other Rosaceae species, including apple, peach, mei, and woodland strawberry. Our data reveal several interesting findings for this particular retrotransposon family: 1) A large number of the intact copies contain three, four, or five long terminal repeats (LTRs) (∼20% in pear); 2) intact copies and solo LTRs with or without target site duplications are both common (∼80% vs. 20%) in each genome; 3) the elements exhibit an overall unbiased distribution among the chromosomes; 4) the elements are most successfully amplified in pear (5,032 copies); and 5) the evolutionary relationships of these elements vary among different lineages, species, and evolutionary time. These results indicate that Cassandra retrotransposons contain more complex structures (elements with multiple LTRs) than what we have known previously, and that frequent interelement unequal recombination followed by transposition may play a critical role in shaping and reshaping host genomes. Thus this study provides insights into the property, propensity, and molecular mechanisms governing the formation and amplification of Cassandra retrotransposons, and enhances our understanding of the structural variation, evolutionary history, and transposition process of LTR retrotransposons in plants. PMID:24899073

Electrochemical biosensor based on functional composite nanofibers for detection of K-ras gene via multiple signal amplification strategy.

PubMed

Wang, Xiaoying; Shu, Guofang; Gao, Chanchan; Yang, Yu; Xu, Qian; Tang, Meng

2014-12-01

An electrochemical biosensor based on functional composite nanofibers for hybridization detection of specific K-ras gene that is highly associated with colorectal cancer via multiple signal amplification strategy has been developed. The carboxylated multiwalled carbon nanotubes (MWCNTs) doped nylon 6 (PA6) composite nanofibers (MWCNTs-PA6) was prepared using electrospinning, which served as the nanosized backbone for thionine (TH) electropolymerization. The functional composite nanofibers [MWCNTs-PA6-PTH, where PTH is poly(thionine)] used as supporting scaffolds for single-stranded DNA1 (ssDNA1) immobilization can dramatically increase the amount of DNA attachment and the hybridization sensitivity. Through the hybridization reaction, a sandwich format of ssDNA1/K-ras gene/gold nanoparticle-labeled ssDNA2 (AuNPs-ssDNA2) was fabricated, and the AuNPs offered excellent electrochemical signal transduction. The signal amplification was further implemented by forming network-like thiocyanuric acid/gold nanoparticles (TA/AuNPs). A significant sensitivity enhancement was obtained; the detection limit was down to 30fM, and the discriminations were up to 54.3 and 51.9% between the K-ras gene and the one-base mismatched sequences including G/C and A/T mismatched bases, respectively. The amenability of this method to the analyses of K-ras gene from the SW480 colorectal cancer cell lysates was demonstrated. The results are basically consistent with those of the K-ras Kit (HRM: high-resolution melt). The method holds promise for the diagnosis and management of cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

Linkage mechanisms in the vertebrate skull: Structure and function of three-dimensional, parallel transmission systems.

PubMed

Olsen, Aaron M; Westneat, Mark W

2016-12-01

Many musculoskeletal systems, including the skulls of birds, fishes, and some lizards consist of interconnected chains of mobile skeletal elements, analogous to linkage mechanisms used in engineering. Biomechanical studies have applied linkage models to a diversity of musculoskeletal systems, with previous applications primarily focusing on two-dimensional linkage geometries, bilaterally symmetrical pairs of planar linkages, or single four-bar linkages. Here, we present new, three-dimensional (3D), parallel linkage models of the skulls of birds and fishes and use these models (available as free kinematic simulation software), to investigate structure-function relationships in these systems. This new computational framework provides an accessible and integrated workflow for exploring the evolution of structure and function in complex musculoskeletal systems. Linkage simulations show that kinematic transmission, although a suitable functional metric for linkages with single rotating input and output links, can give misleading results when applied to linkages with substantial translational components or multiple output links. To take into account both linear and rotational displacement we define force mechanical advantage for a linkage (analogous to lever mechanical advantage) and apply this metric to measure transmission efficiency in the bird cranial mechanism. For linkages with multiple, expanding output points we propose a new functional metric, expansion advantage, to measure expansion amplification and apply this metric to the buccal expansion mechanism in fishes. Using the bird cranial linkage model, we quantify the inaccuracies that result from simplifying a 3D geometry into two dimensions. We also show that by combining single-chain linkages into parallel linkages, more links can be simulated while decreasing or maintaining the same number of input parameters. This generalized framework for linkage simulation and analysis can accommodate linkages of differing geometries and configurations, enabling novel interpretations of the mechanics of force transmission across a diversity of vertebrate feeding mechanisms and enhancing our understanding of musculoskeletal function and evolution. J. Morphol. 277:1570-1583, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Highly Sensitive Colorimetric Cancer Cell Detection Based on Dual Signal Amplification.

PubMed

Yu, Tao; Dai, Pan-Pan; Xu, Jing-Juan; Chen, Hong-Yuan

2016-02-01

Facile and efficient detection of cancer cells at their preclinical stages is one of the central challenges in cancer diagnostics. A direct, rapid, highly sensitive and specific biosensor for detection of cancer biomarkers is desirable in early diagnosis and prognosis of cancer. In this work, we developed, for the first time, an easy and intuitive dispersion-dominated colorimetric strategy for cancer cell detection based on combining multi-DNA released from an aptamer scaffold with cyclic enzymatic amplification, which was triggered by aptamer DNA conformational switch and demonstrated by non-cross-linking gold nanoparticles (Au NPs) aggregation. First, five kinds of messenger DNAs (mDNAs) were aligned on the cancer cell aptamers modified on magnetic beads (MBs) to form mDNAs-Apt-MBs biocompatible nanosensors. In the presence of target cells, the aptamer would bind to the receptors on the cell membranes, and mDNAs would be released, resulting in the first amplification that one biological binding event would cause the release of multiple kinds of mDNAs simultaneously. After magnetic separation, the released mDNAs were introduced into the cyclic enzymatic amplification to cleave more single strand DNA (ssDNA) fragments. Instead of modification of Au NPs, these fragments and mDNAs could be adsorbed on the surface of Au NPs to prevent particle aggregation and ensure the stability and color of solution in high salt environments. The linear response for HL-60 cells in a concentration range from 10 to 10(4) cells was obtained with a detection limit of four cells in buffer solution. Moreover, the feasibility of the proposed strategy was demonstrated in a diluted serum sample. This dual signal amplification method can be extended to other types of cancer cells, which has potential application in point-of-care cancer diagnosis.

Gravitational lensing in observational cosmology

NASA Astrophysics Data System (ADS)

Nottale, L.

