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Sample records for resolution melting application

  1. Application of a dual target PCR-high resolution melting (HRM) method for rapid nontuberculous mycobacteria identification.

    PubMed

    Chen, Jonathan Hk; Cheng, Vincent Cc; She, Kevin Kk; Yam, Wing-Cheong; Yuen, Kwok-Yung

    2017-01-01

    Species differentiation of nontuberculous mycobacteria (NTM) has long been a difficult task in clinical laboratories. This study demonstrated and evaluated a simple and cost-effective method using the real-time PCR with high-resolution melting (PCR-HRM) analysis technique, which could differentiate at least 14 different medically related NTM.

  2. Application of a 16S rRNA PCR-high-resolution melt analysis assay for rapid detection of Salmonella Bacteremia.

    PubMed

    Jeng, Kevin; Yang, Samuel; Won, Helen; Gaydos, Charlotte A; Hsieh, Yu-Hsiang; Kecojevic, Alex; Carroll, Karen C; Hardick, Justin; Rothman, Richard E

    2012-03-01

    Current culture and phenotypic protocols for diagnosing Salmonella infections can be time-consuming. Here, we describe the application of a 16S rRNA PCR coupled to high-resolution melt analysis (HRMA) for species and serotype identification within 6 h of blood sample collection from a patient with Salmonella enterica serotype Enteritidis bacteremia.

  3. High-resolution melt analysis to detect sequence variations in highly homologous gene regions: application to CYP2B6.

    PubMed

    Twist, Greyson P; Gaedigk, Roger; Leeder, J Steven; Gaedigk, Andrea

    2013-06-01

    High-resolution melt (HRM) analysis using 'release-on-demand' dyes, such as EvaGreen(®) has the potential to resolve complex genotypes in situations where genotype interpretation is complicated by the presence of pseudogenes or allelic variants in close proximity to the locus of interest. We explored the utility of HRM to genotype a SNP (785A>G, K262R, rs2279343) that is located within exon 5 of the CYP2B6 gene, which contributes to the metabolism of a number of clinically used drugs. Testing of 785A>G is challenging, but crucial for accurate genotype determination. This SNP is part of multiple known CYP2B6 haplotypes and located in a region that is identical to CYP2B7, a nonfunctional pseudogene. Because small CYP2B6-specific PCR amplicons bracketing 785A>G cannot be generated, we simultaneously amplified both genes. A panel of 235 liver tissue DNAs and five Coriell samples were assessed. Eight CYP2B6/CYP2B7 diplotype combinations were found and a novel variant 769G>A (D257N) was discovered. The frequency of 785G corresponded to those reported for Caucasians and African-Americans. Assay performance was confirmed by CYP2B6 and/or CYP2B7 sequence analysis in a subset of samples, using a preamplified CYP2B6-specific long-range-PCR amplicon as HRM template. Inclusion rather than exclusion of a homologous pseudogene allowed us to devise a sensitive, reliable and affordable assay to test this CYP2B6 SNP. This assay design may be utilized to overcome the challenges and limitations of other methods. Owing to the flexibility of HRM, this assay design can easily be adapted to other gene loci of interest.

  4. Application of high resolution melt (HRM) analysis for duplex detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) in shrimp.

    PubMed

    Senapin, Saengchan; Molthathong, Sudkhate; Phiwsaiya, Kornsunee; Jaengsanong, Chatlada; Chuchird, Niti

    2010-10-01

    In this work, a probe-free, multiplex RT-PCR was combined with high resolution melt (HRM) analysis for the simultaneous detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) infection in the freshwater prawn Macrobrachium rosenbergii. This first application of HRM multiplex RT-PCR in shrimp reveals a new potential for rapid and sensitive detection of multiple pathogens. In addition, sequence variation in XSV could be observed from the high resolution melt peaks, as confirmed by sequence analysis. In 19 field samples of the freshwater prawn M. rosenbergii the technique revealed samples negative for both viruses, positive for both viruses or positive for MrNV alone. No sample was found positive for XSV alone. Comparison of these results to those obtained using the same samples in analysis by traditional nested RT-PCR combined with gel electrophoresis revealed that HRM multiplex RT-PCR was more sensitive. Thus, the latter technique allows for rapid and sensitive, simultaneous detection of MrNV and XSV and also has the potential to be adapted for simultaneous detection of other mixed viral infections in shrimp.

  5. High Resolution Melting (HRM) applied to wine authenticity.

    PubMed

    Pereira, Leonor; Gomes, Sónia; Castro, Cláudia; Eiras-Dias, José Eduardo; Brazão, João; Graça, António; Fernandes, José R; Martins-Lopes, Paula

    2017-02-01

    Wine authenticity methods are in increasing demand mainly in Denomination of Origin designations. The DNA-based methodologies are a reliable means of tracking food/wine varietal composition. The main aim of this work was the study of High Resolution Melting (HRM) application as a screening method for must and wine authenticity. Three sample types (leaf, must and wine) were used to validate the three developed HRM assays (Vv1-705bp; Vv2-375bp; and Vv3-119bp). The Vv1 HRM assay was only successful when applied to leaf and must samples. The Vv2 HRM assay successfully amplified all sample types, allowing genotype discrimination based on melting temperature values. The smallest amplicon, Vv3, produced a coincident melting curve shape in all sample types (leaf and wine) with corresponding genotypes. This study presents sensitive, rapid and efficient HRM assays applied for the first time to wine samples suitable for wine authenticity purposes.

  6. Application of the High Resolution Melting analysis for genetic mapping of Sequence Tagged Site markers in narrow-leafed lupin (Lupinus angustifolius L.).

    PubMed

    Kamel, Katarzyna A; Kroc, Magdalena; Święcicki, Wojciech

    2015-01-01

    Sequence tagged site (STS) markers are valuable tools for genetic and physical mapping that can be successfully used in comparative analyses among related species. Current challenges for molecular markers genotyping in plants include the lack of fast, sensitive and inexpensive methods suitable for sequence variant detection. In contrast, high resolution melting (HRM) is a simple and high-throughput assay, which has been widely applied in sequence polymorphism identification as well as in the studies of genetic variability and genotyping. The present study is the first attempt to use the HRM analysis to genotype STS markers in narrow-leafed lupin (Lupinus angustifolius L.). The sensitivity and utility of this method was confirmed by the sequence polymorphism detection based on melting curve profiles in the parental genotypes and progeny of the narrow-leafed lupin mapping population. Application of different approaches, including amplicon size and a simulated heterozygote analysis, has allowed for successful genetic mapping of 16 new STS markers in the narrow-leafed lupin genome.

  7. High-Resolution Melt Curve Analysis in Cancer Mutation Screen.

    PubMed

    Mehrotra, Meenakshi; Patel, Keyur P

    2016-01-01

    High-resolution melt (HRM) curve analysis is a PCR-based assay that identifies sequence alterations based on subtle variations in the melting curves of mutated versus wild-type DNA sequences. HRM analysis is a high-throughput, sensitive, and efficient alternative to Sanger sequencing and is used to assess for mutations in clinically important genes involved in cancer diagnosis. The technique involves PCR amplification of a target sequence in the presence of a fluorescent double-stranded DNA (dsDNA) binding dye, melting of the fluorescent amplicons, and subsequent interpretation of melt curve profiles.

  8. Establishment of a simple and rapid identification method for Listeria spp. by using high-resolution melting analysis, and its application in food industry.

    PubMed

    Ohshima, Chihiro; Takahashi, Hajime; Phraephaisarn, Chirapiphat; Vesaratchavest, Mongkol; Keeratipibul, Suwimon; Kuda, Takashi; Kimura, Bon

    2014-01-01

    Listeria monocytogenes is the causative bacteria of listeriosis, which has a higher mortality rate than that of other causes of food poisoning. Listeria spp., of which L. monocytogenes is a member, have been isolated from food and manufacturing environments. Several methods have been published for identifying Listeria spp.; however, many of the methods cannot identify newly categorized Listeria spp. Additionally, they are often not suitable for the food industry, owing to their complexity, cost, or time consumption. Recently, high-resolution melting analysis (HRMA), which exploits DNA-sequence differences, has received attention as a simple and quick genomic typing method. In the present study, a new method for the simple, rapid, and low-cost identification of Listeria spp. has been presented using the genes rarA and ldh as targets for HRMA. DNA sequences of 9 Listeria species were first compared, and polymorphisms were identified for each species for primer design. Species specificity of each HRM curve pattern was estimated using type strains of all the species. Among the 9 species, 7 were identified by HRMA using rarA gene, including 3 new species. The remaining 2 species were identified by HRMA of ldh gene. The newly developed HRMA method was then used to assess Listeria isolates from the food industry, and the method efficiency was compared to that of identification by 16S rDNA sequence analysis. The 2 methods were in coherence for 92.6% of the samples, demonstrating the high accuracy of HRMA. The time required for identifying Listeria spp. was substantially low, and the process was considerably simplified, providing a useful and precise method for processing multiple samples per day. Our newly developed method for identifying Listeria spp. is highly valuable; its use is not limited to the food industry, and it can be used for the isolates from the natural environment.

  9. Differentiation of Staphylococcus spp. by high-resolution melting analysis.

    PubMed

    Slany, Michal; Vanerkova, Martina; Nemcova, Eva; Zaloudikova, Barbora; Ruzicka, Filip; Freiberger, Tomas

    2010-12-01

    High-resolution melting analysis (HRMA) is a fast (post-PCR) high-throughput method to scan for sequence variations in a target gene. The aim of this study was to test the potential of HRMA to distinguish particular bacterial species of the Staphylococcus genus even when using a broad-range PCR within the 16S rRNA gene where sequence differences are minimal. Genomic DNA samples isolated from 12 reference staphylococcal strains (Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus warneri, and Staphylococcus xylosus) were subjected to a real-time PCR amplification of the 16S rRNA gene in the presence of fluorescent dye EvaGreen™, followed by HRMA. Melting profiles were used as molecular fingerprints for bacterial species differentiation. HRMA of S. saprophyticus and S. xylosus resulted in undistinguishable profiles because of their identical sequences in the analyzed 16S rRNA region. The remaining reference strains were fully differentiated either directly or via high-resolution plots obtained by heteroduplex formation between coamplified PCR products of the tested staphylococcal strain and phylogenetically unrelated strain.

  10. Consequences and Resolution of Lunar Lower Mantle Partial Melt

    NASA Astrophysics Data System (ADS)

    Fuqua, H.; Bremner, P. M.; Diamond, M. R.; Garapic, G.; Lock, S. J.; Mallik, A.; Nishikawa, Y.; Panovska, S.; Shahar, A.; Lognonne, P. H.; Panero, W. R.; Faul, U.; Panning, M. P.; Jimenez-Perez, H.; Schmerr, N. C.; Williams, Q. C.

    2014-12-01

    Existence of a partially molten layer at depth has been proposed to explain the lack of observed farside deep moonquakes, the observation of reflected phases from deep moonquakes, and the dissipation of tidal energy within the lunar interior. However, subsequent models explore the possibility that dissipation due to elevated temperatures alone can explain the observed dissipation factor (Q) and tidal love numbers. We have explored the hypothesis that high titanium melt compositions associated with lunar mantle overturn may sink to the base of the mantle, locally or regionally. We have performed forward calculations varying composition and thickness of layers to evaluate if a partially molten layer at the base of the mantle is well constrained by the observational data. Self-consistent physical parameters are calculated for each compositional model that are then compared against the observed data to determine a subset of permissible models. The data constraints considered by this study include bulk density, moment of inertia, real and imaginary parts of the Love numbers, seismic travel times, and electrical conductivity. Dynamic calculations using ASPECT have also been considered to determine the implications of early lunar mantle convection for the survivability of the partially molten layer. Finally, and as a perspective for a future NASA New Frontiers Geophysical Network, we present 1D synthetic seismograms calculated for each proposed structure of the Moon to investigate the future seismological resolution of these deep lunar structure features. This work was initiated at the CIDER 2014 program.

  11. Droplet Array Platform for High-Resolution Melt Analysis of DNA Methylation Density.

    PubMed

    Athamanolap, Pornpat; Shin, Dong Jin; Wang, Tza-Huei

    2014-06-01

    High-resolution melting (HRM) has garnered significant interest as an analytical technique for a number of applications, including DNA methylation detection, due to its inherent sensitivity and robustness. In this study, we describe a miniaturized assay platform for quantitative methylation density analysis using a microfluidic droplet array cartridge. We demonstrate that the DNA methylation level of the RASSF1A promoter can be directly analyzed using HRM. PCR products were generated by amplifying bisulfite-treated DNA with varying CpG densities using CpG island-flanking primer sets. Subsequent HRM analysis on the miniaturized droplet platform shows distinct melting curve profiles associated with methylation levels, which was verified using a conventional benchtop PCR-HRM system. The characteristic melting temperature (Tm) of the PCR products was used to directly quantify the respective levels of DNA methylation density. Our approach provides a key advantage over current gold standard methods such as methylation-specific PCR (MSP), which are incapable of providing specific information regarding the overall methylation density of the target genes. The miniaturized platform establishes a practical approach to methylation density profiling from multiple DNA samples with a potential application in point-of-care diagnostics.

  12. High-resolution melt analysis without DNA extraction affords rapid genotype resolution and species identification.

    PubMed

    Rugman-Jones, Paul F; Stouthamer, Richard

    2016-09-22

    Extracting and sequencing DNA from specimens can impose major time and monetary costs to studies requiring genotyping, or identification to species, of large numbers of individuals. As such, so-called direct PCR methods have been developed enabling significant savings at the DNA extraction step. Similarly, real-time quantitative PCR techniques (qPCR) offer very cost-effective alternatives to sequencing. High-resolution melt analysis (HRM) is a qPCR method that incorporates an intercalating dye into a double-stranded PCR amplicon. The dye fluoresces brightly, but only when it is bound. Thus, after PCR, raising the temperature of the amplicon while measuring the fluorescence of the reaction results in the generation of a sequence-specific melt curve, allowing discrimination of genotypes. Methods combining HRM (or other qPCR methods) and direct PCR have not previously been reported, most likely due to concerns that any tissue in the reaction tube would interfere with detection of the fluorescent signal. Here, we couple direct PCR with HRM and, by way of three examples, demonstrate a very quick and cost-effective method for genotyping large numbers of specimens, using Rotor-Gene HRM instruments (QIAGEN). In contrast to the heated-block design of most qPCR/HRM instruments, the Rotor-Gene's centrifugal rotor and air-based temperature-regulation system facilitate our method by depositing tissues away from the pathway of the machine's fluorescence detection optics.

  13. High resolution melting analysis as a new approach to discriminate gluten-containing cereals.

    PubMed

    Martín-Fernández, Begoña; Costa, Joana; de-Los-Santos-Álvarez, Noemí; López-Ruiz, Beatriz; Oliveira, M Beatriz P P; Mafra, Isabel

    2016-11-15

    With this work, it is intended to propose a novel approach based on high resolution melting (HRM) analysis to detect wheat and discriminate it from other gluten-containing cereals. The method consisted of a real-time PCR assay targeting the gene encoding for the germ agglutinin isolectin A protein (Tri a 18 allergen), using the fluorescent Evagreen dye combined with HRM analysis. The results enabled wheat differentiation from other phylogenetically related cereals, namely barley, rye and oat with high level of confidence. Additionally, a quantitative real-time PCR approach was proposed, allowing detecting and quantifying wheat down to 20mg/kg in rice flour and 20pg of wheat DNA (∼1.1 DNA copies). Its application was successfully achieved in the analysis of processed foods to verify labelling compliance, being considered as a cost-effective tool for the specific detection of cereals in gluten-free foods.

  14. The WAIS Melt Monitor: An automated ice core melting system for meltwater sample handling and the collection of high resolution microparticle size distribution data

    NASA Astrophysics Data System (ADS)

    Breton, D. J.; Koffman, B. G.; Kreutz, K. J.; Hamilton, G. S.

    2010-12-01

    Paleoclimate data are often extracted from ice cores by careful geochemical analysis of meltwater samples. The analysis of the microparticles found in ice cores can also yield unique clues about atmospheric dust loading and transport, dust provenance and past environmental conditions. Determination of microparticle concentration, size distribution and chemical makeup as a function of depth is especially difficult because the particle size measurement either consumes or contaminates the meltwater, preventing further geochemical analysis. Here we describe a microcontroller-based ice core melting system which allows the collection of separate microparticle and chemistry samples from the same depth intervals in the ice core, while logging and accurately depth-tagging real-time electrical conductivity and particle size distribution data. This system was designed specifically to support microparticle analysis of the WAIS Divide WDC06A deep ice core, but many of the subsystems are applicable to more general ice core melting operations. Major system components include: a rotary encoder to measure ice core melt displacement with 0.1 millimeter accuracy, a meltwater tracking system to assign core depths to conductivity, particle and sample vial data, an optical debubbler level control system to protect the Abakus laser particle counter from damage due to air bubbles, a Rabbit 3700 microcontroller which communicates with a host PC, collects encoder and optical sensor data and autonomously operates Gilson peristaltic pumps and fraction collectors to provide automatic sample handling, melt monitor control software operating on a standard PC allowing the user to control and view the status of the system, data logging software operating on the same PC to collect data from the melting, electrical conductivity and microparticle measurement systems. Because microparticle samples can easily be contaminated, we use optical air bubble sensors and high resolution ice core density

  15. Massively parallel digital high resolution melt for rapid and absolutely quantitative sequence profiling

    PubMed Central

    Velez, Daniel Ortiz; Mack, Hannah; Jupe, Julietta; Hawker, Sinead; Kulkarni, Ninad; Hedayatnia, Behnam; Zhang, Yang; Lawrence, Shelley; Fraley, Stephanie I.

    2017-01-01

    In clinical diagnostics and pathogen detection, profiling of complex samples for low-level genotypes represents a significant challenge. Advances in speed, sensitivity, and extent of multiplexing of molecular pathogen detection assays are needed to improve patient care. We report the development of an integrated platform enabling the identification of bacterial pathogen DNA sequences in complex samples in less than four hours. The system incorporates a microfluidic chip and instrumentation to accomplish universal PCR amplification, High Resolution Melting (HRM), and machine learning within 20,000 picoliter scale reactions, simultaneously. Clinically relevant concentrations of bacterial DNA molecules are separated by digitization across 20,000 reactions and amplified with universal primers targeting the bacterial 16S gene. Amplification is followed by HRM sequence fingerprinting in all reactions, simultaneously. The resulting bacteria-specific melt curves are identified by Support Vector Machine learning, and individual pathogen loads are quantified. The platform reduces reaction volumes by 99.995% and achieves a greater than 200-fold increase in dynamic range of detection compared to traditional PCR HRM approaches. Type I and II error rates are reduced by 99% and 100% respectively, compared to intercalating dye-based digital PCR (dPCR) methods. This technology could impact a number of quantitative profiling applications, especially infectious disease diagnostics. PMID:28176860

  16. Massively parallel digital high resolution melt for rapid and absolutely quantitative sequence profiling

    NASA Astrophysics Data System (ADS)

    Velez, Daniel Ortiz; Mack, Hannah; Jupe, Julietta; Hawker, Sinead; Kulkarni, Ninad; Hedayatnia, Behnam; Zhang, Yang; Lawrence, Shelley; Fraley, Stephanie I.

    2017-02-01

    In clinical diagnostics and pathogen detection, profiling of complex samples for low-level genotypes represents a significant challenge. Advances in speed, sensitivity, and extent of multiplexing of molecular pathogen detection assays are needed to improve patient care. We report the development of an integrated platform enabling the identification of bacterial pathogen DNA sequences in complex samples in less than four hours. The system incorporates a microfluidic chip and instrumentation to accomplish universal PCR amplification, High Resolution Melting (HRM), and machine learning within 20,000 picoliter scale reactions, simultaneously. Clinically relevant concentrations of bacterial DNA molecules are separated by digitization across 20,000 reactions and amplified with universal primers targeting the bacterial 16S gene. Amplification is followed by HRM sequence fingerprinting in all reactions, simultaneously. The resulting bacteria-specific melt curves are identified by Support Vector Machine learning, and individual pathogen loads are quantified. The platform reduces reaction volumes by 99.995% and achieves a greater than 200-fold increase in dynamic range of detection compared to traditional PCR HRM approaches. Type I and II error rates are reduced by 99% and 100% respectively, compared to intercalating dye-based digital PCR (dPCR) methods. This technology could impact a number of quantitative profiling applications, especially infectious disease diagnostics.

  17. Massively parallel digital high resolution melt for rapid and absolutely quantitative sequence profiling.

    PubMed

    Velez, Daniel Ortiz; Mack, Hannah; Jupe, Julietta; Hawker, Sinead; Kulkarni, Ninad; Hedayatnia, Behnam; Zhang, Yang; Lawrence, Shelley; Fraley, Stephanie I

    2017-02-08

    In clinical diagnostics and pathogen detection, profiling of complex samples for low-level genotypes represents a significant challenge. Advances in speed, sensitivity, and extent of multiplexing of molecular pathogen detection assays are needed to improve patient care. We report the development of an integrated platform enabling the identification of bacterial pathogen DNA sequences in complex samples in less than four hours. The system incorporates a microfluidic chip and instrumentation to accomplish universal PCR amplification, High Resolution Melting (HRM), and machine learning within 20,000 picoliter scale reactions, simultaneously. Clinically relevant concentrations of bacterial DNA molecules are separated by digitization across 20,000 reactions and amplified with universal primers targeting the bacterial 16S gene. Amplification is followed by HRM sequence fingerprinting in all reactions, simultaneously. The resulting bacteria-specific melt curves are identified by Support Vector Machine learning, and individual pathogen loads are quantified. The platform reduces reaction volumes by 99.995% and achieves a greater than 200-fold increase in dynamic range of detection compared to traditional PCR HRM approaches. Type I and II error rates are reduced by 99% and 100% respectively, compared to intercalating dye-based digital PCR (dPCR) methods. This technology could impact a number of quantitative profiling applications, especially infectious disease diagnostics.

  18. High-resolution melt analysis of the minisatellite D1S80: a potential forensic screening tool.

    PubMed

    Pomeroy, Robert S; Balamurugan, Kuppareddi; Wong, Helena; Duncan, George

    2014-11-01

    High-resolution melt (HRM) analysis of the VNTR region of the human D1S80 locus, a 16-bp repeat minisatellite from approximately 400 to over 700 bp in length, was investigated. A Qiagen Rotor-Gene Q using the Type-it PCR HRM kit was used to acquire HRM curves for 14 single, and 16 biallelic, dsDNA samples. The HRM analysis was applicable over a range of DNA concentrations; however the characteristics of the melt curve did depend on the forward and reverse primer ratio. Despite the large amplicon size and the similarities of the repeat sequences, it was possible to discriminate different genotypes. Heterozygotes were clearly different from the homozygous variants and even small differences in the repeat sequence could be differentiated. However, the melt analysis requires a high-resolution system with temperature resolution of 0.02°C or better in order to sort out differences in these large amplicons of near identical GC content (in this case 56%). HRM analysis of amplicons with large repeat sequences can be used as a means of comparing DNA fragments. Examination of multiple sequences can be used to differentiate DNA samples and demonstrate the potential of HRM analysis as a rapid and inexpensive prescreening technique in forensic applications.

  19. Silicon purification melting for photovoltaic applications

    SciTech Connect

    VAN DEN AVYLE,JAMES A.; HO,PAULINE; GEE,JAMES M.

    2000-04-01

    The availability of polysilicon feedstock has become a major issue for the photovoltaic (PV) industry in recent years. Most of the current polysilicon feedstock is derived from rejected material from the semiconductor industry. However, the reject material can become scarce and more expensive during periods of expansion in the integrated-circuit industry. Continued rapid expansion of the PV crystalline-silicon industry will eventually require a dedicated supply of polysilicon feedstock to produce solar cells at lower costs. The photovoltaic industry can accept a lower purity polysilicon feedstock (solar-grade) compared to the semiconductor industry. The purity requirements and potential production techniques for solar-grade polysilicon have been reviewed. One interesting process from previous research involves reactive gas blowing of the molten silicon charge. As an example, Dosaj et all reported a reduction of metal and boron impurities from silicon melts using reactive gas blowing with 0{sub 2} and Cl{sub 2}. The same authors later reassessed their data and the literature, and concluded that Cl{sub 2}and 0{sub 2}/Cl{sub 2} gas blowing are only effective for removing Al, Ca, and Mg from the silicon melt. Researchers from Kawasaki Steel Corp. reported removal of B and C from silicon melts using reactive gas blowing with an 0{sub 2}/Ar plasma torch. Processes that purify the silicon melt are believed to be potentially much lower cost compared to present production methods that purify gas species.

  20. Simple models for disequilibrium fractional melting and batch melting with application to REE fractionation in abyssal peridotites

    NASA Astrophysics Data System (ADS)

    Liang, Yan; Liu, Boda

    2016-01-01

    Disequilibrium melting arises when the kinetics of chemical exchange between a residual mineral and partial melt is sluggish compare to the rate of melting. To better understand the role of a finite crystal-melt exchange rate on trace element fractionation during mantle melting, we have developed a disequilibrium melting model for partial melting in an upwelling steady-state column. We use linear kinetics to approximate crystal-melt mass exchange rate and obtain simple analytical solutions for cases of perfect fractional melting and batch melting. A key parameter determining the extent of chemical disequilibrium during partial melting is an element specific dimensionless ratio (ε) defined as the melting rate relative to the solid-melt chemical exchange rate for the trace element of interest. In the case of diffusion in mineral limited chemical exchange, ε is inversely proportional to diffusivity of the element of interest. Disequilibrium melting is important for the trace element when ε is comparable to or greater than the bulk solid-melt partition coefficient for the trace element (k). The disequilibrium fractional melting model is reduced to the equilibrium perfect fractional melting model when ε is much smaller than k. Hence highly incompatible trace elements with smaller mobilities in minerals are more susceptible to disequilibrium melting than moderately incompatible and compatible trace elements. Effect of chemical disequilibrium is to hinder the extent of fractionation between residual solid and partial melt, making the residual solid less depleted and the accumulated melt more depleted in incompatible trace element abundances relative the case of equilibrium melting. Application of the disequilibrium fractional melting model to REE and Y abundances in clinopyroxene in abyssal peridotites from the Central Indian Ridge and the Vema Lithospheric Section, Mid-Atlantic Ridge revealed a positive correlation between the disequilibrium parameter ε and the

  1. High-resolution melting analysis for identification of the Cryptococcus neoformans-Cryptococcus gattii complex.

    PubMed

    Gago, Sara; Zaragoza, Óscar; Cuesta, Isabel; Rodríguez-Tudela, Juan L; Cuenca-Estrella, Manuel; Buitrago, María J

    2011-10-01

    We have developed a two-step method based on high-resolution melting (HRM) that reliably identifies species from the Cryptococcus species complex (Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii). Our results indicate that HRM can provide a fast protocol to identify and distinguish among the main Cryptococcus species.

  2. Hot-melt extrusion--basic principles and pharmaceutical applications.

    PubMed

    Lang, Bo; McGinity, James W; Williams, Robert O

    2014-09-01

    Originally adapted from the plastics industry, the use of hot-melt extrusion has gained favor in drug delivery applications both in academia and the pharmaceutical industry. Several commercial products made by hot-melt extrusion have been approved by the FDA, demonstrating its commercial feasibility for pharmaceutical processing. A significant number of research articles have reported on advances made regarding the pharmaceutical applications of the hot-melt extrusion processing; however, only limited articles have been focused on general principles regarding formulation and process development. This review provides an in-depth analysis and discussion of the formulation and processing aspects of hot-melt extrusion. The impact of physicochemical properties of drug substances and excipients on formulation development using a hot-melt extrusion process is discussed from a material science point of view. Hot-melt extrusion process development, scale-up, and the interplay of formulation and process attributes are also discussed. Finally, recent applications of hot-melt extrusion to a variety of dosage forms and drug substances have also been addressed.

  3. Differentiating between monozygotic twins through DNA methylation-specific high-resolution melt curve analysis.

    PubMed

    Stewart, Leander; Evans, Neil; Bexon, Kimberley J; van der Meer, Dieudonne J; Williams, Graham A

    2015-05-01

    Although short tandem repeat profiling is extremely powerful in identifying individuals from crime scene stains, it is unable to differentiate between monozygotic (MZ) twins. Efforts to address this include mutation analysis through whole genome sequencing and through DNA methylation studies. Methylation of DNA is affected by environmental factors; thus, as MZ twins age, their DNA methylation patterns change. This can be characterized by bisulfite treatment followed by pyrosequencing. However, this can be time-consuming and expensive; thus, it is unlikely to be widely used by investigators. If the sequences are different, then in theory the melting temperature should be different. Thus, the aim of this study was to assess whether high-resolution melt curve analysis can be used to differentiate between MZ twins. Five sets of MZ twins provided buccal swabs that underwent extraction, quantification, bisulfite treatment, polymerase chain reaction amplification and high-resolution melting curve analysis targeting two markers, Alu-E2F3 and Alu-SP. Significant differences were observed between all MZ twins targeting Alu-E2F3 and in four of five MZ twins targeting Alu-SP (P<0.05). Thus, it has been demonstrated that bisulfite treatment followed by high-resolution melting curve analysis could be used to differentiate between MZ twins.

  4. [A applicability of sugar esters in hot-melt technology].

    PubMed

    Szuts, Angéla; Laczkovich, Orsolya; Nassab, Parya Reisi; Aigner, Zoltán; Szabone Révész, Piroska

    2007-01-01

    One of the most important tasks in pharmaceutical technology is the optimization of drug release. The hot-melt technology is an important method with which to modify the bioavailability. Sugar esters (SEs) have a wide range of HLB values (1-16). Due to their low melting points, they are promising carriers for the melting method. The aims of the present work were to study the thermal properties (DSC) and the structures (XRPD) of SEs with low, medium or high HLB values, and to evaluate their applicability in the hot-melt technology. Relationships were found between the HLB value, the structure and the thermal behaviour. After melting and solidification, the SEs have partially amorphous layered structures which slowly crystallize in time; the original structure does not return for SEs with high, moderate, or low HLB values. These results demonstrate that changes in morphology must be considered during research and development. During the examination of meloxicam-SE melted products the SEs influenced the drug release, depending on their HLB values. In the cases of ibuprofen-SE melted products, the SEs did not influence the drug release. Here, a change in the drug distribution was the predominant effect, which was accompanied by movement in the SE structure.

  5. High resolution melting (HRM) analysis of DNA--its role and potential in food analysis.

    PubMed

    Druml, Barbara; Cichna-Markl, Margit

    2014-09-01

    DNA based methods play an increasing role in food safety control and food adulteration detection. Recent papers show that high resolution melting (HRM) analysis is an interesting approach. It involves amplification of the target of interest in the presence of a saturation dye by the polymerase chain reaction (PCR) and subsequent melting of the amplicons by gradually increasing the temperature. Since the melting profile depends on the GC content, length, sequence and strand complementarity of the product, HRM analysis is highly suitable for the detection of single-base variants and small insertions or deletions. The review gives an introduction into HRM analysis, covers important aspects in the development of an HRM analysis method and describes how HRM data are analysed and interpreted. Then we discuss the potential of HRM analysis based methods in food analysis, i.e. for the identification of closely related species and cultivars and the identification of pathogenic microorganisms.

  6. Rapid screening for sickle cell disease by polymerase chain reaction-high resolution melting analysis.

    PubMed

    Yue, Liang; Lin, Min; Chen, Jiang-Tao; Zhan, Xiao-Fen; Zhong, De-Shang; Monte-Nguba, Santiago-M; Liu, Pei-Fen; Pan, Xue-Fen; Huang, Jiang-Hua; Wang, Xi; Ehapo, Juan Carlos Salas; Eyi, Urbano Monsuy; Yang, Hui-Tian; Yang, Li-Ye

    2014-06-01

    Each year, ~300,000 individuals with sickle cell disease (SCD), a hemoglobinopathy caused by β-globin gene mutation, are born, and >75% of those are in Africa. The present study examined 511 individuals on the island of Bioko (Equatorial Guinea) and attempted to establish a method for rapid sickle cell disease screening. Following DNA extraction and polymerase chain reaction (PCR) amplification, high resolution melting (HRM) analysis was used to assess the specificity of fluorescence signals of the PCR products and to differentiate various genotypes of these products. The analytical results of HRM were validated using DNA sequencing. By HRM analysis, 80 out of 511 samples were classified as hemoglobin S (Hb S) heterozygotes, while 431 out of 511 samples were classified as wild-type. No mutant homozygote was identified. DNA sequencing indicated that within the 431 wild-type samples as indicated by HRM analysis, one case was actually a Hb S heterozygote and another case was a rare hemoglobin S-C genotype (sickle-hemoglobin C disease). One out of 80 suspected Hb S heterozygotes as indicated by HRM was confirmed as wild-type by DNA sequencing and the results of residual 508 cases were consistent for HRM analysis and sequencing. In conclusion, HRM analysis is a simple, high-efficiency approach for Hb S screening and is useful for early diagnosis of SCD and particularly suitable for application in the African area.

  7. Differentiation of Solenopsis invicta social forms using high resolution melt PCR.

    PubMed

    Oakey, J; Harris, E; Pease, B; Jennings, C; McCubbin, K

    2011-10-01

    Solenopsis invicta Buren (red imported fire ant) are invasive pests that have the capability of major destructive impacts on lifestyle, ecology and economy. Control of this species is dependent, in part, upon ability to estimate the potential spread from newly discovered nests. The potential for spread and the spread characteristics differ between monogyne and polygyne social forms. Prior to this study, differentiation of the two social forms in laboratory test samples commonly used a method involving restriction endonuclease digestion of an amplified Gp-9 fragment. Success of this assay is limited by the quality of DNA, which in the field-collected insects may be affected by temporary storage in unfavourable conditions. Here, we describe an alternative and highly objective assay based upon a high resolution melt technique following preamplification of a significantly shorter Gp-9 fragment than that required for restriction endonuclease digestion. We demonstrate the application of this assay to a S. invicta incursion in Queensland, Australia, using field samples from which DNA may be partially degraded. The reductions in hands-on requirements and overall duration of the assay underpin its suitability for high-throughput testing.

  8. A novel high-resolution melt PCR assay discriminates Anaplasma phagocytophilum and "Candidatus Neoehrlichia mikurensis".

    PubMed

    Krücken, Jürgen; Schreiber, Cécile; Maaz, Denny; Kohn, Mareen; Demeler, Janina; Beck, Stefanie; Schein, Eberhard; Olias, Philipp; Richter, Dania; Matuschka, Franz-Rainer; Pachnicke, Stefan; Krieger, Klemens; Kohn, Barbara; von Samson-Himmelstjerna, Georg

    2013-06-01

    "Candidatus Neoehrlichia mikurensis" (Anaplasmataceae) is an emerging pathogen transmitted by Ixodes ticks. Conventional PCR and the newly developed high-resolution melt PCR were used to detect and discriminate "Candidatus Neoehrlichia mikurensis" and Anaplasma phagocytophilum. Both bacterial species were frequently found in Ixodes ricinus and Ixodes hexagonus but virtually absent from Dermacentor reticulatus. In rodents, "Candidatus N. mikurensis" was significantly more prevalent than A. phagocytophilum, whereas in cats, only A. phagocytophilum was found.

  9. High-resolution melting analysis for bird sexing: a successful approach to molecular sex identification using different biological samples.

    PubMed

    Morinha, Francisco; Travassos, Paulo; Seixas, Fernanda; Santos, Nuno; Sargo, Roberto; Sousa, Luís; Magalhães, Paula; Cabral, João A; Bastos, Estela

    2013-05-01

    High-resolution melting (HRM) analysis is a very attractive and flexible advanced post-PCR method with high sensitivity/specificity for simple, fast and cost-effective genotyping based on the detection of specific melting profiles of PCR products. Next generation real-time PCR systems, along with improved saturating DNA-binding dyes, enable the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations. Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sex-specific CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed (plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp. The established methodology clearly revealed the advantages (e.g. closed-tube system, high sensitivity and rapidity) of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing of birds. The high sensitivity and resolution relative to previous real-time PCR methods makes HRM analysis an excellent approach for improving advanced molecular methods for bird sexing.

  10. Use of high-resolution melting and melting temperature-shift assays for specific detection and identification of Bacillus anthracis based on single nucleotide discrimination.

    PubMed

    Derzelle, Sylviane; Mendy, Christiane; Laroche, Séverine; Madani, Nora

    2011-11-01

    Single nucleotide polymorphisms (SNPs) are important diagnostic markers for the detection and differentiation of Bacillus anthracis. High-Resolution Melting (HRM) and Melting Temperature (Tm)-shift methods are two approaches that enable SNP detection without the need for expensive labeled probes. We evaluated the potential diagnostic capability of those methods to discriminate B. anthracis from the other members of the B. cereus group. Two assays targeting B. anthracis-specific SNPs in the plcR and gyrA genes were designed for each method and used to genotype a panel of 155 Bacilli strains. All B. anthracis isolates (n=65) were correctly and unambiguously identified. Assays also proved to be appropriate for the direct genotyping of biological samples. They could reliably detect B. anthracis in contaminated organs containing as little as 10(3)CFU/ml, corresponding to a few genome equivalents per reaction. The HRM and Tm-shift applications described here represent valuable tools for specific identification of B. anthracis at reduced cost.

  11. Sensitive quantitative analysis of murine LINE1 DNA methylation using high resolution melt analysis.

    PubMed

    Newman, Michelle; Blyth, Benjamin J; Hussey, Damian J; Jardine, Daniel; Sykes, Pamela J; Ormsby, Rebecca J

    2012-01-01

    We present here the first high resolution melt (HRM) assay to quantitatively analyze differences in murine DNA methylation levels utilizing CpG methylation of Long Interspersed Elements-1 (LINE1 or L1). By calculating the integral difference in melt temperature between samples and a methylated control, and biasing PCR primers for unmethylated CpGs, the assay demonstrates enhanced sensitivity to detect changes in methylation in a cell line treated with low doses of 5-aza-2'-deoxycytidine (5-aza). The L1 assay was confirmed to be a good marker of changes in DNA methylation of L1 elements at multiple regions across the genome when compared with total 5-methyl-cytosine content, measured by Liquid Chromatography-Mass Spectrometry (LC-MS). The assay design was also used to detect changes in methylation at other murine repeat elements (B1 and Intracisternal-A-particle Long-terminal Repeat elements). Pyrosequencing analysis revealed that L1 methylation changes were non-uniform across the CpGs within the L1-HRM target region, demonstrating that the L1 assay can detect small changes in CpG methylation among a large pool of heterogeneously methylated DNA templates. Application of the assay to various tissues from Balb/c and CBA mice, including previously unreported peripheral blood (PB), revealed a tissue hierarchy (from hypermethylated to hypomethylated) of PB > kidney > liver > prostate > spleen. CBA mice demonstrated overall greater methylation than Balb/c mice, and male mice demonstrated higher tissue methylation compared with female mice in both strains. Changes in DNA methylation have been reported to be an early and fundamental event in the pathogenesis of many human diseases, including cancer. Mouse studies designed to identify modulators of DNA methylation, the critical doses, relevant time points and the tissues affected are limited by the low throughput nature and exorbitant cost of many DNA methylation assays. The L1 assay provides a high throughput, inexpensive

  12. Evaluation of PCR and high resolution melt curve analysis for differentiation of Salmonella isolates.

    PubMed

    Saeidabadi, Mohammad Sadegh; Nili, Hassan; Dadras, Habibollah; Sharifiyazdi, Hassan; Connolly, Joanne; Valcanis, Mary; Raidal, Shane; Ghorashi, Seyed Ali

    2016-12-21

    Consumption of poultry products contaminated with Salmonella is one of the major causes of foodborne diseases worldwide and therefore detection and differentiation of Salmonella spp. in poultry is important. In this study, oligonucleotide primers were designed from hem-D gene and a PCR followed by high-resolution melt (HRM) curve analysis was developed for rapid differentiation of Salmonella isolates. Amplicons of 228 bp were generated from 16 different Salmonella reference strains and from 65 clinical field isolates mainly from poultry farms. High resolution melt (HRM) curve analysis of the amplicons differentiated Salmonella isolates and analysis of the nucleotide sequence of the amplicons from selected isolates revealed that each melting curve profile was related to a unique DNA sequence. The relationship between reference strains and tested specimens was also evaluated using a mathematical model without visual interpretation of HRM curves. In addition, the potential of the PCR-HRM curve analysis was evaluated for genotyping of additional Salmonella isolates from different avian species. The findings indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of Salmonella isolates to determine the serovar/serotype.