This paper reviews some previous theoretical and observational results concerning the various effects of gravitational lensing, and also presents still unpublished results in this field. The theoretical section deals with the Optical Scalar Equation (OSE) approach. We recall the form of these equations, which relate the deformations of the cross sectional area of a light beam to the material and energetic distribution it encounters, via the two basic contributions to lensing, the matter or Ricci term and the shear term. The introduction of a new distance, the optical distance, allows to write the OSE in a simplified way from which new solutions are easily derived. We demonstrate here that a general form may be obtained for the amplification formula in the exact relativistic treatment, provided the Universe is assumed to be Friedmannian in the mean. New results are also presented concerning the probability distribution of amplifications, the relation from matter term to shear terms (the first ones give the mean of the second ones) and the problem of energy conservation. We recall how our method let to an analytical formula yielding the amplification by any number of lenses placed anywhere along the line of sight and present new general solutions for lensing by large scale density inhomogeneities. The gravitational redshift effects are also considered, either due to the crossing by photons of inhomogeneities, or intrinsic to them ; generalized solutions to the last problem are given. Some observational evidence concerning various lensing effects, either statistical or applying to individual sources, are considered. We first recall how the dependence of the amplification formula on the various physical parameters points towards the optimisation of lensing by very rich clusters of galaxies lying at redshifts around 0.7, which may give rise to very large amplifications for reasonable values of the density parameter. Recent results concerning a statistical effect of amplification of Brightest Cluster Galaxies by foreground clusters are analysed, including the discussion of a selection effect precisely due to gravitational luminosity amplification. It results in an artificial increase of the deceleration parameter of the Universe measured from the Hubble diagram of these objects. We recall our proposal that the sample of distant 3C radiogalaxies of redshift > 1 is strongly perturbed by lensing effects, mostly by foreground clusters of galaxies (i.e. only the luminosity is changed without image multiplication), but also for some objects by galaxies, producing gravitational mirages. The case of 3C324, for which definite evidence for multiple imaging has been recently obtained, is described, including detailed modelling of the lensing configuration. We present a highly significant statistical effect of lensing on absorption line QSOs due to matter lying at the absorption redshifts. Microlensing is also considered, and we recall our recent proposal that the variability of some among the OVV QSOs turning to BL Lac at maximum brightness, like the eruptive object 0846 +51W1, is a consequence of microlensing by stars or compact objects constituting foreground galaxy halos. Finally, discrepant redshift associations are considered. We recall how the case of anomalous quintets of galaxies have been explained by the gravitational lensing effects of quartets halos on background galaxies. Then we present evidence that the Arp QSO-galaxy associations may be the result of the combined lensing effects of several superposed galaxies, groups and clusters. Cet article présente une revue de certains résultats théoriques et observationnels concernant les divers effets de lentille gravitationnelle, complétée par des résultats nouveaux non encore publiés. Dans la partie théorique, nous considérons essentiellement l'approche relativiste employant les équations scalaires optiques. La forme de ces équations est rappelée : elles relient les diverses déformations subies par la section d'un faisceau lumineux au cours de sa propagation, à la distribution matérielle et énergétique qu'il rencontre sur son trajet entre la source et l'observateur. Les deux contributions aux effets d'optique apparaissant dans ces équations (et leurs solutions) sont les termes de matière et les termes de cisaillement. Nous montrons comment une nouvelle distance peut être introduite, la distance optique, en fonction de laquelle les équations se simplifient et de nouvelles solutions analytiques peuvent être établies. En particulier, nous démontrons que la formule d'amplification gravitationnelle peut s'écrire sous une forme très générale sous la simple hypothèse que l'Univers est en moyenne de type Friedmann-Robertson-Walker. De nouveaux résultats sont aussi présentés concernant la distribution de probabilité des amplifications, les rapports entre termes de matière et termes de cisaillement (les premiers donnant la moyenne des seconds), et le problème de la conservation de l'énergie. Nous rappelons comment une formule analytique peut être obtenue pour des amplifications multiples et présentons des solutions nouvelles au problème de l'amplification par des hétérogénéités à très grande échelle. Enfin, les effets de décalages spectraux gravitationnels sont aussi considérés, qu'ils soient dus à des effets de traversée d'excès de densité, ou intrinsèques à ceux-ci ; dans ce dernier cas, une généralisation de solutions déjà connues est proposée. On considère ensuite le problème de la mise en évidence observationnelle des divers effets d'optique gravitationnelle, qu'ils soient de nature statistique ou qu'ils concernent des objets individuels. A la suite d'une analyse décrivant la dépendance de la formule d'amplification en fonction des différents paramètres y intervenant, nous insistons tout particulièrement sur le rôle cosmologique que jouent probablement les amas de galaxies très riches de décalages spectraux autour de 0,7 qui peuvent donner lieu à des amplifications quasi-infinies. La mise en évidence récente d'un effet statistique d'amplification par des amas d'avant-plan sur les galaxies les plus brillantes des amas est rappelée, ainsi que celle d'un important effet de sélection observationnel, précisément dû aux amplifications, qui augmente artificiellement la valeur de q0 mesurée à partir du diagramme de Hubble. L'extrapolation de cet effet à des objets plus lointains nous a conduit à proposer que les radiogalaxies lointaines du catalogue 3C de z < 1 étaient fortement affectées par les effets de lentille gravitationnelle, essentiellement par les amas (qui les amplifient la plupart du temps sans multiplication d'image), mais aussi dans certains cas par des galaxies qui peuvent alors provoquer des effets de mirage. Le cas de 3C324, pour lequel des preuves observationelles de multiplication d'image ont été récemment obtenues, est analysé en particulier ; un modèle détaillé en est présenté. Un autre effet statistique récemment démontré concerne les quasars à raies d'absorption, dont la luminosité est fortement corrélée à l'amplification prédite par de la matière située aux décalages spectraux d'absorption. On considère également les effets de microlentille : nous rappelons notre proposition récente que la variabilité de certains OVVs tels que le BL Lac éruptif 0846 + 51W1 est une conséquence du passage d'étoiles ou d'objets compacts du halo de galaxies intervenantes devant les régions centrales d'un quasar. Le lien entre effets d'optique gravitationnelle et associations de décalages spectraux discordants est évoqué. Le cas des quintets de galaxies discordant a été récemment expliqué par les effets (amplification, agrandissement, changement de densité superficielle d'objets lointains) des halos de groupes compacts de galaxies. Nous démontrons pour finir que les associations QSOsgalaxies de Arp peuvent s'expliquer par les effets de lentille gravitationnelle combinés de plusieurs galaxies, groupes et amas vus en superposition.

Electrical Engineering and Nontechnical Design Variables of Multiple Inductive Loop Systems for Auditoriums

ERIC Educational Resources Information Center

Alterovitz, Gil

2004-01-01

This research analyzed both engineering and nontechnical issues involved in the use of Induction Loop Amplification (ILA) devices in auditoriums or large gathering places for hard-of-hearing individuals. A variety of parameters need to be taken into account to determine an optimal shape/configuration for the ILA device. In many cases, an optimal…

[MLPA technique--principles and use in practice].

PubMed

Rusu, Cristina; Sireteanu, Adriana; Puiu, Maria; Skrypnyk, Cristina; Tomescu, E; Csep, Katalin; Creţ, Victoria; Barbarii, Ligia

2007-01-01

MLPA (Multiplex Ligation-dependent Probe Amplification) is a recently introduced method, based on PCR principle, useful for the detection of different genetic abnormalities (aneuploidies, gene deletions/duplications, subtelomeric rearrangements, methylation status etc). The technique is simple, reliable and cheap. We present this method to discuss its importance for a modern genetic service and to underline its multiple advantages.

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

Jiang, Yunshan; DeVore, Peter T. S.; Jalali, Bahram

Optical computing accelerators help alleviate bandwidth and power consumption bottlenecks in electronics. In this paper, we show an approach to implementing logarithmic-type analog co-processors in silicon photonics and use it to perform the exponentiation operation and the recovery of a signal in the presence of multiplicative distortion. Finally, the function is realized by exploiting nonlinear-absorption-enhanced Raman amplification saturation in a silicon waveguide.

Optimal design of a smart post-buckled beam actuator using bat algorithm: simulations and experiments

NASA Astrophysics Data System (ADS)

Mallick, Rajnish; Ganguli, Ranjan; Kumar, Ravi

2017-05-01

The optimized design of a smart post-buckled beam actuator (PBA) is performed in this study. A smart material based piezoceramic stack actuator is used as a prime-mover to drive the buckled beam actuator. Piezoceramic actuators are high force, small displacement devices; they possess high energy density and have high bandwidth. In this study, bench top experiments are conducted to investigate the angular tip deflections due to the PBA. A new design of a linear-to-linear motion amplification device (LX-4) is developed to circumvent the small displacement handicap of piezoceramic stack actuators. LX-4 enhances the piezoceramic actuator mechanical leverage by a factor of four. The PBA model is based on dynamic elastic stability and is analyzed using the Mathieu-Hill equation. A formal optimization is carried out using a newly developed meta-heuristic nature inspired algorithm, named as the bat algorithm (BA). The BA utilizes the echolocation capability of bats. An optimized PBA in conjunction with LX-4 generates end rotations of the order of 15° at the output end. The optimized PBA design incurs less weight and induces large end rotations, which will be useful in development of various mechanical and aerospace devices, such as helicopter trailing edge flaps, micro and nano aerial vehicles and other robotic systems.

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

Datta, Kaustuv; Neder, Reinhard B.; Chen, Jun

Revelation of unequivocal structural information at the atomic level for complex systems is uniquely important for deeper and generic understanding of the structure property connections and a key challenge in materials science. Here in this paper we report an experimental study of the local structure by applying total elastic scattering and Raman scattering analyses to an important non-relaxor ferroelectric solid solution exhibiting the so-called composition-induced morphotropic phase boundary (MPB), where concomitant enhancement of physical properties have been detected. The powerful combination of static and dynamic structural probes enabled us to derive direct correspondence between the atomic-level structural correlations and reportedmore » properties. The atomic pair distribution functions obtained from the neutron total scattering experiments were analysed through big-box atom-modelling implementing reverse Monte Carlo method, from which distributions of magnitudes and directions of off-centred cationic displacements were extracted. We found that an enhanced randomness of the displacement-directions for all ferroelectrically active cations combined with a strong dynamical coupling between the A- and B-site cations of the perovskite structure, can explain the abrupt amplification of piezoelectric response of the system near MPB. Finally, altogether this provides a more fundamental basis in inferring structure-property connections in similar systems including important implications in designing novel and bespoke materials.« less

Negative hair-bundle stiffness betrays a mechanism for mechanical amplification by the hair cell.

PubMed

Martin, P; Mehta, A D; Hudspeth, A J

2000-10-24

Hearing and balance rely on the ability of hair cells in the inner ear to sense miniscule mechanical stimuli. In each cell, sound or acceleration deflects the mechanosensitive hair bundle, a tuft of rigid stereocilia protruding from the cell's apical surface. By altering the tension in gating springs linked to mechanically sensitive transduction channels, this deflection changes the channels' open probability and elicits an electrical response. To detect weak stimuli despite energy losses caused by viscous dissipation, a hair cell can use active hair-bundle movement to amplify its mechanical inputs. This amplificatory process also yields spontaneous bundle oscillations. Using a displacement-clamp system to measure the mechanical properties of individual hair bundles from the bullfrog's ear, we found that an oscillatory bundle displays negative slope stiffness at the heart of its region of mechanosensitivity. Offsetting the hair bundle's position activates an adaptation process that shifts the region of negative stiffness along the displacement axis. Modeling indicates that the interplay between negative bundle stiffness and the motor responsible for mechanical adaptation produces bundle oscillation similar to that observed. Just as the negative resistance of electrically excitable cells and of tunnel diodes can be embedded in a biasing circuit to amplify electrical signals, negative stiffness can be harnessed to amplify mechanical stimuli in the ear.

A target-triggered strand displacement reaction cycle: the design and application in adenosine triphosphate sensing.

PubMed

Cheng, Sheng; Zheng, Bin; Wang, Mozhen; Lam, Michael Hon-Wah; Ge, Xuewu

2014-02-01

A strand displacement reaction (SDR) system that runs solely on oligonucleotides has been developed for the amplification detection of adenosine triphosphate (ATP). It involves a target-induced SDR and an entropy-driven catalytic cycle of two SDRs with five oligonucleotides, denoted as substrate, fuel, catalyst, C-1, and C-2. Catalyst, released from the ATP aptamer-catalyst duplex by ATP molecule, catalyzes the SDRs to finally form the substrate-fuel duplex. All of the intermediates in the catalytic SDR processes have been identified by polyacrylamide gel electrophoresis (PAGE) analysis. The introduction of ATP into the SDR system will induce the ATP aptamer to form G-quadruplex conformation so as to release catalyst and trigger the SDR cycle. When the substrate and C-2 oligonucleotides were labeled with a carboxyfluorescein (FAM) fluorophore and a 4-([4-(dimethylamino)phenyl]azo)benzoic acid (DABCYL) quencher, this SDR catalytic system exhibited a "turn-on" response for ATP. The condition for detecting ATP, such as Mg²⁺ concentration, has been optimized to afford a detection limit of 20 nM. This work provides an enzyme-free biosensing strategy and has potential application in aptamer-based biosensing. Copyright © 2013 Elsevier Inc. All rights reserved.

Sub-Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single-Molecule FRET.

PubMed

Baltierra-Jasso, Laura E; Morten, Michael J; Magennis, Steven W

2018-03-05

Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here, we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constant of 10 m -1  s -1 . We also followed the displacement from a DNA three-way junction (3WJ) by ssDNA. The presence of three internal mismatched bases in the middle of the invading strand did not prevent displacement from the 3WJ, but reduced the second-order rate constant by about 50 %. We attribute strand exchange in the dsDNA and 3WJ to a zero-toehold pathway from the blunt-ended duplex arms. The single-molecule approach demonstrated here will be useful for studying complex DNA networks. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Overtreatment of displaced midshaft clavicle fractures

PubMed Central

Ban, Ilija; Nowak, Jan; Virtanen, Kaisa; Troelsen, Anders

2016-01-01

Background and purpose The best treatment for displaced clavicle fractures has been debated for decades. Operative treatment has become more common. However, several randomized trials comparing non-operative and operative treatment have not shown any compelling evidence in favor of surgery. We identified the preferred treatment of displaced midshaft clavicle fractures at public hospitals in 3 countries in Scandinavia. Patients and methods A purpose-made multiple-choice questionnaire in English was sent to all public hospitals in Denmark, Sweden, and Finland. This was addressed to the orthopedic surgeon responsible for treatment of clavicle fractures, and completed questionnaires were obtained from 85 of 118 hospitals. Results In the 3 countries, 69 of the 85 hospitals that responded would treat displaced clavicle fractures operatively. Clear criteria for treatment allocation were used at 58 of the hospitals, with the remaining 27 using individual assessment in collaboration with the patient. Precontoured locking plates were mostly used, placed either superiorly (64/85) or anteriorly (10/85). Interpretation Displaced midshaft clavicle fractures are mainly treated operatively in Sweden, Denmark, and Finland. This treatment is not supported by compelling evidence. PMID:27225678

Searches for light sterile neutrinos with multitrack displaced vertices

NASA Astrophysics Data System (ADS)

Cottin, Giovanna; Helo, Juan Carlos; Hirsch, Martin

2018-03-01

We study discovery prospects for long-lived sterile neutrinos at the LHC with multitrack displaced vertices, with masses below the electroweak scale. We reinterpret current displaced vertex searches making use of publicly available, parametrized selection efficiencies for modeling the detector response to displaced vertices. We focus on the production of right-handed WR bosons and neutrinos N in a left-right symmetric model, and find poor sensitivity. After proposing a different trigger strategy (considering the prompt lepton accompanying the neutrino displaced vertex) and optimized cuts in the invariant mass and track multiplicity of the vertex, we find that the LHC with √{s }=13 TeV and 300 fb-1 is able to probe sterile neutrino masses between 10 GeV

Applications of Ko Displacement Theory to the Deformed Shape Predictions of the Doubly-Tapered Ikhana Wing

NASA Technical Reports Server (NTRS)

Ko, William L.; Richards, W. Lance; Fleischer, Van Tran

2009-01-01

The Ko displacement theory, formulated for weak nonuniform (slowly changing cross sections) cantilever beams, was applied to the deformed shape analysis of the doubly-tapered wings of the Ikhana unmanned aircraft. The two-line strain-sensing system (along the wingspan) was used for sensing the bending strains needed for the wing-deformed shapes (deflections and cross-sectional twist) analysis. The deflection equation for each strain-sensing line was expressed in terms of the bending strains evaluated at multiple numbers of strain-sensing stations equally spaced along the strain-sensing line. For the preflight shape analysis of the Ikhana wing, the strain data needed for input to the displacement equations for the shape analysis were obtained from the nodal-stress output of the finite-element analysis. The wing deflections and cross-sectional twist angles calculated from the displacement equations were then compared with those computed from the finite-element computer program. The Ko displacement theory formulated for weak nonlinear cantilever beams was found to be highly accurate in the deformed shape predictions of the doubly-tapered Ikhana wing.

Overtreatment of displaced midshaft clavicle fractures.

PubMed

Ban, Ilija; Nowak, Jan; Virtanen, Kaisa; Troelsen, Anders

2016-12-01

Background and purpose - The best treatment for displaced clavicle fractures has been debated for decades. Operative treatment has become more common. However, several randomized trials comparing non-operative and operative treatment have not shown any compelling evidence in favor of surgery. We identified the preferred treatment of displaced midshaft clavicle fractures at public hospitals in 3 countries in Scandinavia. Patients and methods - A purpose-made multiple-choice questionnaire in English was sent to all public hospitals in Denmark, Sweden, and Finland. This was addressed to the orthopedic surgeon responsible for treatment of clavicle fractures, and completed questionnaires were obtained from 85 of 118 hospitals. Results - In the 3 countries, 69 of the 85 hospitals that responded would treat displaced clavicle fractures operatively. Clear criteria for treatment allocation were used at 58 of the hospitals, with the remaining 27 using individual assessment in collaboration with the patient. Precontoured locking plates were mostly used, placed either superiorly (64/85) or anteriorly (10/85). Interpretation - Displaced midshaft clavicle fractures are mainly treated operatively in Sweden, Denmark, and Finland. This treatment is not supported by compelling evidence.

Multi-element fiber technology for space-division multiplexing applications.