  13. Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples.

    PubMed

    Fraley, Stephanie I; Hardick, Justin; Masek, Billie J; Jo Masek, Billie; Athamanolap, Pornpat; Rothman, Richard E; Gaydos, Charlotte A; Carroll, Karen C; Wakefield, Teresa; Wang, Tza-Huei; Yang, Samuel

    2013-10-01

    Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.

  14. Heat flow in impact melts - Apollo 17 Station 6 Boulder and some applications to other breccias and xenolith laden melts

    NASA Technical Reports Server (NTRS)

    Onorato, P. I. K.; Uhlmann, D. R.; Simonds, C. H.

    1976-01-01

    The paper presents results of calculations for the cooling of an impact melt, the specific application being the clast-laden sheet sampled in the Apollo 17 Station 6 Boulder. The calculations were carried out using a two-stage cooling model which involves a short initial phase of thermal equilibration between small clasts and the surrounding melt and a second phase of heat loss from the melt sheet to the surroundings.

  15. Evaluation of High Resolution Melting for MTHFR C677T Genotyping in Congenital Heart Disease

    PubMed Central

    Yue, Shuying; Zhang, Kun; Wang, Hui; Dong, Rui; Yang, Xiaomeng; Liu, Yi; Ma, Yanhui

    2016-01-01

    Background High resolution melting (HRM) is a simple, flexible and low-cost mutation screening technique. The methylenetetrahydrofolate reductase (MTHFR) gene encoding a critical enzyme, potentially affects susceptibility to some congenital defects like congenital heart disease (CHD). We evaluate the performance of HRM for genotyping of the MTHFR gene C677T locus in CHD cases and healthy controls of Chinese Han population. Methods A total of 315 blood samples from 147 CHD patients (male72, female 75) and 168 healthy controls (male 92, female 76) were enrolled in the study. HRM was utilized to genotype MTHFR C677T locus of all the samples. The results were compared to that of PCR-RFLP and Sanger sequencing. The association of the MTHFR C677T genotypes and the risk of CHD was analyzed using odds ratio with their 95% confidence interval (CIs) from unconditional logistic regression. Results All the samples were successfully genotyped by HRM within 1 hour and 30 minutes while at least 6 hours were needed for PCR-RFLP and sequencing. The genotypes of MTHFR C677T CC, CT, and TT were 9.52%, 49.66%, and 40.82% in CHD group but 29.17%, 50% and 20.83% in control group, which were identical using both methods of HRM and PCR-RFLP, demonstrating the sensitivity and specificity of HRM were all 100%. Conclusion MTHFR C677T is a potential risk factor for CHD in our local residents of Shandong province in China. HRM is a fast, sensitive, specific and reliable method for clinical application of genotyping. PMID:26990189

  16. DMSO increases mutation-scanning detection sensitivity in clinical samples using high resolution melting

    PubMed Central

    Song, Chen; Castellanos-Rizaldos, Elena; Bejar, Rafael; Ebert, Benjamin L.; Makrigiorgos, G. Mike

    2016-01-01

    BACKGROUND Mutation scanning provides the simplest, lowest cost method for identifying DNA variations on single PCR amplicons, and it may be performed prior to sequencing to avoid screening of non-informative wild type samples. High resolution melting (HRM) is the most commonly used method for mutation scanning. However, by using PCR-HRM mutations below ≈ 3–10% that can still be clinically significant may often be missed. Therefore, enhancing HRM detection sensitivity is important for mutation scanning and its clinical application. METHODS We used serial dilution of TP53 exon 8 mutation containing cell lines to demonstrate the improvement in detection sensitivity for conventional-PCR-HRM in the presence of DMSO. We also conducted full-COLD-PCR to further enrich low-level mutations prior to HRM±DMSO and employed droplet-digital PCR to derive the optimal conditions for mutation enrichment. Both conventional-PCR-HRM and full-COLD-PCR-HRM ±DMSO were used for mutation scanning in TP53 exon 8 in cancer samples containing known mutations and in myelodysplastic syndrome samples with unknown mutations. Mutations in other genes were also examined. RESULTS The detection sensitivity of PCR-HRM-scanning increases 2–5-fold in the presence of DMSO, depending also on mutation type and sequence context, and can typically detect mutation abundance of about 1%. When mutation enrichment is applied during amplification using full-COLD-PCR and followed by HRM in the presence of DMSO, mutations with 0.2–0.3% mutation abundance in TP53 exon 8 can be detected. CONCLUSIONS DMSO improves HRM mutation scanning sensitivity. When full-COLD-PCR is employed, followed by DMSO-HRM, the overall improvement is about 20-fold as compared to conventional PCR-HRM. PMID:26432802

  17. Pharmaceutical applications of hot-melt extrusion: part I.

    PubMed

    Crowley, Michael M; Zhang, Feng; Repka, Michael A; Thumma, Sridhar; Upadhye, Sampada B; Battu, Sunil Kumar; McGinity, James W; Martin, Charles

    2007-09-01

    Interest in hot-melt extrusion techniques for pharmaceutical applications is growing rapidly with well over 100 papers published in the pharmaceutical scientific literature in the last 12 years. Hot-melt extrusion (HME) has been a widely applied technique in the plastics industry and has been demonstrated recently to be a viable method to prepare several types of dosage forms and drug delivery systems. Hot-melt extruded dosage forms are complex mixtures of active medicaments, functional excipients, and processing aids. HME also offers several advantages over traditional pharmaceutical processing techniques including the absence of solvents, few processing steps, continuous operation, and the possibility of the formation of solid dispersions and improved bioavailability. This article, Part I, reviews the pharmaceutical applications of hot-melt extrusion, including equipment, principles of operation, and process technology. The raw materials processed using this technique are also detailed and the physicochemical properties of the resultant dosage forms are described. Part II of this review will focus on various applications of HME in drug delivery such as granules, pellets, immediate and modified release tablets, transmucosal and transdermal systems, and implants.

  18. High resolution melt curve analysis based on methylation status for human semen identification.

    PubMed

    Fachet, Caitlyn; Quarino, Lawrence; Karnas, K Joy

    2017-03-01

    A high resolution melt curve assay to differentiate semen from blood, saliva, urine, and vaginal fluid based on methylation status at the Dapper Isoform 1 (DACT1) gene was developed. Stains made from blood, saliva, urine, semen, and vaginal fluid were obtained from volunteers and DNA was isolated using either organic extraction (saliva, urine, and vaginal fluid) or Chelex(®) 100 extraction (blood and semen). Extracts were then subjected to bisulfite modification in order to convert unmethylated cytosines to uracil, consequently creating sequences whose amplicons have melt curves that vary depending on their initial methylation status. When primers designed to amplify the promoter region of the DACT1 gene were used, DNA from semen samples was distinguishable from other fluids by a having a statistically significant lower melting temperature. The assay was found to be sperm-significant since semen from a vasectomized man produced a melting temperature similar to the non-semen body fluids. Blood and semen stains stored up to 5 months and tested at various intervals showed little variation in melt temperature indicating the methylation status was stable during the course of the study. The assay is a more viable method for forensic science practice than most molecular-based methods for body fluid stain identification since it is time efficient and utilizes instrumentation common to forensic biology laboratories. In addition, the assay is advantageous over traditional presumptive chemical methods for body fluid identification since results are confirmatory and the assay offers the possibility of multiplexing which may test for multiple body fluids simultaneously.

  19. Probing the atomic structure of basaltic melts generated by partial melting of upper mantle peridotite (KLB-1): Insights from high-resolution solid-state NMR study

    NASA Astrophysics Data System (ADS)

    Park, S. Y.; Lee, S. K.

    2015-12-01

    Probing the structural disorder in multi-component silicate glasses and melts with varying composition is essential to reveal the change of macroscopic properties in natural silicate melts. While a number of NMR studies for the structure of multi-component silicate glasses and melts including basaltic and andesitic glasses have been reported (e.g., Park and Lee, Geochim. Cosmochim. Acta, 2012, 80, 125; Park and Lee, Geochim. Cosmochim. Acta, 2014, 26, 42), many challenges still remain. The composition of multi-component basaltic melts vary with temperature, pressure, and melt fraction (Kushiro, Annu. Rev. Earth Planet. Sci., 2001, 71, 107). Especially, the eutectic point (the composition of first melt) of nepheline-forsterite-quartz (the simplest model of basaltic melts) moves with pressure from silica-saturated to highly undersaturated and alkaline melts. The composition of basaltic melts generated by partial melting of upper mantle peridotite (KLB-1, the xenolith from Kilbourne Hole) also vary with pressure. In this study we report experimental results for the effects of composition on the atomic structure of Na2O-MgO-Al2O3-SiO2 (NMAS) glasses in nepheline (NaAlSiO4)-forsterite (Mg2SiO4)-quartz (SiO2) eutectic composition and basaltic glasses generated by partial melting of upper mantle peridotite (KLB-1) using high-resolution multi-nuclear solid-state NMR. The Al-27 3QMAS (triple quantum magic angle spinning) NMR spectra of NMAS glasses in nepheline-forsterite-quartz eutectic composition show only [4]Al. The Al-27 3QMAS NMR spectra of KLB-1 basaltic glasses show mostly [4]Al and a non-negligible fraction of [5]Al. The fraction of [5]Al, the degree of configurational disorder, increases from 0 at XMgO [MgO/(MgO+Al2O3)]=0.55 to ~3% at XMgO=0.79 in KLB-1 basaltic glasses while only [4]Al are observed in nepheline-forsterite-quartz eutectic composition. The current experimental results provide that the fraction of [5]Al abruptly increases by the effect of

  20. Rapid and inexpensive species differentiation using a multiplex real-time polymerase chain reaction high-resolution melt assay.

    PubMed

    Elkins, Kelly M; Perez, Anjelica C U; Sweetin, Katherine C

    2016-05-01

    We demonstrate a method for developing real-time polymerase chain reaction (PCR) high-resolution melt (HRM) assays to identify multiple species present in a mixture simultaneously using LCGreen Plus and melt temperatures. Highly specific PCR primers are designed to yield amplicons with different melt temperatures for simple routine species identification compared with differentiating melt curve kinetics traces or difference plots. This method is robust and automatable, and it leads to savings in time and reagent costs, is easily modified to probe any species of interest, eliminates the need for post-PCR gel or capillary electrophoresis in routine assays, and requires no expensive dye-labeled primers.

  1. Identification case of evidence in timber tracing of Pinus radiate, using high-resolution melting (HRM) analysis.

    PubMed

    Solano, Jaime; Anabalón, Leonardo; Encina, Francisco

    2016-03-01

    Fast, accurate detection of plant species and their hybrids using molecular tools will facilitate assessment and monitoring of timber tracing evidence. In this study the origin of unknown pine samples is determined for a case of timber theft in the region of Araucania southern Chile. We evaluate the utility of the trnL marker region for species identification applied to pine wood based on High Resolution Melting. This efficient tracing methods can be incorporated into forestry applications such as certification of origin. The object of this work was genotype identification using high-resolution melting (HRM) and trnL approaches for Pinus radiata (Don) in timber tracing evidence. Our results indicate that trnL is a very sensitive marker for delimiting species and HRM analysis was used successfully for genotyping Pinus samples for timber tracing purposes. Genotyping samples by HRM analysis with the trnL1 approach allowed us to differentiate two wood samples from the Pinaceae family: Pinus radiata (Don) and Pseudotsuga menziesii (Mirb.) Franco. The same approach with Pinus trnL wood was not able to discriminate between samples of Pinus radiata, indicating that the samples were genetically indistinguishable, possibly because they have the same genotype at this locus. Timber tracing with HRM analysis is expected to contribute to future forest certification schemes, control of illegal trading, and molecular traceability of Pinus spp.

  2. Rapid and efficient zebrafish genotyping using PCR with high-resolution melt analysis.

    PubMed

    Xing, Lingyan; Quist, Tyler S; Stevenson, Tamara J; Dahlem, Timothy J; Bonkowsky, Joshua L

    2014-02-05

    Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA). This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts. Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

  3. Detection of Indel Mutations in Drosophila by High-Resolution Melt Analysis (HRMA).

    PubMed

    Housden, Benjamin E; Perrimon, Norbert

    2016-09-01

    Although CRISPR technology allows specific genome alterations to be created with relative ease, detection of these events can be problematic. For example, CRISPR-induced double-strand breaks are often repaired imprecisely to generate unpredictable short indel mutations. Detection of these events requires the use of molecular screening techniques such as endonuclease assays, restriction profiling, or high-resolution melt analysis (HRMA). Here, we provide detailed protocols for HRMA-based mutation screening in Drosophila and analysis of the resulting data using the online tool HRMAnalyzer.

  4. Assessment of high resolution melting analysis as a potential SNP genotyping technique in forensic casework.

    PubMed

    Venables, Samantha J; Mehta, Bhavik; Daniel, Runa; Walsh, Simon J; van Oorschot, Roland A H; McNevin, Dennis

    2014-11-01

    High resolution melting (HRM) analysis is a simple, cost effective, closed tube SNP genotyping technique with high throughput potential. The effectiveness of HRM for forensic SNP genotyping was assessed with five commercially available HRM kits evaluated on the ViiA™ 7 Real Time PCR instrument. Four kits performed satisfactorily against forensically relevant criteria. One was further assessed to determine the sensitivity, reproducibility, and accuracy of HRM SNP genotyping. The manufacturer's protocol using 0.5 ng input DNA and 45 PCR cycles produced accurate and reproducible results for 17 of the 19 SNPs examined. Problematic SNPs had GC rich flanking regions which introduced additional melting domains into the melting curve (rs1800407) or included homozygotes that were difficult to distinguish reliably (rs16891982; a G to C SNP). A proof of concept multiplexing experiment revealed that multiplexing a small number of SNPs may be possible after further investigation. HRM enables genotyping of a number of SNPs in a large number of samples without extensive optimization. However, it requires more genomic DNA as template in comparison to SNaPshot®. Furthermore, suitably modifying pre-existing forensic intelligence SNP panels for HRM analysis may pose difficulties due to the properties of some SNPs.

  5. Genotyping of classical swine fever virus using high-resolution melt analysis.

    PubMed

    Titov, Ilya; Tsybanov, Sodnom; Malogolovkin, Alexander

    2015-11-01

    Discrimination between different field and vaccine strains of classical swine fever virus (CSFV) is crucial for meaningful disease diagnosis and epidemiological investigation. In this study, a rapid method for differentiating vaccine strains and outbreak CSFV isolates by combined RT-PCR and high-resolution melt (HRM) analysis has been developed. The assay is based on PCR amplification of short fragments from the most variable region of CSFVgene E2, followed by HRM analysis of amplicons. Real-Time PCR/HRM for CSFV detection and differentiation analysis has sensitivity comparable to RT-qPCR and genotyping resolution comparable to E2 nucleotide sequencing. This assay in one step enables rapid and sensitive identification and genotype discrimination of CSFV in field samples, and thus will be valuable for CSF outbreak response and disease control.

  6. DNA-based identification of Peucedanum ostruthium specimens and detection of common adulterants by high-resolution melting curve analysis.

    PubMed

    Schmiderer, Corinna; Ruzicka, Joana; Novak, Johannes

    2015-12-01

    Masterwort (Peucedanum ostruthium, syn. Imperatoria ostruthium, Apiaceae) is an old economic plant in Alpine countries cultivated as ornamental plant and used for spirits and in folk medicine. P. ostruthium is a species that has often been confused with related Apiaceae species or morphologically similar roots or tubers resulting in products of minor quality. Masterwort can be distinguished from other Apiaceae species by nrDNA (ITS1 and ITS2). The analysed chloroplast markers (trnK 5' intron, trnT-trnL, and psbA-trnH), however, showed no species-specific mutations. With the application of two primer pairs amplifying parts of ITS and developed for high-resolution melting curve analysis (HRM) the target species was distinguishable from the other Peucedanum and Apiaceae species of our reference set. A multiplex PCR/HRM was developed to detect adulterations with Gentiana spp., Aconitum napellus and Veratrum album.

  7. High-resolution imaging of crustal melts using 3D full-waveform seismic inversion

    NASA Astrophysics Data System (ADS)

    Warner, M.; Morgan, J. V.

    2013-12-01

    A newly practical seismic imaging technique, 3D full-waveform inversion (FWI), now has the ability to image zones of melt and melt pathways throughout the crust with a better resolution than any other geophysical method. 3D FWI has recently changed practice within the petroleum industry where it is used to obtain high-resolution high-fidelity models of physical properties in the sub-surface that are both interpreted directly and used to improve the migration of deeper reflections. This technology has been spectacularly successful in improving the imaging of reservoirs beneath shallow heterogeneities produced by, for example, gas clouds, buried fluvial channels, carbonate reefs and salt bodies. During FWI, the sub-surface model is recovered principally by using the low-frequency transmitted, refracted portion of the wavefield which is most sensitive to the macro-velocity structure. In the petroleum industry, these inversions are now routinely performed using long-offset surface-streamer and ocean-bottom data to maximum source-receiver offsets of about 15 km, leading to a maximum penetration depth of around 5 km. Using longer offsets, it is possible to extend this technology to image deeper crustal targets. Localised zones of partial melt produce large changes in p-wave and s-wave properties that are restricted in their spatial extent, and that therefore form ideal targets for 3D FWI. We have performed a suite of tests to explore the use of 3D FWI in imaging melt distribution beneath the active volcano of Montserrat. We built a model of the subsurface using a 3D travel-time tomographic model obtained from the SEA CALIPSO experiment. We added two magma chambers in accordance with a model obtained using surface-elevation changes and geochemical data. We used a wide-angle, wide-azimuth acquisition geometry to generate a fully-elastic synthetic seismic dataset, added noise, and inverted the windowed transmitted arrivals only. We used an elastic code for the forward

  8. High-resolution DNA melt curve analysis of the clustered, regularly interspaced short-palindromic-repeat locus of Campylobacter jejuni.

    PubMed

    Price, Erin P; Smith, Helen; Huygens, Flavia; Giffard, Philip M

    2007-05-01

    A novel method for genotyping the clustered, regularly interspaced short-palindromic-repeat (CRISPR) locus of Campylobacter jejuni is described. Following real-time PCR, CRISPR products were subjected to high-resolution melt (HRM) analysis, a new technology that allows precise melt profile determination of amplicons. This investigation shows that the CRISPR HRM assay provides a powerful addition to existing C. jejuni genotyping methods and emphasizes the potential of HRM for genotyping short sequence repeats in other species.

  9. Exploring crystallization kinetics in natural rhyolitic melts using high resolution CT imagery of spherulites

    NASA Astrophysics Data System (ADS)

    Clow, T. W.; Befus, K. S.; Gardner, J. E.

    2014-12-01

    Little of our understanding of crystallization kinetics has been directly derived from studies of natural samples. We examine crystallization of rhyolitic melts by quantifying spherulite sizes and number densities in obsidian collected from Yellowstone caldera using high-resolution x-ray computed tomography (CT) imagery. Spherulites are spherical to ellipsoidal masses of intergrown alkali feldspar and quartz in a radiating, fibrous structure. They are thought to form in response to relatively rapid crystallization of melt in response to large amounts of undercooling. Recent research using compositional gradients that form outside of spherulites has suggested that they nucleate at 700 to 500 ˚C and their growth slows exponentially until it eventually ceases at ~400 ˚C. By quantifying spherulite textures, and using those temperature constraints, we derive new kinetic information regarding crystallization in natural rhyolitic systems. We find that spherulites range from 0.2 to 12.3 mm in diameter, and are 0.004 to 49.5 mm3 in volume. Such values generate number densities of 70 to 185 spherulites cm-3. Histograms of size display positively skewed distributions indicating small spherulites are far more abundant than larger ones. Those distributions imply nucleation rates change as a function of temperature. At higher temperatures where the melt is undercooled by 400-500 ˚C, nucleation is rare and growth is favored. With decreasing temperature, nucleation rates increase rapidly until cold enough temperatures are reached that diffusion limits crystallization and causes it to cease (undercoolings of ~650 ˚C). Assuming a cooling rate for the host obsidian of 10-5 ˚C s-1, then overall spherulite nucleation rates are 0.01 to 0.03 spherulites cm-3 hour-1.

  10. Cancer mutation screening: Comparison of high-resolution melt analysis between two platforms.

    PubMed

    Ebili, Henry O; Ilyas, Mohammad

    2015-01-01

    High-resolution melt analysis (HRMA) is a cheap and reliable post-polymerase chain reaction (PCR) cancer mutation screening technique, which is fast gaining clinical relevance. The HRMA capabilities of the LightScanner (Idaho Technology) have been severally studied. However, the ABI 7500 HRM has not been tested against the purpose-built HRM instrument such as the LightScanner. DNA from formalin-fixed, paraffin-embedded gastric cancer, colorectal cancer, and normal tissue as well as from colorectal cancer cell lines were amplified at exons 2, 3, and 4 of KRAS, and at exons 11 and 15 of BRAF in the ABI 7500 fast real-time PCR machine and subjected to melting both on the ABI and on the LightScanner. HRMA data were analysed with the ABI HRM software v2.0.1 and the LightScanner Call-IT 2.5. We tested the ABI 7500 HRM for internal precision, accuracy, sensitivity, and specificity at mutation screening relative to the LightScanner, using crude percentage concordance, kappa statistics, and the area under the receiver operator characteristics (AUROC) curve on SPSS version 19. The results show that the ABI 7500 HRMA has a high internal precision, and excellent concordance, sensitivity, and specificity at mutation screening compared with the LightScanner. However, in contrast to the LightScanner HRM software analysis, the ABI HRM software v.2.0.1, cannot distinguish real from certain pseudovariations in PCR amplicons that are sometimes brought about by the artefacts of the melting process. In conclusion, the ABI HRM has a comparable performance level with the LightScanner, although in certain respects mentioned previously, the LightScanner has an edge over the ABI.

  11. High-resolution melt PCR analysis for genotyping of Ureaplasma parvum isolates directly from clinical samples.

    PubMed

    Payne, Matthew S; Tabone, Tania; Kemp, Matthew W; Keelan, Jeffrey A; Spiller, O Brad; Newnham, John P

    2014-02-01

    Ureaplasma sp. infection in neonates and adults underlies a variety of disease pathologies. Of the two human Ureaplasma spp., Ureaplasma parvum is clinically the most common. We have developed a high-resolution melt (HRM) PCR assay for the differentiation of the four serovars of U. parvum in a single step. Currently U. parvum strains are separated into four serovars by sequencing the promoter and coding region of the multiple-banded antigen (MBA) gene. We designed primers to conserved sequences within this region for PCR amplification and HRM analysis to generate reproducible and distinct melt profiles that distinguish clonal representatives of serovars 1, 3, 6, and 14. Furthermore, our HRM PCR assay could classify DNA extracted from 74 known (MBA-sequenced) test strains with 100% accuracy. Importantly, HRM PCR was also able to identify U. parvum serovars directly from 16 clinical swabs. HRM PCR performed with DNA consisting of mixtures of combined known serovars yielded profiles that were easily distinguished from those for single-serovar controls. These profiles mirrored clinical samples that contained mixed serovars. Unfortunately, melt curve analysis software is not yet robust enough to identify the composition of mixed serovar samples, only that more than one serovar is present. HRM PCR provides a single-step, rapid, cost-effective means to differentiate the four serovars of U. parvum that did not amplify any of the known 10 serovars of Ureaplasma urealyticum tested in parallel. Choice of reaction reagents was found to be crucial to allow sufficient sensitivity to differentiate U. parvum serovars directly from clinical swabs rather than requiring cell enrichment using microbial culture techniques.

  12. Reliable Discrimination of 10 Ungulate Species Using High Resolution Melting Analysis of Faecal DNA

    PubMed Central

    Ramón-Laca, Ana; Gleeson, Dianne; Yockney, Ivor; Perry, Michael; Nugent, Graham; Forsyth, David M.

    2014-01-01

    Identifying species occupying an area is essential for many ecological and conservation studies. Faecal DNA is a potentially powerful method for identifying cryptic mammalian species. In New Zealand, 10 species of ungulate (Order: Artiodactyla) have established wild populations and are managed as pests because of their impacts on native ecosystems. However, identifying the ungulate species present within a management area based on pellet morphology is unreliable. We present a method that enables reliable identification of 10 ungulate species (red deer, sika deer, rusa deer, fallow deer, sambar deer, white-tailed deer, Himalayan tahr, Alpine chamois, feral sheep, and feral goat) from swabs of faecal pellets. A high resolution melting (HRM) assay, targeting a fragment of the 12S rRNA gene, was developed. Species-specific primers were designed and combined in a multiplex PCR resulting in fragments of different length and therefore different melting behaviour for each species. The method was developed using tissue from each of the 10 species, and was validated in blind trials. Our protocol enabled species to be determined for 94% of faecal pellet swabs collected during routine monitoring by the New Zealand Department of Conservation. Our HRM method enables high-throughput and cost-effective species identification from low DNA template samples, and could readily be adapted to discriminate other mammalian species from faecal DNA. PMID:24637802

  13. High resolution melting analysis for identification of commercially-important Mytilus species.

    PubMed

    Jilberto, Felipe; Araneda, Cristián; Larraín, María Angélica

    2017-08-15

    Mytilus are edible mussels, including commercially-significant species such as M. chilensis, M. galloprovincialis and M. edulis. The scientific name of the species must be indicated on commercial products to satisfy labelling and traceability requirements. Species identification using morphological criteria is difficult due the plasticity of these characteristics and the absence of shells in processed products, and conventional PCR-based methods are laborious and time-intensive. As alternative, we propose high resolution melting (HRM) analysis as a simple tool to detect and identify SNP (single nucleotide polymorphisms) and length polymorphisms in Mytilus spp. We designed HRM-specific primers for the Mytilus genus to identify M. chilensis, M. galloprovincialis, M. edulis and their hybrids through clearly-distinguishable melting curves. HRM analysis showed high sensitivity (0.9639), specificity (1.0000) and precision (1.0000) compared to a conventional PCR-RFLP test. HRM is a fast and low cost method, being a reliable tool for species identification within the Mytilus genus.

  14. Applying high-resolution melting (HRM) technology to identify five commonly used Artemisia species

    PubMed Central

    Song, Ming; Li, Jingjian; Xiong, Chao; Liu, Hexia; Liang, Junsong

    2016-01-01

    Many members of the genus Artemisia are important for medicinal purposes with multiple pharmacological properties. Often, these herbal plants sold on the markets are in processed forms so it is difficult to authenticate. Routine testing and identification of these herbal materials should be performed to ensure that the raw materials used in pharmaceutical products are suitable for their intended use. In this study, five commonly used Artemisia species included Artemisia argyi, Artemisia annua, Artemisia lavandulaefolia, Artemisia indica, and Artemisia atrovirens were analyzed using high resolution melting (HRM) analysis based on the internal transcribed spacer 2 (ITS2) sequences. The melting profiles of the ITS2 amplicons of the five closely related herbal species are clearly separated so that they can be differentiated by HRM method. The method was further applied to authenticate commercial products in powdered. HRM curves of all the commercial samples tested are similar to the botanical species as labeled. These congeneric medicinal products were also clearly separated using the neighbor-joining (NJ) tree. Therefore, HRM method could provide an efficient and reliable authentication system to distinguish these commonly used Artemisia herbal products on the markets and offer a technical reference for medicines quality control in the drug supply chain. PMID:27698485

  15. Sex determination in highly fragmented human DNA by high-resolution melting (HRM) analysis.

    PubMed

    Álvarez-Sandoval, Brenda A; Manzanilla, Linda R; Montiel, Rafael

    2014-01-01

    Sex identification in ancient human remains is a common problem especially if the skeletons are sub-adult, incomplete or damaged. In this paper we propose a new method to identify sex, based on real-time PCR amplification of small fragments (61 and 64 bp) of the third exon within the amelogenin gene covering a 3-bp deletion on the AMELX-allele, followed by a High Resolution Melting analysis (HRM). HRM is based on the melting curves of amplified fragments. The amelogenin gene is located on both chromosomes X and Y, showing dimorphism in length. This molecular tool is rapid, sensitive and reduces the risk of contamination from exogenous genetic material when used for ancient DNA studies. The accuracy of the new method described here has been corroborated by using control samples of known sex and by contrasting our results with those obtained with other methods. Our method has proven to be useful even in heavily degraded samples, where other previously published methods failed. Stochastic problems such as the random allele drop-out phenomenon are expected to occur in a less severe form, due to the smaller fragment size to be amplified. Thus, their negative effect could be easier to overcome by a proper experimental design.

  16. Reliable discrimination of 10 ungulate species using high resolution melting analysis of faecal DNA.

    PubMed

    Ramón-Laca, Ana; Gleeson, Dianne; Yockney, Ivor; Perry, Michael; Nugent, Graham; Forsyth, David M

    2014-01-01

    Identifying species occupying an area is essential for many ecological and conservation studies. Faecal DNA is a potentially powerful method for identifying cryptic mammalian species. In New Zealand, 10 species of ungulate (Order: Artiodactyla) have established wild populations and are managed as pests because of their impacts on native ecosystems. However, identifying the ungulate species present within a management area based on pellet morphology is unreliable. We present a method that enables reliable identification of 10 ungulate species (red deer, sika deer, rusa deer, fallow deer, sambar deer, white-tailed deer, Himalayan tahr, Alpine chamois, feral sheep, and feral goat) from swabs of faecal pellets. A high resolution melting (HRM) assay, targeting a fragment of the 12S rRNA gene, was developed. Species-specific primers were designed and combined in a multiplex PCR resulting in fragments of different length and therefore different melting behaviour for each species. The method was developed using tissue from each of the 10 species, and was validated in blind trials. Our protocol enabled species to be determined for 94% of faecal pellet swabs collected during routine monitoring by the New Zealand Department of Conservation. Our HRM method enables high-throughput and cost-effective species identification from low DNA template samples, and could readily be adapted to discriminate other mammalian species from faecal DNA.

  17. Rapid genotyping of beak and feather disease virus using high-resolution DNA melt curve analysis.

    PubMed

    Sarker, Subir; Ghorashi, Seyed A; Forwood, Jade K; Raidal, Shane R

    2014-11-01

    Beak and feather disease virus (BFDV) is a significant pathogen both for wild and captive psittacine birds globally. Genotypic differentiation of BFDV isolates is crucial to establish effective control strategies for the conservation of endangered species and epidemiological investigations of disease outbreaks. The technique developed in this study is a simple, rapid and inexpensive genotyping method for BFDV using PCR and subsequent high-resolution melt (HRM) curve analysis. This was achieved using PCR amplification of the conserved Rep gene in the presence of a fluorescent DNA intercalating dye (SYTO9). HRM curve analysis of the resultant amplicon could readily differentiate between reference strain (92-SR14) and 18 other BFDV isolates used in this study. Analysis of the nucleotide sequences of the amplicon from each isolate revealed that each melt curve profile was related to a unique DNA sequence. The potential of the PCR-HRM curve analysis to differentiate inter-host genetic variation among critically endangered orange-bellied parrots, lorikeets and cockatoos was also evaluated. Phylogenetic tree topology based on partial Rep gene sequences used in this study showed that BFDV Rep gene sequence patterns were correlated with the results of HRM curve analysis. The results presented in this study indicate that this technique could be used in both clinical research and differentiation of BFDV isolates in a fraction of time without further nucleotide sequencing and provides a novel approach for the genetic screening of BFDV in clinical virology laboratories.

  18. Rapid detection and identification of four major Schistosoma species by high-resolution melt (HRM) analysis.

    PubMed

    Li, Juan; Zhao, Guang-Hui; Lin, RuiQing; Blair, David; Sugiyama, Hiromu; Zhu, Xing-Quan

    2015-11-01

    Schistosomiasis, caused by blood flukes belonging to several species of the genus Schistosoma, is a serious and widespread parasitic disease. Accurate and rapid differentiation of these etiological agents of animal and human schistosomiasis to species level can be difficult. We report a real-time PCR assay coupled with a high-resolution melt (HRM) assay targeting a portion of the nuclear 18S rDNA to detect, identify, and distinguish between four major blood fluke species (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and Schistosoma mekongi). Using this system, the Schistosoma spp. was accurately identified and could also be distinguished from all other trematode species with which they were compared. As little as 10(-5) ng genomic DNA from a Schistosoma sp. could be detected. This process is inexpensive, easy, and can be completed within 3 h. Examination of 21 representative Schistosoma samples from 15 geographical localities in seven endemic countries validated the value of the HRM detection assay and proved its reliability. The melting curves were characterized by peaks of 83.65 °C for S. japonicum and S. mekongi, 85.65 °C for S. mansoni, and 85.85 °C for S. haematobium. The present study developed a real-time PCR coupled with HRM analysis assay for detection and differential identification of S. mansoni, S. haematobium, S. japonicum, and S. mekongi. This method is rapid, sensitive, and inexpensive. It has important implications for epidemiological studies of Schistosoma.

  19. High-resolution melt analysis for rapid comparison of bacterial community compositions.

    PubMed

    Hjelmsø, Mathis Hjort; Hansen, Lars Hestbjerg; Baelum, Jacob; Feld, Louise; Holben, William E; Jacobsen, Carsten Suhr

    2014-06-01

    In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.

  20. Rapid detection of functional gene polymorphisms of TLRs and IL-17 using high resolution melting analysis

    PubMed Central

    Teräsjärvi, Johanna; Hakanen, Antti; Korppi, Matti; Nuolivirta, Kirsi; Gröndahl-Yli-Hannuksela, Kirsi; Mertsola, Jussi; Peltola, Ville; He, Qiushui

    2017-01-01

    Genetic variations in toll-like receptors (TLRs) and IL-17A have been widely connected to different diseases. Associations between susceptibility and resistance to different infections and single nucleotide polymorphisms (SNPs) in TLR1 to TLR4 and IL17A have been found. In this study, we aimed to develop a rapid and high throughput method to detect functional SNPs of above mentioned proteins. The following most studied and clinically important SNPs: TLR1 (rs5743618), TLR2 (rs5743708), TLR3 (rs3775291), TLR4 (rs4986790) and IL17 (rs2275913) were tested. High resolution melting analysis (HRMA) based on real-time PCR combined with melting analysis of a saturating double stranded-DNA binding dye was developed and used. The obtained results were compared to the “standard” sequencing method. A total of 113 DNA samples with known genotypes were included. The HRMA method correctly identified all genotypes of these five SNPs. Co-efficient values of variation of intra- and inter-run precision repeatability ranged from 0.04 to 0.23%. The determined limit of qualification for testing samples was from 0.5 to 8.0 ng/μl. The identical genotyping result was obtained from the same sample with these concentrations. Compared to “standard” sequencing methods HRMA is cost-effective, rapid and simple. All the five SNPs can be analyzed separately or in combination. PMID:28148965

  1. Sex Determination in Highly Fragmented Human DNA by High-Resolution Melting (HRM) Analysis

    PubMed Central

    Álvarez-Sandoval, Brenda A.; Manzanilla, Linda R.; Montiel, Rafael

    2014-01-01

    Sex identification in ancient human remains is a common problem especially if the skeletons are sub-adult, incomplete or damaged. In this paper we propose a new method to identify sex, based on real-time PCR amplification of small fragments (61 and 64 bp) of the third exon within the amelogenin gene covering a 3-bp deletion on the AMELX-allele, followed by a High Resolution Melting analysis (HRM). HRM is based on the melting curves of amplified fragments. The amelogenin gene is located on both chromosomes X and Y, showing dimorphism in length. This molecular tool is rapid, sensitive and reduces the risk of contamination from exogenous genetic material when used for ancient DNA studies. The accuracy of the new method described here has been corroborated by using control samples of known sex and by contrasting our results with those obtained with other methods. Our method has proven to be useful even in heavily degraded samples, where other previously published methods failed. Stochastic problems such as the random allele drop-out phenomenon are expected to occur in a less severe form, due to the smaller fragment size to be amplified. Thus, their negative effect could be easier to overcome by a proper experimental design. PMID:25098828

  2. Review of selective laser melting: Materials and applications

    SciTech Connect

    Yap, C. Y.; Chua, C. K. Liu, Z. H. Zhang, D. Q. Loh, L. E. Sing, S. L.; Dong, Z. L.

    2015-12-15

    Selective Laser Melting (SLM) is a particular rapid prototyping, 3D printing, or Additive Manufacturing (AM) technique designed to use high power-density laser to melt and fuse metallic powders. A component is built by selectively melting and fusing powders within and between layers. The SLM technique is also commonly known as direct selective laser sintering, LaserCusing, and direct metal laser sintering, and this technique has been proven to produce near net-shape parts up to 99.9% relative density. This enables the process to build near full density functional parts and has viable economic benefits. Recent developments of fibre optics and high-power laser have also enabled SLM to process different metallic materials, such as copper, aluminium, and tungsten. Similarly, this has also opened up research opportunities in SLM of ceramic and composite materials. The review presents the SLM process and some of the common physical phenomena associated with this AM technology. It then focuses on the following areas: (a) applications of SLM materials and (b) mechanical properties of SLM parts achieved in research publications. The review is not meant to put a ceiling on the capabilities of the SLM process but to enable readers to have an overview on the material properties achieved by the SLM process so far. Trends in research of SLM are also elaborated in the last section.

  3. Review of selective laser melting: Materials and applications

    NASA Astrophysics Data System (ADS)

    Yap, C. Y.; Chua, C. K.; Dong, Z. L.; Liu, Z. H.; Zhang, D. Q.; Loh, L. E.; Sing, S. L.

    2015-12-01

    Selective Laser Melting (SLM) is a particular rapid prototyping, 3D printing, or Additive Manufacturing (AM) technique designed to use high power-density laser to melt and fuse metallic powders. A component is built by selectively melting and fusing powders within and between layers. The SLM technique is also commonly known as direct selective laser sintering, LaserCusing, and direct metal laser sintering, and this technique has been proven to produce near net-shape parts up to 99.9% relative density. This enables the process to build near full density functional parts and has viable economic benefits. Recent developments of fibre optics and high-power laser have also enabled SLM to process different metallic materials, such as copper, aluminium, and tungsten. Similarly, this has also opened up research opportunities in SLM of ceramic and composite materials. The review presents the SLM process and some of the common physical phenomena associated with this AM technology. It then focuses on the following areas: (a) applications of SLM materials and (b) mechanical properties of SLM parts achieved in research publications. The review is not meant to put a ceiling on the capabilities of the SLM process but to enable readers to have an overview on the material properties achieved by the SLM process so far. Trends in research of SLM are also elaborated in the last section.

  4. Glacier surface melt characterization and trend analysis (1992-2011) in the Russian High Arctic from combined resolution-enhanced scatterometer and passive microwave data

    NASA Astrophysics Data System (ADS)

    Zhao, M.; Ramage, J. M.; Semmens, K. A.