PubMed

Jain, S; Rancaño, V J F; May-Smith, T C; Petropoulos, P; Sahu, J K; Richardson, D J

2014-02-24

A novel technological approach to space division multiplexing (SDM) based on the use of multiple individual fibers embedded in a common polymer coating material is presented, which is referred to as Multi-Element Fiber (MEF). The approach ensures ultralow crosstalk between spatial channels and allows for cost-effective ways of realizing multi-spatial channel amplification and signal multiplexing/demultiplexing. Both the fabrication and characterization of a passive 3-element MEF for data transmission, and an active 5-element erbium/ytterbium doped MEF for cladding-pumped optical amplification that uses one of the elements as an integrated pump delivery fiber is reported. Finally, both components were combined to emulate an optical fiber network comprising SDM transmission lines and amplifiers, and illustrate the compatibility of the approach with existing installed single-mode WDM fiber systems.

Multiscale image contrast amplification (MUSICA)

NASA Astrophysics Data System (ADS)

Vuylsteke, Pieter; Schoeters, Emile P.

1994-05-01

This article presents a novel approach to the problem of detail contrast enhancement, based on multiresolution representation of the original image. The image is decomposed into a weighted sum of smooth, localized, 2D basis functions at multiple scales. Each transform coefficient represents the amount of local detail at some specific scale and at a specific position in the image. Detail contrast is enhanced by non-linear amplification of the transform coefficients. An inverse transform is then applied to the modified coefficients. This yields a uniformly contrast- enhanced image without artefacts. The MUSICA-algorithm is being applied routinely to computed radiography images of chest, skull, spine, shoulder, pelvis, extremities, and abdomen examinations, with excellent acceptance. It is useful for a wide range of applications in the medical, graphical, and industrial area.

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.

Comparison of single cell sequencing data between two whole genome amplification methods on two sequencing platforms.

PubMed

Chen, DaYang; Zhen, HeFu; Qiu, Yong; Liu, Ping; Zeng, Peng; Xia, Jun; Shi, QianYu; Xie, Lin; Zhu, Zhu; Gao, Ya; Huang, GuoDong; Wang, Jian; Yang, HuanMing; Chen, Fang

2018-03-21

Research based on a strategy of single-cell low-coverage whole genome sequencing (SLWGS) has enabled better reproducibility and accuracy for detection of copy number variations (CNVs). The whole genome amplification (WGA) method and sequencing platform are critical factors for successful SLWGS (<0.1 × coverage). In this study, we compared single cell and multiple cells sequencing data produced by the HiSeq2000 and Ion Proton platforms using two WGA kits and then comprehensively evaluated the GC-bias, reproducibility, uniformity and CNV detection among different experimental combinations. Our analysis demonstrated that the PicoPLEX WGA Kit resulted in higher reproducibility, lower sequencing error frequency but more GC-bias than the GenomePlex Single Cell WGA Kit (WGA4 kit) independent of the cell number on the HiSeq2000 platform. While on the Ion Proton platform, the WGA4 kit (both single cell and multiple cells) had higher uniformity and less GC-bias but lower reproducibility than those of the PicoPLEX WGA Kit. Moreover, on these two sequencing platforms, depending on cell number, the performance of the two WGA kits was different for both sensitivity and specificity on CNV detection. The results can help researchers who plan to use SLWGS on single or multiple cells to select appropriate experimental conditions for their applications.

SAR measurements of surface displacements at Augustine Volcano, Alaska from 1992 to 2005

USGS Publications Warehouse

Lee, C.-W.; Lu, Z.; Kwoun, Oh-Ig

2007-01-01

Augustine volcano is an active stratovolcano located at the southwest of Anchorage, Alaska. Augustine volcano had experienced seven significantly explosive eruptions in 1812, 1883, 1908, 1935, 1963, 1976, and 1986, and a minor eruption in January 2006. We measured the surface displacements of the volcano by radar interferometry and GPS before and after the eruption in 2006. ERS-1/2, RADARSAT-1 and ENVISAT SAR data were used for the study. Multiple interferograms were stacked to reduce artifacts caused by different atmospheric conditions. Least square (LS) method was used to reduce atmospheric artifacts. Singular value decomposition (SVD) method was applied for retrieval of time sequential deformations. Satellite radar interferometry helps to understand the surface displacements system of Augustine volcano. ?? 2007 IEEE.

SAR measurements of surface displacements at Augustine Volcano, Alaska from 1992 to 2005

USGS Publications Warehouse

Lee, C.-W.; Lu, Z.; Kwoun, Oh-Ig

2008-01-01

Augustine volcano is an active stratovolcano located at the southwest of Anchorage, Alaska. Augustine volcano had experienced seven significantly explosive eruptions in 1812, 1883, 1908, 1935, 1963, 1976, and 1986, and a minor eruption in January 2006. We measured the surface displacements of the volcano by radar interferometry and GPS before and after the eruption in 2006. ERS-1/2, RADARSAT-1 and ENVISAT SAR data were used for the study. Multiple interferograms were stacked to reduce artifacts caused by different atmospheric conditions. Least square (LS) method was used to reduce atmospheric artifacts. Singular value decomposition (SVD) method was applied for retrieval of time sequential deformations. Satellite radar interferometry helps to understand the surface displacements system of Augustine volcano. ?? 2007 IEEE.

Double jeopardy: assessing the association between internal displacement, housing quality and chronic illness in a low-income neighborhood.

PubMed

Habib, Rima R; Yassin, Nasser; Ghanawi, Joly; Haddad, Pascale; Mahfoud, Ziyad

2011-04-01

PURPOSE: This study analyzed associations between war-related internal displacement, housing quality and the prevalence of chronic illness in Nabaa, a low-income neighborhood on the outskirts of Beirut, Lebanon. METHODS: A cross-sectional survey of sociodemographics, household characteristics and health conditions of the study population was carried out in 2002. Using a structured questionnaire, the research team surveyed 1,151 households representing 4,987 residents of all ages. The survey was administered to a proxy respondent from each household in face-to-face interviews. A multiple logistic regression model using the generalized estimation equation method was constructed to assess the simultaneous effect of displacement and housing quality on reported ill health, while adjusting for potential confounders. RESULTS: Housing quality and internal displacement were strongly associated with occurrences of chronic illness. The most vulnerable respondents were older residents, females and internally displaced people, who reported high rates of chronic illnesses. Residents with high levels of education were less likely to report a chronic illness than those that had elementary education or less. CONCLUSION: Nabaa residents' experience of poor health was associated with inadequate housing quality. Moreover, residents who have been displaced experience worse living conditions and were more likely to experience poor health than those who were not displaced. These results reveal a need for policies to improve housing quality and alleviate war-related consequences in low-income neighborhoods.

Displacement, county social cohesion and depression after a large-scale traumatic event

PubMed Central

Lê, Félice; Tracy, Melissa; Norris, Fran H.; Galea, Sandro

2013-01-01

Background Depression is a common and potentially debilitating consequence of traumatic events. Mass traumatic events cause wide-ranging disruptions to community characteristics, influencing the population risk of depression. In the aftermath of such events, population displacement is common. Stressors associated with displacement may increase risk of depression directly. Indirectly, persons who are displaced may experience erosion in social cohesion, further exacerbating their risk for depression. Methods Using data from a population-based cross-sectional survey of adults living in the 23 southernmost counties of Mississippi (N = 708), we modeled the independent and joint relations of displacement and county-level social cohesion with depression 18–24 months after Hurricane Katrina. Results After adjustment for individual- and county-level sociodemographic characteristics and county-level hurricane exposure, joint exposure to both displacement and low social cohesion was associated with substantially higher log-odds of depression (b = 1.34 [0.86–1.83]). Associations were much weaker for exposure only to low social cohesion (b = 0.28 [−0.35–0.90]) or only to displacement (b = 0.04 [−0.80– 0.88]). The associations were robust to additional adjustment for individually perceived social cohesion and social support. Conclusion Addressing the multiple, simultaneous disruptions that are a hallmark of mass traumatic events is important to identify vulnerable populations and understand the psychological ramifications of these events. PMID:23644724

The influence of current neutralization and multiple Coulomb scattering on the spatial dynamics of resistive sausage instability of a relativistic electron beam propagating in ohmic plasma

NASA Astrophysics Data System (ADS)

Kolesnikov, E. K.; Manuilov, A. S.; Petrov, V. S.; Klyushnikov, G. N.; Chernov, S. V.