    2012-12-01

    Global warming has been pronounced in the remote glacierized archipelagoes (Severnaya Zemlya, Novaya Zemlya and Franz Josef Land) of the Russian High Arctic (RHA) and its effect on the low altitude, high latitude small ice caps needs examination. The timing and spatial variability of snow melt onset, duration and intensity are key factors influencing mass balance and the ice marginal hydrological system as well as important indicators of glacial response to anthropogenic and natural forcings. Characterization and trend analysis of RHA glacier melt behaviors provide insight about assessing the mass loss rate under recent Arctic climate change. However, due to the harsh environment, long term records of glaciological data for RHA are limited, necessitating the application of remotely sensed data to accomplish the research. The high sensitivity to liquid water and the ability to penetrate non-precipitating clouds enables microwave remote sensing to detect glacier surface melt. The appearance of melt water in snow dramatically decreases the returned scatterometer radar signal from active microwave sensors and sharply augments passive microwave emission. Based on this feature, we combined resolution-enhanced ERS-1/2 C-band (1992-2000), QuickSCAT Ku-band (2000-2009), ASCAT C-band (2009-2011) scatterometer data and SSMI 37 GHz (1995-2007) vertically polarized passive microwave products from Brigham Young University and analyzed glacier surface melt trends from 1992 to 2011 with a spatial resolution downscaled to 4.45km. We concatenated scatterometer derived melt behaviors by overlapping years and refined the results based on passive microwave data. Cross-validation shows that melt timing to be consistent between the active and passive sensors. Trend analysis (α < 0.005) reveals that the average glacier surface melt onset date occurs earlier by approximately 0.85 days/year in Severnaya Zemlya which outpaced the mean advancing rate in the pan-Arctic. Surrounded by ocean

  5. Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria.

    PubMed

    Daniels, Rachel; Hamilton, Elizabeth J; Durfee, Katelyn; Ndiaye, Daouda; Wirth, Dyann F; Hartl, Daniel L; Volkman, Sarah K

    2015-11-10

    Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs. One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures. SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status. While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele. In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs

  6. A Rapid Multiplex Real-Time PCR High-Resolution Melt Curve Assay for the Simultaneous Detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in Food.

    PubMed

    Forghani, Fereidoun; Wei, Shuai; Oh, Deog-Hwan

    2016-05-01

    Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, are of great concern for food safety. They may also coexist in food matrices and, in the case of B. cereus and S. aureus, the resulting illnesses can resemble each other owing to similar symptoms. Therefore, their simultaneous detection may have advantages in terms of cost savings and rapidity. Given this context, a rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of these three pathogens in food was developed. The assay successfully detected B. cereus (gyrB), L. monocytogenes (hly), and S. aureus (nuc) in a single reaction, and the average melting temperatures were 76.23, 80.19, and 74.01°C, respectively. The application of SYTO9 dye and a slow melt curve analysis ramp rate (0.1°C/s) enabled the production of sharp, high-resolution melt curve peaks that were easily distinguishable from each other. The detection limit in food (milk, rice, and lettuce) was 3.7 × 10(3) CFU/g without an enrichment step and 3.7 × 10(1) CFU/g following the 10-h enrichment. Hence, the assay developed here is specific and sensitive, providing an efficient tool for implementation in food for the simultaneous detection of B. cereus, L. monocytogenes, and S. aureus .

  7. Refining DNA Barcoding Coupled High Resolution Melting for Discrimination of 12 Closely Related Croton Species

    PubMed Central

    Osathanunkul, Maslin; Suwannapoom, Chatmongkon; Ounjai, Sarawut; Rora, Jantarika A.; Madesis, Panagiotis; de Boer, Hugo

    2015-01-01

    DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Croton (Euphorbiaceae), one of the largest genera of plants with over 1,200 species. Seven primer pairs were evaluated (matK, rbcL1, rbcL2, rbcL3, rpoC, trnL and ITS1) from four plastid regions, matK, rbcL, rpoC, and trnL, and the nuclear ribosomal marker ITS1. The primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL1 primer pair gave the lowest resolution. It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Our Bar-HRM results here also provide further support for the hypothesis that both sequence and base composition affect DNA duplex stability. PMID:26406615

  8. Rapid identification of Borrelia by high resolution melting analysis of the groEL gene.

    PubMed

    Koś, Władysław; Wodecka, Beata; Anklewicz, Marek; Skotarczak, Bogumiła

    2013-01-01

    This study examined the possibility of applying a new diagnostic method, high resolution analysis of DNA denaturation curve (high resolution melting - HRM), for identification of Borrelia species. DNA samples were obtained from Ixodes ricinus ticks collected from vegetation and removed from hunted roe deer. For differentiation of Borrelia species, the HRM protocol based on the analysis of the groEL gene was applied. A product characteristic for Borrelia was obtained in 19/123 samples (15.4%). The studied isolates were classified as four species: B. garinii, B. valaisiana, B. afzelii and B. miyamotoi. Two separate groups of isolates within the B. afzelii species were also found. The results show that the groEL gene is useful for rapid differentiation of B. burgdorferi sensu lato with the HRM method from different extracts of DNA and it also allows precise differentiation of Borrelia species and strains. The HRM method shortened and simplified detection and differentiation of Borrelia species from different biological sources.

  9. Refining DNA Barcoding Coupled High Resolution Melting for Discrimination of 12 Closely Related Croton Species.

    PubMed

    Osathanunkul, Maslin; Suwannapoom, Chatmongkon; Ounjai, Sarawut; Rora, Jantarika A; Madesis, Panagiotis; de Boer, Hugo

    2015-01-01

    DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Croton (Euphorbiaceae), one of the largest genera of plants with over 1,200 species. Seven primer pairs were evaluated (matK, rbcL1, rbcL2, rbcL3, rpoC, trnL and ITS1) from four plastid regions, matK, rbcL, rpoC, and trnL, and the nuclear ribosomal marker ITS1. The primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL1 primer pair gave the lowest resolution. It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Our Bar-HRM results here also provide further support for the hypothesis that both sequence and base composition affect DNA duplex stability.

  10. High resolution melt analysis to track infections due to ribotype 027 Clostridium difficile.

    PubMed

    Grando, Danilla; Said, Mohamed M; Mayall, Barrie C; Gurtler, Volker

    2012-05-01

    The increased prevalence of hypervirulent ribotype 027 Clostridium difficile requires rapid identification of isolates in order to implement timely infection control strategies. High resolution melt (HRM) analysis of PCR products can identify strain variation amongst genera of bacteria. The intergenic (16S-23S rDNA) spacer region contains sequence regions conserved within genera and other sequence region variables between species within genera. We wished to investigate whether HRM analysis of PCR ribotyping products could identify ribotype 027 C. difficile. Ribotyping was performed on 93 clinical isolates and five control strains and band patterns were analysed using GelCompar II (Applied Maths, USA). Real-time PCR using ribotyping primers was performed and normalised melt curves were generated. The HRM data was then imported into ScreenClust software (QIAGEN) to generate principal component analysis graphs depicting clustered relationships of strains. Ribotyping produced clear PCR bands for 88/98 isolates tested. Dendrograms generated by GelCompar showed a diversity of ribotype patterns amongst these 88 isolates with 18 groups identified with 70% homology. One clinical isolate showed 100% homology with the control 027 strains. ScreenClust analysis of the same 88 HRM results showed clustering of isolates, with 027 strains identifiable as a unique cluster. HRM analysis correctly identified the control 027 stains and the clinical isolate shown to be 027. HRM combined with ScreenClust analysis of real-time PCR products of the 16S-23S rDNA spacer region successfully identified ribotype 027 strains. For infection control purposes this was achieved within 2-3 h of colony isolation.

  11. A novel high-resolution melting analysis-based method for Yersinia pseudotuberculosis genotyping.

    PubMed

    Souza, Roberto A; Falcão, Juliana P

    2012-12-01

    Yersinia pseudotuberculosis is an enteric pathogen that is environmentally widespread and is known to cause human and animal infections. The development of a fast and inexpensive typing system is necessary to facilitate epidemiological studies of Y. pseudotuberculosis infections. In this study, we aimed to develop a method of Y. pseudotuberculosis genotyping based on determining differences in single-nucleotide polymorphisms (SNPs) using a high-resolution melting analysis (HRMA). Using a set of nine primer pairs, ten SNPs were screened from sequences in the 16S rRNA, glnA, gyrB and recA sequences of 12 Y. pseudotuberculosis strains that were deposited in the GenBank database. The genetic diversity of a collection of 40 clinical Y. pseudotuberculosis strains was determined using the HRMA method and the multilocus sequence typing (MLST) technique was used for comparison. Different melting profiles were found in five out of a total of nine analyzed fragments. A phylogenetic tree was constructed from the nucleotides that were identified in the nine analyzed fragments, and the tree demonstrated that Y. pseudotuberculosis strains were separated into two groups. The first cluster was composed of strains from the 1/O:1a serogroup and the second of strains from the 2/O:3 serogroup. The separation into two clusters based on distinct bio-serogroups of Y. pseudotuberculosis was consistent with the results in the MLST database. The simple and highly reproducible HRMA assay developed by us may be used as a rapid and cost-effective method to genotype Y. pseudotuberculosis strains of O:1 and O:3 serogroups and it can complement sequence-based methods facilitating epidemiological studies of this Yersinia species.

  12. Performance and applications of quench melt-growth bulk magnets

    NASA Astrophysics Data System (ADS)

    Nariki, S.; Teshima, H.; Morita, M.

    2016-03-01

    This paper describes the progress in quench melt-growth (QMG) bulk magnets, developed by the Nippon Steel & Sumitomo Metal Corporation, which consist of single crystalline RE123 phase and finely dispersed RE211 particles. QMG bulks can trap high magnetic fields. The field-trapping ability of QMG bulks is largely increased with an improvement in its J c and size, promising the realization of various applications such as flywheel energy-storage systems, ship motors, NMR/MRI spectrometers, wind-power generators and so on. Intensive research has revealed that the optimal RE element is different depending on application requirements. Gd-QMG bulk is the most promising material for several high-field engineering applications. The trapped magnetic field of Gd-QMG bulk 60 mm in diameter at 77 K is twice as large as that of Y-QMG bulk with a similar size due to its excellent J c properties. The large Gd-based QMG bulks up to 150 mm in diameter are fabricated by incorporating the RE compositional gradient method. Compact NMR/MRI spectrometers are one of the promising applications of bulk superconductors. Eu-QMG bulks are suitable for NMR magnets. NMR applications require extremely homogeneous magnetic fields. In the Eu-system, the small paramagnetic moment of a Eu ion compared to a Gd ion improves the field homogeneity in the bulk. For the application of current leads, Dy-based QMG is available by utilizing a low thermal conductivity.

  13. Rapid Diagnosis of Old World Leishmaniasis by High-Resolution Melting Analysis of the 7SL RNA Gene▿ †

    PubMed Central

    Nasereddin, Abedelmajeed; Jaffe, Charles L.

    2010-01-01

    High-resolution melt analysis PCR (HRM PCR) for diagnosis of Old World Leishmania was developed using the 7SL RNA gene. Cutaneous leishmaniasis samples were analyzed. Sensitivity and specificity of HRM PCR were significantly better (P < 0.001) than those of internal transcribed spacer 1 PCR and similar to those of kinetoplast DNA PCR. PMID:20392923

  14. A PCR–High-Resolution Melt Assay for Rapid Differentiation of Nontypeable Haemophilus influenzae and Haemophilus haemolyticus

    PubMed Central

    Binks, Michael J.; Beissbarth, Jemima; Hare, Kim M.; Kirkham, Lea-Ann S.; Smith-Vaughan, Heidi

    2014-01-01

    We have developed a PCR–high-resolution melt (PCR-HRM) assay to discriminate nontypeable Haemophilus influenzae (NTHi) colonies from Haemophilus haemolyticus. This method is rapid and robust, with 96% sensitivity and 92% specificity compared to the hpd#3 assay. PCR-HRM is ideal for high-throughput screening for NTHi surveillance and clinical trials. PMID:24478508

  15. A PCR-high-resolution melt assay for rapid differentiation of nontypeable Haemophilus influenzae and Haemophilus haemolyticus.

    PubMed

    Pickering, Janessa; Binks, Michael J; Beissbarth, Jemima; Hare, Kim M; Kirkham, Lea-Ann S; Smith-Vaughan, Heidi

    2014-02-01

    We have developed a PCR-high-resolution melt (PCR-HRM) assay to discriminate nontypeable Haemophilus influenzae (NTHi) colonies from Haemophilus haemolyticus. This method is rapid and robust, with 96% sensitivity and 92% specificity compared to the hpd#3 assay. PCR-HRM is ideal for high-throughput screening for NTHi surveillance and clinical trials.

  16. Rapid determination of lymphogranuloma venereum serovars of Chlamydia trachomatis by quantitative high-resolution melt analysis (HRMA).

    PubMed

    Twin, Jimmy; Stevens, Matthew P; Garland, Suzanne M; Zaia, Angelo M; Tabrizi, Sepehr N

    2012-11-01

    A quantitative high-resolution melt analysis assay was developed to differentiate lymphogranuloma venereum-causing serovars of Chlamydia trachomatis (L1 to L3) from other C. trachomatis serovars (D to K). The detection limit of this assay is approximately 10 copies per reaction, comparable to the limits of other quantitative-PCR-based methods.

  17. High-resolution DNA melt-curve analysis for cost-effective mass screening of pairwise species interactions.

    PubMed

    McCarthy, James K; Didham, Raphael K; Brockerhoff, Eckehard G; van Bysterveldt, Katherine A; Varsani, Arvind

    2013-09-01

    Ecological studies of pairwise interactions are constrained by the methods available for rapid species identification of the interacting organisms. The resolution of data required to characterize species interaction networks at multiple spatio-temporal scales can be intensive, and therefore laborious and costly to collect. We explore the utility of high-resolution DNA melt-curve analysis (HRM) as a rapid species identification method. An approach was developed to identify organisms at the pairwise interaction level, with particular application to cryptic species interactions that are traditionally difficult to study. Here, we selected a challenging application; to identify the presence/absence of pathogenic fungi (Sporothrix inflata, Ophiostoma nigrocarpum and Ophiostoma galeiforme) transported by bark beetle vectors (Hylastes ater and Hylurgus ligniperda). The technique was able to distinguish between different species of DNA within a single, pooled sample. In test applications, HRM was effective in the mass screening and identification of pathogenic fungal species carried by many individual bark beetle vectors (n = 455 beetles screened) across large geographic scales. For two of the fungal species, there was no difference in the frequency of association with either of their vectors, but for the third fungal species there was a shift in vector-pathogen associations across locations. This technique allows rapid, mass screening and characterization of species interactions at a fraction of the time and cost of traditional methods. It is anticipated that this method can be readily applied to explore other cryptic species interactions, or other studies requiring rapid generation of large data sets and/or high-throughput efficiency.

  18. High Resolution Melting Analysis for JAK2 Exon 14 and Exon 12 Mutations

    PubMed Central

    Rapado, Inmaculada; Grande, Silvia; Albizua, Enriqueta; Ayala, Rosa; Hernández, José-Angel; Gallardo, Miguel; Gilsanz, Florinda; Martinez-Lopez, Joaquin

    2009-01-01

    JAK2 mutations are important criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess JAK2 exon 14 and exon 12 mutations by high-resolution melting (HRM) analysis, which allows variation screening. The exon 14 analysis included 163 patients with polycythemia vera, secondary erythrocytoses, essential thrombocythemia, or secondary thrombocytoses, and 126 healthy subjects. The study of exon 12 included 40 JAK2 V617F-negative patients (nine of which had polycythemia vera, and 31 with splanchnic vein thrombosis) and 30 healthy subjects. HRM analyses of JAK2 exons 14 and 12 gave analytical sensitivities near 1% and both intra- and interday coefficients of variation of less than 1%. For HRM analysis of JAK2 exon 14 in polycythemia vera and essential thrombocythemia, clinical sensitivities were 93.5% and 67.9%, clinical specificities were 98.8% and 97.0%, positive predictive values were 93.5% and 79.2%, and negative predictive values were 98.8% and 94.6, respectively. Correlations were observed between the results from HRM and three commonly used analytical methods. The JAK2 exon 12 HRM results agreed completely with those from sequencing analysis, and the three mutations in exon 12 were detected by both methods. Hence, HRM analysis of exons 14 and 12 in JAK2 shows better diagnostic values than three other routinely used methods against which it was compared. In addition, HRM analysis has the advantage of detecting unknown mutations. PMID:19225136

  19. Analysis of strain relatedness using High Resolution Melting in a case of recurrent candiduria

    PubMed Central

    2013-01-01

    Background Several genotyping protocols have been described to study Candida albicans strains with different sensitivity values. In this study we have analyzed the genetic relatedness and the antifungal susceptibility of several Candida albicans strains isolated from a patient who from suffered recurrent candiduria for a period of five years. Strains were genotyped using Microsatellite Length Polymorphism (MLP) with three microsatellite markers (HIS 3, EF 3 and CDC 3), and a new method based on high resolution melting (HRM) was developed to analyze the microsatellite region. This method was compared with the conventional technique that uses capillary electrophoresis. Results MICs of the isolates showed the existence of fluconazole susceptible and resistant strains. An inter-colony test using single concentration (8 and 16 mg/l) of fluconazole revealed the coexistence of both fluconazole susceptible and resistant strains. Both genotyping analysis methods showed that all the patient’s isolates had a clonal origin. HRM analysis method developed was able to accurately establish strain relatedness and presented a discriminatory power of 0.77. Conclusions Although HRM analysis method presented a lower discriminatory power compared to methods based on capillary electrophoresis, it provided a more cost-effective and suitable alternative for genotyping C. albicans in a clinical laboratory. PMID:23343107

  20. Differential effect of three base modifications on DNA thermostability revealed by high resolution melting.

    PubMed

    López, Carlos M Rodríguez; Lloyd, Amanda J; Leonard, Kate; Wilkinson, Mike J

    2012-09-04

    High resolution melting (HRM) can detect and quantify the presence of 5-methylcytosine (5mC) in DNA samples, but the ability of HRM to diagnose other DNA modifications remains unexplored. The DNA bases N6-methyladenine and 5-hydroxymethylcytosine occur across almost all phyla. While their function remains controversial, their presence perturbs DNA structure. Such modifications could affect gene regulation, chromatin condensation and DNA packaging. Here, we reveal that DNA containing N6-methyladenine or 5-hydroxymethylcytosine exhibits reduced thermal stability compared to cytosine-methylated DNA. These thermostability changes are sufficiently divergent to allow detection and quantification by HRM analysis. Thus, we report that HRM distinguishes between sequence-identical DNA differing only in the modification type of one base. This approach is also able to distinguish between two DNA fragments carrying both N6-methyladenine and 5-methylcytosine but differing only in the distance separating the modified bases. This finding provides scope for the development of new methods to characterize DNA chemically and to allow for low cost screening of mutant populations of genes involved in base modification. More fundamentally, contrast between the thermostabilizing effects of 5mC on dsDNA compared with the destabilizing effects of N6-methyladenine (m6A) and 5-hydroxymethylcytosine (5hmC) raises the intriguing possibility of an antagonistic relationship between modification types with functional significance.

  1. Development of High Resolution Melting Analysis for the Diagnosis of Human Malaria

    PubMed Central

    Chua, Kek Heng; Lim, Siew Chee; Ng, Ching Ching; Lee, Ping Chin; Lim, Yvonne Ai Lian; Lau, Tze Pheng; Chai, Hwa Chia

    2015-01-01

    Molecular detection has overcome limitations of microscopic examination by providing greater sensitivity and specificity in Plasmodium species detection. The objective of the present study was to develop a quantitative real-time polymerase chain reaction coupled with high-resolution melting (qRT-PCR-HRM) assay for rapid, accurate and simultaneous detection of all five human Plasmodium spp. A pair of primers targeted the 18S SSU rRNA gene of the Plasmodium spp. was designed for qRT-PCR-HRM assay development. Analytical sensitivity and specificity of the assay were evaluated. Samples collected from 229 malaria suspected patients recruited from Sabah, Malaysia were screened using the assay and results were compared with data obtained using PlasmoNexTM, a hexaplex PCR system. The qRT-PCR-HRM assay was able to detect and discriminate the five Plasmodium spp. with lowest detection limits of 1–100 copy numbers without nonspecific amplifications. The detection of Plasmodium spp. in clinical samples using this assay also achieved 100% concordance with that obtained using PlasmoNexTM. This indicated that the diagnostic sensitivity and specificity of this assay in Plasmodium spp. detection is comparable with those of PlasmoNexTM. The qRT-PCR-HRM assay is simple, produces results in two hours and enables high-throughput screening. Thus, it is an alternative method for rapid and accurate malaria diagnosis. PMID:26507008

  2. High-resolution melting system to perform multilocus sequence typing of Campylobacter jejuni.

    PubMed

    Lévesque, Simon; Michaud, Sophie; Arbeit, Robert D; Frost, Eric H

    2011-01-24

    Multi-locus sequence typing (MLST) has emerged as the state-of-the-art method for resolving bacterial population genetics but it is expensive and time consuming. We evaluated the potential of high resolution melting (HRM) to identify known MLST alleles of Campylobacter jejuni at reduced cost and time. Each MLST locus was amplified in two or three sub fragments, which were analyzed by HRM. The approach was investigated using 47 C. jejuni isolates, previously characterized by classical MLST, representing isolates from diverse environmental, animal and clinical sources and including the six most prevalent sequence types (ST) and the most frequent alleles. HRM was then applied to a validation set of 84 additional C. jejuni isolates from chickens; 92% of the alleles were resolved in 35 hours of laboratory time and the cost of reagents per isolate was $20 compared with $100 for sequence-based typing. HRM has the potential to complement sequence-based methods for resolving SNPs and to facilitate a wide range of genotyping studies.

  3. High resolution melting: improvements in the genetic diagnosis of hypertrophic cardiomyopathy in a Portuguese cohort

    PubMed Central

    2012-01-01

    Background Hypertrophic Cardiomyopathy (HCM) is a complex myocardial disorder with a recognized genetic heterogeneity. The elevated number of genes and mutations involved in HCM limits a gene-based diagnosis that should be considered of most importance for basic research and clinical medicine. Methodology In this report, we evaluated High Resolution Melting (HRM) robustness, regarding HCM genetic testing, by means of analyzing 28 HCM-associated genes, including the most frequent 4 HCM-associated sarcomere genes, as well as 24 genes with lower reported HCM-phenotype association. We analyzed 80 Portuguese individuals with clinical phenotype of HCM allowing simultaneously a better characterization of this disease in the Portuguese population. Results HRM technology allowed us to identify 60 mutated alleles in 72 HCM patients: 49 missense mutations, 3 nonsense mutations, one 1-bp deletion, one 5-bp deletion, one in frame 3-bp deletion, one insertion/deletion, 3 splice mutations, one 5'UTR mutation in MYH7, MYBPC3, TNNT2, TNNI3, CSRP3, MYH6 and MYL2 genes. Significantly 22 are novel gene mutations. Conclusions HRM was proven to be a technique with high sensitivity and a low false positive ratio allowing a rapid, innovative and low cost genotyping of HCM. In a short return, HRM as a gene scanning technique could be a cost-effective gene-based diagnosis for an accurate HCM genetic diagnosis and hopefully providing new insights into genotype/phenotype correlations. PMID:22429680

  4. High-resolution melt analysis of DNA methylation to discriminate semen in biological stains.

    PubMed

    Antunes, Joana; Silva, Deborah S B S; Balamurugan, Kuppareddi; Duncan, George; Alho, Clarice S; McCord, Bruce

    2016-02-01

    The goal of this study was to develop a method for the detection of semen in biological stains using high-resolution melt (HRM) analysis and DNA methylation. To perform this task, we used an epigenetic locus that targets a tissue-specific differentially methylated region for semen. This specific locus, ZC3H12D, contains methylated CpG sites that are hypomethylated in semen and hypermethylated in blood and saliva. Using this procedure, DNA from forensic stains can be isolated, processed using bisulfite-modified polymerase chain reaction (PCR), and detected by real-time PCR with HRM capability. The method described in this article is robust; we were able to obtain results from samples with as little as 1 ng of genomic DNA. Samples inhibited by humic acid still produced reliable results. Furthermore, the procedure is specific and will not amplify non-bisulfite-modified DNA. Because this process can be performed using real-time PCR and is quantitative, it fits nicely within the workflow of current forensic DNA laboratories. As a result, it should prove to be a useful technique for processing trace evidence samples for serological analysis.

  5. High-resolution melting (HRM) for genotyping bovine ephemeral fever virus (BEFV).

    PubMed

    Erster, Oran; Stram, Rotem; Menasherow, Shopia; Rubistein-Giuni, Marisol; Sharir, Binyamin; Kchinich, Evgeni; Stram, Yehuda

    2017-02-02

    In recent years there have been several major outbreaks of bovine ephemeral disease in the Middle East, including Israel. Such occurrences raise the need for quick identification of the viruses responsible for the outbreaks, in order to rapidly identify the entry of viruses that do not belong to the Middle-East BEFV lineage. This challenge was met by the development of a high-resolution melt (HRM) assay. The assay is based on the viral G gene sequence and generation of an algorithm that calculates and evaluates the GC content of various fragments. The algorithm was designed to scan 50- to 200-base-long segments in a sliding-window manner, compare and rank them using an Order of Technique of Preference by Similarity to Ideal Solution (TOPSIS) the technique for order preference by similarity to ideal solution technique, according to the differences in GC content of homologous fragments. Two fragments were selected, based on a match to the analysis criteria, in terms of size and GC content. These fragments were successfully used in the analysis to differentiate between different virus lineages, thus facilitating assignment of the viruses' geographical origins. Moreover, the assay could be used for differentiating infected from vaccinated animales (DIVA). The new algorithm may therefore be useful for development of improved genotyping studies for other viruses and possibly other microorganisms.

  6. Assessing contamination of microalgal astaxanthin producer Haematococcus cultures with high-resolution melting curve analysis.

    PubMed

    Dawidziuk, Adam; Popiel, Delfina; Luboinska, Magda; Grzebyk, Michal; Wisniewski, Maciej; Koczyk, Grzegorz

    2016-11-26

    Due to its superior antioxidant capabilities and higher activity than other carotenoids, astaxanthin is used widely in the nutraceutical and medicine industries. The most prolific natural producer of astaxanthin is the unicellular green microalga Haematococcus pluvialis. The correct identification of any contaminants in H. pluvialis cultures is both essential and nontrivial for several reasons. Firstly, while it is possible to distinguish the main microalgal contaminant Coelastrella sp. (in H. pluvialis cultures), in practice, it is frequently a daunting and error-prone task for personnel without extensive experience in the microscopic identification of algal species. Secondly, the undetected contaminants may decrease or stop production of astaxanthin. Lastly, the presence of other contaminants such as fungi can eventually infect and destroy the whole algae collection. In this study, high-resolution melting (HRM) analysis was developed to detect microalgal and fungal contamination. The developed diagnostic procedure allowed to distinguish pure H. pluvialis samples from cultures contaminated with low amounts (1.25 ng/ml) of microalgal DNA and fungal DNA (2.5 ng/ml). Such discrimination is not possible with the use of microscopy observations and allows fast and efficient collection testing.

  7. Nested Machine Learning Facilitates Increased Sequence Content for Large-Scale Automated High Resolution Melt Genotyping.

    PubMed

    Fraley, Stephanie I; Athamanolap, Pornpat; Masek, Billie J; Hardick, Justin; Carroll, Karen C; Hsieh, Yu-Hsiang; Rothman, Richard E; Gaydos, Charlotte A; Wang, Tza-Huei; Yang, Samuel

    2016-01-18

    High Resolution Melt (HRM) is a versatile and rapid post-PCR DNA analysis technique primarily used to differentiate sequence variants among only a few short amplicons. We recently developed a one-vs-one support vector machine algorithm (OVO SVM) that enables the use of HRM for identifying numerous short amplicon sequences automatically and reliably. Herein, we set out to maximize the discriminating power of HRM + SVM for a single genetic locus by testing longer amplicons harboring significantly more sequence information. Using universal primers that amplify the hypervariable bacterial 16 S rRNA gene as a model system, we found that long amplicons yield more complex HRM curve shapes. We developed a novel nested OVO SVM approach to take advantage of this feature and achieved 100% accuracy in the identification of 37 clinically relevant bacteria in Leave-One-Out-Cross-Validation. A subset of organisms were independently tested. Those from pure culture were identified with high accuracy, while those tested directly from clinical blood bottles displayed more technical variability and reduced accuracy. Our findings demonstrate that long sequences can be accurately and automatically profiled by HRM with a novel nested SVM approach and suggest that clinical sample testing is feasible with further optimization.

  8. Pyrazinamide susceptibility testing of Mycobacterium tuberculosis by high resolution melt analysis.

    PubMed

    Pholwat, Suporn; Stroup, Suzanne; Gratz, Jean; Trangan, Varittha; Foongladda, Suporn; Kumburu, Happiness; Juma, Saumu Pazia; Kibiki, Gibson; Houpt, Eric

    2014-01-01

    Pyrazinamide (PZA) plays the important role in shortening the tuberculosis treatment period and in treating MDR-TB. Phenotypic PZA susceptibility methods are limited because they require specialized acidified media, which increases costs and complexity. In this study we developed a genotypic high resolution melt (HRM) analysis technique to detect pncA mutations associated with PZA resistant Mycobacterium tuberculosis. Seven overlapping primer pairs were designed to cover the entire pncA gene and upstream regions. Each gene segment was individually amplified by real-time PCR followed by HRM analysis. The assay was evaluated on 98 clinical M. tuberculosis isolates (41 PZA susceptible by MGIT method, 55 PZA resistant, 2 undetermined). HRM was 94% concordant to full-length sequencing results, with most discrepancies attributable to mixed populations per HRM or transversions. Sequencing and HRM yielded 82% and 84% concordance, respectively, to phenotypic PZA susceptibilities by MGIT, with most discrepancies attributable to isolates with wild-type pncA but phenotypic PZA resistance. This HRM technique is a simple and high-throughput method for screening clinical M. tuberculosis samples for PZA resistance.

  9. Nested Machine Learning Facilitates Increased Sequence Content for Large-Scale Automated High Resolution Melt Genotyping

    PubMed Central

    Fraley, Stephanie I.; Athamanolap, Pornpat; Masek, Billie J.; Hardick, Justin; Carroll, Karen C.; Hsieh, Yu-Hsiang; Rothman, Richard E.; Gaydos, Charlotte A.; Wang, Tza-Huei; Yang, Samuel

    2016-01-01

    High Resolution Melt (HRM) is a versatile and rapid post-PCR DNA analysis technique primarily used to differentiate sequence variants among only a few short amplicons. We recently developed a one-vs-one support vector machine algorithm (OVO SVM) that enables the use of HRM for identifying numerous short amplicon sequences automatically and reliably. Herein, we set out to maximize the discriminating power of HRM + SVM for a single genetic locus by testing longer amplicons harboring significantly more sequence information. Using universal primers that amplify the hypervariable bacterial 16 S rRNA gene as a model system, we found that long amplicons yield more complex HRM curve shapes. We developed a novel nested OVO SVM approach to take advantage of this feature and achieved 100% accuracy in the identification of 37 clinically relevant bacteria in Leave-One-Out-Cross-Validation. A subset of organisms were independently tested. Those from pure culture were identified with high accuracy, while those tested directly from clinical blood bottles displayed more technical variability and reduced accuracy. Our findings demonstrate that long sequences can be accurately and automatically profiled by HRM with a novel nested SVM approach and suggest that clinical sample testing is feasible with further optimization. PMID:26778280

  10. High resolution melt analysis (HRMA); a viable alternative to agarose gel electrophoresis for mouse genotyping.

    PubMed

    Thomsen, Nicole; Ali, Radiya G; Ahmed, Jehangir N; Arkell, Ruth M

    2012-01-01

    Most mouse genetics laboratories maintain mouse strains that require genotyping in order to identify the genetically modified animals. The plethora of mutagenesis strategies and publicly available mouse alleles means that any one laboratory may maintain alleles with random or targeted insertions of orthologous or unrelated sequences as well as random or targeted deletions and point mutants. Many experiments require that different strains be cross bred conferring the need to genotype progeny at more than one locus. In contrast to the range of new technologies for mouse mutagenesis, genotyping methods have remained relatively static with alleles typically discriminated by agarose gel electrophoresis of PCR products. This requires a large amount of researcher time. Additionally it is susceptible to contamination of future genotyping experiments because it requires that tubes containing PCR products be opened for analysis. Progress has been made with the genotyping of mouse point mutants because a range of new high-throughput techniques have been developed for the detection of Single Nucleotide Polymorphisms. Some of these techniques are suitable for genotyping point mutants but do not detect insertion or deletion alleles. Ideally, mouse genetics laboratories would use a single, high-throughput platform that enables closed-tube analysis to genotype the entire range of possible insertion and deletion alleles and point mutants. Here we show that High Resolution Melt Analysis meets these criteria, it is suitable for closed-tube genotyping of all allele types and current genotyping assays can be converted to this technology with little or no effort.

  11. Diagnosis of genetic predisposition for lactose intolerance by high resolution melting analysis.

    PubMed

    Delacour, Hervé; Leduc, Amandine; Louçano-Perdriat, Andréa; Plantamura, Julie; Ceppa, Franck

    2017-02-01

    Lactose, the principle sugar in milk, is a disaccharide hydrolyzed by intestinal lactase into glucose and galactose, which are absorbed directly by diffusion in the intestine. The decline of lactase expression (or hypolactasia) in intestinal microvilli after weaning is a normal phenomenon in mammals known as lactase deficiency. It is observed in nearly 75% of the world population and is an inherited autosomal recessive trait with incomplete penetrance. It is caused by SNPs in a regulatory element for lactase gene. In Indo-European, lactase deficiency is associated with rs4982235 SNP (or -13910C>T). The aim of this study is to describe a method based on high resolution melting for rapidly detecting genetic predisposition to lactose intolerance. Analytical performance of the assay was assessed by evaluating within and betwwen-run precision and by comparing the results (n = 50 patients) obtained with the HRM assay to those obtained with the gold standard (Sanger sequencing of the region of interest). In silico prediction of HRM curves was performed to evaluate the potential impact of the other SNPs described within the PCR product on the HRM analytical performances. The assay has good performance (CV <0.2% during the between-run study). A perfect agreement with the gold standard method was observed. The presence of other polymorphisms within the amplified sequence is detected, the misclassification risk is low. This assay can be used for rapidly diagnosing genetic predisposition to lactose intolerance.

  12. Limited resolution of 16S rDNA DGGE caused by melting properties and closely related DNA sequences.

    PubMed

    Kisand, Veljo; Wikner, Johan

    2003-08-01

    The phylogenetic affiliation of 91 operational taxonomic units, randomly sampled from three aquatic microcosm experiments, was investigated by two PCR based and one culture dependent method. The occurrence of multiple melting domains and poor coupling between Tm and DGGE retardation was demonstrated to cause poor resolution at the species level in PCR-DGGE analysis of microbial communities. We also showed that the problem of multiple melting domains was particularly prone for brackish water bacterioplankton in the Flavobacterium genus, providing characteristic band morphology for this genus. Banding patterns from DGGE analysis may therefore be misinterpreted in terms of the species richness in natural bacterial communities, when using commonly applied universal primers.

  13. Applications of nonequilibrium melting concept to damage-accumulation processes

    SciTech Connect

    Lam, N.Q.; Okamoto, P.R.

    1998-01-01

    The authors recent study of crystalline-to-amorphous transformation led to the successful development of a unified thermodynamic description of disorder-induced amorphization and heat-induced melting, based on a generalized version of the Lindemann melting criterion. The generalized criterion requires that the melting temperature of a defective crystal decreases with increasing static atomic disorder. Hence, any crystal can melt at temperatures below the melting point of its perfect crystalline state when driven far from equilibrium by introducing critical amounts of misfitting solute atoms and lattice imperfections, radiation damage, and/or tensile stresses. This conceptual approach to nonequilibrium melting provides new insight into long-standing materials problems such as brittle fracture, embrittlement, and environmentally-induced cracking, for example irradiation-assisted stress corrosion cracking.

  14. Rapid detection of HLA-B*51 by real-time polymerase chain reaction and high-resolution melting analysis.

    PubMed

    Imperiali, C; Alía-Ramos, P; Padró-Miquel, A

    2015-08-01

    HLA-B*51, a class I human leukocyte antigen (HLA) molecule, is the strongest known genetic risk factor for Behçet disease. However, there are only few articles reporting methods to determine the presence or absence of HLA-B51. For this reason, we designed and developed an easy, fast, and inexpensive real-time high-resolution melting (HRM) assay to detect HLA-B*51. We genotyped 61 samples by our HRM assay and by conventional polymerase chain reaction, and no discrepancies were found between results. Besides, a subgroup of 25 samples was also genotyped in a different laboratory, and another subgroup of 16 samples was obtained from the International Histocompatibility Working Group DNA Bank, and a full concordance of results was observed with those obtained by HRM. Regarding the identifying system evaluated, we obtained 100% of specificity, sensibility, and repeatability, and 0% of false positive and false negative rates. Therefore, this HRM analysis is easily applicable to the rapid detection of HLA-B*51, exhibits a high speed, and requires a very low budget.

  15. Development of a high-resolution melting-based approach for efficient differentiation among Bacillus cereus group isolates.

    PubMed

    Antolinos, Vera; Fernández, Pablo S; Ros-Chumillas, María; Periago, Paula M; Weiss, Julia

    2012-09-01

    Strains belonging to Bacillus cereus Group include six different species, among which are Bacillus thuringiensis, Bacillus weihenstephanensis, and Bacillus cereus sensu stricto, a causative agent of food poisoning. Sequence of the panC-housekeeping gene is used for B. cereus Group affiliation to seven major phylogenetic groups (I-VII) with different ecological niches and variations in thermal growth range and spore heat resistance of B. cereus Group microorganisms varies among phylogenetic groups. We assigned a selection of B. cereus sensu stricto strains related to food poisoning from the Spanish cultivar Collection (Valencia) to Group IV strains based on panC gene sequence. Thermal inactivation assays revealed variability of spore heat resistance within these Group IV strains. Adequate food sanitizing treatments therefore require fast and reliable identification of particular strains. In the present study, feasibility of genotyping via high-resolution melting (HRM) analysis was examined. HRM analysis of amplified polymorphic 16S-23 intergenic spacer region (ISR) region proved to be discriminatory for B. cereus sensu stricto strain typing, while two other polymorphic regions within the bacterial rRNA operon allowed differentiation between Bacillus species, demonstrating its applicability for discrimination on the species and strain level within B. cereus Group.

  16. Trainable high resolution melt curve machine learning classifier for large-scale reliable genotyping of sequence variants.

    PubMed

    Athamanolap, Pornpat; Parekh, Vishwa; Fraley, Stephanie I; Agarwal, Vatsal; Shin, Dong J; Jacobs, Michael A; Wang, Tza-Huei; Yang, Samuel

    2014-01-01

    High resolution melt (HRM) is gaining considerable popularity as a simple and robust method for genotyping sequence variants. However, accurate genotyping of an unknown sample for which a large number of possible variants may exist will require an automated HRM curve identification method capable of comparing unknowns against a large cohort of known sequence variants. Herein, we describe a new method for automated HRM curve classification based on machine learning methods and learned tolerance for reaction condition deviations. We tested this method in silico through multiple cross-validations using curves generated from 9 different simulated experimental conditions to classify 92 known serotypes of Streptococcus pneumoniae and demonstrated over 99% accuracy with 8 training curves per serotype. In vitro verification of the algorithm was tested using sequence variants of a cancer-related gene and demonstrated 100% accuracy with 3 training curves per sequence variant. The machine learning algorithm enabled reliable, scalable, and automated HRM genotyping analysis with broad potential clinical and epidemiological applications.

  17. Hot-melt extrusion technology and pharmaceutical application.

    PubMed

    Wilson, Matthew; Williams, Marcia A; Jones, David S; Andrews, Gavin P

    2012-06-01

    The use of hot-melt extrusion (HME) within the pharmaceutical industry is steadily increasing, due to its proven ability to efficiently manufacture novel products. The process has been utilized readily in the plastics industry for over a century and has been used to manufacture medical devices for several decades. The development of novel drugs with poor solubility and bioavailability brought the application of HME into the realm of drug-delivery systems. This has specifically been shown in the development of drug-delivery systems of both solid dosage forms and transdermal patches. HME involves the application of heat, pressure and agitation through an extrusion channel to mix materials together, and subsequently forcing them out through a die. Twin-screw extruders are most popular in solid dosage form development as it imparts both dispersive and distributive mixing. It blends materials while also imparting high shear to break-up particles and disperse them. HME extrusion has been shown to molecularly disperse poorly soluble drugs in a polymer carrier, increasing dissolution rates and bioavailability. The most common difficulty encountered in producing such dispersions is stabilization of amorphous drugs, which prevents them from recrystallization during storage. Pharmaceutical industrial suppliers, of both materials and equipment, have increased their development of equipment and chemicals for specific use with HME. Clearly, HME has been identified as an important and significant process to further enhance drug solubility and solid-dispersion production.

  18. High-throughput identification and quantification of Candida species using high resolution derivative melt analysis of panfungal amplicons.

    PubMed

    Mandviwala, Tasneem; Shinde, Rupali; Kalra, Apoorv; Sobel, Jack D; Akins, Robert A

    2010-01-01

    Fungal infections pose unique challenges to molecular diagnostics; fungal molecular diagnostics consequently lags behind bacterial and viral counterparts. Nevertheless, fungal infections are often life-threatening, and early detection and identification of species is crucial to successful intervention. A high throughput PCR-based method is needed that is independent of culture, is sensitive to the level of one fungal cell per milliliter of blood or other tissue types, and is capable of detecting species and resistance mutations. We introduce the use of high resolution melt analysis, in combination with more sensitive, inclusive, and appropriately positioned panfungal primers, to address these needs. PCR-based amplification of the variable internal transcribed regions of the rDNA genes generates an amplicon whose sequence melts with a shape that is characteristic and therefore diagnostic of the species. Simple analysis of the differences between test and reference melt curves generates a single number that calls the species. Early indications suggest that high resolution melt analysis can distinguish all eight major species of Candida of clinical significance without interference from excess human DNA. Candida species, including mixed and novel species, can be identified directly in vaginal samples. This tool can potentially detect, count, and identify fungi in hundreds of samples per day without further manipulation, costs, or delays, offering a major step forward in fungal molecular diagnostics.

  19. Molecular Identification of Broomrape Species from a Single Seed by High Resolution Melting Analysis.

    PubMed

    Rolland, Mathieu; Dupuy, Aurélie; Pelleray, Aude; Delavault, Philippe

    2016-01-01

    Broomrapes are holoparasitic plants spreading through seeds. Each plant produces hundreds of thousands of seeds which remain viable in the soils for decades. To limit their spread, drastic measures are being taken and the contamination of a commercial seed lot by a single broomrape seed can lead to its rejection. Considering that broomrapes species identification from a single seed is extremely difficult even for trained botanists and that among all the described species, only a few are really noxious for the crops, numerous seed lots are rejected because of the contamination by seeds of non-noxious broomrape species. The aim of this study was to develop and evaluate a High Resolution Melting assay identifying the eight most noxious and common broomrape species (Phelipanche aegyptiaca, Orobanche cernua, O. crenata, O. cumana, O. foetida, O. hederae, O. minor, and P. ramosa) from a single seed. Based on trnL and rbcL plastidial genes amplification, the designed assay successfully identifies O. cumana, O. cernua, O. crenata, O. minor, O. hederae, and O. foetida; P. ramosa, and P. aegyptiaca can be differentiated from other species but not from each other. Tested on 50 seed lots, obtained results perfectly matched identifications performed by sequencing. Through the analysis of common seed lots by different analysts, the reproducibility of the assay was evaluated at 90%. Despite an original sample preparation process it was not possible to extract enough DNA from some seeds (10% of the samples). The described assay fulfills its objectives and allows an accurate identification of the targeted broomrape species. It can be used to identify contaminants in commercial seed lots or for any other purpose. The assay might be extended to vegetative material.

  20. Putative hybrids between two Anisakis cryptic species: molecular genotyping using High Resolution Melting.

    PubMed

    Cavallero, S; Costa, A; Caracappa, S; Gambetta, B; D'Amelio, S

    2014-11-01

    The genus Anisakis includes nine recognized species and the complex of cryptic species Anisakis simplex s. l. is often associated with the human disease known as anisakiasis. During the last decades the use of nuclear ribosomal ITS allowed the identification and description of numerous anisakid nematodes and the discovery of recombinant genotypes or putative hybrids even in other parasitic helminths, such as those between A. simplex sensu stricto and A. pegreffii. The existence of pure hybrids of the two sibling species has been long debated due to the large recovery of larval forms from sympatric areas and the rare observation of adult hybrids. The aims of the present report were to identify anisakid nematodes collected from Stenella coeruleoalba using PCR-RFLP of ITS and to focus the interest on hybrid forms using a High Resolution Melting (HRM) and direct sequencing analyses, since the new record of putative hybrid at adult stage. The PCR-RFLP analysis enabled to identify A. simplex s.s., A. pegreffii, the heterozygous genotype of the two species and A. physeteris. The use of the genotyping approach based on HRM confirmed the profiles of the two species A. simplex s.s. and A. pegreffii, and of the hybrid individuals. The new record of adult hybrids in definitive hosts rekindles the long debate about their existence and their evolutionary meaning. Since the reproductive isolation between A. simplex s.s. and A. pegreffii is the assumption for their existence as separated species, the use of alternative molecular markers and population genetic studies on adult anisakids are recommended.

  1. Molecular Identification of Broomrape Species from a Single Seed by High Resolution Melting Analysis

    PubMed Central

    Rolland, Mathieu; Dupuy, Aurélie; Pelleray, Aude; Delavault, Philippe

    2016-01-01

    Broomrapes are holoparasitic plants spreading through seeds. Each plant produces hundreds of thousands of seeds which remain viable in the soils for decades. To limit their spread, drastic measures are being taken and the contamination of a commercial seed lot by a single broomrape seed can lead to its rejection. Considering that broomrapes species identification from a single seed is extremely difficult even for trained botanists and that among all the described species, only a few are really noxious for the crops, numerous seed lots are rejected because of the contamination by seeds of non-noxious broomrape species. The aim of this study was to develop and evaluate a High Resolution Melting assay identifying the eight most noxious and common broomrape species (Phelipanche aegyptiaca, Orobanche cernua, O. crenata, O. cumana, O. foetida, O. hederae, O. minor, and P. ramosa) from a single seed. Based on trnL and rbcL plastidial genes amplification, the designed assay successfully identifies O. cumana, O. cernua, O. crenata, O. minor, O. hederae, and O. foetida; P. ramosa, and P. aegyptiaca can be differentiated from other species but not from each other. Tested on 50 seed lots, obtained results perfectly matched identifications performed by sequencing. Through the analysis of common seed lots by different analysts, the reproducibility of the assay was evaluated at 90%. Despite an original sample preparation process it was not possible to extract enough DNA from some seeds (10% of the samples). The described assay fulfills its objectives and allows an accurate identification of the targeted broomrape species. It can be used to identify contaminants in commercial seed lots or for any other purpose. The assay might be extended to vegetative material. PMID:28018378

  2. Rapid and high throughput molecular identification of diverse mosquito species by high resolution melting analysis.

    PubMed

    Ajamma, Yvonne Ukamaka; Mararo, Enock; Omondi, David; Onchuru, Thomas; Muigai, Anne W T; Masiga, Daniel; Villinger, Jandouwe

    2016-01-01

    Mosquitoes are a diverse group of invertebrates, with members that are among the most important vectors of diseases. The correct identification of mosquitoes is paramount to the control of the diseases that they transmit. However, morphological techniques depend on the quality of the specimen and often unavailable taxonomic expertise, which may still not be able to distinguish mosquitoes among species complexes (sibling and cryptic species). High resolution melting (HRM) analyses, a closed-tube, post-polymerase chain reaction (PCR) method used to identify variations in nucleic acid sequences, has been used to differentiate species within the Anopheles gambiae and Culex pipiens complexes. We validated the use of PCR-HRM analyses to differentiate species within Anopheles and within each of six genera of culicine mosquitoes, comparing primers targeting cytochrome b ( cyt b), NADH dehydrogenase subunit 1 (ND1), intergenic spacer region (IGS) and cytochrome c oxidase subunit 1 ( COI) gene regions. HRM analyses of amplicons from all the six primer pairs successfully differentiated two or more mosquito species within one or more genera ( Aedes ( Ae. vittatus from Ae. metallicus), Culex ( Cx. tenagius from Cx. antennatus, Cx. neavei from Cx. duttoni, cryptic Cx. pipiens species), Anopheles ( An. gambiae s.s. from An. arabiensis) and Mansonia ( Ma. africana from Ma. uniformis)) based on their HRM profiles. However, PCR-HRM could not distinguish between species within Aedeomyia ( Ad. africana and Ad. furfurea), Mimomyia ( Mi. hispida and Mi. splendens) and Coquillettidia ( Cq. aurites, Cq. chrysosoma, Cq. fuscopennata, Cq. metallica, Cq. microannulatus, Cq. pseudoconopas and Cq. versicolor) genera using any of the primers. The IGS and COI barcode region primers gave the best and most definitive separation of mosquito species among anopheline and culicine mosquito genera, respectively, while the other markers may serve to confirm identifications of closely related sub

  3. High-Resolution Melting Analysis of the TPMT Gene: A Study in the Polish Population

    PubMed Central

    Borun, Pawel; Milanowska, Katarzyna; Jakubowska-Burek, Ludwika; Zakerska, Oliwia; Dobrowolska-Zachwieja, Agnieszka; Plawski, Andrzej; Froster, Ursula G.; Szalata, Marlena; Slomski, Ryszard

    2013-01-01

    The thiopurine S-methyltransferase (TPMT) gene encoding thiopurine methyltransferase is a crucial enzyme in metabolism of thiopurine drugs: azathioprine and 6-mercoptopurine, which are used in the treatment of leukemia or inflammatory bowel diseases. Genetic polymorphism of the TPMT gene correlates with activity of this enzyme, individual reaction, and dosing of thiopurines. Thirty-one variants of the TPMT gene with low enzymatic activity have been described with three major alleles: TPMT*2 (c.238G>C), *3A (c.460 G>A, c.719A>G), and *3C (c.719A>G), accounting for 80% to 95% of inherited TPMT deficiency in different populations in the world. The aim of the study was to establish a rapid and highly sensitive method of analysis for the complete coding sequence of the TPMT gene and to determine the spectrum and prevalence of the TPMT gene sequence variations in the Polish population. Recently, high-resolution melting analysis (HRMA) has become a highly sensitive, automated, and economical technique for mutation screening or genotyping. We applied HRMA for the first time to TPMT gene scanning. In total, we analyzed 548 alleles of the Polish population. We found 11 different sequence variations, where two are novel changes: c.200T>C (p.P67S, TPMT*30) and c.595G>A (p.V199I, TPMT*31). Detection of these new rare alleles TPMT*30 and *31 in the Polish population suggests the need to analyze the whole TPMT gene and maybe also the extension of routinely used tests containing three major alleles, TPMT*2, *3A, and *3C. Identification of sequence variants using HRMA is highly sensitive and less time consuming compared to standard sequencing. We conclude that HRMA can be easy integrated into genetic testing of the TPMT gene in patients treated with thiopurines. PMID:23252704

  4. High-resolution melting analysis of the TPMT gene: a study in the Polish population.

    PubMed

    Skrzypczak-Zielinska, Marzena; Borun, Pawel; Milanowska, Katarzyna; Jakubowska-Burek, Ludwika; Zakerska, Oliwia; Dobrowolska-Zachwieja, Agnieszka; Plawski, Andrzej; Froster, Ursula G; Szalata, Marlena; Slomski, Ryszard

    2013-02-01

    The thiopurine S-methyltransferase (TPMT) gene encoding thiopurine methyltransferase is a crucial enzyme in metabolism of thiopurine drugs: azathioprine and 6-mercoptopurine, which are used in the treatment of leukemia or inflammatory bowel diseases. Genetic polymorphism of the TPMT gene correlates with activity of this enzyme, individual reaction, and dosing of thiopurines. Thirty-one variants of the TPMT gene with low enzymatic activity have been described with three major alleles: TPMT*2 (c.238G>C), *3A (c.460 G>A, c.719A>G), and *3C (c.719A>G), accounting for 80% to 95% of inherited TPMT deficiency in different populations in the world. The aim of the study was to establish a rapid and highly sensitive method of analysis for the complete coding sequence of the TPMT gene and to determine the spectrum and prevalence of the TPMT gene sequence variations in the Polish population. Recently, high-resolution melting analysis (HRMA) has become a highly sensitive, automated, and economical technique for mutation screening or genotyping. We applied HRMA for the first time to TPMT gene scanning. In total, we analyzed 548 alleles of the Polish population. We found 11 different sequence variations, where two are novel changes: c.200T>C (p.P67S, TPMT*30) and c.595G>A (p.V199I, TPMT*31). Detection of these new rare alleles TPMT*30 and *31 in the Polish population suggests the need to analyze the whole TPMT gene and maybe also the extension of routinely used tests containing three major alleles, TPMT*2, *3A, and *3C. Identification of sequence variants using HRMA is highly sensitive and less time consuming compared to standard sequencing. We conclude that HRMA can be easy integrated into genetic testing of the TPMT gene in patients treated with thiopurines.

  5. Rapid and high throughput molecular identification of diverse mosquito species by high resolution melting analysis

    PubMed Central

    Ajamma, Yvonne Ukamaka; Mararo, Enock; Omondi, David; Onchuru, Thomas; Muigai, Anne W. T.; Masiga, Daniel; Villinger, Jandouwe

    2016-01-01

    Mosquitoes are a diverse group of invertebrates, with members that are among the most important vectors of diseases. The correct identification of mosquitoes is paramount to the control of the diseases that they transmit. However, morphological techniques depend on the quality of the specimen and often unavailable taxonomic expertise, which may still not be able to distinguish mosquitoes among species complexes (sibling and cryptic species). High resolution melting (HRM) analyses, a closed-tube, post-polymerase chain reaction (PCR) method used to identify variations in nucleic acid sequences, has been used to differentiate species within the Anopheles gambiae and Culex pipiens complexes. We validated the use of PCR-HRM analyses to differentiate species within Anopheles and within each of six genera of culicine mosquitoes, comparing primers targeting cytochrome b ( cyt b), NADH dehydrogenase subunit 1 (ND1), intergenic spacer region (IGS) and cytochrome c oxidase subunit 1 ( COI) gene regions. HRM analyses of amplicons from all the six primer pairs successfully differentiated two or more mosquito species within one or more genera ( Aedes ( Ae. vittatus from Ae. metallicus), Culex ( Cx. tenagius from Cx. antennatus, Cx. neavei from Cx. duttoni, cryptic Cx. pipiens species), Anopheles ( An. gambiae s.s. from An. arabiensis) and Mansonia ( Ma. africana from Ma. uniformis)) based on their HRM profiles. However, PCR-HRM could not distinguish between species within Aedeomyia ( Ad. africana and Ad. furfurea), Mimomyia ( Mi. hispida and Mi. splendens) and Coquillettidia ( Cq. aurites, Cq. chrysosoma, Cq. fuscopennata, Cq. metallica, Cq. microannulatus, Cq. pseudoconopas and Cq. versicolor) genera using any of the primers. The IGS and COI barcode region primers gave the best and most definitive separation of mosquito species among anopheline and culicine mosquito genera, respectively, while the other markers may serve to confirm identifications of closely related sub

  6. A Melting Layer Model for Passive/Active Microwave Remote Sensing Applications. Part 1; Model Formulation and Comparison with Observations

    NASA Technical Reports Server (NTRS)

    Olson, William S.; Bauer, Peter; Viltard, Nicolas F.; Johnson, Daniel E.; Tao, Wei-Kuo

    2000-01-01

    In this study, a 1-D steady-state microphysical model which describes the vertical distribution of melting precipitation particles is developed. The model is driven by the ice-phase precipitation distributions just above the freezing level at applicable gridpoints of "parent" 3-D cloud-resolving model (CRM) simulations. It extends these simulations by providing the number density and meltwater fraction of each particle in finely separated size categories through the melting layer. The depth of the modeled melting layer is primarily determined by the initial material density of the ice-phase precipitation. The radiative properties of melting precipitation at microwave frequencies are calculated based upon different methods for describing the dielectric properties of mixed phase particles. Particle absorption and scattering efficiencies at the Tropical Rainfall Measuring Mission Microwave Imager frequencies (10.65 to 85.5 GHz) are enhanced greatly for relatively small (approx. 0.1) meltwater fractions. The relatively large number of partially-melted particles just below the freezing level in stratiform regions leads to significant microwave absorption, well-exceeding the absorption by rain at the base of the melting layer. Calculated precipitation backscatter efficiencies at the Precipitation Radar frequency (13.8 GHz) increase in proportion to the particle meltwater fraction, leading to a "bright-band" of enhanced radar reflectivities in agreement with previous studies. The radiative properties of the melting layer are determined by the choice of dielectric models and the initial water contents and material densities of the "seeding" ice-phase precipitation particles. Simulated melting layer profiles based upon snow described by the Fabry-Szyrmer core-shell dielectric model and graupel described by the Maxwell-Garnett water matrix dielectric model lead to reasonable agreement with radar-derived melting layer optical depth distributions. Moreover, control profiles

  7. A Melting Layer Model for Passive/Active Microwave Remote Sensing Applications. Part 2; Simulation of TRMM Observations

    NASA Technical Reports Server (NTRS)

    Olson, William S.; Bauer, Peter; Kummerow, Christian D.; Tao, Wei-Kuo

    2000-01-01

    The one-dimensional, steady-state melting layer model developed in Part I of this study is used to calculate both the microphysical and radiative properties of melting precipitation, based upon the computed concentrations of snow and graupel just above the freezing level at applicable horizontal gridpoints of 3-dimensional cloud resolving model simulations. The modified 3-dimensional distributions of precipitation properties serve as input to radiative transfer calculations of upwelling radiances and radar extinction/reflectivities at the TRMM Microwave Imager (TMI) and Precipitation Radar (PR) frequencies, respectively. At the resolution of the cloud resolving model grids (approx. 1 km), upwelling radiances generally increase if mixed-phase precipitation is included in the model atmosphere. The magnitude of the increase depends upon the optical thickness of the cloud and precipitation, as well as the scattering characteristics of ice-phase precipitation aloft. Over the set of cloud resolving model simulations utilized in this study, maximum radiance increases of 43, 28, 18, and 10 K are simulated at 10.65, 19.35 GHz, 37.0, and 85.5 GHz, respectively. The impact of melting on TMI-measured radiances is determined not only by the physics of the melting particles but also by the horizontal extent of the melting precipitation, since the lower-frequency channels have footprints that extend over 10''s of kilometers. At TMI resolution, the maximum radiance increases are 16, 15, 12, and 9 K at the same frequencies. Simulated PR extinction and reflectivities in the melting layer can increase dramatically if mixed-phase precipitation is included, a result consistent with previous studies. Maximum increases of 0.46 (-2 dB) in extinction optical depth and 5 dBZ in reflectivity are simulated based upon the set of cloud resolving model simulations.

  8. Primate genotyping via high resolution melt analysis: rapid and reliable identification of color vision status in wild lemurs.

    PubMed

    Jacobs, Rachel L; Spriggs, Amanda N; MacFie, Tammie S; Baden, Andrea L; Irwin, Mitchell T; Wright, Patricia C; Louis, Edward E; Lawler, Richard R; Mundy, Nicholas I; Bradley, Brenda J

    2016-10-01

    Analyses of genetic polymorphisms can aid our understanding of intra- and interspecific variation in primate sociality, ecology, and behavior. Studies of primate opsin genes are prime examples of this, as single nucleotide variants (SNVs) in the X-linked opsin gene underlie variation in color vision. For primate species with polymorphic trichromacy, genotyping opsin SNVs can generally indicate whether individual primates are red-green color-blind (denoted homozygous M or homozygous L) or have full trichromatic color vision (heterozygous ML). Given the potential influence of color vision on behavior and fitness, characterizing the color vision status of study subjects is becoming commonplace for many primate field projects. Such studies traditionally involve a multi-step sequencing-based method that can be costly and time-consuming. Here we present a new reliable, rapid, and relatively inexpensive method for characterizing color vision in primate populations using high resolution melt analysis (HRMA). Using lemurs as a case study, we characterized variation at exons 3 and/or 5 of the X-linked opsin gene for 87 individuals representing nine species. We scored opsin genotypes and color vision status using both traditional sequencing-based methods as well as our novel melting-curve based HRMA protocol. For each species, the melting curves of varying genotypes (homozygous M, homozygous L, heterozygous ML) differed in melting temperature and/or shape. Melting curves for each sample were consistent across replicates, and genotype-specific melting curves were consistent across DNA sources (blood vs. feces). We show that opsin genotypes can be quickly and reliably scored using HRMA once lab-specific reference curves have been developed based on known genotypes. Although the protocol presented here focuses on genotyping lemur opsin loci, we also consider the larger potential for applying this approach to various types of genetic studies of primate populations.

  9. High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting Curve Analysis

    PubMed Central

    Thomas, Holly R.; Percival, Stefanie M.; Yoder, Bradley K.; Parant, John M.

    2014-01-01

    With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high

  10. Refining lunar impact chronology through high spatial resolution (40)Ar/(39)Ar dating of impact melts.

    PubMed

    Mercer, Cameron M; Young, Kelsey E; Weirich, John R; Hodges, Kip V; Jolliff, Bradley L; Wartho, Jo-Anne; van Soest, Matthijs C

    2015-02-01

    Quantitative constraints on the ages of melt-forming impact events on the Moon are based primarily on isotope geochronology of returned samples. However, interpreting the results of such studies can often be difficult because the provenance region of any sample returned from the lunar surface may have experienced multiple impact events over the course of billions of years of bombardment. We illustrate this problem with new laser microprobe (40)Ar/(39)Ar data for two Apollo 17 impact melt breccias. Whereas one sample yields a straightforward result, indicating a single melt-forming event at ca. 3.83 Ga, data from the other sample document multiple impact melt-forming events between ca. 3.81 Ga and at least as young as ca. 3.27 Ga. Notably, published zircon U/Pb data indicate the existence of even older melt products in the same sample. The revelation of multiple impact events through (40)Ar/(39)Ar geochronology is likely not to have been possible using standard incremental heating methods alone, demonstrating the complementarity of the laser microprobe technique. Evidence for 3.83 Ga to 3.81 Ga melt components in these samples reinforces emerging interpretations that Apollo 17 impact breccia samples include a significant component of ejecta from the Imbrium basin impact. Collectively, our results underscore the need to quantitatively resolve the ages of different melt generations from multiple samples to improve our current understanding of the lunar impact record, and to establish the absolute ages of important impact structures encountered during future exploration missions in the inner Solar System.

  11. Rapid identification of biothreat and other clinically relevant bacterial species by use of universal PCR coupled with high-resolution melting analysis.

    PubMed

    Yang, Samuel; Ramachandran, Padmini; Rothman, Richard; Hsieh, Yu-Hsiang; Hardick, Andrew; Won, Helen; Kecojevic, Aleksandar; Jackman, Joany; Gaydos, Charlotte

    2009-07-01

    A rapid assay for eubacterial species identification is described using high-resolution melt analysis to characterize PCR products. Unique melt profiles generated from multiple hypervariable regions of the 16S rRNA gene for 100 clinically relevant bacterial pathogens, including category A and B biothreat agents and their surrogates, allowed highly specific species identification.

  12. The development and validation of a rapid genetic method for species identification and genotyping of medically important fungal pathogens using high-resolution melting curve analysis.

    PubMed

    Alnuaimi, A D; Wiesenfeld, D; O'Brien-Simpson, N M; Reynolds, E C; Peng, B; McCullough, M J

    2014-06-01

    Accurate, rapid and economical fungal species identification has been a major aim in mycology. In this study, our goal was to examine the feasibility of a high-resolution melting curve analysis (HRMA) of internal transcribed regions ITS1 and ITS2 in ribosomal DNA (rDNA) for a rapid, simple and inexpensive differentiation of eight clinically relevant Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis, Candida guilliermondii, Candida dubliniensis and Candida lusitaniae). In addition, for the first time, we tested the applicability of HRMA to classify C. albicans strains into four previously described genotypes (A, B, C and D) using a primer set that spans the transposable intron region of 25S of rDNA. Type and unknown clinical oral isolates were used in this study and the melting curve analysis was compared with both amplicons' sequencing and agarose gel electrophoresis analysis. Real-time PCR and subsequent HRMA of the two described rDNA regions generated distinct melting curve profiles that were in accord with sequencing and gel electrophoresis analysis, highly reproducible, and characteristic of each of the eight Candida species and C. albicans genotypes. Moreover, results were obtained in 4 h and without the need for any post-amplification handling, so reducing time and cost. Owing to its simplicity and speed, this technique is a good fit for genotypic analysis of hundreds of clinical strains in large epidemiological settings.

  13. Application of direct laser melting to restore damaged steel dies

    NASA Astrophysics Data System (ADS)

    Jang, Jeong Hwan; Joo, Byeong Don; Mun, Sung Min; Sung, Min Young; Moon, Young Hoon

    2011-02-01

    Direct laser melting (DLM) technology can be applied to restore damaged steel dies. To understand the effects of DLM process parameters such as the laser power and scan rate, a series of experiments was conducted to determine the optimal operating parameters. To investigate the laser melting characteristics, the depth/height ratio, depth/width ratio and micro-hardness as a function of the laser energy density were analyzed. Fe-Cr and Fe-Ni layers were deposited on a steel die with 11.38 J/mm2 of energy input. The wear-resistance and the friction coefficient of the deposited layer were investigated by a pin-on-disk test. The penetration depth decreased as the scan rate increased as a consequence of the shorter interaction time. The depth/height ratio of the deposited layer decreased with an increase in the scan rate. The depth/width ratio increased as laser power increased and the scan rate decreased. The deposition shape of the Fe-Ni powder was relatively shallow and wide compared with that of the Fe-Cr powder. The scan rate had a substantial effect upon the deposition height, with the Fe-Cr powder melting more than the Fe-Ni powder. The micro-hardness of the layer melted from the powders is higher than that of the substrate, and the hardness of the laser-surface-melted layer without any metal powder is higher compared to that of the metal-powder-melted layer. The direct laser melting process with Fe-Ni powder represents a superior method when restoring a steel die when the bead shape and hardness of the restored surface are important outcome considerations.

  14. Dynamic time warping assessment of high-resolution melt curves provides a robust metric for fungal identification

    PubMed Central

    Phatak, Sayali S.; Li, Dongmei; Luka, Janos; Calderone, Richard A.

    2017-01-01

    Fungal infections are a global problem imposing considerable disease burden. One of the unmet needs in addressing these infections is rapid, sensitive diagnostics. A promising molecular diagnostic approach is high-resolution melt analysis (HRM). However, there has been little effort in leveraging HRM data for automated, objective identification of fungal species. The purpose of these studies was to assess the utility of distance methods developed for comparison of time series data to classify HRM curves as a means of fungal species identification. Dynamic time warping (DTW), first introduced in the context of speech recognition to identify temporal distortion of similar sounds, is an elastic distance measure that has been successfully applied to a wide range of time series data. Comparison of HRM curves of the rDNA internal transcribed spacer (ITS) region from 51 strains of 18 fungal species using DTW distances allowed accurate classification and clustering of all 51 strains. The utility of DTW distances for species identification was demonstrated by matching HRM curves from 243 previously identified clinical isolates against a database of curves from standard reference strains. The results revealed a number of prior misclassifications, discriminated species that are not resolved by routine phenotypic tests, and accurately identified all 243 test strains. In addition to DTW, several other distance functions, Edit Distance on Real sequence (EDR) and Shape-based Distance (SBD), showed promise. It is concluded that DTW-based distances provide a useful metric for the automated identification of fungi based on HRM curves of the ITS region and that this provides the foundation for a robust and automatable method applicable to the clinical setting. PMID:28264030

  15. Dynamic time warping assessment of high-resolution melt curves provides a robust metric for fungal identification.

    PubMed

    Lu, Sha; Mirchevska, Gordana; Phatak, Sayali S; Li, Dongmei; Luka, Janos; Calderone, Richard A; Fonzi, William A

    2017-01-01

    Fungal infections are a global problem imposing considerable disease burden. One of the unmet needs in addressing these infections is rapid, sensitive diagnostics. A promising molecular diagnostic approach is high-resolution melt analysis (HRM). However, there has been little effort in leveraging HRM data for automated, objective identification of fungal species. The purpose of these studies was to assess the utility of distance methods developed for comparison of time series data to classify HRM curves as a means of fungal species identification. Dynamic time warping (DTW), first introduced in the context of speech recognition to identify temporal distortion of similar sounds, is an elastic distance measure that has been successfully applied to a wide range of time series data. Comparison of HRM curves of the rDNA internal transcribed spacer (ITS) region from 51 strains of 18 fungal species using DTW distances allowed accurate classification and clustering of all 51 strains. The utility of DTW distances for species identification was demonstrated by matching HRM curves from 243 previously identified clinical isolates against a database of curves from standard reference strains. The results revealed a number of prior misclassifications, discriminated species that are not resolved by routine phenotypic tests, and accurately identified all 243 test strains. In addition to DTW, several other distance functions, Edit Distance on Real sequence (EDR) and Shape-based Distance (SBD), showed promise. It is concluded that DTW-based distances provide a useful metric for the automated identification of fungi based on HRM curves of the ITS region and that this provides the foundation for a robust and automatable method applicable to the clinical setting.

  16. Strain-level characterization of nonstarter lactic acid bacteria in Norvegia cheese by high-resolution melt analysis.

    PubMed

    Porcellato, D; Østlie, H M; Liland, K H; Rudi, K; Isaksson, T; Skeie, S B

    2012-09-01

    The nonstarter lactic acid bacteria (NSLAB) constitute an important microbial group found during cheese ripening and they are thought to be fundamental to the quality of cheese. Rapid and accurate diagnostic tests for NSLAB are important for cheese quality control and in understanding the cheese ripening process. Here, we present a novel rapid approach for strain-level characterization through combined 16S rRNA gene and repetitive sequence-based high-resolution melt analysis (HRM). The approach was demonstrated through the characterization of 94 isolates from Norvegia, a Gouda-type cheese. The HRM profiles of the V1 and V3 variable regions of the 16S rRNA gene of the isolates were compared with the HRM profiles of 13 reference strains. The HRM profile comparison of the V1 and V3 regions of the 16S rRNA gene allowed discrimination of isolates and reference strains. Among the cheese isolates, Lactobacillus casei/paracasei (62 isolates) and Lactobacillus plantarum/Lactobacillus pentosus (27 isolates) were the dominant species, whereas Lactobacillus curvatus/Lactobacillus sakei were found occasionally (5 isolates). The HRM profiling of repetitive sequence-based PCR using the (GTG)(5) primer was developed for strain-level characterization. The clustering analysis of the HRM profiles showed high discriminatory power, similar to that of cluster analysis based on the gel method. In conclusion, the HRM approach in this study may be applied as a fast, accurate, and reproducible method for characterization of the NSLAB microflora in cheese and may be applicable to other microbial environments following selective plate culturing.

  17. Refining lunar impact chronology through high spatial resolution 40Ar/39Ar dating of impact melts

    PubMed Central

    Mercer, Cameron M.; Young, Kelsey E.; Weirich, John R.; Hodges, Kip V.; Jolliff, Bradley L.; Wartho, Jo-Anne; van Soest, Matthijs C.

    2015-01-01

    Quantitative constraints on the ages of melt-forming impact events on the Moon are based primarily on isotope geochronology of returned samples. However, interpreting the results of such studies can often be difficult because the provenance region of any sample returned from the lunar surface may have experienced multiple impact events over the course of billions of years of bombardment. We illustrate this problem with new laser microprobe 40Ar/39Ar data for two Apollo 17 impact melt breccias. Whereas one sample yields a straightforward result, indicating a single melt-forming event at ca. 3.83 Ga, data from the other sample document multiple impact melt–forming events between ca. 3.81 Ga and at least as young as ca. 3.27 Ga. Notably, published zircon U/Pb data indicate the existence of even older melt products in the same sample. The revelation of multiple impact events through 40Ar/39Ar geochronology is likely not to have been possible using standard incremental heating methods alone, demonstrating the complementarity of the laser microprobe technique. Evidence for 3.83 Ga to 3.81 Ga melt components in these samples reinforces emerging interpretations that Apollo 17 impact breccia samples include a significant component of ejecta from the Imbrium basin impact. Collectively, our results underscore the need to quantitatively resolve the ages of different melt generations from multiple samples to improve our current understanding of the lunar impact record, and to establish the absolute ages of important impact structures encountered during future exploration missions in the inner Solar System. PMID:26601128

  18. Identification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by righ-resolution melting analysis

    PubMed Central

    2017-01-01

    The objective of this study was to standardize the high-resolution melting method for identification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by amplification of 18S ribosomal DNA (rDNA) using a single primer pair. The analyses were performed on individual reactions (containing DNA from a single species of a protozoan), on duplex reactions (containing DNA from two species of protozoa in each reaction), and on a multiplex reaction (containing DNA of four parasites in a single reaction). The proposed method allowed us to identify and discriminate the four species by analyzing the derivative, normalized, and difference melting curves, with high reproducibility among and within the experiments, as demonstrated by low coefficients of variation (less than 2.2% and 2.0%, respectively). This is the first study where this method is used for discrimination of these four species of protozoa in a single reaction. PMID:28346485

  19. Detection and differentiation of classical swine fever virus strains C and Shimen by high-resolution melt analysis.

    PubMed

    Ning, Pengbo; Li, Helin; Liang, Wulong; Guo, Kangkang; Tan, Xuechao; Cao, Weiwei; Cheng, Liang; Zhang, Yanming

    2013-12-01

    Differentiation of classical swine fever virus (CSFV) strains is crucial for the development of effective vaccination programs and in epidemiological investigations. Most of current detection methods do not discriminate between wild-type CSFV strains and those used in vaccines. In this study, method involving high-resolution melt (HRM) curve analysis for the simultaneous detection and differentiation of the C and Shimen strains of CSFV was developed. A specific fragment of the NS2 gene was amplified from various CSFV strains and subjected to HRM curve analysis. Analysis of the melt curve profile for the amplicons of each strain allowed the differentiation of CSFV strains in blood samples taken from the field, or from vaccinated commercial flocks. These findings indicate that HRM curve analysis is a rapid and practical technique for discriminating CSFV isolates/strains; it can contribute to epidemiological studies of CSFV and effective control of classical swine fever.

  20. Rapid real-time PCR and high resolution melt analysis in a self-filling thermoplastic chip.

    PubMed

    Sposito, A; Hoang, V; DeVoe, D L

    2016-09-21

    A microfluidic platform designed for point-of-care PCR-based nucleic acid diagnostics is described. Compared to established microfluidic PCR technologies, the system is unique in its ability to achieve exceptionally rapid PCR amplification in a low cost thermoplastic format, together with high temperature accuracy enabling effective validation of reaction product by high resolution melt analysis performed in the same chamber as PCR. In addition, the system employs capillary pumping for automated loading of sample into the reaction chamber, combined with an integrated hydrophilic valve for precise self-metering of sample volumes into the device. Using the microfluidic system to target a mutation in the G6PC gene, efficient PCR from human genomic DNA template is achieved with cycle times as low as 14 s, full amplification in 8.5 min, and final melt analysis accurately identifying the desired amplicon.

  1. Molecular Differentiation of Schistosoma japonicum and Schistosoma mekongi by Real-Time PCR with High Resolution Melting Analysis

    PubMed Central

    Kongklieng, Amornmas; Kaewkong, Worasak; Intapan, Pewpan M.; Sanpool, Oranuch; Janwan, Penchom; Thanchomnang, Tongjit; Lulitanond, Viraphong; Sri-Aroon, Pusadee; Limpanont, Yanin

    2013-01-01

    Human schistosomiasis caused by Schistosoma japonicum and Schistosoma mekongi is a chronic and debilitating helminthic disease still prevalent in several countries of Asia. Due to morphological similarities of cercariae and eggs of these 2 species, microscopic differentiation is difficult. High resolution melting (HRM) real-time PCR is developed as an alternative tool for the detection and differentiation of these 2 species. A primer pair was designed for targeting the 18S ribosomal RNA gene to generate PCR products of 156 base pairs for both species. The melting points of S. japonicum and S. mekongi PCR products were 84.5±0.07℃ and 85.7±0.07℃, respectively. The method permits amplification from a single cercaria or an egg. The HRM real-time PCR is a rapid and simple tool for differentiation of S. japonicum and S. mekongi in the intermediate and final hosts. PMID:24516269

  2. Rapid discrimination between Anopheles gambiae s.s. and Anopheles arabiensis by High-Resolution Melt (HRM) analysis.

    PubMed

    Zianni, Michael R; Nikbakhtzadeh, Mahmood R; Jackson, Bryan T; Panescu, Jenny; Foster, Woodbridge A

    2013-04-01

    There is a need for more cost-effective options to more accurately discriminate among members of the Anopheles gambiae complex, particularly An. gambiae and Anopheles arabiensis. These species are morphologically indistinguishable in the adult stage, have overlapping distributions, but are behaviorally and ecologically different, yet both are efficient vectors of malaria in equatorial Africa. The method described here, High-Resolution Melt (HRM) analysis, takes advantage of minute differences in DNA melting characteristics, depending on the number of incongruent single nucleotide polymorphisms in an intragenic spacer region of the X-chromosome-based ribosomal DNA. The two species in question differ by an average of 13 single-nucleotide polymorphisms giving widely divergent melting curves. A real-time PCR system, Bio-Rad CFX96, was used in combination with a dsDNA-specific dye, EvaGreen, to detect and measure the melting properties of the amplicon generated from leg-extracted DNA of selected mosquitoes. Results with seven individuals from pure colonies of known species, as well as 10 field-captured individuals unambiguously identified by DNA sequencing, demonstrated that the method provided a high level of accuracy. The method was used to identify 86 field mosquitoes through the assignment of each to the two common clusters with a high degree of certainty. Each cluster was defined by individuals from pure colonies. HRM analysis is simpler to use than most other methods and provides comparable or more accurate discrimination between the two sibling species but requires a specialized melt-analysis instrument and software.

  3. Analysis of HIV Using a High Resolution Melting (HRM) Diversity Assay: Automation of HRM Data Analysis Enhances the Utility of the Assay for Analysis of HIV Incidence

    PubMed Central

    Cousins, Matthew M.; Swan, David; Magaret, Craig A.; Hoover, Donald R.; Eshleman, Susan H.

    2012-01-01

    Background HIV diversity may be a useful biomarker for discriminating between recent and non-recent HIV infection. The high resolution melting (HRM) diversity assay was developed to quantify HIV diversity in viral populations without sequencing. In this assay, HIV diversity is expressed as a single numeric HRM score that represents the width of a melting peak. HRM scores are highly associated with diversity measures obtained with next generation sequencing. In this report, a software package, the HRM Diversity Assay Analysis Tool (DivMelt), was developed to automate calculation of HRM scores from melting curve data. Methods DivMelt uses computational algorithms to calculate HRM scores by identifying the start (T1) and end (T2) melting temperatures for a DNA sample and subtracting them (T2–T1 = HRM score). DivMelt contains many user-supplied analysis parameters to allow analyses to be tailored to different contexts. DivMelt analysis options were optimized to discriminate between recent and non-recent HIV infection and to maximize HRM score reproducibility. HRM scores calculated using DivMelt were compared to HRM scores obtained using a manual method that is based on visual inspection of DNA melting curves. Results HRM scores generated with DivMelt agreed with manually generated HRM scores obtained from the same DNA melting data. Optimal parameters for discriminating between recent and non-recent HIV infection were identified. DivMelt provided greater discrimination between recent and non-recent HIV infection than the manual method. Conclusion DivMelt provides a rapid, accurate method of determining HRM scores from melting curve data, facilitating use of the HRM diversity assay for large-scale studies. PMID:23240016

  4. Simple calculation of ab initio melting curves: Application to aluminum

    NASA Astrophysics Data System (ADS)

    Robert, Grégory; Legrand, Philippe; Arnault, Philippe; Desbiens, Nicolas; Clérouin, Jean

    2015-03-01

    We present a simple, fast, and promising method to compute the melting curves of materials with ab initio molecular dynamics. It is based on the two-phase thermodynamic model of Lin et al [J. Chem. Phys. 119, 11792 (2003), 10.1063/1.1624057] and its improved version given by Desjarlais [Phys. Rev. E 88, 062145 (2013), 10.1103/PhysRevE.88.062145]. In this model, the velocity autocorrelation function is utilized to calculate the contribution of the nuclei motion to the entropy of the solid and liquid phases. It is then possible to find the thermodynamic conditions of equal Gibbs free energy between these phases, defining the melting curve. The first benchmark on the face-centered cubic melting curve of aluminum from 0 to 300 GPa demonstrates how to obtain an accuracy of 5%-10%, comparable to the most sophisticated methods, for a much lower computational cost.