2017-06-01

The influence of the current neutralization process, the phase mixing of the trajectories of electrons and multiple Coulomb scattering of electrons beam on the atoms of the background medium on the spatial increment of the growth of sausage instability of a relativistic electron beam propagating in ohmic plasma channel has been considered. It has been shown that the amplification of the current neutralization leads to a significant increase in this instability, and phase mixing and the process of multiple scattering of electrons beam on the atoms of the background medium are the stabilizing factor.

Directional Site Amplification Effect on Tarzana Hill, California

NASA Astrophysics Data System (ADS)

Graizer, V.; Shakal, A.

2003-12-01

Significantly amplified ground accelerations at the Tarzana Hill station were recorded during the 1987 Mw 5.9 Whittier Narrows and the 1994 Mw 6.7 Northridge earthquakes. Peak horizontal ground acceleration at the Tarzana station during the 1999 Mw 7.1 Hector Mine earthquake was almost twice as large as the accelerations recorded at nearby stations. The Tarzana site was drilled to a depth of 100 m. A low shear-wave velocity near the surface of 100 m/sec increasing to near 750 m/sec at 100 m depth was measured. The 20 m high hill was found to be well drained with a water table near 17 m. Modelo formation (extremely weathered at the surface to fresh at depth) underlies the hill. The subsurface geology and velocities obtained allow classification of this location as a soft-rock site. After the Northridge earthquake the California Strong Motion Instrumentation Program significantly increased instrumentation at Tarzana to study the unusual site amplification effect. Current instrumentation at Tarzana consists of an accelerograph at the top of Tarzana hill (Tarzana - Cedar Hill B), a downhole instrument at 60 m depth, and an accelerograph at the foot of the hill (Tarzana - Clubhouse), 180 m from the Cedar Hill B station. The original station, Tarzana - Cedar Hill Nursery A, was lost in 1999 due to construction. More than twenty events, including the Hector Mine earthquake, were recorded by all these instruments at Tarzana. Comparison of recordings and response spectra demonstrates strong directional resonance on the top of the hill in a direction perpendicular to the strike of the hill in the period range from 0.04 to 0.8 sec (1.2 to 25 Hz). There is practically no amplification from the bottom to the top of the hill for the component parallel to the strike of the hill. In contrast to accelerations recorded during the Hector Mine earthquake (high frequency part of seismic signal), displacements (relatively low frequency part of seismic signal) demonstrate almost no site amplification from the bottom of the hole to the surface at periods greater than 1.5 sec, in either direction. The directional effect at Tarzana hill seems to be azimuth dependent. Relatively higher amplification at the perpendicular component is produced for the earthquake sources located north of the station. We were not able to see any differences in hill response before and after development (a relatively small part of the hill was developed). The source of the site amplification that produces large motions at Tarzana is still under investigation with "the usual suspects" like topography and shear wave velocity profile not providing the explanation. New data recorded at Tarzana in recent years clearly show that the Tarzana effect is a very localized high-frequency effect observed only at the top of the hill. Drilling at Tarzana was co-funded by CSMIP and by the National Science Foundation through the Resolution of Site Response Issues from the Northridge Earthquake Project (ROSRINE).

Anticipatory and compensatory postural adjustments in individuals with multiple sclerosis in response to external perturbations.

PubMed

Aruin, Alexander S; Kanekar, Neeta; Lee, Yun-Ju

2015-03-30

Deficit in balance control is a common and often an initial disabling symptom of multiple sclerosis (MS). The aim of the study was to investigate the organization of anticipatory and compensatory postural adjustments in individuals with MS dealing with external perturbations. Ten individuals with MS and ten age-and-gender matched healthy controls were exposed to external perturbations applied at the shoulder level. The perturbations were either predictable or unpredictable as subjects stood with eyes open or closed. Electrical activity of six leg and trunk muscles as well as displacements of the center of pressure (COP) were recorded and quantified within the time intervals typical of anticipatory (APAs) and compensatory (CPAs) postural adjustments. Individuals with MS demonstrated delayed anticipatory onsets of muscle activity and smaller anticipatory COP displacements as compared to healthy control subjects. The deficiency of the APAs was associated with increased displacements of the COP during the balance restoration phase. The results demonstrate the underlying impairment in anticipatory postural control of individuals with MS. The study outcome provides a background for development of rehabilitation strategies focused on balance restoration in people with MS. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Derivation and application of an analytical rock displacement solution on rectangular cavern wall using the inverse mapping method.

PubMed

Gao, Mingzhong; Yu, Bin; Qiu, Zhiqiang; Yin, Xiangang; Li, Shengwei; Liu, Qiang

2017-01-01

Rectangular caverns are increasingly used in underground engineering projects, the failure mechanism of rectangular cavern wall rock is significantly different as a result of the cross-sectional shape and variations in wall stress distributions. However, the conventional computational method always results in a long-winded computational process and multiple displacement solutions of internal rectangular wall rock. This paper uses a Laurent series complex method to obtain a mapping function expression based on complex variable function theory and conformal transformation. This method is combined with the Schwarz-Christoffel method to calculate the mapping function coefficient and to determine the rectangular cavern wall rock deformation. With regard to the inverse mapping concept, the mapping relation between the polar coordinate system within plane ς and a corresponding unique plane coordinate point inside the cavern wall rock is discussed. The disadvantage of multiple solutions when mapping from the plane to the polar coordinate system is addressed. This theoretical formula is used to calculate wall rock boundary deformation and displacement field nephograms inside the wall rock for a given cavern height and width. A comparison with ANSYS numerical software results suggests that the theoretical solution and numerical solution exhibit identical trends, thereby demonstrating the method's validity. This method greatly improves the computing accuracy and reduces the difficulty in solving for cavern boundary and internal wall rock displacements. The proposed method provides a theoretical guide for controlling cavern wall rock deformation failure.

Derivation and application of an analytical rock displacement solution on rectangular cavern wall using the inverse mapping method

PubMed Central

Gao, Mingzhong; Qiu, Zhiqiang; Yin, Xiangang; Li, Shengwei; Liu, Qiang

2017-01-01

Rectangular caverns are increasingly used in underground engineering projects, the failure mechanism of rectangular cavern wall rock is significantly different as a result of the cross-sectional shape and variations in wall stress distributions. However, the conventional computational method always results in a long-winded computational process and multiple displacement solutions of internal rectangular wall rock. This paper uses a Laurent series complex method to obtain a mapping function expression based on complex variable function theory and conformal transformation. This method is combined with the Schwarz-Christoffel method to calculate the mapping function coefficient and to determine the rectangular cavern wall rock deformation. With regard to the inverse mapping concept, the mapping relation between the polar coordinate system within plane ς and a corresponding unique plane coordinate point inside the cavern wall rock is discussed. The disadvantage of multiple solutions when mapping from the plane to the polar coordinate system is addressed. This theoretical formula is used to calculate wall rock boundary deformation and displacement field nephograms inside the wall rock for a given cavern height and width. A comparison with ANSYS numerical software results suggests that the theoretical solution and numerical solution exhibit identical trends, thereby demonstrating the method’s validity. This method greatly improves the computing accuracy and reduces the difficulty in solving for cavern boundary and internal wall rock displacements. The proposed method provides a theoretical guide for controlling cavern wall rock deformation failure. PMID:29155892

Rectus Pulley Displacements without Abnormal Oblique Contractility Explain Strabismus in Superior Oblique Palsy.