  5. Identification of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis.

    PubMed

    Mohamed Zahidi, Jama'ayah; Bee Yong, Tay; Hashim, Rohaidah; Mohd Noor, Azura; Hamzah, Siti Hawa; Ahmad, Norazah

    2015-04-01

    Molecular approaches have been investigated to overcome difficulties in identification and differentiation of Brucella spp. using conventional phenotypic methods. In this study, high-resolution melt (HRM) analysis was used for rapid identification and differentiation of members of Brucella genus. A total of 41 Brucella spp. isolates from human brucellosis were subjected to HRM analysis using 4 sets of primers, which identified 40 isolates as Brucella melitensis and 1 as Brucella canis. The technique utilized low DNA concentration and was highly reproducible. The assay is shown to be a useful diagnostic tool, which can rapidly differentiate Brucella up to species level.

  6. Antimicrobial thermoplastic materials for biomedical applications prepared by melt processing

    NASA Astrophysics Data System (ADS)

    Botta, L.; Scaffaro, R.; Ceraulo, M.; Gallo, G.

    2014-05-01

    In this work thermoplastic polymers with antimicrobial properties were prepared by incorporating an antibiotic, i.e., ciprofloxacin (CFX), by melt processing. Two different polymers were used as matrices, i.e., polypropylene (PP) and poly(lactid acid) (PLA) and different concentrations of CFX have been incorporated. The antimicrobial properties, the release kinetic and the mechanical performances of the prepared materials were evaluated.

  7. High Resolution Melting Analysis Is a More Sensitive and Effective Alternative to Gel-Based Platforms in Analysis of SSR – An Example in Citrus

    PubMed Central

    Distefano, Gaetano; Caruso, Marco; La Malfa, Stefano; Gentile, Alessandra; Wu, Shu-Biao

    2012-01-01

    High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel-electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE) analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost-effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to analyse SSR

  8. High resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR--an example in citrus.

    PubMed

    Distefano, Gaetano; Caruso, Marco; La Malfa, Stefano; Gentile, Alessandra; Wu, Shu-Biao

    2012-01-01

    High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel-electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE) analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost-effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to analyse SSR

  9. Lower Mantle melting model and it's geodynamical applications

    NASA Astrophysics Data System (ADS)

    Fomin, I.; Tackley, P. J.

    2014-12-01

    Model of solid-liquid equilibrium laws and substances properties in lower mantle conditions is important to understand the early stages of evolution of terrestrial planets, such as core formation and magma ocean crystallization. This model is also necessary to prove theories on some modern seismic features of the Earth (e.g. ultra-low velocity zones) and petrological observations (e.g. lower mantle mineral assemblage inclusions in diamonds). Numerous experimental and numerical studies of the lower mantle phases provide sufficient amount of data to build up a thermodynamic model, which can be used in geophysical fluid dynamics research. Experimental studies are the direct source of soliduses values, but other thermodynamic parameters stay unclear. Molecular Dynamics modeling provides data on thermodynamic properties of solids and liquids (density, heat capacity, latent heat of melting etc.). But absence of minor components (iron, alkali etc.) and some numerical issues (e.g. [Belonoshko, 2001]) make it to overestimate melting temperatures significantly (up to 20-30%). Our approach is to develop a model based on MD data by [de Koker et al., 2013] with evaluation of all important parameters according to classical thermodynamic equations. But melting temperatures (especially at eutectic points) are corrected along Clausius-Clapeyron slopes to agree with modern experimental data ([Andrault et al., 2011], [Andrault et al., 2014], [Fiquet et al., 2010], [Hirose et al., 1999], [Mosenfelder et al., 2007], [Nomura et al., 2014],[Ozawa et al., 2011], [Zerr et al., 1998]). Notable effect on melt and solid densities has iron partitioning, so KD value reported by [Andrault et al., 2012] was used. Proposed model was implemented into StagYY software (e.g. [Tackley, 2008]). It is a finite-volume discretization code for advection of solid and liquid in a planetary scale. CMB temperature was set to be 4000-4400 K. Calculations predict appearing and disappearing batches containing up

  10. High-resolution melting analysis: a new molecular approach for the early detection of Diplodia pinea in Austrian pine.

    PubMed

    Luchi, Nicola; Pratesi, Nicola; Simi, Lisa; Pazzagli, Mario; Capretti, Paolo; Scala, Aniello; Slippers, Bernard; Pinzani, Pamela

    2011-08-01

    The differentiation of Diplodia pinea from closely related species, such as Diplodia scrobiculata and Diplodia seriata, and its detection in plant tissue, represented a critical issue for a long time. Molecular screening tools have recently been developed to address this topic. In this study we applied one of the most sensitive and rapid diagnostic screening method so far developed, called High-Resolution Melting Analysis (HRMA), to detect D. pinea in Austrian pine (Pinus nigra). HRMA exploits differences in the melting behaviour of PCR products to rapidly identify DNA sequence variants without the need for cumbersome post-PCR methods. We developed a HRMA method to detect specific fungal sequences in the mitochondrial small subunit ribosome gene (mt SSU rDNA). The reliability of this technique was firstly assessed on DNA extracted from pure cultures of D. pinea and closely related species. Amplicon differences were screened by HRMA and the results confirmed by direct DNA sequencing. Subsequently, HRMA was tested on DNA from symptomatic and symptomless pine shoots, and the presence of the fungus was also confirmed by both conventional and molecular quantitative approaches. The HRMA allowed the distinction of D. pinea from closely related species, showing specific melting profiles for the each pathogen. This new molecular technique, here tested in a plant-fungus pathosystem for the first time, was very reliable in both symptomatic and symptomless shoots. HRMA is therefore a highly effective and accurate technique that permits the rapid screening of pathogens in the host.

  11. Rapid Detection and Identification of Nontuberculous Mycobacterial Pathogens in Fish by Using High-Resolution Melting Analysis

    PubMed Central

    Phung, Thu Nguyet; Caruso, Domenico; Godreuil, Sylvain; Keck, Nicolas; Vallaeys, Tatiana

    2013-01-01

    Mycobacterial infections in fish are commonly referred to as piscine mycobacteriosis, irrespectively of the specific identity of the causal organism. They usually cause a chronic disease and sometimes may result in high mortalities and severe economic losses. Nearly 20 species of Mycobacterium have been reported to infect fish. Among them, Mycobacterium marinum, M. fortuitum, and M. chelonae are generally considered the major agents responsible for fish mycobacteriosis. As no quick and inexpensive diagnostic test exists, we tested the potential of high-resolution melting analysis (HRMA) to rapidly identify and differentiate several Mycobacterium species involved in fish infections. By analyzing both the melting temperature and melting profile of the 16S-23S rRNA internal transcribed spacer (ITS), we were able to discriminate 12 different species simultaneously. Sensitivity tests conducted on purified M. marinum and M. fortuitum DNA revealed a limit of detection of 10 genome equivalents per reaction. The primers used in this procedure did not lead to any amplification signal with 16 control non-Mycobacterium species, thereby demonstrating their specificity for the genus Mycobacterium. PMID:24123734

  12. Differentiation of infectious bursal disease virus strains using real-time RT-PCR and high resolution melt curve analysis.

    PubMed

    Ghorashi, Seyed A; O'Rourke, Denise; Ignjatovic, Jagoda; Noormohammadi, Amir H

    2011-01-01

    Differentiation of infectious bursal disease virus (IBDV) strains is crucial for effective vaccination programs and epidemiological investigations. In this study, a combination of real-time RT-PCR and high resolution melt (HRM) curve analysis was developed for simultaneous detection and differentiation of IBDV strains/isolates. The hypervariable region of VP2 gene was amplified from several IBDV strains and subjected to HRM curve analysis. The method could readily differentiate between classical vaccines/isolates and variants. Analysis of the nucleotide sequence of the amplicons from each strain revealed that each melt curve profile was related to a unique DNA sequence. The real-time RT-PCR HRM curve analysis was also able to differentiate IBDV strains/isolates directly in bursal tissues from field submissions and from vaccinated commercial flocks. The differences between melting peaks generated from IBDV strains were significantly different (P<0.0001) demonstrating the high discriminatory power of this technique. The results presented in this study indicated that real-time RT-PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping IBDV isolates/strains and can contribute to effective control of IBDV outbreaks.

  13. High-resolution melting analysis using unlabeled probe and amplicon scanning simultaneously detects several lactase persistence variants.

    PubMed

    Janukonyté, Jurgita; Vestergaard, Else M; Ladefoged, Søren A; Nissen, Peter H

    2010-12-01

    Lactase persistence and thereby tolerance to lactose is a common trait in people of Northern European descent. It is linked to the LCT -13910C>T variant located in intron 13 of the MCM6 gene 13.9 kb upstream of the lactase (LCT) gene. In people of African and Middle Eastern descent, lactase persistence can be associated with other variants nearby the -13910C>T variant, limiting the use of the -13910C>T-based SNP analysis, e.g. TaqMan assays for the diagnosis of lactose intolerance. Using high-resolution melting analysis, we identified five samples that were heterozygous for the -13915T>G variant among 78 patients genotyped as -13910C/C by a TaqMan assay. All samples originated from patients of probable Middle Eastern descent. In order to detect the -13910 and -13915 variants simultaneously, we developed a new high-resolution melting (HRM) analysis assay based on unlabeled probe genotyping and simultaneous amplicon scanning analysis. By using this assay we were able to distinguish the -13910 and -13915 genotypes clearly. Furthermore, we identified two rare variants, the -13907C>G and -13913T>C. With this method, based on an inexpensive unlabeled probe, it is possible to simultaneously detect the -13910C>T and -13915T>G variants in addition to rarer variants surrounding the -13910 site. This new method may contribute to improve the diagnostic performance of the genetic analysis for lactose intolerance.

  14. Rapid High-Resolution Melt Analysis of Cytauxzoon felis Cytochrome b To Aid in the Prognosis of Cytauxzoonosis.

    PubMed

    Schreeg, Megan E; Marr, Henry S; Tarigo, Jaime L; Cohn, Leah A; Levy, Michael G; Birkenheuer, Adam J

    2015-08-01

    Cytauxzoon felis is a virulent, tick-transmitted, protozoan parasite that infects felines. Cytauxzoonosis was previously thought to be uniformly fatal in domestic cats. Treatment combining atovaquone and azithromycin (A&A) has been associated with survival rates of over 60%. Atovaquone, a ubiquinone analogue, targets C. felis cytochrome b (cytb), of which 30 unique genotypes have been identified. The C. felis cytb genotype cytb1 is associated with increased survival rates in cats treated with A&A. The purpose of this study was to design a PCR panel that could distinguish C. felis cytb1 from other cytochrome b genotypes. Primer pairs were designed to span five different nucleotide positions at which single-nucleotide polymorphisms in the C. felis cytb gene had been identified. Through the use of high-resolution melt analysis, this panel was predicted to distinguish cytb1 from other cytb genotypes. Assays were validated using samples from 69 cats with cytauxzoonosis for which the C. felis cytb genotypes had been characterized previously. The PCR panel identified C. felis cytb1 with 100% sensitivity and 98.2% specificity. High-resolution melt analysis can rapidly provide prognostic information for clients considering A&A treatment in cats with cytauxzoonosis.

  15. High-resolution melt analysis for species identification of coagulase-negative staphylococci derived from bovine milk.

    PubMed

    Ajitkumar, Praseeda; Barkema, Herman W; Zadoks, Ruth N; Morck, Douglas W; van der Meer, Frank J U M; De Buck, Jeroen

    2013-03-01

    Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens isolated from bovine milk. In this study, we report a rapid assay for species identification of CNS using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time polymerase chain reaction amplification of 16S rRNA gene fragment, spanning the variable region V1 and V2, was performed with a resulting amplicon of 215 bp. A library of distinct melt curves of reference strains of 13 common CNS species was created using HRMA. Sequencing of 16S rRNA and rpoB genes, and, when needed, tuf gene, of 100 CNS isolates obtained from Canadian Bovine Mastitis Research Network was done to determine their species identity, allowing for subsequent evaluation of the performance of HRMA for field isolates of bovine CNS. A combination of HRMA and sequencing revealed that Staphylococcus chromogenes, S. xylosus, S. simulans, and S. sciuri had multiple genotypes, complicating their resolution by HRMA. As the 3 genotypes of S. chromogenes had distinct melt curves, the 3 distinct genotypes were employed as reference strains in a blinded trial of 156 CNS isolates to identify S. chromogenes. HRMA correctly identified all S. chromogenes isolates which were later confirmed by sequencing. Staphylococcus chromogenes (68%) was most frequently found among the CNS isolates, followed by S. haemolyticus (10%) and S. xylosus (6%). The present study revealed that HRMA of 16S rRNA gene (V1-V2) could be used as a rapid, efficient, low-cost, and minimally cumbersome technique for S. chromogenes identification, the most common CNS derived from bovine milk.

  16. Rapid identification of bovine mastitis pathogens by high-resolution melt analysis of 16S rDNA sequences.

    PubMed

    Ajitkumar, Praseeda; Barkema, Herman W; De Buck, Jeroen

    2012-03-23

    Accurate identification of mastitis pathogens is often compromised when using conventional culture-based methods. Here, we report a novel, rapid assay tested for speciation of bacterial mastitis pathogens using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time PCR amplification of 16S rRNA gene fragment, spanning the variable region V5 and V6 was performed with a resulting amplicon of 290bp. First, a library was generated of melt curves of 9 common pathogens that are implicated in bovine mastitis. Six of the isolates, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus uberis, Staphylococcus aureus and Mycoplasma bovis, were type strains while the other 3, Arcanobacterium pyogenes, Corynebacterium bovis and Streptococcus dysgalactiae, were bovine mastitis field isolates. Four of the type strains, E. coli, S. agalactiae, K. pneumoniae and S. aureus, were found to be of human origin, while the other 3 type strains were isolated from bovine infections. Secondly, the melt curves and corresponding amplicon sequences of A. pyogenes, E. coli, S. agalactiae, S. dysgalactiae, K. pneumoniae, S. uberis and S. aureus were compared with 10 bovine mastitis field isolates of each pathogen. Based on the distinct differences in melt curves and sequences between human and bovine isolates of E. coli and K. pneumoniae, it was deemed necessary to select a set of bovine strains for these pathogens to be used as reference strains in the HRMA. Next, the HRMA was validated by three interpreters analyzing the differential clustering pattern of melt curves of 60 bacterial cultures obtained from mastitis milk samples. The three test interpreters were blinded to the culture and sequencing results of the isolates. Overall accuracy of the validation assay was 95% as there was difficulty in identifying the streptococci due to heterogeneity observed in the PCR amplicons of S. uberis. The present study revealed that broad-range real-time PCR with

  17. Viscosity of Carbonate-Silicate Melts Using Ultra-High Resolution Falling Sphere Viscometry

    NASA Astrophysics Data System (ADS)

    Hummer, D. R.; Kavner, A.; Manning, C. E.; Park, C.; Kono, Y.; Kenney-Benson, C.

    2014-12-01

    Carbonatites are carbon-rich magmas that participate in the deep-Earth carbon cycle, and may be precursors to many types of terrestrial magmatism. [1] Viscosity is a crucial parameter in determining migration rates and behavior of melts from the upper mantle to Earth's surface, but very little is known about the viscosity of carbonate-silicate liquids at conditions relevant to Earth's interior. To examine the viscosity of carbonate-silicate liquids as a function of composition, we performed in situ falling sphere viscosity experiments using a high speed X-ray camera and Paris-Edinburgh press at the HPCAT beamline (Advanced Photon Source, Argonne National Laboratory). Mixtures from the CaCO3-CaSiO3 (calcite-wollastonite) binary system were used to simulate mantle silicate carbonatites. Samples were loaded using the experimental setup of Yamada et al [2], held at 3 GPa, and heated until the sample was fully molten (between 1350-1650 oC). The high speed camera recorded the falling rate of a platinum sphere placed near the top of the sample chamber, enabling the calculation of terminal velocity and hence viscosity. Results indicate that pure CaCO3 at upper mantle conditions has a very low viscosity of ~0.006 Pa-s, only a little higher than that of water. This viscosity is 2-6 times lower than that of potassium carbonates at similar pressures (2.5-4.0 GPa) but at somewhat lower temperatures (800-1200 oC). [3] Our measured viscosity as a function of increasing silicate content increases along a log-linear trend, reaching 0.256 Pa-s for CaSiO3 liquid. This heavy dependence of viscosity on composition has implications for melt migration processes at different depths, suggesting either viscosity-driven or porosity-driven migration depending on both extent of melting and carbonate content. [1] Dasgupta, R. et al. (2013) Nature 493, 211-215. [2] Yamada, A. et al. (2011) Rev. Sci. Instr. 82, 015103. [3] Dobson, D. et al. (1996) Earth Plan. Sci. Lett. 143, 207-215.

  18. Development of a quantitation approach for total human and male DNA based on real time PCR followed by high resolution melting analysis.

    PubMed

    Ginart, Santiago; Caputo, Mariela; Alechine, Evguenia; Corach, Daniel; Sala, Andrea

    2016-10-01

    We developed and validated a total human DNA quantitation technique that simultaneously allows male DNA detection. This assay, called Amel-Y, is a duplex Real Time PCR followed by HRM (high resolution melting) analysis using the intercalating dye SYTO9. Amel-Y duplex produces two amplicons, one for the amelogenin gene (106/112 bp, female/male) and another (84 bp) corresponding to human Y chromosome-specific fragment to detect male DNA. After HRM analysis, two melting peaks differing in 5.3°C-5.5°C are detected if both male and female DNA are present and only one if only female DNA is present. For specificity assessment, the inclusion of high concentrations of bacterial and fungal DNA in the quantitation reactions allowed discarding species cross-reactivity. A set of crime scene evidence from forensic casework has been quantified with commercial kits and compared with Amel-Y duplex. Our method detected male DNA from a concentration of 18 pg/μL and supports autosomal/Y DNA detection ratio up to 200:1. A limitation of the technique is its inability to quantify male and female donnors in a mixed sample. The Amel-Y duplex demonstrated to be an efficient system for quantifying total human DNA being a specific, rapid, sensitive, and cost-effective method suitable for mixed DNA samples and applicable to any field where human DNA quantification is required, such as molecular diagnosis, population genetics, and forensic identification.

  19. Melt spreading code assessment, modifications, and application to the EPR core catcher design.

    SciTech Connect

    Farmer, M. T .; Nuclear Engineering Division

    2009-03-30

    The Evolutionary Power Reactor (EPR) is under consideration by various utilities in the United States to provide base load electrical production, and as a result the design is undergoing a certification review by the U.S. Nuclear Regulatory Commission (NRC). The severe accident design philosophy for this reactor is based upon the fact that the projected power rating results in a narrow margin for in-vessel melt retention by external cooling of the reactor vessel. As a result, the design addresses ex-vessel core melt stabilization using a mitigation strategy that includes: (1) an external core melt retention system to temporarily hold core melt released from the vessel; (2) a layer of 'sacrificial' material that is admixed with the melt while in the core melt retention system; (3) a melt plug in the lower part of the retention system that, when failed, provides a pathway for the mixture to spread to a large core spreading chamber; and finally, (4) cooling and stabilization of the spread melt by controlled top and bottom flooding. The overall concept is illustrated in Figure 1.1. The melt spreading process relies heavily on inertial flow of a low-viscosity admixed melt to a segmented spreading chamber, and assumes that the melt mass will be distributed to a uniform height in the chamber. The spreading phenomenon thus needs to be modeled properly in order to adequately assess the EPR design. The MELTSPREAD code, developed at Argonne National Laboratory, can model segmented, and both uniform and nonuniform spreading. The NRC is thus utilizing MELTSPREAD to evaluate melt spreading in the EPR design. MELTSPREAD was originally developed to support resolution of the Mark I containment shell vulnerability issue. Following closure of this issue, development of MELTSPREAD ceased in the early 1990's, at which time the melt spreading database upon which the code had been validated was rather limited. In particular, the database that was utilized for initial validation consisted

  20. High resolution melting curve analysis as a new tool for rapid identification of canine parvovirus type 2 strains.

    PubMed

    Bingga, Gali; Liu, Zhicheng; Zhang, Jianfeng; Zhu, Yujun; Lin, Lifeng; Ding, Shuangyang; Guo, Pengju

    2014-01-01

    A high resolution melting (HRM) curve method was developed to identify canine parvovirus type 2 (CPV-2) strains by nested PCR. Two sets of primers, CPV-426F/426R and CPV-87R/87F, were designed that amplified a 52 bp and 53 bp product from the viral VP2 capsid gene. The region amplified by CPV-426F/426R included the A4062G and T4064A mutations in CPV-2a, CPV-2b and CPV-2c. The region amplified by CPV-87F/87R included the A3045T mutation in the vaccine strains of CPV-2 and CPV-2a, CPV-2b and CPV-2c. Faecal samples were obtained from 30 dogs that were CPV antigen-positive. The DNA was isolated from the faecal samples and PCR-amplified using the two sets of primers, and genotyped by HRM curve analysis. The PCR-HRM assay was able to distinguish single nucleotide polymorphisms between CPV-2a, CPV-2b and CPV-2c using CPV-426F/426R. CPV-2a was distinguished from CPV-2b and CPV-2c by differences in the melting temperature. CPV-2b and CPV-2c could be distinguished based on the shape of the melting curve after generating heteroduplexes using a CPV-2b reference sample. The vaccine strains of CPV-2 were identified using CPV-87F/87R. Conventional methods for genotyping CPV strains are labor intensive, expensive or time consuming; the present PCR-based HRM assay might be an attractive alternative.

  1. Impact of Mutation Type and Amplicon Characteristics on Genetic Diversity Measures Generated Using a High-Resolution Melting Diversity Assay

    PubMed Central

    Cousins, Matthew M.; Donnell, Deborah; Eshleman, Susan H.

    2013-01-01

    We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion < 1-bp change < 3-bp insertion < 9-bp insertion). The impact of mutations on HRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impact at 50%. The G/C content altered melting temperature values of amplicons but had no impact on HRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology. PMID:23178437

  2. High Resolution Melting Analysis for Rapid Mutation Screening in Gyrase and Topoisomerase IV Genes in Quinolone-Resistant Salmonella enterica

    PubMed Central

    Thong, Kwai Lin

    2014-01-01

    The increased Salmonella resistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian region. The objective of this study is to develop a high resolution melt curve (HRM) assay to rapidly screen for mutations in quinolone-resistant determining region (QRDR) of gyrase and topoisomerase IV genes. DNA sequencing was performed on 62 Salmonella strains to identify mutations in the QRDR of gyrA, gyrB, parC, and parE genes. Mutations were detected in QRDR of gyrA (n = 52; S83F, S83Y, S83I, D87G, D87Y, and D87N) and parE (n = 1; M438I). Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16 > 256 μg/mL). Mutations were uncommon within the QRDR of gyrB, parC, and parE genes. In the HRM assay, mutants can be distinguished from the wild-type strains based on the transition of melt curves, which is more prominent when the profiles are displayed in difference plot. In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes in Salmonella. This assay markedly reduced the sequencing effort involved in mutational studies of quinolone-resistance genes. PMID:25371903

  3. A high-resolution melting (HRM) assay for the differentiation between Israeli field and Neethling vaccine lumpy skin disease viruses.

    PubMed

    Menasherow, Sophia; Erster, Oran; Rubinstein-Giuni, Marisol; Kovtunenko, Anita; Eyngor, Evgeny; Gelman, Boris; Khinich, Evgeny; Stram, Yehuda

    2016-06-01

    Lumpy skin disease (LSD) is a constant threat to the Middle East including the State of Israel. During vaccination programs it is essential for veterinary services and farmers to be able to distinguish between animals affected by the cattle-borne virulent viruses and vaccinated animals, subsequently affected by the vaccine strain. This study describes an improved high resolution-melting (HRM) test that exploits a 27 base pair (bp) fragment of the LSDV126 extracellular enveloped virion (EEV) gene that is present in field viruses but is absent from the Neethling vaccine strain. This difference leads to ∼0.5 °C melting point change in the HRM assay, when testing the quantitative PCR (qPCR) products generated from the virulent field viruses compared to the attenuated vaccine. By exploiting this difference, it could be shown using the newly developed HRM assay that virus isolated from vaccinated cattle that developed disease symptoms behave similarly to vaccine virus control, indicating that the vaccine virus can induce disease symptoms. This assay is not only in full agreement with the previously published PCR gradient and restriction fragment length polymorphism (RFLP) tests but it is faster with, fewer steps, cheaper and dependable.

  4. Molecular differentiation of Opisthorchis viverrini and Clonorchis sinensis eggs by multiplex real-time PCR with high resolution melting analysis.

    PubMed

    Kaewkong, Worasak; Intapan, Pewpan M; Sanpool, Oranuch; Janwan, Penchom; Thanchomnang, Tongjit; Laummaunwai, Porntip; Lulitanond, Viraphong; Doanh, Pham Ngoc; Maleewong, Wanchai

    2013-12-01

    Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4±0.09℃ and 85.9±0.08℃ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.

  5. Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis.

    PubMed

    Banowary, Banya; Dang, Van Tuan; Sarker, Subir; Connolly, Joanne H; Chenu, Jeremy; Groves, Peter; Ayton, Michelle; Raidal, Shane; Devi, Aruna; Vanniasinkam, Thiru; Ghorashi, Seyed A

    2015-01-01

    Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates.

  6. Differential diagnosis of Goatpox virus in Taiwan by multiplex polymerase chain reaction assay and high-resolution melt analysis.

    PubMed

    Chan, Kun-Wei; Lee, Ming-Liang; Yang, Wei-Cheng; Wong, Min-Liang; Hsu, Wei-Li; Ho, Chia-Fang; Hsieh, Yao-Ching; Wang, Chi-Young

    2014-03-01

    The A32L gene from a Goatpox virus (GTPV) strain isolated from a goat in Yunlin County (Taiwan) displays several substitutions compared with the sequence of the Kenyan GTPV vaccine strain SGP0240 and the Pellor GTPV strain. Samples from the skin lesions on 6 goats with GTPV infection or from goats with Orf virus (ORFV) infection were tested in a multiplex polymerase chain reaction (PCR) system that used primers GPF, GPR1, and GPR2 as well as previously published primers specific for ORFV. These primers were able to amplify either GTPV or ORFV without cross-reactivity. A high-resolution melt analysis (HRMA) was carried out on amplified DNA from the skin lesions of 6 goats with GTPV infection and with the GTPV SGP0240 strain. The results indicated that the melting temperature profiles amplified from samples with Yunlin GTPV infection can be differentiated from the GTPV SGP0240 strain. The findings showed that a successful differential assay for these GTPVs had been developed. Accordingly, both methods can be used to detect and differentiate GTPV isolated from animals that may have either been vaccinated or been infected with a wild strain. The multiplex PCR and HRMA could be used on skin samples of suspected cases to serve as the front-line and confirmative assays, respectively, which will be beneficial to the eradication of GTPV.

  7. Rapid molecular identification of Listeria species by use of real-time PCR and high-resolution melting analysis.

    PubMed

    Jin, Dazhi; Luo, Yun; Zhang, Zheng; Fang, Weijia; Ye, Julian; Wu, Fang; Ding, Gangqiang

    2012-05-01

    Identification of Listeria species via a molecular method is critical for food safety and clinical diagnosis. In this study, an assay integrating real-time quantitative PCR (Q-PCR) with high-resolution melting (HRM) curve analysis was developed and assessed for rapid identification of six Listeria species. The ssrA gene, which encodes a transfer-messenger RNA (tmRNA) is conserved and common to all bacterial phyla, contains a variable domain in Listeria spp. Therefore, Q-PCR and a HRM profile were applied to characterize this gene. Fifty-three Listeria species and 45 non-Listeria species were detected using one primer set, with an accuracy of 100% in reference to conventional methods. There was a 93.3% correction rate to 30 artificially contaminated samples. Thus, Q-PCR with melting profiling analysis proved able to identify Listeria species accurately. Consequently, this study demonstrates that the assay we developed is a functional tool for rapidly identifying six Listeria species, and has the potential for discriminating novel species food safety and epidemiological research.

  8. Melt-infiltrated Sic Composites for Gas Turbine Engine Applications

    NASA Technical Reports Server (NTRS)

    Morscher, Gregory N.; Pujar, Vijay V.

    2004-01-01

    SiC-SiC ceramic matrix composites (CMCs) manufactured by the slurry -cast melt-infiltration (MI) process are leading candidates for many hot-section turbine engine components. A collaborative program between Goodrich Corporation and NASA-Glenn Research Center is aimed at determining and optimizing woven SiC/SiC CMC performance and reliability. A variety of composites with different fiber types, interphases and matrix compositions have been fabricated and evaluated. Particular focus of this program is on the development of interphase systems that will result in improved intermediate temperature stressed-oxidation properties of this composite system. The effect of the different composite variations on composite properties is discussed and, where appropriate, comparisons made to properties that have been generated under NASA's Ultra Efficient Engine Technology (UEET) Program.

  9. A method to distinguish morphologically similar Peromyscus species using extracellular RNA and high-resolution melt analysis.

    PubMed

    Seifert, Veronica A; Clarke, Benjamin L; Crossland, Janet P; Bemis, Lynne T

    2016-09-01

    A method applying high-resolution melt (HRM) analysis to PCR products copied and amplified from extracellular RNA (exRNA) has been developed to distinguish two morphologically similar Peromyscus species: Peromyscus leucopus and Peromyscus maniculatus. P. leucopus is considered the primary reservoir host of Borrelia burgdorferi, the causative agent for Lyme disease in North America. In northern Minnesota the habitat ranges of P. leucopus overlaps with that of P. maniculatus. Serum samples from live mice of both species were collected from cheek bleeds, total extracellular RNA (exRNA) was extracted, copied using reverse transcription and amplified by PCR followed by HRM analysis. A circulating ribosomal RNA (rRNA) was identified which differed at seven nucleotides between the two species and a method of HRM analysis was developed allowing rapid species confirmation. In the future, this HRM based method may be adapted for additional species.

  10. Simultaneous Identification of Four "Legal High" Plant Species in a Multiplex PCR High-Resolution Melt Assay().

    PubMed

    Elkins, Kelly M; Perez, Anjelica C U; Quinn, Alicia A

    2016-12-13

    The international prevalence of "legal high" drugs necessitates the development of a method for their detection and identification. Herein, we describe the development and validation of a tetraplex multiplex real-time polymerase chain reaction (PCR) assay used to simultaneously identify morning glory, jimson weed, Hawaiian woodrose, and marijuana detected by high-resolution melt using LCGreen Plus(®) . The PCR assay was evaluated based on the following: (i) specificity and selectivity-primers were tested on DNA extracted from 30 species and simulated forensic samples, (ii) sensitivity-serial dilutions of the target DNA were prepared, and (iii) reproducibility and reliability-sample replicates were tested and remelted on different days. The assay is ideal for cases in which inexpensive assays are needed to quickly detect and identify trace biological material present on drug paraphernalia that is too compromised for botanical microscopic identification and for which analysts are unfamiliar with the morphology of the emerging "legal high" species.

  11. Implementation of a cost-effective unlabeled probe high-resolution melt assay for genotyping of Factor V Leiden.

    PubMed

    Svensson, Annika M; Chou, Lan-Szu; Meadows, Cindy; Miller, Christine E; Palais, Robert; Sumner, Kelli; Wayman, Tyler C; Mao, Rong; Lyon, Elaine

    2011-04-01

    The Factor V Leiden mutation (FVL; c.1601G>A, p.Arg534Gln), the most common aberration underlying activated Protein C resistance, results in disruption of a major anticoagulation pathway and is a leading cause of inherited thrombophilia. A high-throughput assay for FVL mutation detection was developed using a single unlabeled probe on a high-resolution platform, the 96-well Roche 480 LightCycler (LC480) instrument. This method replaced the U.S. Food and Drug Administration-approved Roche Factor V Leiden kit assay on the LightCycler PCR instrument, decreasing total cost by 48%. The analytical sensitivity and specificity of the LC480 high-resolution assay approached 100% for the FVL mutation. Factor V mutations in proximity to the FVL locus may influence probe binding efficiency and melt characteristics. One out of three very rare variants tested in a separate study, 1600delC, was not distinguishable from FVL using the described high-resolution assay. However, a c.1598G>A variant, which changes the amino acid sequence from arginine to lysine at position 533, was detected by this high-resolution assay and confirmed by bidirectional sequencing. In the labeled probe LightCycler assay, the c.1598G>A variant was indistinguishable from the heterozygous FVL control. The c.1598G>A variant has not been described previously and its clinical significance is uncertain. In conclusion, the LC480 FVL assay is cost effective in a high-throughput setting, with capability to detect both previously described and novel FV variants.

  12. Melt Infiltrated Ceramic Composites (Hipercomp) for Gas Turbine Engine Applications

    SciTech Connect

    Gregory Corman; Krishan Luthra

    2005-09-30

    This report covers work performed under the Continuous Fiber Ceramic Composites (CFCC) program by GE Global Research and its partners from 1994 through 2005. The processing of prepreg-derived, melt infiltrated (MI) composite systems based on monofilament and multifilament tow SiC fibers is described. Extensive mechanical and environmental exposure characterizations were performed on these systems, as well as on competing Ceramic Matrix Composite (CMC) systems. Although current monofilament SiC fibers have inherent oxidative stability limitations due to their carbon surface coatings, the MI CMC system based on multifilament tow (Hi-Nicalon ) proved to have excellent mechanical, thermal and time-dependent properties. The materials database generated from the material testing was used to design turbine hot gas path components, namely the shroud and combustor liner, utilizing the CMC materials. The feasibility of using such MI CMC materials in gas turbine engines was demonstrated via combustion rig testing of turbine shrouds and combustor liners, and through field engine tests of shrouds in a 2MW engine for >1000 hours. A unique combustion test facility was also developed that allowed coupons of the CMC materials to be exposed to high-pressure, high-velocity combustion gas environments for times up to {approx}4000 hours.

  13. Detection of Sequence Polymorphism in Rubus Occidentalis L. Monomorphic Microsatellite Markers by High Resolution Melting

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microsatellite, or simple sequence repeat (SSR) markers, are valuable as co-dominant genetic markers with a variety of applications such as DNA fingerprinting, linkage mapping, and population structure analysis. Development of microsatellite primers through the identification of appropriate repeate...

  14. Geophysical controls on C band polarimetric backscatter from melt pond covered Arctic first-year sea ice: Assessment using high-resolution scatterometry

    NASA Astrophysics Data System (ADS)

    Scharien, R. K.; Yackel, J. J.; Barber, D. G.; Asplin, M.; Gupta, M.; Isleifson, D.

    2012-08-01

    Geophysical controls on C band polarimetric backscatter from the discrete surface cover types which comprise advanced melt first-year sea ice (FYI): snow covered ice, bare ice, and melt pond; are assessed using polarimetric radar scatterometry from test sites representing high Arctic and marginal ice zones in the Canadian Arctic. Surface characterization data is used to evaluate the interaction of polarized radiation with each feature, and dominant scattering mechanisms are assessed in a regional context. High-resolution time series (diurnal) scatterometry and coincident atmospheric boundary layer profile data are used to explain linkages between ice-atmosphere interactions and polarimetric backscatter in a marginal ice zone. The co-polarization ratio for FYI melt ponds is shown to be distinct from snow covered ice or bare ice during early and peak phases of advanced melt, making it a candidate parameter for the unambiguous detection of pond formation and the inversion of melt pond fraction. The ratio displays an increasing trend with radar incidence angle in a manner consistent with Bragg surface scattering theory, though it is not predictable by a Bragg model. Cross-polarization backscatter intensity shows potential for discriminating the onset and duration of freeze events in a marginal ice zone, due to dominant backscatter from the snow cover adjacent to melt ponds. Preliminary results here outline the potential of covariance matrix derived polarimetric measurements for the inversion of advanced melt sea ice geophysical parameters, and provide a basis for the investigation of distributed targets in late season spaceborne polarimetric SAR scenes.

  15. High resolution melting detects sequence polymorphism in rubus occidentalis L. monomorphic microsatellite markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microsatellite, or simple sequence repeat (SSR) markers, are valuable as co-dominant genetic markers with a variety of applications such as DNA fingerprinting, linkage mapping, and population structure analysis. However, primer pairs designed from the regions that flank SSRs often generate fragment...

  16. Melt inclusion record of immiscibility between silicate, hydrosaline, and carbonate melts: Applications to skarn genesis at Mount Vesuvius

    NASA Astrophysics Data System (ADS)

    Fulignati, Paolo; Kamenetsky, Vadim S.; Marianelli, Paola; Sbrana, Alessandro; Mernagh, Terrence P.

    2001-11-01

    Foid-bearing syenites and endoskarn xenoliths of the A.D. 472 Vesuvius eruption represent the magma chamber carbonate wall-rock interface. Melt inclusions hosted in crystals from these rocks offer a rare opportunity to depict the formation and the composition of metasomatic skarn-forming fluids at the peripheral part of a growing K-alkaline magma chamber disrupted by an explosive eruption. Four principal types of melt inclusions represent highly differentiated phonolite (type 1), hydrosaline melt (type 3), unmixed silicate salt melts (type 2), and a complex chloride-carbonate melt with minor sulfates (type 4). The high-temperature (700 800 °C) magmatic-derived hydrosaline melt is considered to be the main metasomatic agent for the skarn-forming reactions. The interaction between this melt (fluid) and carbonate wall rocks produces a Na-K-Ca carbonate-chloride melt that shows immiscibility between carbonate and chloride constituents at ˜700 °C in 1 atm experiments. This unmixing can be viewed as a possible mechanism for the origin of carbonatites associated with intrusion-related skarn systems.

  17. Detection of Schistosoma mansoni and Schistosoma haematobium by Real-Time PCR with High Resolution Melting Analysis.

    PubMed

    Sady, Hany; Al-Mekhlafi, Hesham M; Ngui, Romano; Atroosh, Wahib M; Al-Delaimy, Ahmed K; Nasr, Nabil A; Dawaki, Salwa; Abdulsalam, Awatif M; Ithoi, Init; Lim, Yvonne A L; Chua, Kek Heng; Surin, Johari

    2015-07-16

    The present study describes a real-time PCR approach with high resolution melting-curve (HRM) assay developed for the detection and differentiation of Schistosoma mansoni and S. haematobium in fecal and urine samples collected from rural Yemen. The samples were screened by microscopy and PCR for the Schistosoma species infection. A pair of degenerate primers were designed targeting partial regions in the cytochrome oxidase subunit I (cox1) gene of S. mansoni and S. haematobium using real-time PCR-HRM assay. The overall prevalence of schistosomiasis was 31.8%; 23.8% of the participants were infected with S. haematobium and 9.3% were infected with S. mansoni. With regards to the intensity of infections, 22.1% and 77.9% of S. haematobium infections were of heavy and light intensities, respectively. Likewise, 8.1%, 40.5% and 51.4% of S. mansoni infections were of heavy, moderate and light intensities, respectively. The melting points were distinctive for S. mansoni and S. haematobium, categorized by peaks of 76.49 ± 0.25 °C and 75.43 ± 0.26 °C, respectively. HRM analysis showed high detection capability through the amplification of Schistosoma DNA with as low as 0.0001 ng/µL. Significant negative correlations were reported between the real-time PCR-HRM cycle threshold (Ct) values and microscopic egg counts for both S. mansoni in stool and S. haematobium in urine (p < 0.01). In conclusion, this closed-tube HRM protocol provides a potentially powerful screening molecular tool for the detection of S. mansoni and S. haematobium. It is a simple, rapid, accurate, and cost-effective method. Hence, this method is a good alternative approach to probe-based PCR assays.

  18. Direct detection of Rifampicin and Isoniazid resistance in sputum samples from tuberculosis patients by High Resolution Melt curve analysis.

    PubMed

    Anthwal, Divya; Gupta, Rakesh Kumar; Bhalla, Manpreet; Bhatnagar, Shinjini; Tyagi, Jaya Sivaswami; Haldar, Sagarika

    2017-03-22

    Drug-resistant tuberculosis (TB) is a major threat for TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of High Resolution Melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis (M. tb) in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance determining regions of rpoB, katG and inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR-M. tb clinical isolates. The assay was evaluated on DNA isolated from 99 M. tb culture-positive sputum samples that included 84 smear-negative sputum, using DNA sequencing as gold standard. Mutants were discriminated from wild-type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampicin (RIF) and isoniazid (INH) mono-resistance were detected in 11 and 21 specimens, respectively by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. HRM-RIF, INH-katG and INH-inhA assay had 89% (52;100), 85% (62;97) and 100% (74;100) sensitivity, respectively in smear-negative samples; while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97%-100% (κ value;0.9-1) was noted between sequencing and HRM. Hetero-resistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (INR400/US$6), rapid and closed-tube method for direct detection of MDR-TB in sputum, especially for direct smear negative cases.

  19. Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis.

    PubMed

    Hanson, Erin K; Ballantyne, Jack

    2013-01-01

    Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive

  20. High-resolution melt PCR analysis for rapid identification of Chlamydia abortus live vaccine strain 1B among C. abortus strains and field isolates.

    PubMed

    Vorimore, Fabien; Cavanna, Noémie; Vicari, Nadia; Magnino, Simone; Willems, Hermann; Rodolakis, Annie; Siarkou, Victoria I; Laroucau, Karine

    2012-09-01

    We describe a novel high-resolution melt assay that clearly differentiates Chlamydia abortus live vaccine strain 1B from field C. abortus strains and field wild-type isolates based on previously described single nucleotide polymorphisms. This modern genotyping technique is inexpensive, easy to use, and less time-consuming than PCR-RFLP.

  1. Molecular simultaneous detection of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus by real-time RT-PCR and high resolution melting analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, real-time RT-PCR assays were combined with high resolution melting (HRM) analysis for the simultaneous detection of Cherry necrotic rusty mottle virus (CNRMV) and Cherry green ring mottle virus (CGRMV) infection in sweet cherry trees. Detection of CNRMV and CGRMV was performed using a...

  2. High resolution SAR applications and instrument design

    NASA Technical Reports Server (NTRS)

    Dionisio, C.; Torre, A.

    1993-01-01

    The Synthetic Aperture Radar (SAR) has viewed, in the last two years, a huge increment of interest from many preset and potential users. The good spatial resolution associated to the all weather capability lead to considering SAR not only a scientific instrument but a tool for verifying and controlling the daily human relationships with the Earth Environment. New missions were identified for SAR as spatial resolution became lower than three meters: disasters, pollution, ships traffic, volcanic eruptions, earthquake effect are only a few of the possible objects which can be effectively detected, controlled and monitored by SAR mounted on satellites. High resolution radar design constraints and dimensioning are discussed.

  3. High-resolution melting analysis of 15 genes in 60 patients with cytochrome-c oxidase deficiency.

    PubMed

    Vondrackova, Alzbeta; Vesela, Katerina; Hansikova, Hana; Docekalova, Dagmar Zajicova; Rozsypalova, Eva; Zeman, Jiri; Tesarova, Marketa

    2012-07-01

    Cytochrome-c oxidase (COX) deficiency is one of the common childhood mitochondrial disorders. Mutations in genes for the assembly factors SURF1 and SCO2 are prevalent in children with COX deficiency in the Slavonic population. Molecular diagnosis is difficult because of the number of genes involved in COX biogenesis and assembly. The aim of this study was to screen for mutations in 15 nuclear genes that encode the 10 structural subunits, their isoforms and two assembly factors of COX in 60 unrelated Czech children with COX deficiency. Nine novel variants were identified in exons and adjacent intronic regions of COX4I2, COX6A1, COX6A2, COX7A1, COX7A2 and COX10 using high-resolution melting (HRM) analysis. Online bioinformatics servers were used to predict the importance of the newly identified amino-acid substitutions. The newly characterized variants updated the contemporary spectrum of known genetic sequence variations that are present in the Czech population, which will be important for further targeted mutation screening in Czech COX-deficient children. HRM and predictive bioinformatics methodologies are advantageous because they are low-cost screening tools that complement large-scale genomic studies and reduce the required time and effort.

  4. Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene.

    PubMed

    Costa, Jean-Marc; Cabaret, Odile; Moukoury, Sandrine; Bretagne, Stéphane

    2011-09-01

    Genetic studies of the protozoan parasite Toxoplasma gondii have identified three main distinct types according to virulence in some hosts. Several methods have been developed to differentiate genotypes currently dominated by microsatellite markers targeting single-copy loci. We analyzed the possibility of using the 35-fold repetitive B1 gene via high-resolution melting (HRM) curve analysis. Sequencing of the B1 gene of 14 reference strains (four Type I, six Type II, and four Type III strains) identified 18 single nucleotide polymorphisms (SNP). Primers were designed to amplify eight of them for HRM analysis and for relative quantification of each nucleotide variation using SNaPshot mini-sequencing. Genotyping with five microsatellite markers was performed for comparison. Two to four HRM profiles were obtained depending on the SNP tested. The differences observed relied on the different ratios of nucleotides at the SNP locus as evidenced via SNaPshot mini-sequencing. The three main lineages could be distinguished by using several HRM profiles. Some HRM profiles proved more informative than the analysis based on five microsatellite markers, showing additional differences in Type I and Type II strains. Using HRM analysis, we obtained at least an equally good discrimination of the main lineages than that based on five microsatellite markers.

  5. Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini using real-time PCR and high resolution melting analysis.

    PubMed

    Cai, Xian-Quan; Yu, Hai-Qiong; Li, Rong; Yue, Qiao-Yun; Liu, Guo-Hua; Bai, Jian-Shan; Deng, Yan; Qiu, De-Yi; Zhu, Xing-Quan

    2014-01-01

    Clonorchis sinensis and Opisthorchis viverrini are both important fish-borne pathogens, causing serious public health problem in Asia. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the specific detection and rapid identification of C. sinensis and O. viverrini. Primers targeting COX1 gene were highly specific for these liver flukes, as evidenced by the negative amplification of closely related trematodes. Assays using genomic DNA extracted from the two flukes yielded specific amplification and their identity was confirmed by sequencing, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit below 1 pg of purified genomic DNA, 5 EPG, or 1 metacercaria of C. sinensis. Moreover, C. sinensis and O. viverrini were able to be differentiated by their HRM profiles. The method can reduce labor of microscopic examination and the contamination of agarose electrophoresis. Moreover, it can differentiate these two flukes which are difficult to be distinguished using other methods. The established method provides an alternative tool for rapid, simple, and duplex detection of C. sinensis and O. viverrini.

  6. First report of Tasmanian sheep strain (G2) genotype isolated from Iranian goat using the high resolution melting (HRM) analysis

    PubMed Central

    Hosseini-Safa, Ahmad; Mohag, hegh, Mohammad Ali; Pestechian, Nader; Ganji, Maryam; Mohammadi, Rasoul; Mahmoudi Lamouki, Reza; Rostami-Nejad, Mohammad

    2016-01-01

    Aim: The present study was aimed to evaluate E. granulosus genotypes isolated from goats using HRM analysis in Isfahan province. Background: Cystic echincoccosis, so-called hydatidosis, is widespread infection caused by the larval stage of Echinococcus granulosus. This is an important zoonotic disease worldwide, especially in the developing countries such as Iran. To date, molecular studies mainly based on the mitochondrial DNA sequences have identified distinct genotypes termed G1-G10 which can differ in some characteristics such as the growth and infectivity to different intermediate hosts or the survival rate in the definitive hosts that are important for the development of control strategies. Methods: From August to December 2014, 1341 goats were investigated and hydatid cysts were collected from the liver and lungs of 43 infected goats in Isfahan province abattoirs, Isfahan, Iran. Total genomic DNA was extracted from each sample, amplified for the presence of polymorphism of mitochondrial gene coding for cytochrome c oxidase subunit 1 (CO1), using high resolution melting curve (HRM) method. Results: the results of HRM analysis using the sequence of CO1 gene for 43 Echinococcus granulosus isolates from goats showed 31, 2 and 10 isolates were identified as G1, G2, and G3 genotypes, respectively. Conclusion: G1 is the predominant genotype in the isolated goat samples in Isfahan province, and the presence of G2 strain was reported for the first time in goat in Iran. PMID:28224031

  7. Interferon λ 3 and 4 Genotyping Using High-Resolution Melt Curve Analysis Suitable for Multiple Clinical Sample Types.

    PubMed

    Lamoury, François M J; Bartlett, Sofia; Jacka, Brendan; Hajarizadeh, Behzad; Grebely, Jason; Matthews, Gail V; Dore, Gregory J; Applegate, Tanya L

    2015-09-01

    Many people living with hepatitis C virus (HCV) infection will continue to rely on interferon-based regimens until effective strategies to minimize the cost of directly acting antivirals (DAAs) and to improve treatment access are implemented. Host single-nucleotide polymorphisms related to IFNL3 and IFNL4 are associated with spontaneous clearance of HCV, and pegylated interferon- and DAA-based treatment outcomes. We describe a simple and rapid genotyping method for IFNL rs12979860, rs8099917, and rs368234815 using high-resolution melting analysis for DNA extracted from whole blood, buffy coat, plasma, serum, and dried blood spots. This assay successfully detected all three polymorphisms on DNA extracted by the automated platform easyMAG from all samples when compared to sequenced amplicons. Analysis of 126 participants with recent HCV infection from the Australian Trial in Acute Hepatitis C study demonstrated the prevalence of favorable single-nucleotide polymorphisms were 62%, 51%, and 45% for rs8099917 TT, rs12979860 CC, and rs368234815 TT/TT, respectively. The genotyping assay described here provides a rapid and affordable IFNL3 and IFNL4 genotyping method for a range of clinical sample types. Until global access to DAAs is achieved, IFNL3 and IFNL4 genotyping could identify those likely to clear naturally and in whom treatment could be delayed, or help prioritize DAA treatment to those less likely to respond to interferon-containing regimens.

  8. Genotyping of dairy Bacillus licheniformis isolates by high resolution melt analysis of multiple variable number tandem repeat loci.

    PubMed

    Dhakal, Rajat; Chauhan, Kanika; Seale, R Brent; Deeth, Hilton C; Pillidge, Christopher J; Powell, Ian B; Craven, Heather; Turner, Mark S

    2013-06-01

    In dairy foods, the sporeformer Bacillus licheniformis can be the cause of spoilage or specification compliance issues. Currently used methods for genotyping B. licheniformis have limited discrimination with only 2 or 3 different subgroups being identified. Here, we have developed a multi-locus variable number tandem repeat analysis (MLVA) method and combined it with high resolution melt analysis (MLV-HRMA) for genotyping B. licheniformis. Five repetitive loci were identified and used as markers for genotyping 52 isolates from two milk powder processing plants and retail samples. Nineteen genotypes could be identified using both MLVA and MLV-HRMA leading to Hunter-Gaston discrimination indices (D-value) of 0.93 each. It was found that all 5 MLVA loci were stable following 10 days of sub-culturing of 8 representative isolates. All isolates were also genotyped using previously used methods including randomly amplified polymorphic DNA-PCR (RAPD) and partial rpoB sequencing. Five different RAPD profiles and 5 different partial rpoB sequence types were identified resulting in corresponding D-values of 0.6 and 0.46, respectively. Analysis of the genotypes from dairy samples revealed that dairy B. licheniformis isolates are more heterogeneous than previously thought and that this new method can potentially allow for more discriminatory tracking and monitoring of specific genotypes.

  9. Development of a High-Resolution Melting Analysis Method for CYP2C19*17 Genotyping in Healthy Volunteers

    PubMed Central

    Ghasemi, Zahra; Hashemi, Mehrdad; Ejabati, Mahsa; Ebrahimi, Seyyed Meisam; Kheiri Manjili, Hamidreza; Sharafi, Ali; Ramazani, Ali

    2016-01-01

    Background: Genetic polymorphisms of drug metabolisms by cytochrome P450 (P450s) could affect drug response, attracting particular interest in the pharmacogenetics. Due to the importance of CYP2C19* 17 allele and its capability of super- fast metabolism and also lack of information about distribution of the alleles in Iranian population, this research aimed to use High Resolution Melting (HRM) method compared to PCR-RFLP for genotyping healthy Iranian population. Methods: Blood samples were collected from 100 healthy Iranian volunteers. DNA was extracted by salting out method. Real-time PCR was used for amplification of the CYP2C19 gene and the alleles were identified by HRM. Sequencing was used to confirm the amplified DNA fragments and data were analyzed using SPSS software ver.18. Results: The frequency of alleles CYP2C19*1/*1, CYP2C19*1/*17 and CYP2C19*17/*17 were estimated as 58.33, 29.1 and 11.1%, respectively. Specificity and sensitivity of HRM method were 90% and 100%, with respect to PCR-RFLP. Also, HRM analysis has been evaluated as a faster and more effective approach. Conclusion: Comparison of our results based on HRM analysis with PCR-RFLP showed that our developed method is rapid, accurate, fast and economic to study the CYP2C19*17 allele and it is appropriate for other similar population genetic studies. PMID:27920888

  10. Identification of molecular markers associated with Verticillium wilt resistance in alfalfa (Medicago sativa L.) using high-resolution melting.

    PubMed

    Zhang, Tiejun; Yu, Long-Xi; McCord, Per; Miller, David; Bhamidimarri, Suresh; Johnson, David; Monteros, Maria J; Ho, Julie; Reisen, Peter; Samac, Deborah A

    2014-01-01

    Verticillium wilt, caused by the soilborne fungus, Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To identify loci associated with resistance to Verticillium wilt, a bulk segregant analysis was conducted in susceptible or resistant pools constructed from 13 synthetic alfalfa populations, followed by association mapping in two F1 populations consisted of 352 individuals. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were used for genotyping. Phenotyping was done by manual inoculation of the pathogen to replicated cloned plants of each individual and disease severity was scored using a standard scale. Marker-trait association was analyzed by TASSEL. Seventeen SNP markers significantly associated with Verticillium wilt resistance were identified and they were located on chromosomes 1, 2, 4, 7 and 8. SNP markers identified on chromosomes 2, 4 and 7 co-locate with regions of Verticillium wilt resistance loci reported in M. truncatula. Additional markers identified on chromosomes 1 and 8 located the regions where no Verticillium resistance locus has been reported. This study highlights the value of SNP genotyping by high resolution melting to identify the disease resistance loci in tetraploid alfalfa. With further validation, the markers identified in this study could be used for improving resistance to Verticillium wilt in alfalfa breeding programs.

  11. Evaluation of low melting halide systems for battery applications

    NASA Astrophysics Data System (ADS)

    Mamantov, G.; Perrovic, C.

    1981-03-01

    This three year program involves evaluation of selected low temperature molten salt solvent systems containing inorganic and/or organic chlorides and bromides for battery applications. The research involves determination of the liquidus temperatures, the specific electrical conductivity, and the electrochemical span of selected halide systems. Characterization of the solvent species by Raman spectroscopy, vapor pressure measurements, and the electrochemical study of a few cathode and anode systems will be undertaken for the most promising solvent systems. The research during the second year of this project involved the determination of liquidus temperatures and/or specific electrical conductivities for a number of binary and ternary molten salt systems containing AlCl3, AlBr3, SbCl3, FeCl3, and GaCl3.

  12. A Melting-Layer Model for Passive/Active Microwave Remote Sensing Applications. Part I: Model Formulation and Comparison with Observations.

    NASA Astrophysics Data System (ADS)

    Olson, William S.; Bauer, Peter; Viltard, Nicolas F.; Johnson, Daniel E.; Tao, Wei-Kuo; Meneghini, Robert; Liao, Liang

    2001-07-01

    In this study, a 1D steady-state microphysical model that describes the vertical distribution of melting precipitation particles is developed. The model is driven by the ice-phase precipitation distributions just above the freezing level at applicable grid points of `parent' 3D cloud-resolving model (CRM) simulations. It extends these simulations by providing the number density and meltwater fraction of each particle in finely separated size categories through the melting layer. The depth of the modeled melting layer is primarily determined by the initial material density of the ice-phase precipitation. The radiative properties of melting precipitation at microwave frequencies are calculated based upon different methods for describing the dielectric properties of mixed-phase particles. Particle absorption and scattering efficiencies at the Tropical Rainfall Measuring Mission Microwave Imager frequencies (10.65-85.5 GHz) are enhanced greatly for relatively small (0.1) meltwater fractions. The relatively large number of partially melted particles just below the freezing level in stratiform regions leads to significant microwave absorption, well exceeding the absorption by rain at the base of the melting layer. Calculated precipitation backscatter efficiencies at the precipitation radar frequency (13.8 GHz) increase with particle meltwater fraction, leading to a `bright band' of enhanced radar reflectivities in agreement with previous studies. The radiative properties of the melting layer are determined by the choice of dielectric models and the initial water contents and material densities of the `seeding' ice-phase precipitation particles. Simulated melting-layer profiles based upon snow described by the Fabry-Szyrmer core-shell dielectric model and graupel described by the Maxwell-Garnett water matrix dielectric model lead to reasonable agreement with radar-derived melting-layer optical depth distributions. Moreover, control profiles that do not contain mixed

  13. Detection and characterization of Leishmania (Leishmania) and Leishmania (Viannia) by SYBR green-based real-time PCR and high resolution melt analysis targeting kinetoplast minicircle DNA.

    PubMed

    Ceccarelli, Marcello; Galluzzi, Luca; Migliazzo, Antonella; Magnani, Mauro

    2014-01-01

    Leishmaniasis is a neglected disease with a broad clinical spectrum which includes asymptomatic infection. A thorough diagnosis, able to distinguish and quantify Leishmania parasites in a clinical sample, constitutes a key step in choosing an appropriate therapy, making an accurate prognosis and performing epidemiological studies. Several molecular techniques have been shown to be effective in the diagnosis of leishmaniasis. In particular, a number of PCR methods have been developed on various target DNA sequences including kinetoplast minicircle constant regions. The first aim of this study was to develop a SYBR green-based qPCR assay for Leishmania (Leishmania) infantum detection and quantification, using kinetoplast minicircle constant region as target. To this end, two assays were compared: the first used previously published primer pairs (qPCR1), whereas the second used a nested primer pairs generating a shorter PCR product (qPCR2). The second aim of this study was to evaluate the possibility to discriminate among subgenera Leishmania (Leishmania) and Leishmania (Viannia) using the qPCR2 assay followed by melting or High Resolution Melt (HRM) analysis. Both assays used in this study showed good sensitivity and specificity, and a good correlation with standard IFAT methods in 62 canine clinical samples. However, the qPCR2 assay allowed to discriminate between Leishmania (Leishmania) and Leishmania (Viannia) subgenera through melting or HRM analysis. In addition to developing assays, we investigated the number and genetic variability of kinetoplast minicircles in the Leishmania (L.) infantum WHO international reference strain (MHOM/TN/80/IPT1), highlighting the presence of minicircle subclasses and sequence heterogeneity. Specifically, the kinetoplast minicircle number per cell was estimated to be 26,566±1,192, while the subclass of minicircles amplifiable by qPCR2 was estimated to be 1,263±115. This heterogeneity, also observed in canine clinical samples

  14. Practical Applications Using A High Resolution Infrared Imaging System

    NASA Astrophysics Data System (ADS)

    Baraniak, David W.

    1981-01-01

    Infrared imaging systems can be classified into three general categories, low resolution, medium resolution and high resolution. It is the purpose of this paper to highlight specific applications best suited to high resolution, television capatable, infrared data acquisition techniques. The data was collected from both ground loped andoaerial based mobile positions where the temperature differentials varied from 15 C to 25 C. Specific applications include scanning building complexes from the exterior using a ground based moving vehicle, scanning buildings, concrete bridge decks and terrain from the air using a helicopter and scanning building interiors using a mobile hand truck.

  15. A novel method for simultaneous Enterococcus species identification/typing and van genotyping by high resolution melt analysis.

    PubMed

    Gurtler, Volker; Grando, Danilla; Mayall, Barrie C; Wang, Jenny; Ghaly-Derias, Shahbano

    2012-09-01

    In order to develop a typing and identification method for van gene containing Enterococcus faecium, two multiplex PCR reactions were developed for use in HRM-PCR (High Resolution Melt-PCR): (i) vanA, vanB, vanC, vanC23 to detect van genes from different Enterococcus species; (ii) ISR (intergenic spacer region between the 16S and 23S rRNA genes) to detect all Enterococcus species and obtain species and isolate specific HRM curves. To test and validate the method three groups of isolates were tested: (i) 1672 Enterococcus species isolates from January 2009 to December 2009; (ii) 71 isolates previously identified and typed by PFGE (pulsed-field gel electrophoresis) and MLST (multi-locus sequence typing); and (iii) 18 of the isolates from (i) for which ISR sequencing was done. As well as successfully identifying 2 common genotypes by HRM from the Austin Hospital clinical isolates, this study analysed the sequences of all the vanB genes deposited in GenBank and developed a numerical classification scheme for the standardised naming of these vanB genotypes. The identification of Enterococcus faecalis from E. faecium was reliable and stable using ISR PCR. The typing of E. faecium by ISR PCR: (i) detected two variable peaks corresponding to different copy numbers of insertion sequences I and II corresponding to peak I and II respectively; (ii) produced 7 melt profiles for E. faecium with variable copy numbers of sequences I and II; (iii) demonstrated stability and instability of peak heights with equal frequency within the patient sample (36.4±4.5 days and 38.6±5.8 days respectively for 192 patients); (iv) detected ISR-HRM types with as much discrimination as PFGE and more than MLST; and (v) detected ISR-HRM types that differentiated some isolates that were identical by PFGE and MLST. In conjunction with the rapid and accurate van genotyping method described here, this ISR-HRM typing and identification method can be used as a stable identification and typing method with

  16. High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley.

    PubMed

    Hofinger, Bernhard J; Jing, Hai-Chun; Hammond-Kosack, Kim E; Kanyuka, Kostya

    2009-09-01

    An original method has been established for the identification of novel alleles of eukaryotic translation initiation factor 4E (eIF4E) gene, which is required for resistance to agronomically important bymoviruses, in barley germplasm. This method involves scanning for sequence variations in cDNA-derived PCR amplicons using High-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM is a simple, cost-effective, rapid and high-throughput assay, which so far has only been widely used in clinical pathology for molecular diagnostic of diseases and patient genotyping. Application of HRM allowed significant reduction in the amount of expensive Sanger sequencing required for allele mining in plants. The method described here involved an investigation of total cDNA rather than genomic DNA, thus permitting the analyses of shorter (up to 300-bp) and fewer overlapping amplicons to cover the coding sequence. This strategy further reduced the allele mining costs. The sensitivity and accuracy of HRM for predicting genotypes carrying a wide range of nucleotide polymorphisms in eIF4E approached 100%. Results of the current study are promising and suggest that this method could also potentially be applied to the discovery of superior alleles controlling other important traits in barley as well in other model and crop plant species.

  17. Chalcophile element partitioning between sulfide phases and hydrous mantle melt: Applications to mantle melting and the formation of ore deposits

    NASA Astrophysics Data System (ADS)

    Li, Yuan

    2014-11-01

    Understanding the geochemical behavior of chalcophile elements in magmatic processes is hindered by the limited partition coefficients between sulfide phases and silicate melt, in particular at conditions relevant to partial melting of the hydrated, metasomatized upper mantle. In this study, the partitioning of elements Co, Ni, Cu, Zn, As, Mo, Ag, and Pb between sulfide liquid, monosulfide solid solution (MSS), and hydrous mantle melt has been investigated at 1200 °C/1.5 GPa and oxygen fugacity ranging from FMQ-2 to FMQ+1 in a piston-cylinder apparatus. The determined partition coefficients between sulfide liquid and hydrous mantle melt are: 750-1500 for Cu; 600-1200 for Ni; 35-42 for Co; 35-53 for Pb; and 1-2 for Zn, As, and Mo. The partition coefficients between MSS and hydrous mantle melt are: 380-500 for Cu; 520-750 for Ni; ∼50 for Co; <0.5 for Zn; 0.3-6 for Pb; 0.1-2 for As; 1-2 for Mo; and >34 for Ag. The variation of the data is primarily due to differences in oxygen fugacity. These partitioning data in conjunction with previous data are applied to partial melting of the upper mantle and the formation of magmatic-hydrothermal Cu-Au deposits and magmatic sulfide deposits. I show that the metasomatized arc mantle may no longer contain sulfide after >10-14% melt extraction but is still capable of producing the Cu concentrations in the primitive arc basalts, and that the comparable Cu concentrations in primitive arc basalts and in MORB do not necessarily imply similar oxidation states in their source regions. Previous models proposed for producing Cu- and/or Au-rich magmas have been reassessed, with the conclusions summarized as follows. (1) Partial melting of the oxidized (fO2 > FMQ), metasomatized arc mantle with sulfide exhaustion at degrees >10-14% may not generate Cu-rich, primitive arc basalts. (2) Partial melting of sulfide-bearing cumulates in the root of thickened lower continental crust or lithospheric mantle does not typically generate Cu- and

  18. Preliminary validation of a novel high-resolution melt-based typing method based on the multilocus sequence typing scheme of Streptococcus pyogenes.

    PubMed

    Richardson, L J; Tong, S Y C; Towers, R J; Huygens, F; McGregor, K; Fagan, P K; Currie, B J; Carapetis, J R; Giffard, P M

    2011-09-01

    The major limitation of current typing methods for Streptococcus pyogenes, such as emm sequence typing and T typing, is that these are based on regions subject to considerable selective pressure. Multilocus sequence typing (MLST) is a better indicator of the genetic backbone of a strain but is not widely used due to high costs. The objective of this study was to develop a robust and cost-effective alternative to S. pyogenes MLST. A 10-member single nucleotide polymorphism (SNP) set that provides a Simpson's Index of Diversity (D) of 0.99 with respect to the S. pyogenes MLST database was derived. A typing format involving high-resolution melting (HRM) analysis of small fragments nucleated by each of the resolution-optimized SNPs was developed. The fragments were 59-119 bp in size and, based on differences in G+C content, were predicted to generate three to six resolvable HRM curves. The combination of curves across each of the 10 fragments can be used to generate a melt type (MelT) for each sequence type (ST). The 525 STs currently in the S. pyogenes MLST database are predicted to resolve into 298 distinct MelTs and the method is calculated to provide a D of 0.996 against the MLST database. The MelTs are concordant with the S. pyogenes population structure. To validate the method we examined clinical isolates of S. pyogenes of 70 STs. Curves were generated as predicted by G+C content discriminating the 70 STs into 65 distinct MelTs.

  19. Combined molecular gram typing and high-resolution melting analysis for rapid identification of a syndromic panel of bacteria responsible for sepsis-associated bloodstream infection.

    PubMed

    Ozbak, Hani; Dark, Paul; Maddi, Satyanarayana; Chadwick, Paul; Warhurst, Geoffrey

    2012-01-01

    Effective diagnosis and treatment of bloodstream infections are often hampered by a lack of time-critical information from blood cultures. Molecular techniques aimed at the detection of circulating pathogen DNA have the potential to dramatically improve the timeliness of infection diagnosis. Our aim in this study was to establish a rapid, low-cost PCR approach using high-resolution melting analysis to identify a syndromic panel of 21 pathogens responsible for most bloodstream bacterial infections encountered in critical care environments. A broad-range, real-time PCR technique that combines primers for molecular Gram classification and high-resolution melting analysis in a single run was established. The differentiation of bacterial species was achieved using a multiparameter, decision-tree approach that was based on Gram type, grouping according to melting temperature, and sequential comparisons of melting profiles against multiple reference organisms. A preliminary validation study was undertaken by blinded analysis of 53 consecutive bloodstream isolates from a clinical microbiology laboratory. Fifty isolates contained organisms that were present in the panel, and 96% of these were identified correctly at the genus or species level. A correct Gram classification was reported for all 53 isolates. This technique shows promise as a cost-effective tool for the timely identification of bloodstream pathogens, allowing clinicians to make informed decisions on appropriate antibiotic therapies at an earlier stage.

  20. Quantitative evaluation of DNMT3B promoter methylation in breast cancer patients using differential high resolution melting analysis.

    PubMed

    Naghitorabi, M; Mohammadi Asl, J; Mir Mohammad Sadeghi, H; Rabbani, M; Jafarian-Dehkordi, A; Javanmard, Haghjooye S

    2013-07-01

    DNA methylation plays an important role in carcinogenesis through epigenetic silencing of tumor suppressor genes. Aberrant methylation usually results from changes in the activity of DNA methyltransferases (DNMTs). Some studies show that the overexpression of the DNMTs may lead to aberrant methylation of tumor suppressor genes. Also the overexpression of DNMTs may be related to methylation status of their genes. Due to limited number of studies on DNMT3B promoter methylation, this study was performed to quantitatively measure the methylation level of DNMT3B gene in archival formalin fixed paraffin embedded (FFPE) tissues from breast cancer patients. Using differential high resolution melting analysis (D-HRMA) technology, the methylation level of DNMT3B gene promoter was quantified in 98 breast cancer FFPE tissues and also 10 fresh frozen normal tissue samples. Statistical analyses used for analyzing the correlation between the methylation and clinical variables. All the normal samples were found to be methylated at the DNMT3B promoter (the average methylation level 3.34%). Patients were identified as hypo-methylated (mean methylation level 0.8%), methylated (mean methylation level 2.48%) and hyper-methylated (mean methylation level 10.5%). Statistical analysis showed a significant correlation between the methylation status and the sample type, cancer type and tumor size. Also the methylation level was significantly associated with histologic grade. It is concluded that quantification of DNMT3B promoter methylation might be used as a reliable and sensitive diagnostic and prognostic tool in breast cancer. Also D-HRMA is demonstrated as a rapid and cost effective method for quantitative evaluation of promoter methylation.

  1. Comparison of a High-Resolution Melting Assay to Next-Generation Sequencing for Analysis of HIV Diversity

    PubMed Central

    Cousins, Matthew M.; Ou, San-San; Wawer, Maria J.; Munshaw, Supriya; Swan, David; Magaret, Craig A.; Mullis, Caroline E.; Serwadda, David; Porcella, Stephen F.; Gray, Ronald H.; Quinn, Thomas C.; Donnell, Deborah; Eshleman, Susan H.

    2012-01-01

    Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those obtained using a diversity assay based on high-resolution melting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n = 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The amplicon pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence-based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less expensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution. PMID:22785188

  2. Arbovirus and insect-specific virus discovery in Kenya by novel six genera multiplex high-resolution melting analysis.

    PubMed

    Villinger, Jandouwe; Mbaya, Martin K; Ouso, Daniel; Kipanga, Purity N; Lutomiah, Joel; Masiga, Daniel K

    2016-08-02

    A broad diversity of arthropod-borne viruses (arboviruses) of global health concern are endemic to East Africa, yet most surveillance efforts are limited to just a few key viral pathogens. Additionally, estimates of arbovirus diversity in the tropics are likely to be underestimated as their discovery has lagged significantly over past decades due to limitations in fast and sensitive arbovirus identification methods. Here, we developed a nearly pan-arbovirus detection assay that uses high-resolution melting (HRM) analysis of RT-PCR products from highly multiplexed assays to differentiate broad diversities of arboviruses. We differentiated 15 viral culture controls and seven additional synthetic viral DNA sequence controls, within Flavivirus, Alphavirus, Nairovirus, Phlebovirus, Orthobunyavirus and Thogotovirus genera. Among Bunyamwera, sindbis, dengue and Thogoto virus serial dilutions, detection by multiplex RT-PCR-HRM was comparable to the gold standard Vero cell plaque assays. We applied our low-cost method for enhanced broad-range pathogen surveillance from mosquito samples collected in Kenya and identified diverse insect-specific viruses, including a new clade in anopheline mosquitoes, and Wesselsbron virus, an arbovirus that can cause viral haemorrhagic fever in humans and has not previously been isolated in Kenya, in Culex spp. and Anopheles coustani mosquitoes. Our findings demonstrate how multiplex RT-PCR-HRM can identify novel viral diversities and potential disease threats that may not be included in pathogen detection panels of routine surveillance efforts. This approach can be adapted to other pathogens to enhance disease surveillance and pathogen discovery efforts, as well as the study of pathogen diversity and viral evolutionary ecology.

  3. High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes

    PubMed Central

    2011-01-01

    Background Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. Results We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Conclusions Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service. PMID:22152063

  4. High-resolution melt analysis does not reveal mutagenic risk in sexed sperm and in vitro-derived bovine embryos.

    PubMed

    Pozzi, A; Previtali, C; Lukaj, A; Galli, A; Bongioni, G; Puglisi, R

    2014-08-01

    The objectives of the present work were to verify whether simultaneous exposure to Hoechst 33342 and UV irradiation during sorting by flow cytometry may induce gene point mutations in bovine sperm and to assess whether the dye incorporated in the sperm may imply a mutagenic effect during the embryonic development. To this aim, high-resolution melt analysis (HRMA) was used to discriminate variations of single nucleotides in sexed vs. non-sexed control samples. Three batches of sorted and non-sorted commercial semen of seven bulls (42 samples) were subjected to HRMA. A set of 139 genes located on all the chromosomes was selected, and 407 regions of the genome covering a total of 83 907 bases were analyzed. Thereafter, sperm of one sexed and one non-sexed batch of each bull was used in in vitro fertilization, and the derived embryos were analyzed (n = 560). One hundred and thirty-three regions of the bovine genome, located in 40 genes, were screened for a total coverage of 23 397 bases. The comparison between the frequencies of variations, with respect to the sequences deposited, observed in the sexed and non-sexed sperm (843 vs. 770) and embryos (246 vs. 212) showed no significant differences (P > 0.05), as measured by chi-square tests. It can be concluded that staining with Hoechst 33342 and exposure to UV during sorting does not lead to significant changes in the frequencies of variants in the commercial sexed semen and in embryos produced in vitro with the same treated sperm.

  5. Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae

    PubMed Central

    Donà, Valentina; Kasraian, Sara; Lupo, Agnese; Guilarte, Yuvia N.; Hauser, Christoph; Furrer, Hansjakob; Unemo, Magnus; Low, Nicola

    2016-01-01

    Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 103 to 104 genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing. PMID:27225407

  6. Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

    PubMed

    Donà, Valentina; Kasraian, Sara; Lupo, Agnese; Guilarte, Yuvia N; Hauser, Christoph; Furrer, Hansjakob; Unemo, Magnus; Low, Nicola; Endimiani, Andrea

    2016-08-01

    Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 10(3) to 10(4) genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing.

  7. High time-resolution photodetectors for PET applications

    DOE PAGES

    Ronzhin, Anatoly

    2016-02-01

    This paper describes recent developments aiming at the improvement of the time resolution of photodetectors used in positron emission tomography (PET). Promising photodetector candidates for future PET-time-of-flight (TOF) applications are also discussed.

  8. Sensitive detection and serovar differentiation of typhoidal and nontyphoidal Salmonella enterica species using 16S rRNA Gene PCR coupled with high-resolution melt analysis.

    PubMed

    Masek, Billie J; Hardick, Justin; Won, Helen; Yang, Samuel; Hsieh, Yu-Hsiang; Rothman, Richard E; Gaydos, Charlotte A

    2014-03-01

    Salmonella enterica species infections are a significant public health problem causing high morbidity rates worldwide and high mortality rates in the developing world. These infections are not always rapidly diagnosed as a cause of bloodstream infections because of the limitations of blood culture, which greatly affects clinical care as a result of treatment delays. A molecular diagnostic assay that could rapidly detect and identify S. enterica species infections as a cause of sepsis is needed. Nine typhoidal and nontyphoidal S. enterica serovars were used to establish the limit of detection (LOD) of a previously published 16S rRNA gene PCR (16S PCR) in mock whole blood specimens. In addition, 16 typhoidal and nontyphoidal S. enterica serovars were used to evaluate the serovar differentiation capability of 16S PCR coupled with high-resolution melt analysis. The overall LOD of 16S PCR for the nine typhoidal and nontyphoidal S. enterica serovars analyzed was <10 colony-forming units per milliliter (CFU/mL) in mock whole blood specimens, with the lowest and highest LOD at <1 CFU/mL and 9 CFU/mL, respectively. By high-resolution melt analysis, the typhoidal and nontyphoidal S. enterica serovar groups analyzed each generated a unique grouping code, allowing for serovar-level identification. 16S PCR coupled with high-resolution melt analysis could be a useful molecular diagnostic that could enhance the current diagnostic, treatment, and surveillance methods of S. enterica bloodstream infections.

  9. Structure and disorder in iron-bearing sodium silicate glasses and melts: High-resolution 29Si and 17O solid-state NMR study

    NASA Astrophysics Data System (ADS)

    Kim, H.; Lee, S.

    2012-12-01

    Understanding of the effect of iron content on the structure (Si coordination environment and the degree of polymerization) of iron-bearing silicate melts and glasses is essential for studying their macroscopic properties and diverse geological processes in Earth's interior. Although the recent advances in high-resolution solid-state NMR techniques provide detailed structural information of a diverse iron-free oxide glasses with varying composition (e.g., Lee, P. Natl. Acad. Sci. USA., 2011, 108, 6847; Lee and Sung, Chem. Geol., 2008, 256, 326; Park and Lee, Geochim. Cosmochim. Acta, 2012, 80, 125; Lee et al., Phys. Rev., 103, 095501, 2009), their application to iron-bearing silicate glasses has a limited usefulness in resolving atomic configurations due to the effect of paramagnetic cation (i.e., Fe) on the NMR spectra. Here, we report the first ^{29}Si and ^{17}O NMR spectra for sodium-iron silicate glasses with varying iron content (Na_{2}O-Fe_{2}O_{3}-SiO_{2} glasses, up to 34.60 wt% Fe_{2}O_{3}), revealing previously unknown details of iron-induced changes in structure and disorder. While signal intensity decreases and peak width increases exponentially with increasing iron content [=Fe_{2}O_{3}/(Na_{2}O+Fe_{2}O_{3})], ^{29}Si MAS NMR spectra for sodium-iron silicate glasses present the slight peak shift and an asymmetrical peak broadening toward higher Q^{n} species with increasing iron content. This result implies an increase in the degree of polymerization with increasing iron content. Additionally, ^{29}Si spin-relaxation time (T_{1}) for the glasses decreases with increasing of iron content by several orders of magnitude. ^{17}O 3QMAS NMR spectra for the glasses show well-resolved non-bridging oxygen (NBO, Na-O-Si) and bridging oxygen (BO, Si-O-Si) even at relatively high iron content, providing the first direct experimental estimation of the degree of polymerization. In sodium-iron silicate glasses, the fraction of NBO decreases with increasing iron

  10. Identification and Evaluation of Single-Nucleotide Polymorphisms in Allotetraploid Peanut (Arachis hypogaea L.) Based on Amplicon Sequencing Combined with High Resolution Melting (HRM) Analysis

    PubMed Central

    Hong, Yanbin; Pandey, Manish K.; Liu, Ying; Chen, Xiaoping; Liu, Hong; Varshney, Rajeev K.; Liang, Xuanqiang; Huang, Shangzhi

    2015-01-01

    The cultivated peanut (Arachis hypogaea L.) is an allotetraploid (AABB) species derived from the A-genome (Arachis duranensis) and B-genome (Arachis ipaensis) progenitors. Presence of two versions of a DNA sequence based on the two progenitor genomes poses a serious technical and analytical problem during single nucleotide polymorphism (SNP) marker identification and analysis. In this context, we have analyzed 200 amplicons derived from expressed sequence tags (ESTs) and genome survey sequences (GSS) to identify SNPs in a panel of genotypes consisting of 12 cultivated peanut varieties and two diploid progenitors representing the ancestral genomes. A total of 18 EST-SNPs and 44 genomic-SNPs were identified in 12 peanut varieties by aligning the sequence of A. hypogaea with diploid progenitors. The average frequency of sequence polymorphism was higher for genomic-SNPs than the EST-SNPs with one genomic-SNP every 1011 bp as compared to one EST-SNP every 2557 bp. In order to estimate the potential and further applicability of these identified SNPs, 96 peanut varieties were genotyped using high resolution melting (HRM) method. Polymorphism information content (PIC) values for EST-SNPs ranged between 0.021 and 0.413 with a mean of 0.172 in the set of peanut varieties, while genomic-SNPs ranged between 0.080 and 0.478 with a mean of 0.249. Total 33 SNPs were used for polymorphism detection among the parents and 10 selected lines from mapping population Y13Zh (Zhenzhuhei × Yueyou13). Of the total 33 SNPs, nine SNPs showed polymorphism in the mapping population Y13Zh, and seven SNPs were successfully mapped into five linkage groups. Our results showed that SNPs can be identified in allotetraploid peanut with high accuracy through amplicon sequencing and HRM assay. The identified SNPs were very informative and can be used for different genetic and breeding applications in peanut. PMID:26697032

  11. Identification and Evaluation of Single-Nucleotide Polymorphisms in Allotetraploid Peanut (Arachis hypogaea L.) Based on Amplicon Sequencing Combined with High Resolution Melting (HRM) Analysis.