PubMed

Suh, Soh Youn; Le, Alan; Clark, Robert A; Demer, Joseph L

2016-06-01

Using high-resolution magnetic resonance imaging (MRI), we investigated whether rectus pulleys are significantly displaced in superior oblique (SO) palsy and whether displacements account for strabismus patterns. Prospective case-control study. Twenty-four patients diagnosed with SO palsy based on atrophy of the SO muscle on MRI and 19 age-matched orthotropic control subjects. High-resolution, surface coil MRI scans were obtained in multiple, contiguous, quasicoronal planes during monocular central gaze fixation. Pulley locations in oculocentric coordinates in the following subgroups of patients with SO palsy were compared with normal results in subgroups of patients with SO palsy: unilateral versus bilateral, congenital versus acquired, and isotropic (round) versus anisotropic (elongated) SO atrophy. Expected effects of pulley displacements were modeled using Orbit 1.8 (Eidactics, San Francisco, CA) computational simulation. Rectus pulley positions and ocular torsion. Rectus pulleys typically were displaced in SO palsy. In unilateral SO palsy, on average the medial rectus (MR) pulley was displaced 1.1 mm superiorly, the superior rectus (SR) pulley was displaced 0.8 mm temporally, and the inferior rectus (IR) pulley was displaced 0.6 mm superiorly and 0.9 mm nasally from normal. Displacements were similar in bilateral SO palsy, with the SR pulley additionally displaced 0.9 mm superiorly. However, the lateral rectus pulley was not displaced in either unilateral or bilateral SO palsy. The SR and MR pulleys were displaced in congenital SO palsy, whereas the IR and MR pulleys were displaced in acquired palsy. Pulley positions did not differ between isotropic and anisotropic palsy or between patients with cyclotropia of less than 7° versus cyclotropia of 7° or more. Simulations predicted that the observed pulley displacements alone could cause patterns of incomitant strabismus typical of SO palsy, without requiring any abnormality of SO or inferior oblique strength. Rectus pulley displacements alone, without abnormal oblique muscle contractility, can create the clinical patterns of incomitant strabismus in SO palsy. This finding supports accumulating evidence that clinical binocular misalignment patterns are not reliable indicators of contractile function of the SO muscle. Ocular torsion does not correlate with and thus cannot account for pulley displacements in SO palsy. Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

Tracking heavy water (D 2O) incorporation for identifying and sorting active microbial cells

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

Berry, David; Mader, Esther; Lee, Tae Kwon

Here, microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. Here in this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D 2O) combined with Raman microspectroscopy. Incorporation of D 2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labelingmore » pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D 2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Raman-based cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/or glucosamine were identified, demonstrating the potential of the nondestructive D 2O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics.« less

Tracking heavy water (D 2O) incorporation for identifying and sorting active microbial cells

DOE PAGES

Berry, David; Mader, Esther; Lee, Tae Kwon; ...

2014-12-30

Here, microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. Here in this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D 2O) combined with Raman microspectroscopy. Incorporation of D 2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labelingmore » pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D 2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Raman-based cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/or glucosamine were identified, demonstrating the potential of the nondestructive D 2O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics.« less

Next-Level ShakeZoning for Earthquake Hazard Definition in Nevada

NASA Astrophysics Data System (ADS)

Louie, J. N.; Savran, W. H.; Flinchum, B. A.; Dudley, C.; Prina, N.; Pullammanappallil, S.; Pancha, A.

2011-12-01

We are developing "Next-Level ShakeZoning" procedures tailored for defining earthquake hazards in Nevada. The current Federally sponsored tools- the USGS hazard maps and ShakeMap, and FEMA HAZUS- were developed as statistical summaries to match earthquake data from California, Japan, and Taiwan. The 2008 Wells and Mogul events in Nevada showed in particular that the generalized statistical approach taken by ShakeMap cannot match actual data on shaking from earthquakes in the Intermountain West, even to first order. Next-Level ShakeZoning relies on physics and geology to define earthquake shaking hazards, rather than statistics. It follows theoretical and computational developments made over the past 20 years, to capitalize on detailed and specific local data sets to more accurately model the propagation and amplification of earthquake waves through the multiple geologic basins of the Intermountain West. Excellent new data sets are now available for Las Vegas Valley. Clark County, Nevada has completed the nation's very first effort to map earthquake hazard class systematically through an entire urban area using Optim's SeisOpt° ReMi technique, which was adapted for large-scale data collection. Using the new Parcel Map in computing shaking in the Valley for scenario earthquakes is crucial for obtaining realistic predictions of ground motions. In an educational element of the project, a dozen undergraduate students have been computing 50 separate earthquake scenarios affecting Las Vegas Valley, using the Next-Level ShakeZoning process. Despite affecting only the upper 30 meters, the Vs30 geotechnical shear-velocity from the Parcel Map shows clear effects on 3-d shaking predictions computed so far at frequencies from 0.1 Hz up to 1.0 Hz. The effect of the Parcel Map on even the 0.1-Hz waves is prominent even with the large mismatch of wavelength to geotechnical depths. Amplifications and de-amplifications affected by the Parcel Map exceed a factor of two, and are highly dependent on the particular scenario. As well, Parcel Map amplification effects extend into areas not characterized in the Parcel Map. The fully 3-d Next-Level ShakeZoning scenarios show many areas of shaking amplification and de-amplification that USGS ShakeMap scenarios cannot predict. For example, the Frenchman Mountain scenario shows PGV of the two approaches within 15% of each other near the source, but upwards of 200% relative amplification or de-amplification, depending on location, throughout Las Vegas Valley.

The histone acetyltransferases CBP and Chameau integrate developmental and DNA replication programs in Drosophila ovarian follicle cells

PubMed Central

McConnell, Kristopher H.; Dixon, Michael; Calvi, Brian R.

2012-01-01

DNA replication origin activity changes during development. Chromatin modifications are known to influence the genomic location of origins and the time during S phase that they initiate replication in different cells. However, how chromatin regulates origins in concert with cell differentiation remains poorly understood. Here, we use developmental gene amplification in Drosophila ovarian follicle cells as a model to investigate how chromatin modifiers regulate origins in a developmental context. We find that the histone acetyltransferase (HAT) Chameau (Chm) binds to amplicon origins and is partially required for their function. Depletion of Chm had relatively mild effects on origins during gene amplification and genomic replication compared with previous knockdown of its ortholog HBO1 in human cells, which has severe effects on origin function. We show that another HAT, CBP (Nejire), also binds amplicon origins and is partially required for amplification. Knockdown of Chm and CBP together had a more severe effect on nucleosome acetylation and amplicon origin activity than knockdown of either HAT alone, suggesting that these HATs collaborate in origin regulation. In addition to their local function at the origin, we show that Chm and CBP also globally regulate the developmental transition of follicle cells into the amplification stages of oogenesis. Our results reveal a complexity of origin epigenetic regulation by multiple HATs during development and suggest that chromatin modifiers are a nexus that integrates differentiation and DNA replication programs. PMID:22951641

Gene amplification confers glyphosate resistance in Amaranthus palmeri

PubMed Central

Gaines, Todd A.; Zhang, Wenli; Wang, Dafu; Bukun, Bekir; Chisholm, Stephen T.; Shaner, Dale L.; Nissen, Scott J.; Patzoldt, William L.; Tranel, Patrick J.; Culpepper, A. Stanley; Grey, Timothy L.; Webster, Theodore M.; Vencill, William K.; Sammons, R. Douglas; Jiang, Jiming; Preston, Christopher; Leach, Jan E.; Westra, Philip

2009-01-01

The herbicide glyphosate became widely used in the United States and other parts of the world after the commercialization of glyphosate-resistant crops. These crops have constitutive overexpression of a glyphosate-insensitive form of the herbicide target site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Increased use of glyphosate over multiple years imposes selective genetic pressure on weed populations. We investigated recently discovered glyphosate-resistant Amaranthus palmeri populations from Georgia, in comparison with normally sensitive populations. EPSPS enzyme activity from resistant and susceptible plants was equally inhibited by glyphosate, which led us to use quantitative PCR to measure relative copy numbers of the EPSPS gene. Genomes of resistant plants contained from 5-fold to more than 160-fold more copies of the EPSPS gene than did genomes of susceptible plants. Quantitative RT-PCR on cDNA revealed that EPSPS expression was positively correlated with genomic EPSPS relative copy number. Immunoblot analyses showed that increased EPSPS protein level also correlated with EPSPS genomic copy number. EPSPS gene amplification was heritable, correlated with resistance in pseudo-F2 populations, and is proposed to be the molecular basis of glyphosate resistance. FISH revealed that EPSPS genes were present on every chromosome and, therefore, gene amplification was likely not caused by unequal chromosome crossing over. This occurrence of gene amplification as an herbicide resistance mechanism in a naturally occurring weed population is particularly significant because it could threaten the sustainable use of glyphosate-resistant crop technology. PMID:20018685

ChIP-chip.