    PubMed

    Hong, Yanbin; Pandey, Manish K; Liu, Ying; Chen, Xiaoping; Liu, Hong; Varshney, Rajeev K; Liang, Xuanqiang; Huang, Shangzhi

    2015-01-01

    The cultivated peanut (Arachis hypogaea L.) is an allotetraploid (AABB) species derived from the A-genome (Arachis duranensis) and B-genome (Arachis ipaensis) progenitors. Presence of two versions of a DNA sequence based on the two progenitor genomes poses a serious technical and analytical problem during single nucleotide polymorphism (SNP) marker identification and analysis. In this context, we have analyzed 200 amplicons derived from expressed sequence tags (ESTs) and genome survey sequences (GSS) to identify SNPs in a panel of genotypes consisting of 12 cultivated peanut varieties and two diploid progenitors representing the ancestral genomes. A total of 18 EST-SNPs and 44 genomic-SNPs were identified in 12 peanut varieties by aligning the sequence of A. hypogaea with diploid progenitors. The average frequency of sequence polymorphism was higher for genomic-SNPs than the EST-SNPs with one genomic-SNP every 1011 bp as compared to one EST-SNP every 2557 bp. In order to estimate the potential and further applicability of these identified SNPs, 96 peanut varieties were genotyped using high resolution melting (HRM) method. Polymorphism information content (PIC) values for EST-SNPs ranged between 0.021 and 0.413 with a mean of 0.172 in the set of peanut varieties, while genomic-SNPs ranged between 0.080 and 0.478 with a mean of 0.249. Total 33 SNPs were used for polymorphism detection among the parents and 10 selected lines from mapping population Y13Zh (Zhenzhuhei × Yueyou13). Of the total 33 SNPs, nine SNPs showed polymorphism in the mapping population Y13Zh, and seven SNPs were successfully mapped into five linkage groups. Our results showed that SNPs can be identified in allotetraploid peanut with high accuracy through amplicon sequencing and HRM assay. The identified SNPs were very informative and can be used for different genetic and breeding applications in peanut.

  12. Application of Markov Chain Monte Carlo Method to Mantle Melting: An Example from REE Abundances in Abyssal Peridotites

    NASA Astrophysics Data System (ADS)

    LIU, B.; Liang, Y.

    2015-12-01

    Markov chain Monte Carlo (MCMC) simulation is a powerful statistical method in solving inverse problems that arise from a wide range of applications, such as nuclear physics, computational biology, financial engineering, among others. In Earth sciences applications of MCMC are primarily in the field of geophysics [1]. The purpose of this study is to introduce MCMC to geochemical inverse problems related to trace element fractionation during concurrent melting, melt transport and melt-rock reaction in the mantle. MCMC method has several advantages over linearized least squares methods in inverting trace element patterns in basalts and mantle rocks. First, MCMC can handle equations that have no explicit analytical solutions which are required by linearized least squares methods for gradient calculation. Second, MCMC converges to global minimum while linearized least squares methods may be stuck at a local minimum or converge slowly due to nonlinearity. Furthermore, MCMC can provide insight into uncertainties of model parameters with non-normal trade-off. We use MCMC to invert for extent of melting, amount of trapped melt, and extent of chemical disequilibrium between the melt and residual solid from REE data in abyssal peridotites from Central Indian Ridge and Mid-Atlantic Ridge. In the first step, we conduct forward calculation of REE evolution with melting models in a reasonable model space. We then build up a chain of melting models according to Metropolis-Hastings algorithm to represent the probability of specific model. We show that chemical disequilibrium is likely to play an important role in fractionating LREE in residual peridotites. In the future, MCMC will be applied to more realistic but also more complicated melting models in which partition coefficients, diffusion coefficients, as well as melting and melt suction rates vary as functions of temperature, pressure and mineral compositions. [1]. Sambridge & Mosegarrd [2002] Rev. Geophys.

  13. Programmable resolution imager for imaging applications

    NASA Astrophysics Data System (ADS)

    Roca, Elisenda; Soriano, German; Espejo, Servando; Dominguez-Castro, Rafael; Linan, Gustavo; Rodriguez-Vazquez, Angel

    2000-05-01

    In this paper a programmable imager with averaging capabilities will be described which is intended for averaging of different groups or sets of pixels formed by n X n kernels, n X m kernels or any group of randomly- selected pixels across the array. This imager is a 64 X 64 array which uses passive pixels with electronic shutter and anti-blooming structure that can be randomly accessed. The read-out stage includes a sole charge amplifier with programmable gain, a sample-and-hold structure and an analog buffer. This read-out structure is different from other existing imagers with variable resolution since it uses a sole charge amplifier, whereas the conventional structure employs an opamp per column plus another global opamp. this architecture allows a reduction of the fixed-pattern noise observed in standard imagers. The prototype also includes an analog to digital converter which provides the digital output of the images.

  14. Diagnosis of ABCB11 gene mutations in children with intrahepatic cholestasis using high resolution melting analysis and direct sequencing

    PubMed Central

    HU, GUORUI; HE, PING; LIU, ZHIFENG; CHEN, QIAN; ZHENG, BIXIA; ZHANG, QIHUA

    2014-01-01

    Intrahepatic cholestasis represents a heterogeneous group of disorders that begin during childhood, most commonly manifesting as neonatal cholestasis, and lead to ongoing liver dysfunction in children and adults. For children, inherited pathogenic factors of cholestasis have gained increasing attention owing to the rapid development of molecular biology technology. However, these methods have their advantages and disadvantages in terms of simplicity, sensitivity, specificity, time required and expense. In the present study, an effective, sensitive and economical method is recommended, termed high-resolution melting (HRM) analysis and direct sequencing, based on general polymerase chain reaction, to detect mutations in disease-causing genes. As one type of inherited intrahepatic cholestasis, progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by pathogenic mutations in the ABCB11 gene, HRM was used to detect mutations in the ABCB11 gene in the present study, and the diagnosis for PFIC2 was made by comprehensive analysis of genetic findings and clinical features. Furthermore, the characteristics of mutations and single nucleotide polymorphisms (SNPs) in the ABCB11 gene were elucidated. A total of 14 types of mutations/polymorphisms were identified in 20 patients from mainland China, including six missense mutations (p.Y337H, p.Y472C, p.R696W, p.Q931P, p.D1131V and p.H1198R), one nonsense mutation (p.R928X) and seven SNPs (p.D36D/rs3815675, p.F90F/rs4148777, p.Y269Y/rs2287616, p.I416I/rs183390670, p.V444A/rs2287622, p.A865V/rs118109635 and p.A1028A/rs497692). Five mutations were novel. The majority of the mutations were different from those detected in other population groups. A total of 4/20 patients (1/5) were diagnosed to be PFIC2 by combining genetic findings with the clinical features. Polymorphisms V444A and A1028A, with an allele frequency of 74.5 and 67.2%, respectively, were highly prevalent in the mainland Chinese subjects. No differences

  15. Comparative study of IDH1 mutations in gliomas by high resolution melting analysis, immunohistochemistry and direct DNA sequencing.

    PubMed

    Li, Juan; Zhang, Haiyan; Wang, Li; Yang, Chuanhong; Lai, Huangwen; Zhang, Wei; Chen, Xiaodong; Wang, Jie

    2015-09-01

    Patients with glioblastomas with a specific mutation in the isocitrate dehydrogenase 1 (IDH1) gene have a better prognosis than those with gliomas with wild‑type IDH1. IDH1 analysis has become part of the standard diagnostic procedure and a promising tool used for stratification in clinical trials. The present study aimed to compare high resolution melting (HRM) analysis, immunohistochemistry (IHC) and direct DNA sequencing for the detection of IDH mutations in gliomas. Fifty‑one formalin‑fixed paraffin‑embedded tumor samples were selected. For the HRM analysis and direct DNA sequencing, DNA was extracted from the tissues. For IHC, sections were stained with an anti‑IDH1‑R132H specific antibody. The HRM analysis method identified 33 cases of IDH1 gene mutations, and all mutations occurred at the R132H site. There were 33 cases of IDH1 gene mutations found by IHC, which was consistent with that identified using the HRM analysis method. However, only 30 IDH1 samples were confirmed by sequencing, in which mutations occurred at the IDH1 exon 4 R132H site. No mutation was detected in the other three of these 33 cases (two grade II oligodendroglioma and one grade II diffuse astrocytoma) by sequencing, while IHC was positive for IDH1‑R132H. The results showed that the mutation detection rate was not identified to be significantly different (P=0.250) when determined by the HRM analysis method or by direct DNA sequencing, as the concordant rate between the two methods was high (κ=0.866). The HRM analysis method in glioma IDH1 gene mutation detection has advantages of high sensitivity, good repeatability, simple operation and accurate results. It provides a novel method for detecting mutations of the IDH1 gene in paraffin embedded tissue samples of clinical glioma. Related to a small amount of sample, there was no evidence showing that HRM analysis method is superior to IHC. Direct DNA sequencing, HRM analysis and IHC results were consistent; however, HRM and

  16. Littrow spectrographs for moderate resolution infrared applications

    NASA Astrophysics Data System (ADS)

    Warren, David W.; Lampen, Sara

    2016-09-01

    The Littrow form of spectrograph has a long and storied history in astronomical spectroscopy since its presentation in 1862 by Otto von Littrow. Light from an input slit traverses the same optical elements in reaching the dispersing element (prism or grating) and returning to a focused, dispersed image at the focal plane. This 1:1 symmetry helps cancel aberrations in the reimaging optics while presenting the dispersing element with the geometry most favorable to dispersion, efficiency and anamorphic scale change. Historically, Littrow spectrographs have not been pushed to high throughputs (fast f/ratios). However in the short- and mid-wave infrared particularly, high index, low dispersion materials like silicon and germanium can be combined effectively into compact, high throughput (resolution spectrographic space missions such as atmospheric sounders. We present some high throughput Littrow spectrograph concepts designed for infrared atmospheric sounding missions and incorporating both plane and immersion gratings.

  17. High spatial resolution probes for neurobiology applications

    NASA Astrophysics Data System (ADS)

    Gunning, D. E.; Kenney, C. J.; Litke, A. M.; Mathieson, K.

    2009-06-01

    Position-sensitive biological neural networks, such as the brain and the retina, require position-sensitive detection methods to identify, map and study their behavior. Traditionally, planar microelectrodes have been employed to record the cell's electrical activity with device limitations arising from the electrode's 2-D nature. Described here is the development and characterization of an array of electrically conductive micro-needles aimed at addressing the limitations of planar electrodes. The capability of this array to penetrate neural tissue improves the electrode-cell electrical interface and allows more complicated 3-D networks of neurons, such as those found in brain slices, to be studied. State-of-the-art semiconductor fabrication techniques were used to etch and passivate conformally the metal coat and fill high aspect ratio holes in silicon. These are subsequently transformed into needles with conductive tips. This process has enabled the fabrication of arrays of unprecedented dimensions: 61 hexagonally close-packed electrodes, ˜200 μm tall with 60 μm spacing. Electroplating the tungsten tips with platinum ensure suitable impedance values (˜600 kΩ at 1 kHz) for the recording of neuronal signals. Without compromising spatial resolution of the neuronal recordings, this array adds a new and exciting dimension to the study of biological neural networks.

  18. High-resolution melting analysis (HRM) for differentiation of four major Taeniidae species in dogs Taenia hydatigena, Taenia multiceps, Taenia ovis, and Echinococcus granulosus sensu stricto.

    PubMed

    Dehghani, Mansoureh; Mohammadi, Mohammad Ali; Rostami, Sima; Shamsaddini, Saeedeh; Mirbadie, Seyed Reza; Harandi, Majid Fasihi

    2016-07-01

    Tapeworms of the genus Taenia include several species of important parasites with considerable medical and veterinary significance. Accurate identification of these species in dogs is the prerequisite of any prevention and control program. Here, we have applied an efficient method for differentiating four major Taeniid species in dogs, i.e., Taenia hydatigena, T. multiceps, T. ovis, and Echinococcus granulosus sensu stricto. High-resolution melting (HRM) analysis is simpler, less expensive, and faster technique than conventional DNA-based assays and enables us to detect PCR amplicons in a closed system. Metacestode samples were collected from local abattoirs from sheep. All the isolates had already been identified by PCR-sequencing, and their sequence data were deposited in the GenBank. Real-time PCR coupled with HRM analysis targeting mitochondrial cox1 and ITS1 genes was used to differentiate taeniid species. Distinct melting curves were obtained from ITS1 region enabling accurate differentiation of three Taenia species and E. granulosus in dogs. The HRM curves of Taenia species and E .granulosus were clearly separated at Tm of 85 to 87 °C. In addition, double-pick melting curves were produced in mixed infections. Cox1 melting curves were not decisive enough to distinguish four taeniids. In this work, the efficiency of HRM analysis to differentiate four major taeniid species in dogs has been demonstrated using ITS1 gene.

  19. A real-time ARMS PCR/high-resolution melt curve assay for the detection of the three primary mitochondrial mutations in Leber’s hereditary optic neuropathy

    PubMed Central

    Ryan, Fergus; O’Dwyer, Veronica; Neylan, Derek

    2016-01-01

    Purpose Approximately 95% of patients who are diagnosed with Leber’s hereditary optic neuropathy (LHON) have one of three mitochondrial point mutations responsible for the disease, G3460A, G11778A, and T14484C. The purpose of this study was to develop a novel multiplex real-time amplification-refractory mutation system (ARMS) PCR combined with high-resolution melt curves to identify the individual mutations involved. The study aimed to provide a more robust, cost- and time-effective mutation detection strategy than that offered with currently available methods. The assay reported in this study will allow diagnostic laboratories to avoid costly next-generation sequencing (NGS) assays for most patients with LHON and to focus resources on patients with unknown mutations that require further analysis. Methods The test uses a combination of multiplex allele-specific PCR (ARMS PCR) in combination with a high-resolution melt curve analysis to detect the presence of the mutations in G3460A, G11778A, and T14484C. PCR primer sets were designed to produce a control PCR product and PCR products only in the presence of the mutations in 3460A, 11778A, and 14484C in a multiplex single tube format. Products produce discrete well-separated melt curves to clearly detect the mutations. Results This novel real-time ARMS PCR/high-resolution melt curve assay accurately detected 95% of the mutations that cause LHON. The test has proved to be robust, cost- and time-effective with the real-time closed tube system taking approximately 1 h to complete. Conclusions A novel real-time ARMS PCR/high-resolution melt curve assay is described for the detection of the three primary mitochondrial mutations in LHON. This test provides a simple, robust, easy-to-read output that is cost- and time-effective, thus providing an alternative method to individual endpoint PCR-restriction fragment length polymorphism (RFLP), PCR followed by Sanger sequencing or pyrosequencing, and next-generation sequencing

  20. Melt-processed bulk superconductors: Fabrication and characterization for power and space applications

    NASA Technical Reports Server (NTRS)

    Hojaji, Hamid; Barkatt, Aaron; Hu, Shouxiang; Thorpe, Arthur N.; Ware, Matthew F.; Davis, David; Alterescu, Sidney

    1991-01-01

    Melt-process bulk superconducting materials based on variations on the base YBa2Cu3O(x) were produced in a variety of shapes and forms. Very high values of both zero-field and high-field magnetization were observed. These are useful for levitation and power applications. Magnetic measurements show that the effects of field direction and intensity, temperature and time are consistent with an aligned grain structure with multiple pinning sites and with models of thermally activated flux motion.

  1. Detection of plant oil DNA using high resolution melting (HRM) post PCR analysis: a tool for disclosure of olive oil adulteration.

    PubMed

    Vietina, Michelangelo; Agrimonti, Caterina; Marmiroli, Nelson

    2013-12-15

    Extra virgin olive oil is frequently subjected to adulterations with addition of oils obtained from plants other than olive. DNA analysis is a fast and economic tool to identify plant components in oils. Extraction and amplification of DNA by PCR was tested in olives, in milled seeds and in oils, to investigate its use in olive oil traceability. DNA was extracted from different oils made of hazelnut, maize, sunflower, peanut, sesame, soybean, rice and pumpkin. Comparing the DNA melting profiles in reference plant materials and in the oils, it was possible to identify any plant components in oils and mixtures of oils. Real-Time PCR (RT-PCR) platform has been added of the new methodology of high resolution melting (HRM), both were used to analyse olive oils mixed with different percentage of other oils. Results showed HRM a cost effective method for efficient detection of adulterations in olive oils.

  2. Detection and identification of vegetative insecticidal proteins vip3 genes of Bacillus thuringiensis strains using polymerase chain reaction-high resolution melt analysis.

    PubMed

    Li, Haitao; Shu, Changlong; He, Xiaoming; Gao, JiGuo; Liu, Rongmei; Huang, Dafang

    2012-05-01

    In this study, vegetative insecticidal proteins vip3 genes from Bacillus thuringiensis strains were detected based on polymerase chain reaction-high resolution melt (PCR-HRM) analysis. A pair of primers was designed according to the conservative sequences in 150 bp region of the known vip3 subfamily. The 150 bp regions of difference vip3 genes have only a few nucleotide difference vip3 genes were detected in 8 of 11 standard B. thuringiensis strains, and vip3Aa genes, vip3Af genes and vip3Ba gene can be distinguished as different melting curves by this method. The results demonstrate the utility of the HRM assay for mutant screening using vip3 gene. The PCR-HRM method may be a valuable and reliable tool for specific detection and identification of vip3 genes.

  3. Hybrid analysis (barcode-high resolution melting) for authentication of Thai herbal products, Andrographis paniculata (Burm.f.) Wall.ex Nees

    PubMed Central

    Osathanunkul, Maslin; Suwannapoom, Chatmongkon; Khamyong, Nuttaluck; Pintakum, Danupol; Lamphun, Santisuk Na; Triwitayakorn, Kanokporn; Osathanunkul, Kitisak; Madesis, Panagiotis

    2016-01-01

    Background: Andrographis paniculata Nees is a medicinal plant with multiple pharmacological properties. It has been used over many centuries as a household remedy. A. paniculata products sold on the markets are in processed forms so it is difficult to authenticate. Therefore buying the herbal products poses a high-risk of acquiring counterfeited, substituted and/or adulterated products. Due to these issues, a reliable method to authenticate products is needed. Materials and Methods: High resolution melting analysis coupled with DNA barcoding (Bar-HRM) was applied to detect adulteration in commercial herbal products. The rbcL barcode was selected to use in primers design for HRM analysis to produce standard melting profile of A. paniculata species. DNA of the tested commercial products was isolated and their melting profiles were then generated and compared with the standard A. paniculata. Results: The melting profiles of the rbcL amplicons of the three closely related herbal species (A. paniculata, Acanthus ebracteatus and Rhinacanthus nasutus) are clearly separated so that they can be distinguished by the developed method. The method was then used to authenticate commercial herbal products. HRM curves of all 10 samples tested are similar to A. paniculata which indicated that all tested products were contained the correct species as labeled. Conclusion: The method described in this study has been proved to be useful in aiding identification and/or authenticating A. paniculata. This Bar-HRM analysis has allowed us easily to determine the A. paniculata species in herbal products on the markets even they are in processed forms. SUMMARY We propose the use of DNA barcoding combined with High Resolution Melting analysis for authenticating of Andrographis paniculata products.The developed method can be used regardless of the type of the DNA template (fresh or dried tissue, leaf, and stem).rbcL region was chosen for the analysis and work well with our samplesWe can easily

  4. Stratigraphic analysis of an ice core from the Prince of Wales Icefield, Ellesmere Island, Arctic Canada, using digital image analysis: High-resolution density, past summer warmth reconstruction, and melt effect on ice core solid conductivity

    NASA Astrophysics Data System (ADS)

    Kinnard, Christophe; Koerner, Roy M.; Zdanowicz, Christian M.; Fisher, David A.; Zheng, Jiancheng; Sharp, Martin J.; Nicholson, Lindsey; Lauriol, Bernard

    2008-12-01

    High-resolution (1 mm) stratigraphic information was derived from digital image analysis of an ice core from the Prince of Wales (POW) Icefield, Central Ellesmere Island, Canada. Following careful image processing, a profile of ice core transmitted light was derived from the greyscale images and used to reconstruct high-resolution density variations for the unfractured sections of the core. Images were further classified into infiltration and glacier ice using an automatic thresholding procedure, and were converted to a high-resolution melt percentage index. The mean annual melt percentage over the last 580 years was 9%, and melting occurred in 8 years out of 10. Melting obliterated most of the original depositional sequence, and seasonal density cycles were mostly unrecognizable. The ice core solid conductivity was greater and more variable in melt features than in glacier ice, owing to washout of strong acids by meltwater (elution) and chemical enrichment upon refreezing. This hindered the identification of acid volcanic layers and further compromised dating by annual layer counting. Comparison of the melt record with those from other Arctic ice caps shows that the melt-temperature relationship on POW Icefield is site-specific. We speculate that this is due to the peculiar position of the icefield, which rests on the periphery of the Baffin Bay maritime climate zone, and to the proximity of the North Open Water polynya, which controls snow accumulation variability on the icefield and affects the melt percentage index.

  5. Rapid and accurate typing of Bordetella pertussis targeting genes encoding acellular vaccine antigens using real time PCR and High Resolution Melt analysis.

    PubMed

    Chan, Wai-Fong; Maharjan, Ram P; Reeves, Peter R; Sintchenko, Vitali; Gilbert, Gwendolyn L; Lan, Ruiting

    2009-06-01

    Real Time-PCR (RT-PCR) and high resolution melt (HRM) analyses were used for rapid typing of genes encoding components of the pertussis acellular vaccine, namely prn, ptxA, fhaB, fim2 and fim3. The length polymorphisms in prn were detected by RT-PCR followed by HRM; single nucleotide polymorphisms in prn and other genes were detected by hairpin primer RT-PCR. These rapid methods are suitable for large-scale studies of vaccine-driven evolution of Bordetella pertussis.

  6. Additive manufacturing of 316L stainless steel by electron beam melting for nuclear fusion applications

    NASA Astrophysics Data System (ADS)

    Zhong, Yuan; Rännar, Lars-Erik; Liu, Leifeng; Koptyug, Andrey; Wikman, Stefan; Olsen, Jon; Cui, Daqing; Shen, Zhijian

    2017-04-01

    A feasibility study was performed to fabricate ITER In-Vessel components by one of the metal additive manufacturing methods, Electron Beam Melting® (EBM®). Solid specimens of SS316L with 99.8% relative density were prepared from gas atomized precursor powder granules. After the EBM® process the phase remains as austenite and the composition has practically not been changed. The RCC-MR code used for nuclear pressure vessels provides guidelines for this study and tensile tests and Charpy-V tests were carried out at 22 °C (RT) and 250 °C (ET). This work provides the first set of mechanical and microstructure data of EBM® SS316L for nuclear fusion applications. The mechanical testing shows that the yield strength, ductility and toughness are well above the acceptance criteria and only the ultimate tensile strength of EBM® SS316L is below the RCC-MR code. Microstructure characterizations reveal the presence of hierarchical structures consisting of solidified melt pools, columnar grains and irregular shaped sub-grains. Lots of precipitates enriched in Cr and Mo are observed at columnar grain boundaries while no sign of element segregation is shown at the sub-grain boundaries. Such a unique microstructure forms during a non-equilibrium process, comprising rapid solidification and a gradient 'annealing' process due to anisotropic thermal flow of accumulated heat inside the powder granule matrix. Relations between process parameters, specimen geometry (total building time) and sub-grain structure are discussed. Defects are formed mainly due to the large layer thickness (100 μm) which generates insufficient bonding between a few of the adjacently formed melt pools during the process. Further studies should focus on adjusting layer thickness to improve the strength of EBM® SS316L and optimizing total building time.

  7. Hot-melt extrusion of polyvinyl alcohol for oral immediate release applications.

    PubMed

    De Jaeghere, W; De Beer, T; Van Bocxlaer, J; Remon, J P; Vervaet, C

    2015-08-15

    The primary purpose of this study was to process partially hydrolyzed PVOH grades (degree of hydroxylation (DH): 33-88%) via HME and to evaluate them as carrier for oral immediate release dosage forms in order to improve the release rate of poorly water soluble drugs (i.e., HCT and CEL) via the formulation of solid dispersions. PVOH grades (DH >70%) were able to solubilize HCT and CEL up to 15%, but required higher extrusion temperature, due to the crystalline nature of PVOH. The highest drug release rate was observed from hot-melt extruded PVOH samples with a high DH. While drug release from extrudates consisting of PVOH with a low DH was affected by ionic strength, there was no influence of pH and ionic strength on HCT release from PVOH samples with a higher DH. However, PVOH (DH >70%) required higher extrusion temperatures, which could hamper its application for thermosensitive drugs. Therefore, the secondary purpose was to investigate the effect of sorbitol, a water-soluble plasticizer, on the thermal properties of hot-melt extruded PVOH (DH >70%). The melting of PVOH/sorbitol mixture was required to establish molecular interactions between PVOH and sorbitol. These molecular interactions were reflected in the HME behavior: whereas an extrusion temperature of 180 °C was necessary to process physical mixtures of PVOH (DH >70%) and sorbitol, only 140 °C was necessary during re-extrusion (after quench cooling and cryomilling) of the PVOH/sorbitol mixture. In addition, the in vitro and in vivo dug release of plasticized PVOH was examined; whereas the CEL/PVO/sorbitol system was able to maintain supersaturation during in vitro dissolution (0.1N HCl) compared to Celebrex(®), the in vivo pharmacokinetic parameters (AUC0-24h, Cmax and Tmax) were highly comparable.

  8. Rapid detection and identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in mosquito vectors and blood samples by high resolution melting real-time PCR.

    PubMed

    Thanchomnang, Tongjit; Intapan, Pewpan M; Tantrawatpan, Chairat; Lulitanond, Viraphong; Chungpivat, Sudchit; Taweethavonsawat, Piyanan; Kaewkong, Worasak; Sanpool, Oranuch; Janwan, Penchom; Choochote, Wej; Maleewong, Wanchai

    2013-12-01

    A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

  9. Rapid Detection and Identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in Mosquito Vectors and Blood Samples by High Resolution Melting Real-Time PCR

    PubMed Central

    Thanchomnang, Tongjit; Intapan, Pewpan M.; Tantrawatpan, Chairat; Lulitanond, Viraphong; Chungpivat, Sudchit; Taweethavonsawat, Piyanan; Kaewkong, Worasak; Sanpool, Oranuch; Janwan, Penchom; Choochote, Wej

    2013-01-01

    A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors. PMID:24516268

  10. Hot-Melt Extrusion: from Theory to Application in Pharmaceutical Formulation.

    PubMed

    Patil, Hemlata; Tiwari, Roshan V; Repka, Michael A

    2016-02-01

    Hot-melt extrusion (HME) is a promising technology for the production of new chemical entities in the developmental pipeline and for improving products already on the market. In drug discovery and development, industry estimates that more than 50% of active pharmaceutical ingredients currently used belong to the biopharmaceutical classification system II (BCS class II), which are characterized as poorly water-soluble compounds and result in formulations with low bioavailability. Therefore, there is a critical need for the pharmaceutical industry to develop formulations that will enhance the solubility and ultimately the bioavailability of these compounds. HME technology also offers an opportunity to earn intellectual property, which is evident from an increasing number of patents and publications that have included it as a novel pharmaceutical formulation technology over the past decades. This review had a threefold objective. First, it sought to provide an overview of HME principles and present detailed engineered extrusion equipment designs. Second, it included a number of published reports on the application of HME techniques that covered the fields of solid dispersions, microencapsulation, taste masking, targeted drug delivery systems, sustained release, films, nanotechnology, floating drug delivery systems, implants, and continuous manufacturing using the wet granulation process. Lastly, this review discussed the importance of using the quality by design approach in drug development, evaluated the process analytical technology used in pharmaceutical HME monitoring and control, discussed techniques used in HME, and emphasized the potential for monitoring and controlling hot-melt technology.

  11. Improved group contribution parameter set for the application of solubility parameters to melt extrusion.

    PubMed

    Just, Susann; Sievert, Frank; Thommes, Markus; Breitkreutz, Jörg

    2013-11-01

    Hot-melt extrusion is gaining importance for the production of amorphous solid solutions; in parallel, predictive tools for estimating drug solubility in polymers are increasingly demanded. The Hansen solubility parameter (SP) approach is well acknowledged for its predictive power of the miscibility of liquids as well as the solubility of some amorphous solids in liquid solvents. By solely using the molecular structure, group contribution (GC) methods allow the calculation of Hansen SPs. The GC parameter sets available were derived from liquids and polymers which conflicts with the object of prediction, the solubility of solid drugs. The present study takes a step from the liquid based SPs toward their application to solid solutes. On the basis of published experimental Hansen SPs of solid drugs and excipients only, a new GC parameter set was developed. In comparison with established parameter sets by van Krevelen/Hoftyzer, Beerbower/Hansen, Breitkreutz and Stefanis/Panayiotou, the new GC parameter set provides the highest overall predictive power for solubility experiments (correlation coefficient r = -0.87 to -0.91) as well as for literature data on melt extrudates and casted films (r = -0.78 to -0.96).

  12. High-resolution melting analysis for detection of a single-nucleotide polymorphism and the genotype of the myostatin gene in warmblood horses.

    PubMed

    Serpa, Priscila B S; Garbade, Petra; Natalini, Cláudio C; Pires, Ananda R; Tisotti, Tainor M

    2017-01-01

    OBJECTIVE To develop a high-resolution melting (HRM) assay to detect the g.66493737C>T polymorphism in the myostatin gene (MSTN) and determine the frequency of 3 previously defined g.66493737 genotypes (T/T, T/C, and C/C) in warmblood horses. SAMPLES Blood samples from 23 horses. PROCEDURES From each blood sample, DNA was extracted and analyzed by standard PCR methods and an HRM assay to determine the MSTN genotype. Three protocols (standard protocol, protocol in which a high-salt solution was added to the reaction mixture before the first melting cycle, and protocol in which an unlabeled probe was added to the reaction mixture before analysis) for the HRM assay were designed and compared. Genotype results determined by the HRM protocol that generated the most consistent melting curves were compared with those determined by sequencing. RESULTS The HRM protocol in which an unlabeled probe was added to the reaction mixture generated the most consistent melting curves. The genotypes of the g.66493737C>T polymorphism were determined for 22 horses (16 by HRM analysis and 20 by sequencing); 14, 7, and 1 had the T/T, T/C, and C/C genotypes, respectively. The genotype determined by HRM analysis agreed with that determined by sequencing for 14 of 16 horses. The frequency of alleles T and C was 79.5% and 20.5%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that HRM analysis may be a faster and more economical alternative than PCR methods for genotyping. Genotyping results might be useful as predictors of athletic performance for horses.

  13. Rapid detection and statistical differentiation of KPC gene variants in Gram-negative pathogens by use of high-resolution melting and ScreenClust analyses.

    PubMed

    Roth, Amanda L; Hanson, Nancy D

    2013-01-01

    In the United States, the production of the Klebsiella pneumoniae carbapenemase (KPC) is an important mechanism of carbapenem resistance in Gram-negative pathogens. Infections with KPC-producing organisms are associated with increased morbidity and mortality; therefore, the rapid detection of KPC-producing pathogens is critical in patient care and infection control. We developed a real-time PCR assay complemented with traditional high-resolution melting (HRM) analysis, as well as statistically based genotyping, using the Rotor-Gene ScreenClust HRM software to both detect the presence of bla(KPC) and differentiate between KPC-2-like and KPC-3-like alleles. A total of 166 clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii with various β-lactamase susceptibility patterns were tested in the validation of this assay; 66 of these organisms were known to produce the KPC β-lactamase. The real-time PCR assay was able to detect the presence of bla(KPC) in all 66 of these clinical isolates (100% sensitivity and specificity). HRM analysis demonstrated that 26 had KPC-2-like melting peak temperatures, while 40 had KPC-3-like melting peak temperatures. Sequencing of 21 amplified products confirmed the melting peak results, with 9 isolates carrying bla(KPC-2) and 12 isolates carrying bla(KPC-3). This PCR/HRM assay can identify KPC-producing Gram-negative pathogens in as little as 3 h after isolation of pure colonies and does not require post-PCR sample manipulation for HRM analysis, and ScreenClust analysis easily distinguishes bla(KPC-2-like) and bla(KPC-3-like) alleles. Therefore, this assay is a rapid method to identify the presence of bla(KPC) enzymes in Gram-negative pathogens that can be easily integrated into busy clinical microbiology laboratories.

  14. Simultaneous detection and quantitation of Chikungunya, dengue and West Nile viruses by multiplex RT-PCR assays and dengue virus typing using high resolution melting.

    PubMed

    Naze, F; Le Roux, K; Schuffenecker, I; Zeller, H; Staikowsky, F; Grivard, P; Michault, A; Laurent, P

    2009-12-01

    Chikungunya (CHIKV), Dengue (DENV) and West Nile (WNV) viruses are arthropod-borne viruses that are able to emerge or re-emerge in many regions due to climatic changes and increase in travel. Since these viruses produce similar clinical signs it is important for physicians and epidemiologists to differentiate them rapidly. A molecular method was developed for their detection and quantitation in plasma samples and a DENV typing technique were developed. The method consisted in performing two multiplex real-time one-step RT-PCR assays, to detect and quantify the three viruses. Both assays were conducted in a single run, from a single RNA extract containing a unique coextracted and coamplified composite internal control. The quantitation results were close to the best detection thresholds obtained with simplex RT-PCR techniques. The differentiation of DENV types was performed using a High Resolution Melting technique. The assays enable the early diagnosis of the three arboviruses during viremia, including cases of coinfection. The method is rapid, specific and highly sensitive with a potential for clinical diagnosis and epidemiological surveillance. A DENV positive sample can be typed conveniently using the High Resolution Melting technique using the same apparatus.

  15. High-resolution melt curve analysis to confirm the presence of co-circulating isolates of avian nephritis virus in commercial chicken flocks.

    PubMed

    Chamings, A; Hewson, K A; O'Rourke, D; Ignjatovic, J; Noormohammadi, A H

    2015-01-01

    Avian Nephritis Virus (ANV) has been implicated in poor growth and renal disease of young chickens. This paper describes the development of a reverse-transcriptase polymerase chain reaction for the detection of ANV in commercial meat chickens and the use of high-resolution melt curves to detect the presence of genetically different ANVs. Pooled cloacal swabs from both healthy and ill commercial chicken broiler flocks were tested for the presence of ANV using a combination of polymerase chain reaction, molecular cloning, high-resolution melt curve analysis and sequencing. Except for one, all specimens were found to contain two genetically different ANVs. Phylogenetic analysis of the capsid amino acid sequences revealed the presence of four of six groups of ANV identified previously in other countries as well as in two novel groups of ANV. Phylogenetic analysis of nucleotide sequences of partial polymerase, capsid and 3' untranslated regions reveal that the genes of individual ANV virus isolates have different ancestors. This was shown to be due to a template-switching event in the capsid gene that resulted in the 3' end of the capsid gene and the 3' untranslated region of one ANV isolate being transferred to another ANV. These results reveal that infection of chicken flocks with multiple ANV isolates is common and this needs to be taken into consideration in diagnosis of ANV using molecular techniques and in future epidemiological investigations.

  16. Application of hot melt extrusion for poorly water-soluble drugs: limitations, advances and future prospects.

    PubMed

    Lu, Ming; Guo, Zhefei; Li, Yongcheng; Pang, Huishi; Lin, Ling; Liu, Xu; Pan, Xin; Wu, Chuanbin

    2014-01-01

    Hot melt extrusion (HME) is a powerful technology to enhance the solubility and bioavailability of poorly water-soluble drugs by producing amorphous solid dispersions. Although the number of articles and patents about HME increased dramatically in the past twenty years, there are very few commercial products by far. The three main obstacles limiting the commercial application of HME are summarized as thermal degradation of heat-sensitive drugs at high process temperature, recrystallization of amorphous drugs during storage and dissolving process, and difficulty to obtain products with reproducible physicochemical properties. Many efforts have been taken in recent years to understand the basic mechanism underlying these obstacles and then to overcome them. This article reviewed and summarized the limitations, recent advances, and future prospects of HME.

  17. Polymeric formulations for drug release prepared by hot melt extrusion: application and characterization.

    PubMed

    Stanković, Milica; Frijlink, Henderik W; Hinrichs, Wouter L J

    2015-07-01

    Over the past few decades hot melt extrusion (HME) has emerged as a powerful processing technology for the production of pharmaceutical solid dosage forms in which an active pharmaceutical ingredient (API) is dispersed into polymer matrices. It has been shown that formulations using HME can provide time-controlled, sustained and targeted drug delivery, and improved bioavailability of poorly soluble drugs. In this review, the basic principles of the HME process are described together with an overview of some of the most common biodegradable and nonbiodegradable polymers used for the preparation of different formulations using this method. Further, the applications of HME in drug delivery and analytical techniques employed to characterize HME products are addressed.

  18. Amundsen Sea sector ice shelf thickness, melt rates, and inland response from annual high-resolution DEM mosaics

    NASA Astrophysics Data System (ADS)

    Shean, D. E.; Joughin, I. R.; Smith, B. E.; Alexandrov, O.; Moratto, Z.; Porter, C. C.; Morin, P. J.