PubMed

Kim, Tae Hoon; Dekker, Job

2018-05-01

ChIP-chip can be used to analyze protein-DNA interactions in a region-wide and genome-wide manner. DNA microarrays contain PCR products or oligonucleotide probes that are designed to represent genomic sequences. Identification of genomic sites that interact with a specific protein is based on competitive hybridization of the ChIP-enriched DNA and the input DNA to DNA microarrays. The ChIP-chip protocol can be divided into two main sections: Amplification of ChIP DNA and hybridization of ChIP DNA to arrays. A large amount of DNA is required to hybridize to DNA arrays, and hybridization to a set of multiple commercial arrays that represent the entire human genome requires two rounds of PCR amplifications. The relative hybridization intensity of ChIP DNA and that of the input DNA is used to determine whether the probe sequence is a potential site of protein-DNA interaction. Resolution of actual genomic sites bound by the protein is dependent on the size of the chromatin and on the genomic distance between the probes on the array. As with expression profiling using gene chips, ChIP-chip experiments require multiple replicates for reliable statistical measure of protein-DNA interactions. © 2018 Cold Spring Harbor Laboratory Press.

Mechanical seal assembly

DOEpatents

Kotlyar, Oleg M.

2001-01-01

An improved mechanical seal assembly is provided for sealing rotating shafts with respect to their shaft housings, wherein the rotating shafts are subject to substantial axial vibrations. The mechanical seal assembly generally includes a rotating sealing ring fixed to the shaft, a non-rotating sealing ring adjacent to and in close contact with the rotating sealing ring for forming an annular seal about the shaft, and a mechanical diode element that applies a biasing force to the non-rotating sealing ring by means of hemispherical joint. The alignment of the mechanical diode with respect to the sealing rings is maintained by a series of linear bearings positioned axially along a desired length of the mechanical diode. Alternative embodiments include mechanical or hydraulic amplification components for amplifying axial displacement of the non-rotating sealing ring and transferring it to the mechanical diode.

Mechanical seal assembly

DOEpatents

Kotlyar, Oleg M.

2002-01-01

An improved mechanical seal assembly is provided for sealing rotating shafts with respect to their shaft housings, wherein the rotating shafts are subject to substantial axial vibrations. The mechanical seal assembly generally includes a rotating sealing ring fixed to the shaft, a non-rotating sealing ring adjacent to and in close contact with the rotating sealing ring for forming an annular seal about the shaft, and a mechanical diode element that applies a biasing force to the non-rotating sealing ring by means of hemispherical joint. The alignment of the mechanical diode with respect to the sealing rings is maintained by a series of linear bearings positioned axially along a desired length of the mechanical diode. Alternative embodiments include mechanical or hydraulic amplification components for amplifying axial displacement of the non-rotating sealing ring and transfering it to the mechanical diode.

Global transport calculations with an equivalent barotropic system

NASA Technical Reports Server (NTRS)

Salby, Murry L.; O'Sullivan, Donal; Garcia, Rolando R.; Tribbia, Joseph

1990-01-01

Transport properties of the two-dimensional equations governing equivalent barotropic motion are investigated on the sphere. This system has ingredients such as forcing, equivalent depth, and thermal dissipation explicitly represented, and takes into account compression effects associated with vertical motion along isentropic surfaces. Horizontal transport properties of this system are investigated under adiabatic and diabatic conditions for different forms of dissipation, and over a range of resolutions. It is shown that forcing represetative of time-mean and amplified conditions at 10 mb leads to the behavior typical of observations at this level. The displacement of the polar night vortex and its distortion into a comma shape are evident, as is irreversible mixing under sufficiently strong forcing amplitude. It is shown that thermal dissipation influences the behavior significantly by inhibiting the amplification of unstable eddies and thereby the horizontal stirring of air.

Parametric amplification and bidirectional invisibility in PT -symmetric time-Floquet systems

NASA Astrophysics Data System (ADS)

Koutserimpas, Theodoros T.; Alù, Andrea; Fleury, Romain

2018-01-01

Parity-time (PT )-symmetric wave devices, which exploit balanced interactions between material gain and loss, exhibit extraordinary properties, including lasing and flux-conserving scattering processes. In a seemingly different research field, periodically driven systems, also known as time-Floquet systems, have been widely studied as a relevant platform for reconfigurable active wave control and manipulation. In this article, we explore the connection between PT -symmetry and parametric time-Floquet systems. Instead of relying on material gain, we use parametric amplification by considering a time-periodic modulation of the refractive index at a frequency equal to twice the incident signal frequency. We show that the scattering from a simple parametric slab, whose dynamics follows the Mathieu equation, can be described by a PT -symmetric scattering matrix, whose PT -breaking threshold corresponds to the Mathieu instability threshold. By combining different parametric slabs modulated out of phase, we create PT -symmetric time-Floquet systems that feature exceptional scattering properties, such as coherent perfect absorption (CPA)-laser operation and bidirectional invisibility. These bidirectional properties, rare for regular PT -symmetric systems, are related to a compensation of parametric amplification due to multiple scattering between two parametric systems modulated with a phase difference.

The dynamics of diverse segmental amplifications in populations of Saccharomyces cerevisiae adapting to strong selection.

PubMed

Payen, Celia; Di Rienzi, Sara C; Ong, Giang T; Pogachar, Jamie L; Sanchez, Joseph C; Sunshine, Anna B; Raghuraman, M K; Brewer, Bonita J; Dunham, Maitreya J

2014-03-20

Population adaptation to strong selection can occur through the sequential or parallel accumulation of competing beneficial mutations. The dynamics, diversity, and rate of fixation of beneficial mutations within and between populations are still poorly understood. To study how the mutational landscape varies across populations during adaptation, we performed experimental evolution on seven parallel populations of Saccharomyces cerevisiae continuously cultured in limiting sulfate medium. By combining quantitative polymerase chain reaction, array comparative genomic hybridization, restriction digestion and contour-clamped homogeneous electric field gel electrophoresis, and whole-genome sequencing, we followed the trajectory of evolution to determine the identity and fate of beneficial mutations. During a period of 200 generations, the yeast populations displayed parallel evolutionary dynamics that were driven by the coexistence of independent beneficial mutations. Selective amplifications rapidly evolved under this selection pressure, in particular common inverted amplifications containing the sulfate transporter gene SUL1. Compared with single clones, detailed analysis of the populations uncovers a greater complexity whereby multiple subpopulations arise and compete despite a strong selection. The most common evolutionary adaptation to strong selection in these populations grown in sulfate limitation is determined by clonal interference, with adaptive variants both persisting and replacing one another.

The Dynamics of Diverse Segmental Amplifications in Populations of Saccharomyces cerevisiae Adapting to Strong Selection

PubMed Central

Payen, Celia; Di Rienzi, Sara C.; Ong, Giang T.; Pogachar, Jamie L.; Sanchez, Joseph C.; Sunshine, Anna B.; Raghuraman, M. K.; Brewer, Bonita J.; Dunham, Maitreya J.

2014-01-01

Population adaptation to strong selection can occur through the sequential or parallel accumulation of competing beneficial mutations. The dynamics, diversity, and rate of fixation of beneficial mutations within and between populations are still poorly understood. To study how the mutational landscape varies across populations during adaptation, we performed experimental evolution on seven parallel populations of Saccharomyces cerevisiae continuously cultured in limiting sulfate medium. By combining quantitative polymerase chain reaction, array comparative genomic hybridization, restriction digestion and contour-clamped homogeneous electric field gel electrophoresis, and whole-genome sequencing, we followed the trajectory of evolution to determine the identity and fate of beneficial mutations. During a period of 200 generations, the yeast populations displayed parallel evolutionary dynamics that were driven by the coexistence of independent beneficial mutations. Selective amplifications rapidly evolved under this selection pressure, in particular common inverted amplifications containing the sulfate transporter gene SUL1. Compared with single clones, detailed analysis of the populations uncovers a greater complexity whereby multiple subpopulations arise and compete despite a strong selection. The most common evolutionary adaptation to strong selection in these populations grown in sulfate limitation is determined by clonal interference, with adaptive variants both persisting and replacing one another. PMID:24368781

Two Polo-like kinase 4 binding domains in Asterless perform distinct roles in regulating kinase stability

PubMed Central

Klebba, Joseph E.; Galletta, Brian J.; Nye, Jonathan; Plevock, Karen M.; Buster, Daniel W.; Hollingsworth, Natalie A.; Slep, Kevin C.

2015-01-01

Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4’s tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl–Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification. PMID:25688134

Cross-subtype detection of HIV-1 using reverse transcription and recombinase polymerase amplification.

PubMed

Lillis, Lorraine; Lehman, Dara A; Siverson, Joshua B; Weis, Julie; Cantera, Jason; Parker, Mathew; Piepenburg, Olaf; Overbaugh, Julie; Boyle, David S

2016-04-01

A low complexity diagnostic test that rapidly and reliably detects HIV infection in infants at the point of care could facilitate early treatment, improving outcomes. However, many infant HIV diagnostics can only be performed in laboratory settings. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that can rapidly amplify proviral DNA from multiple subtypes of HIV-1 in under twenty minutes without complex equipment. In this study we added reverse transcription (RT) to RPA to allow detection of both HIV-1 RNA and DNA. We show that this RT-RPA HIV-1 assay has a limit of detection of 10-30 copies of an exact sequence matched DNA or RNA, respectively. In addition, at 100 copies of RNA or DNA, the assay detected 171 of 175 (97.7%) sequence variants that represent all the major subtypes and recombinant forms of HIV-1 Groups M and O. This data suggests that the application of RT-RPA for the combined detection of HIV-1 viral RNA and proviral DNA may prove a highly sensitive tool for rapid and accurate diagnosis of infant HIV. Copyright © 2016 Elsevier B.V. All rights reserved.

Rapid and Sensitive Detection of Shigella spp. and Salmonella spp. by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique

PubMed Central

Wang, Yi; Wang, Yan; Luo, Lijuan; Liu, Dongxin; Luo, Xia; Xu, Yanmei; Hu, Shoukui; Niu, Lina; Xu, Jianguo; Ye, Changyun

2015-01-01

Shigella and Salmonella are frequently isolated from various food samples and can cause human gastroenteritis. Here, a novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully established and validated for simultaneous detection of Shigella strains and Salmonella strains in only a single reaction. Two sets of MERT-LAMP primers for 2 kinds of pathogens were designed from ipaH gene of Shigella spp. and invA gene of Salmonella spp., respectively. Under the constant condition at 63°C, the positive results were yielded in as short as 12 min with the genomic DNA extracted from the 19 Shigella strains and 14 Salmonella strains, and the target pathogens present in a sample could be simultaneously identified based on distinct fluorescence curves in real-time format. Accordingly, the multiplex detection assay significantly reduced effort, materials and reagents used, and amplification and differentiation were conducted at the same time, obviating the use of postdetection procedures. The analytical sensitivity of MERT-LAMP was found to be 62.5 and 125 fg DNA/reaction with genomic templates of Shigella strains and Salmonella strains, which was consist with normal LAMP assay, and at least 10- and 100-fold more sensitive than that of qPCR and conventional PCR approaches. The limit of detection of MERT-LAMP for Shigella strains and Salmonella strains detection in artificially contaminated milk samples was 5.8 and 6.4 CFU per vessel. In conclusion, the MERT-LAMP methodology described here demonstrated a potential and valuable means for simultaneous screening of Shigella and Salmonella in a wide variety of samples. PMID:26697000

Postoperative fever after hemiarthroplasty in elderly patients over 70 years of age with displaced femoral neck fracture: Necessity of routine workup?

PubMed

Yoo, Je-Hyun; Kim, Ki-Tae; Kim, Tae-Young; Hwang, Ji-Hyo; Chang, Jun-Dong

2017-02-01

Displaced femoral neck fracture in elderly patients has been treated with hemiarthroplasty as the treatment of choice. Fever following HA is common in these elderly patients. The aim of this study was to determine which post-HA fever workup could be beneficial in this group of patients. A total of 272 consecutive patients aged ≥70 years undergoing HA for displaced femoral neck fracture were retrospectively investigated. Postoperative fever (POF) was defined as any recorded body temperature ≥38°C in the early postoperative period. POF in each patient was characterized by the maximum temperature, the day of the first fever, and frequency of fever, stratified as either single or multiple fever spikes. Medical records were reviewed to identify positive fever workups and febrile complications. Of 272 patients, 135 (49.6%) developed POF. A total of 428 routine diagnostic tests were performed in all patients with POF, of which only 57 tests (13.3%) were positive. Urinalysis showed the highest positive rate (21.9%), followed by urine culture (14.3%), chest x-ray (12.6%), and blood culture (1.1%). The most common febrile complication was pneumonia (12.6%), followed by urinary tract infection (8.1%). On multivariate logistic regression for positive workups, only fever after postoperative day (POD) 2 was a risk factor for positive chest x-ray (OR 3.86, p=0.016) and urine culture (OR 5.04, p=0.019). Moreover, fever after POD 2 (OR 6.93, p<0.0001) and multiple fever spikes (OR 2.92, p=0.026) were independent predictors of infectious febrile complications. Routine workup for POF following hemiarthroplasty in elderly patients with displaced femoral neck fracture is not warranted. However, for fever after POD 2 and multiple fever spikes, chest x-ray and urinalysis would be necessary to rule out the two most common febrile complications such as pneumonia and urinary tract infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

The role of long-term strain history on the generation and amplification of inherited heterogeneities in continental lithosphere extensional settings

NASA Astrophysics Data System (ADS)

Morena Salerno, V.; Capitanio, Fabio A.

2017-04-01

The Earth's lithosphere is characters by various types of heterogeneities, at different scales and located at variable depth. They can be represented at crustal level by remnants of earlier tectonics evolution, such as previous orogenetic structures, remains of passive margins and magmatic bodies intrusion, or at deeper level by mantle anisotropies. These heterogeneities can severely affect the stress and strain localization in subsequent continental lithospheric extension and rift basins evolution, hence contributing to the formation of diverse and complex rift basin types and architectures. In order to explain the difference in rift basin and passive margin types, their subsidence patterns and melt production, previous studies have exanimated the role of initial heterogeneities, rheological layering, geothermal gradients, and extension rates during a single rifting event. However, this approach does not consider the previous strain history of many basins that are characterized by multiple rifting events. In this study we use numerical models of a pristine lithosphere undergoing two rifting events separated by cooling, to show the effect of early events on later evolution. The strain histories are controlled by the variation of velocity of boundary displacement during two rifting events. We use both fast and slow first rifting events, followed by a cooling period, producing diverse mechanical heterogeneities at Moho level that represent inherited initial conditions for the second rifting event. These inherited heterogeneities range from several small perturbations distributed along the numerical domain at the end of the slowest first rifting event, to a single large perturbation at the end of first fastest rifting event. In the second rifting event, the inherited heterogeneities are amplified at different degree and time, depending on the velocity of boundary displacement used. To highlight the role of previous strain history, we parametrize the inherited heterogeneities by calculating localization indexes for all the models at the onset of the second rifting event. This calculation embeds the inherited rheology from the previous rift event. We show that the lithosphere progressively localises along the inherited heterogeneities leading to the formation of various rift basin types, ranging from narrow to wide to hyperextended and with variation degrees of symmetry. Our result show that rift basin types and structural styles are strongly affected by inherited heterogeneities generated from previous rifting events, showing cases in which the previous strain history cannot be neglected. The subsidence patterns and melt production result to be very sensitive to the strain history, the type of inherited heterogeneities and their interplay with variation of boundary displacement velocity. Our numerical simulations replicate the first-order features of rift basins and provide a general framework to assess the inherited heterogeneities' role in the interpretation of extensional basins and their evolution.

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

PubMed

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

2016-01-01

MicroRNAs (miRNAs) play an important role in many biological processes, and have been regarded as potential targets and biomarkers in cancer diagnosis and therapy. Also, to meet the big challenge imposed by the characteristics of miRNAs, such as small size and vulnerability to enzymatic digestion, it is of great importance to develop accurate, sensitive and simple miRNA assays. Herein, we developed a label-free fluorescence strategy for sensitive miRNA detection by combining isothermal exponential amplification and the unique features of SYBR Green I (SG) and graphene oxide (GO), in which SG gives significantly enhanced fluorescence upon intercalation into double-stranded DNAs (dsDNAs), and GO selectively adsorbs miRNA, single-stranded DNA and SG, to protect miRNA from enzymatic digestion, and to quench the fluorescence of the adsorbed SG. In the presence of the target miRNA, the ingeniously designed hairpin probe (HP) is unfolded and the subsequent polymerization and strand displacement reaction takes place to initiate the target recycling process. The newly formed dsDNAs are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNAs are formed to generate significantly enhanced fluorescence upon the intercalation of SG. Thus sensitive and selective fluorescence miRNA detection is realized, and the detection limit of 3 fM is obtained. Besides, this method exhibits additional advantages of simplicity and low cost, since expensive and tedious labeling process is avoided. Therefore, the as-proposed label-free fluorescence strategy has great potential in the applications in miRNA-related clinical practices and biochemical researches. Copyright © 2015 Elsevier B.V. All rights reserved.