    2014-12-01

    Significant grounding line retreat, acceleration, and thinning have occurred along the Amundsen Sea sector of West Antarctica in recent decades. These changes are driven primarily by ice-ocean interaction beneath ice shelves, but existing observations of the spatial distribution, timing, and magnitude of ice shelf melt are limited. Using the NASA Ames Stereo Pipeline, we generated digital elevation models (DEMs) with ~2 m posting from all ~450 available WorldView-1/2 along-track stereopairs for the Amundsen Sea sector. A novel iterative closest point algorithm was used to coregister DEMs to filtered Operation IceBridge ATM/LVIS data and ICESat-1 GLAS data, offering optimal sub-meter horizontal/vertical accuracy. The corrected DEMs were used to produce annual mosaics for the entire ~500x700 km region with focused, sub-annual products for ice shelves and grounding zones. These mosaics provide spatially-continuous measurements of ice shelf topography with unprecedented detail. Using these data, we derive estimates of ice shelf thickness for regions in hydrostatic equilibrium and map networks of sub-shelf melt channels for the Pine Island (PIG), Thwaites, Crosson, and Dotson ice shelves. We also document the break-up of the Thwaites ice shelf and PIG rift evolution leading up to the 2013 calving event. Eulerian difference maps document 2010-2014 thinning over fast-flowing ice streams and adjacent grounded ice. These data reveal the greatest thinning rates over the Smith Glacier ice plain and slopes beyond the margins of the fast-flowing PIG trunk. Difference maps also highlight the filling of at least two subglacial lakes ~30 km upstream of the PIG grounding line in 2011. Lagrangian difference maps reveal the spatial distribution of ice shelf thinning, which can primarily be attributed to basal melt. Preliminary results show focused ice shelf thinning within troughs and large basal channels, especially along the western margin of the Dotson ice shelf. These new data

  19. Probing the structural disorder of basalts and slab-driven andesite melts: Insights from high-resolution solid-state NMR study

    NASA Astrophysics Data System (ADS)

    Park, S.; Lee, S.

    2012-12-01

    Whereas the structure of multi-component silicate melts has strong implication for the properties of natural silicate melts and relevant magmatic processes in Earth's mantle and crust, little is known about their atomic structures due to lack of suitable experimental probes of multi-component amorphous oxides. Although most of the progress in melt structure has been made for relatively simple binary and ternary silicate glasses, recent advances in high-resolution solid-state NMR (nuclear magnetic resonance) unveil previously unknown structural details of multi-component silicate melts (Lee, S. K. and Sung, S., Chem. Geol., 2008, 256, 326; Lee et al., P. Natl. Acad. Sci. USA., 2011, 108, 6847; Park and Lee, Geochim. Cosmochim. Acta, 2012, 80, 125). In this study we report experimental results on the effects of composition. atomic structure of CaO-MgO-Al_{2} O_{3} -SiO_{2} (CMAS) glasses in diopside (CaMgSi_{2}O_{6}) and Ca-tschermakite (CaAl_{2}SiO_{6}) join and glass in the diopside-anorthite eutectic composition (Di_{64}An_{36})—model systems for basaltic melts—using solid-state NMR. We also report the first high-resolution experimental results on the atomic structure of CaO-MgO-Na_{2}O-Al_{2}O_{3}-SiO_{2} (CMNAS) glasses in diopside and jadeite (NaAlSi_{2}O_{6}) join, and glass in the natural phonolite composition (CaO: MgO: Na_{2}O: K_{2}O: Al_{2}O_{3}: SiO_{2}= 1.4: 8.0: 9.0: 3.8: 13: 64 mol%), a model system for slab driven andesite melts. The Al-27 3QMAS (triple quantum magic angle spinning) NMR spectra of CMAS glasses in diopside-Ca-tschermakite join show predominant ^{[4]}Al and a non-negligible fraction of ^{[5]}Al. Approximately 3.3% of ^{[5]}Al is observed for Di_{64}An_{36} glass. The Al-27 3QMAS NMR spectra of CMNAS glasses in diopside and jadeite join show mostly ^{[4]}Al and a non-negligible fraction of ^{[5]}Al (X_{Diopside}=0.75, the mole fraction of diopside content). While the C_{q} (quadrupolar coupling constant) of ^{[4]}Al for glasses in

  20. SIMS Calibration of Nitrogen in Silicate Glasses and Applications to Melt Inclusions

    NASA Astrophysics Data System (ADS)

    Regier, M. E.; Hervig, R. L.; Wallace, P. J.; Myers, M.; Wilson, C. J. N.

    2015-12-01

    Previous attempts to constrain N fluxes between the mantle, crust, and atmospheric reservoirs over geologic time have been inhibited due to the difficulty of detecting trace amounts of nitrogen, especially as triply-bonded N2 in oxidized magmas. Secondary ion mass spectrometry (SIMS) can detect nitrogen as N+, and as the molecular ions SiN-, NO-, and CN-. However, there are few matrix-matched, bulk-analyzed standards for N, which makes quantification of its signal challenging. Here, we use the implantation of known amounts of nitrogen into rhyolitic glasses containing a range of H2O, and subsequent depth-profile analysis of these samples to derive N calibration factors1,2. We use a primary beam of O- and detection of N+ ions. Results demonstrate that the useful yield (ions detected per atom sputtered) of N increases with H2O content, whereas the useful yield of the measured matrix ion (30Si) decreases with higher H2O. Quantification is also complicated by variable N background that scales inversely with observed higher sputtering rates in hydrated glasses. The former issue can be resolved by fitting a curve to the H2O vs. calibration factor plot and the latter by varying the primary beam density on low-H2O materials. Together, these approaches allow us to quantify the N content in variably hydrated rhyolitic glasses. Application of these calibrations to quartz-hosted melt inclusions from the Bishop3, 4 and Huckleberry Ridge Tuffs4 reveals un-degassed N contents of melts at depth. We show that N can be used in concert with other volatiles to tease out magmatic processes, such as recharge events and magma mixing. We conclude that unless these inclusions do not represent the bulk concentration of N in the melt, large silicic eruptions have not released enough N to significantly impact the atmospheric reservoir over geologic time. 1Burnett DS et al. (2015) Geostand Geoanalytical Res 39:265-276; 2Wilson RG et al. (1989) Secondary ion mass spectrometry. Wiley (New York

  1. Development and Validation of High-Resolution Melting Markers Derived from Rysto STS Markers for High-Throughput Marker-Assisted Selection of Potato Carrying Rysto.

    PubMed

    Nie, Xianzhou; Sutherland, Darcy; Dickison, Virginia; Singh, Mathuresh; Murphy, Agnes M; De Koeyer, David

    2016-11-01

    Sequence analysis of the chromosome region harboring the sequence-tagged site (STS) markers YES3-3A and YES3-3B for Rysto, a gene responsible for extreme resistance to Potato virus Y (PVY) in potato, was performed in tetraploid potato 'Barbara' (Rrrr) and 'AC Chaleur' (rrrr) as well as their progeny selections. Three and two sequence variants were identified in Barbara resistant (R) selections and AC Chaleur susceptible (S) selections, respectively. Further analysis indicates that the variant with a 21-nucleotide (nt) deletion is likely the chromosome copy harboring the STS markers. Two primer pairs, one targeting the region containing a 20-nt deletion and the other targeting the region anchoring the YES3-3A reverse primer, were designed. As anticipated, pair one produced two visible fragments in Barbara-R bulk and one visible fragment in AC Chaleur-S bulk; pair two produced one visible fragment in all samples. When subjected to high-resolution melting (HRM) analysis, two distinct melting profiles for R and S samples were observed. Analysis of 147 progeny of Barbara × AC Chaleur revealed 72 and 75 progeny with R and S melting profiles, respectively, which was consistent with YES3-3A and YES3-3B assays and phenotyping analysis, thus demonstrating the potential of HRM profiles as novel molecular markers for Rysto. The efficacy of the newly developed HRM markers for high-throughput marker-assisted selection for Rysto-conferred resistance to PVY was validated further with three populations involving Barbara as the R parent.

  2. Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations.

    PubMed

    Chassanidis, Christos; Boutou, Effrossyni; Voskaridou, Ersi; Balassopoulou, Angeliki

    2016-01-01

    Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease's high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics' assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for β globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a High-Resolution Melting Analysis (HRMA) approach, suitable for scanning the particularly heterogeneous beta globin gene mutations present in the Greek population, and thus adaptable to the Mediterranean and other areas where these mutations have been identified. Within this context, β globin gene regions containing mutations frequently identified in the Greek population were divided in ten overlapping amplicons. Our reactions' setup allowed for the simultaneous amplification of multiple primer sets and partial multiplexing, thereby resulting in significant reduction of the experimental time. DNA samples from β-thalassaemia patients/carriers with defined genotypes were tested. Distinct genotypes displayed distinguishable melting curves, enabling accurate detection of mutations. The described HRMA can be adapted to a high-throughput level. It represents a rapid, simple, cost-effective, reliable, highly feasible and sensitive method for β-thalassaemia gene scanning.

  3. A fast and accurate method for controlling the correct labeling of products containing buffalo meat using High Resolution Melting (HRM) analysis.

    PubMed

    Sakaridis, Ioannis; Ganopoulos, Ioannis; Argiriou, Anagnostis; Tsaftaris, Athanasios

    2013-05-01

    The substitution of high priced meat with low cost ones and the fraudulent labeling of meat products make the identification and traceability of meat species and their processed products in the food chain important. A polymerase chain reaction followed by a High Resolution Melting (HRM) analysis was developed for species specific detection of buffalo; it was applied in six commercial meat products. A pair of specific 12S and universal 18S rRNA primers were employed and yielded DNA fragments of 220bp and 77bp, respectively. All tested products were found to contain buffalo meat and presented melting curves with at least two visible inflection points derived from the amplicons of the 12S specific and 18S universal primers. The presence of buffalo meat in meat products and the adulteration of buffalo products with unknown species were established down to a level of 0.1%. HRM was proven to be a fast and accurate technique for authentication testing of meat products.

  4. A comparison of PCR assays for beak and feather disease virus and high resolution melt (HRM) curve analysis of replicase associated protein and capsid genes.

    PubMed

    Das, Shubhagata; Sarker, Subir; Ghorashi, Seyed Ali; Forwood, Jade K; Raidal, Shane R

    2016-11-01

    Beak and feather disease virus (BFDV) threatens a wide range of endangered psittacine birds worldwide. In this study, we assessed a novel PCR assay and genetic screening method using high-resolution melt (HRM) curve analysis for BFDV targeting the capsid (Cap) gene (HRM-Cap) alongside conventional PCR detection as well as a PCR method that targets a much smaller fragment of the virus genome in the replicase initiator protein (Rep) gene (HRM-Rep). Limits of detection, sensitivity, specificity and discriminatory power for differentiating BFDV sequences were compared. HRM-Cap had a high positive predictive value and could readily differentiate between a reference genotype and 17 other diverse BFDV genomes with more discriminatory power (genotype confidence percentage) than HRM-Rep. Melt curve profiles generated by HRM-Cap correlated with unique DNA sequence profiles for each individual test genome. The limit of detection of HRM-Cap was lower (2×10(-5)ng/reaction or 48 viral copies) than that for both HRM-Rep and conventional BFDV PCR which had similar sensitivity (2×10(-6)ng or 13 viral copies/reaction). However, when used in a diagnostic setting with 348 clinical samples there was strong agreement between HRM-Cap and conventional PCR (kappa=0.87, P<0.01, 98% specificity) and HRM-Cap demonstrated higher specificity (99.9%) than HRM-Rep (80.3%).

  5. Melt-growth bulk superconductors and application to an axial-gap-type rotating machine

    NASA Astrophysics Data System (ADS)

    Zhang, Yufeng; Zhou, Difan; Ida, Tetsuya; Miki, Motohiro; Izumi, Mitsuru

    2016-04-01

    The present manuscript addresses key issues in the course of our study of materials processing of bulk high-temperature superconductors, trapped flux and its application to a prototype axial-gap-type rotating machine. The TUMSAT group has conducted a series of studies since 2003 on the growth of GdBa2Cu3O7-δ bulk material and its application in a compact low-speed high-torque rotating machine. In the stage of material growth, gaining the advantage of a large motive torque density requires large integrated flux in the motor/generators. A large grain surface might be required with sophisticated techniques for the melt-growth texture in the bulk with optimal flux pinning. In the second stage, the in situ magnetization procedure for bulk superconductors in the applied machine is a crucial part of the technology. Pulsed current excitation by using an armature copper winding has magnetized field pole bulks on the rotor. The axial-gap flux synchronous machine studied in the past decade is a condensed technology and indicates that further scientific development is required for a future compact machine to be superior to conventional ones in accordance with the cryogenic periphery and flux stabilization.

  6. A new identification method for five species of oysters in genus Crassostrea from China based on high-resolution melting analysis

    NASA Astrophysics Data System (ADS)

    Wang, Jiafeng; Xu, Fei; Li, Li; Zhang, Guofan

    2014-03-01

    The high phenotypic plasticity in the shell of oysters presents a challenge during taxonomic and phylogenetic studies of these economically important bivalves. However, because DNA can exhibit marked differences among morphologically similar species, DNA barcoding offers a potential means for oyster identification. We analyzed the complete sequences of the cytochrome oxidase subunit I (COI) of five common Crassostrea species in China (including Hong Kong oyster C. hongkongensis, Jinjiang oyster C. ariakensis, Portuguese oyster C. angulata, Kumamoto oyster C. sikamea, and Pacific oyster C. gigas) and screened for distinct fragments. Using these distinct fragments on a high-resolution melting analysis platform, we developed an identification method that does not rely on species-specific PCR or fragment length polymorphism and is efficient, reliable, and easy to visualize. Using a single pair of primers (Oyster-COI-1), we were able to successfully distinguish among the five oyster species. This new method provides a simple and powerful tool for the identification of oyster species.

  7. Genotyping of Candida albicans using length fragment and high-resolution melting analyses together with minisequencing of a polymorphic microsatellite locus.

    PubMed

    Costa, Jean-Marc; Garcia-Hermoso, Dea; Olivi, Martine; Cabaret, Odile; Farrugia, Cécile; Lecellier, Gaël; Dromer, Françoise; Bretagne, Stéphane

    2010-03-01

    Microsatellite length polymorphism (MLP) typing is a PCR-based method used for genotyping of the diploid yeast Candida albicans. However, MLP is subject to homoplasia which can hamper the accuracy of the results. We combined fragment length analysis, high-resolution DNA melting (HRM) analysis, and SNaPshot minisequencing after a single amplification of the CDC3 locus to study 95 epidemiologically independent C. albicans isolates. HRM analysis for a given electrophoretic group led to a maximum of three different curves due to the presence of a SNP upstream of the tandem repeat which could be characterized using the SNaPshot assay. The combination of the three methods had a discriminatory index of 0.88 in complete congruence with previous MLP typing (Mantel test R=0.99, P<10(-)(4)). HRM is a useful tool of adding resolving power to MLP genotyping in identifying SNPs.

  8. Development of a Rapid High-Throughput Method for High-Resolution Melting Analysis for Routine Detection and Genotyping of Noroviruses▿

    PubMed Central

    Tajiri-Utagawa, Etsuko; Hara, Masayuki; Takahashi, Kuniaki; Watanabe, Mayumi; Wakita, Takaji

    2009-01-01

    We developed a simple, rapid, high-throughput detection and genotyping method for noroviruses using real-time reverse transcription-PCR (RT-PCR) and high-resolution melting (HRM) analysis to create a difference plot. The capsid gene was amplified by real-time RT-PCR in the presence of ResoLight HRM dye, a saturating DNA dye. Following optimization of the HRM assay conditions, the major norovirus genotypes were selected. Because we had only small quantities of the patient stool samples used in this study, we constructed plasmids for each genotype and used these to optimize the HRM assay. We selected six stool samples, each positive for one of the six dominant subtypes of noroviruses that have been circulating in Japan, namely, genotypes 4, 8, and 9 from genogroup 1 and genotypes 3, 4, and 10 from genogroup 2. The specific high-resolution derivate plot of the HRM assay for each plasmid was constructed by subtracting the melting-curve shape of the plasmid from the reference or base curve. The RNAs extracted from 14 clinical samples positive for small round structured viruses were then directly analyzed using the HRM assay. The HRM data from the clinical RNA samples corresponded with the genotype results obtained by RT-PCR and sequencing of the clinical samples. In addition, the HRM data from the clinical RNA samples corresponded with the HRM data from the six reference plasmid DNAs, indicating that this assay is useful for the direct detection and genotyping of noroviruses in clinical samples. This assay requires no multiplexing or hybridization probes and provides a new approach to the genetic screening of noroviruses in clinical virology laboratories. PMID:19073870

  9. Unraveling Host-Vector-Arbovirus Interactions by Two-Gene High Resolution Melting Mosquito Bloodmeal Analysis in a Kenyan Wildlife-Livestock Interface

    PubMed Central

    Omondi, David; Masiga, Daniel K.; Ajamma, Yvonne Ukamaka; Fielding, Burtram C.; Njoroge, Laban; Villinger, Jandouwe

    2015-01-01

    The blood-feeding patterns of mosquitoes are directly linked to the spread of pathogens that they transmit. Efficient identification of arthropod vector bloodmeal hosts can identify the diversity of vertebrate species potentially involved in disease transmission cycles. While molecular bloodmeal analyses rely on sequencing of cytochrome b (cyt b) or cytochrome oxidase 1 gene PCR products, recently developed bloodmeal host identification based on high resolution melting (HRM) analyses of cyt b PCR products is more cost-effective. To resolve the diverse vertebrate hosts that mosquitoes may potentially feed on in sub-Saharan Africa, we utilized HRM profiles of both cyt b and 16S ribosomal RNA genes. Among 445 blood-fed Aedeomyia, Aedes, Anopheles, Culex, Mansonia, and Mimomyia mosquitoes from Kenya’s Lake Victoria and Lake Baringo regions where many mosquito-transmitted pathogens are endemic, we identified 33 bloodmeal hosts including humans, eight domestic animal species, six peridomestic animal species and 18 wildlife species. This resolution of vertebrate host species was only possible by comparing profiles of both cyt b and 16S markers, as melting profiles of some pairs of species were similar for either marker but not both. We identified mixed bloodmeals in a Culex pipiens from Mbita that had fed on a goat and a human and in two Mansonia africana mosquitoes from Baringo that each had fed on a rodent (Arvicanthis niloticus) in addition to a human or baboon. We further detected Sindbis and Bunyamwera viruses in blood-fed mosquito homogenates by Vero cell culture and RT-PCR in Culex, Aedeomyia, Anopheles and Mansonia mosquitoes from Baringo that had fed on humans and livestock. The observed mosquito feeding on both arbovirus amplifying hosts (including sheep and goats) and possible arbovirus reservoirs (birds, porcupine, baboons, rodents) informs arbovirus disease epidemiology and vector control strategies. PMID:26230507

  10. Improved capacitive melting curve measurements

    NASA Astrophysics Data System (ADS)

    Sebedash, Alexander; Tuoriniemi, Juha; Pentti, Elias; Salmela, Anssi

    2009-02-01

    Sensitivity of the capacitive method for determining the melting pressure of helium can be enhanced by loading the empty side of the capacitor with helium at a pressure nearly equal to that desired to be measured and by using a relatively thin and flexible membrane in between. This way one can achieve a nanobar resolution at the level of 30 bar, which is two orders of magnitude better than that of the best gauges with vacuum reference. This extends the applicability of melting curve thermometry to lower temperatures and would allow detecting tiny anomalies in the melting pressure, which must be associated with any phenomena contributing to the entropy of the liquid or solid phases. We demonstrated this principle in measurements of the crystallization pressure of isotopic helium mixtures at millikelvin temperatures by using partly solid pure 4He as the reference substance providing the best possible universal reference pressure. The achieved sensitivity was good enough for melting curve thermometry on mixtures down to 100 μK. Similar system can be used on pure isotopes by virtue of a blocked capillary giving a stable reference condition with liquid slightly below the melting pressure in the reference volume. This was tested with pure 4He at temperatures 0.08-0.3 K. To avoid spurious heating effects, one must carefully choose and arrange any dielectric materials close to the active capacitor. We observed some 100 pW loading at moderate excitation voltages.

  11. Atmospheric Plasma Spraying of High Melting Temperature Complex Perovskites for TBC Application

    NASA Astrophysics Data System (ADS)

    Jarligo, M. O.; Mack, D. E.; Mauer, G.; Vaßen, R.; Stöver, D.

    2010-01-01

    High melting materials have always been very attractive candidates for materials development in thermal barrier coating (TBC) applications. Among these materials, complex perovskites with Ba(Mg1/3Ta2/3)O3 and La(Al1/4Mg1/2T1/4)O3 compositions have been developed and deposited in TBC systems by atmospheric plasma spraying. Spray parameters were optimized and in-flight particle temperatures were recorded using Accuraspray-g3 and DPV 2000. Plasma sprayed coatings were found to undergo non-stoichiometric decomposition of components which could have contributed to early failure of the coatings. Particle temperature diagnostics suggest that gun power of ~15 kW or lower where majority of the particles have already solidified upon impact to the substrate could probably prevent the decomposition of phases. Additionally, it has been found that the morphology of the powder feedstock plays a critical role during atmospheric plasma spraying of complex perovskites.

  12. Specific heat treatment of selective laser melted Ti-6Al-4V for biomedical applications

    NASA Astrophysics Data System (ADS)

    Huang, Qianli; Liu, Xujie; Yang, Xing; Zhang, Ranran; Shen, Zhijian; Feng, Qingling

    2015-12-01

    The ductility of as-fabricated Ti-6Al-4V falls far short of the requirements for biomedical titanium alloy implants and the heat treatment remains the only applicable option for improvement of their mechanical properties. In the present study, the decomposition of as-fabricated martensite was investigated to provide a general understanding on the kinetics of its phase transformation. The decomposition of asfabricated martensite was found to be slower than that of water-quenched martensite. It indicates that specific heat treatment strategy is needed to be explored for as-fabricated Ti-6Al-4V. Three strategies of heat treatment were proposed based on different phase transformation mechanisms and classified as subtransus treatment, supersolvus treatment and mixed treatment. These specific heat treatments were conducted on selective laser melted samples to investigate the evolutions of microstructure and mechanical properties. The subtransus treatment leaded to a basket-weave structure without changing the morphology of columnar prior β grains. The supersolvus treatment resulted in a lamellar structure and equiaxed β grains. The mixed treatment yielded a microstructure that combines both features of the subtransus treatment and supersolvus treatment. The subtransus treatment is found to be the best choice among these three strategies for as-fabricated Ti-6Al-4V to be used as biomedical implants.

  13. Process analytical techniques for hot-melt extrusion and their application to amorphous solid dispersions.

    PubMed

    Hitzer, Patrick; Bäuerle, Tim; Drieschner, Tobias; Ostertag, Edwin; Paulsen, Katharina; van Lishaut, Holger; Lorenz, Günter; Rebner, Karsten

    2017-03-25

    Newly developed active pharmaceutical ingredients (APIs) are often poorly soluble in water. As a result the bioavailability of the API in the human body is reduced. One approach to overcome this restriction is the formulation of amorphous solid dispersions (ASDs), e.g., by hot-melt extrusion (HME). Thus, the poorly soluble crystalline form of the API is transferred into a more soluble amorphous form. To reach this aim in HME, the APIs are embedded in a polymer matrix. The resulting amorphous solid dispersions may contain small amounts of residual crystallinity and have the tendency to recrystallize. For the controlled release of the API in the final drug product the amount of crystallinity has to be known. This review assesses the available analytical methods that have been recently used for the characterization of ASDs and the quantification of crystalline API content. Well-established techniques like near- and mid-infrared spectroscopy (NIR and MIR, respectively), Raman spectroscopy, and emerging ones like UV/VIS, terahertz, and ultrasonic spectroscopy are considered in detail. Furthermore, their advantages and limitations are discussed with regard to general practical applicability as process analytical technology (PAT) tools in industrial manufacturing. The review focuses on spectroscopic methods which have been proven as most suitable for in-line and on-line process analytics. Further aspects are spectroscopic techniques that have been or could be integrated into an extruder.

  14. An innovative energy-saving in-flight melting technology and its application to glass production.

    PubMed

    Yao, Yaochun; Watanabe, Takayuki; Yano, Tetsuji; Iseda, Toru; Sakamoto, Osamu; Iwamoto, Masanori; Inoue, Satoru

    2008-04-01

    The conventional method used for glass melting is air-fuel firing, which is inefficient, energy-intensive and time-consuming. In this study, an innovative in-flight melting technology was developed and applied to glass production for the purposes of energy conservation and environmental protection. Three types of heating sources, radio-frequency (RF) plasma, a 12-phase alternating current (ac) arc and an oxygen burner, were used to investigate the in-flight melting behavior of granulated powders. Results show that the melted particles are spherical with a smooth surface and compact structure. The diameter of the melted particles is about 50% of that of the original powders. The decomposition and vitrification degrees of the prepared powders decrease in the order of powders prepared by RF plasma, the 12-phase ac arc and the oxygen burner. The largest heat transfer is from RF plasma to particles, which results in the highest particle temperature (1810 °C) and the greatest vitrification degree of the raw material. The high decomposition and vitrification degrees, which are achieved in milliseconds, shorten the melting and fining times of the glass considerably. Our results indicate that the proposed in-flight melting technology is a promising method for use in the glass industry.

  15. An innovative energy-saving in-flight melting technology and its application to glass production

    NASA Astrophysics Data System (ADS)

    Yao, Yaochun; Watanabe, Takayuki; Yano, Tetsuji; Iseda, Toru; Sakamoto, Osamu; Iwamoto, Masanori; Inoue, Satoru

    2008-04-01

    The conventional method used for glass melting is air-fuel firing, which is inefficient, energy-intensive and time-consuming. In this study, an innovative in-flight melting technology was developed and applied to glass production for the purposes of energy conservation and environmental protection. Three types of heating sources, radio-frequency (RF) plasma, a 12-phase alternating current (ac) arc and an oxygen burner, were used to investigate the in-flight melting behavior of granulated powders. Results show that the melted particles are spherical with a smooth surface and compact structure. The diameter of the melted particles is about 50% of that of the original powders. The decomposition and vitrification degrees of the prepared powders decrease in the order of powders prepared by RF plasma, the 12-phase ac arc and the oxygen burner. The largest heat transfer is from RF plasma to particles, which results in the highest particle temperature (1810 °C) and the greatest vitrification degree of the raw material. The high decomposition and vitrification degrees, which are achieved in milliseconds, shorten the melting and fining times of the glass considerably. Our results indicate that the proposed in-flight melting technology is a promising method for use in the glass industry.

  16. A novel closed-tube method based on high resolution melting (HRM) analysis for authenticity testing and quantitative detection in Greek PDO Feta cheese.

    PubMed

    Ganopoulos, Ioannis; Sakaridis, Ioannis; Argiriou, Anagnostis; Madesis, Panagiotis; Tsaftaris, Athanasios

    2013-11-15

    Animal species identification of milk and dairy products has received increasing attention concerning food composition, traceability, allergic pathologies and accurate consumer information. Here we sought to develop an easy to use and robust method for species identification in cheese with emphasis on an authenticity control of PDO Feta cheese products. We used specific mitochondrial DNA regions coupled with high resolution melting (HRM) a closed-tube method allowing us to detect bovine, ovine and caprine species and authenticate Greek PDO Feta cheese. The primers successfully amplified DNA isolated from milk and cheese and showed a high degree of specificity. HRM was proven capable of accurately identifying the presence of bovine milk (not allowed in Feta) down to 0.1% and also of quantifying the ratio of sheep to goat milk mixture in different Feta cheese commercial products. In conclusion, HRM analysis can be a faster, with higher resolution and a more cost effective alternative method to authenticate milk and dairy products including PDO Feta cheese and to quantitatively detect its sheep milk adulterations.

  17. Enhancing the resolution of gpr spectra for pavement engineering applications

    NASA Astrophysics Data System (ADS)

    Benedetto, F.; Benedetto, A.

    2012-04-01

    Ground Penetrating Radar (GPR) is a geophysical method that uses radar pulses to image the subsurface. This non-destructive method uses electromagnetic radiation and detects the reflected signals from subsurface structures. It can detect objects, changes in material, and voids and cracks. GPR has many applications in a number of fields. In the field of civil engineering one of the most advanced technologies used for road pavement monitoring is based on the deployment of advanced GPR systems. One of the most relevant causes of road pavement damage is often referable to water intrusion in structural layers. In this context, GPR has been recently proposed as a method to estimate moisture content in a porous medium without preventive calibration. Hence, the development of methods to obtain an estimate of the moisture content is a crucial research field involving economic, social and strategic aspects in road safety for a great number of public and private Agencies. In particular, a recent new approach was proposed to estimate moisture content in a porous medium basing on the theory of Rayleigh scattering, showing a shift of the frequency peak of the GPR spectrum towards lower frequencies as the moisture content increases in the soil. The weakness characterizing this approach is represented by the needs of high resolution signals, whereas GPR spectra are affected by low resolution. Hence, the rising requirement for high resolution leads to specific demands for improved prediction methods. Recently, a new technique combining the response of the conventional fast Fourier transform (FFT, well known for its high-precision receiving signal level) with that of the MUSIC (multiple signal classification) algorithm, well known for its super-resolution capacity has been proposed. This combined method has been proved to obtain a high precision level in quantifying the shift of the frequency peak of the GPR spectrum. This combined method can perform a reliable coarse estimate of

  18. Analysis of impact melt and vapor production in CTH for planetary applications

    DOE PAGES

    Quintana, S. N.; Crawford, D. A.; Schultz, P. H.

    2015-05-19

    This study explores impact melt and vapor generation for a variety of impact speeds and materials using the shock physics code CTH. The study first compares the results of two common methods of impact melt and vapor generation to demonstrate that both the peak pressure method and final temperature method are appropriate for high-speed impact models (speeds greater than 10 km/s). However, for low-speed impact models (speeds less than 10 km/s), only the final temperature method is consistent with laboratory analyses to yield melting and vaporization. Finally, a constitutive model for material strength is important for low-speed impacts because strengthmore » can cause an increase in melting and vaporization.« less

  19. Analysis of impact melt and vapor production in CTH for planetary applications

    SciTech Connect

    Quintana, S. N.; Crawford, D. A.; Schultz, P. H.

    2015-05-19

    This study explores impact melt and vapor generation for a variety of impact speeds and materials using the shock physics code CTH. The study first compares the results of two common methods of impact melt and vapor generation to demonstrate that both the peak pressure method and final temperature method are appropriate for high-speed impact models (speeds greater than 10 km/s). However, for low-speed impact models (speeds less than 10 km/s), only the final temperature method is consistent with laboratory analyses to yield melting and vaporization. Finally, a constitutive model for material strength is important for low-speed impacts because strength can cause an increase in melting and vaporization.

  20. Congruent melting of gallium nitride at 6 GPa and its application to single-crystal growth.

    PubMed

    Utsumi, Wataru; Saitoh, Hiroyuki; Kaneko, Hiroshi; Watanuki, Tetsu; Aoki, Katsutoshi; Shimomura, Osamu

    2003-11-01

    The synthesis of large single crystals of GaN (gallium nitride) is a matter of great importance in optoelectronic devices for blue-light-emitting diodes and lasers. Although high-quality bulk single crystals of GaN suitable for substrates are desired, the standard method of cooling its stoichiometric melt has been unsuccessful for GaN because it decomposes into Ga and N(2) at high temperatures before its melting point. Here we report that applying high pressure completely prevents the decomposition and allows the stoichiometric melting of GaN. At pressures above 6.0 GPa, congruent melting of GaN occurred at about 2,220 degrees C, and decreasing the temperature allowed the GaN melt to crystallize to the original structure, which was confirmed by in situ X-ray diffraction. Single crystals of GaN were formed by cooling the melt slowly under high pressures and were recovered at ambient conditions.

  1. Rheology melts and magmatic suspensions. I - Design and calibration of concentric cylinder viscometer with application to rhyolitic magma

    NASA Technical Reports Server (NTRS)

    Spera, Frank J.; Borgia, Andrea; Strimple, James; Feigenson, Mark

    1988-01-01

    The design and calibration of concentric cylinder viscometer for rhyolitic magma applications are described together with the methods of data reduction and error analysis. Experimental data are presented on two rhyolitic magmas (melt plus a small fraction of vapor) under conditions of varying temperature (1100-1350 C) and shear rate (0.05-13.0/sec) at 100 kPa total pressure. Data obtained include a first reported measurement of a normal stress coefficient for magma.

  2. Stability assessment of hypromellose acetate succinate (HPMCAS) NF for application in hot melt extrusion (HME).

    PubMed

    Sarode, Ashish L; Obara, Sakae; Tanno, Fumie K; Sandhu, Harpreet; Iyer, Raman; Shah, Navnit

    2014-01-30

    HPMCAS is a widely used polymer in the pharmaceutical industry as an excipient. In this work, the physicochemical stability of HPMCAS was investigated for hot melt extrusion (HME) application. The reduction in zero rate viscosity (η0) of the polymer with the increase in temperature was determined using rheological evaluation prior to HME processing. The energy of activation for AS-MF determined by fitting Arrhenius model to the temperature dependent reduction in η0 was found to be slightly lower than that for the other grades of HPMCAS. Glassy yellowish HMEs were obtained using Haake Mini-Lab MicroCompounder operated at 160, 180, and 200°C and 100, 200, and 300 rpm for all the grades at each temperature. Various physicochemical properties of HPMCAS such as glass transition temperature, semi-crystalline nature, solid state functional group properties, moisture content, and solution viscosity were not significantly affected by the HME processing. The most significant change was the release of acetic and succinic acid with the increase in HME temperature and speed. The free acid content release due to HME was directly proportional to the speed at lower operating temperatures. AS-LF was found to be the most stable with the lowest increase in total free acid content even at higher HME temperature and speed. Although the dissolution time was not affected due to HME for AS-LF and AS-MF grades, it was notably increased for AS-HF, perhaps due to significant reduction of succinoyl content. In conclusion, the HME processing conditions for solid dispersions of HPMCAS should be based on the acceptance levels of free acid for the drug and the drug product.

  3. Applications of Atomic Resolution Atomic Force Microscopy to Nanoscience & Nanotechnology

    NASA Astrophysics Data System (ADS)

    Rhodin, Thor

    2001-03-01

    New developments in nanophysical scanning probe microscopy in terms of its unique relatonship to nanoscience, together with specific applications to nanoelectronic and biotechnology, will be discussed(1).Innovative examples of chemical physics at interfaces are analyzed where state-of-the-art non contact atomic force microscopy(nc-AFM) measurement of a specific physical or chemical property is correlated with position, orientation and/or location with atomic resolution. Analysis of specific current as well as future applications of nc-AFM to the detection, manipulation and fabrication of nanostructures on the molecular scale will be presented.Design features of nano-instrumentation based on carbon nanotube technology, high frequency solid state micro-oscillators and variable temperature applications will be presented.Specific examples pertaining to, (1) chemical bonding interaction on a semiconductor,(2) surface structure of an ionic insulator,(3) structural features in a biological interface and (4) nanofabrication of a quantum electron device, will be reviewed in terms of their innovativeness and significance to nanoscience and nanotechnology. 1 ``Scanning Probe Microscopies,Nanoscience & Nanotechnology" T.N. Rhodin, Proceedings of nc-AFM Workshop, July 2000, Hamburg, Germany. Springer Verlag U. Schwarz, H. Hoelscher and M. Wiesendanger, guest editors.

  4. Thermodynamics of Oligonucleotide Duplex Melting

    ERIC Educational Resources Information Center

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-01-01

    Melting temperatures of oligonucleotides are useful for a number of molecular biology applications, such as the polymerase chain reaction (PCR). Although melting temperatures are often calculated with simplistic empirical equations, application of thermodynamics provides more accurate melting temperatures and an opportunity for students to apply…

  5. High resolution solar observations from first principles to applications

    NASA Astrophysics Data System (ADS)

    Verdoni, Angelo P.

    2009-10-01

    polarization optics for the Visible-light Imaging Magnetograph (VIM) is presented. VIM uses a set of two Liquid Crystal Variable Retarders (LCVRs) as the main components of its Stokes analyzer. Calibration of these components is a crucial step in providing reliable polarimetric measurements of the Sun using VIM. On 2007 July 15, using the Dunn Solar Telescope (DST) at the National Solar Observatory at Sacramento Peak (NSO/SP), New Mexico, the first polarimetric measurements using VIM were made. As a final step, illustrating an application of high-resolution solar observations, the results of a two-dimensional time-series acquired on 2006 June 11, using the DST at NSOP is presented. The data is used in a study of upflow events that are observed to occur in the Halpha 656.3 nm and Na D2 589.0 nm chromospheric absorption lines.

  6. Taxonomic identification of mediterranean pines and their hybrids based on the high resolution melting (HRM) and trnL approaches: from cytoplasmic inheritance to timber tracing.

    PubMed

    Ganopoulos, Ioannis; Aravanopoulos, Filippos; Madesis, Panagiotis; Pasentsis, Konstantinos; Bosmali, Irene; Ouzounis, Christos; Tsaftaris, Athanasios

    2013-01-01

    Fast and accurate detection of plant species and their hybrids using molecular tools will facilitate the assessment and monitoring of local biodiversity in an era of climate and environmental change. Herein, we evaluate the utility of the plastid trnL marker for species identification applied to Mediterranean pines (Pinus spp.). Our results indicate that trnL is a very sensitive marker for delimiting species biodiversity. Furthermore, High Resolution Melting (HRM) analysis was exploited as a molecular fingerprint for fast and accurate discrimination of Pinus spp. DNA sequence variants. The trnL approach and the HRM analyses were extended to wood samples of two species (Pinus nigra and Pinus sylvestris) with excellent results, congruent to those obtained using leaf tissue. Both analyses demonstrate that hybrids from the P. brutia (maternal parent) × P. halepensis (paternal parent) cross, exhibit the P. halepensis profile, confirming paternal plastid inheritance in Group Halepensis pines. Our study indicates that a single one-step reaction method and DNA marker are sufficient for the identification of Mediterranean pines, their hybrids and the origin of pine wood. Furthermore, our results underline the potential for certain DNA regions to be used as novel biological information markers combined with existing morphological characters and suggest a relatively reliable and open taxonomic system that can link DNA variation to phenotype-based species or hybrid assignment status and direct taxa identification from recalcitrant tissues such as wood samples.

  7. ITS2 barcoding DNA region combined with high resolution melting (HRM) analysis of Hyoscyami Semen, the mature seed of Hyoscyamus niger.

    PubMed

    Xiong, Chao; Hu, Zhi-Gang; Tu, Yuan; Liu, He-Gang; Wang, Ping; Zhao, Ming-Ming; SHIi, Yu-Hua; Wu, Lan; Sun, Wei; Chen, Shi-Lin

    2016-12-01

    Hyoscyami Semen, the mature dried seed of Hyoscyamus niger L., has long been used as a traditional Chinese medicine to treat human diseases. Hyoscyami Semen is found in local markets in China. In markets, sellers and buyers commonly inadvertently mix the seeds of H. niger with the seeds of related species such as Hygrophila salicifolia (Vahl) Nees, Astragalus complanatus R. Br., Cuscuta australis R. Br., Cuscuta chinensis Lam., and Impatiens balsamina L. because of their similar morphologies or similar names. Thus, developing a reliable method for discriminating H. niger seeds from its adulterants is necessary to reduce confusion and ensure the safe use of Hyoscyami Semen. The present study was designed to evaluate the efficiency of high-resolution melting analysis combined with DNA barcoding (Bar-HRM) with internal transcribed spacer 2 to discriminate H. niger. Our results show that Bar-HRM successfully identified the adulterants and detected the proportion of H. niger DNA extract within an admixture. In particular, HRM detected H. niger DNA extract in A. complanatus DNA extract at concentrations as low as 1%. In conclusion, the Bar-HRM method developed in the present study for authenticating H. niger is rapid and cost-effective. It can be used in the future to guarantee the purity of Hyoscyami Semen for the clinical use.

  8. Taxonomic Identification of Mediterranean Pines and Their Hybrids Based on the High Resolution Melting (HRM) and trnL Approaches: From Cytoplasmic Inheritance to Timber Tracing

    PubMed Central

    Ganopoulos, Ioannis; Aravanopoulos, Filippos; Madesis, Panagiotis; Pasentsis, Konstantinos; Bosmali, Irene; Ouzounis, Christos; Tsaftaris, Athanasios

    2013-01-01

    Fast and accurate detection of plant species and their hybrids using molecular tools will facilitate the assessment and monitoring of local biodiversity in an era of climate and environmental change. Herein, we evaluate the utility of the plastid trnL marker for species identification applied to Mediterranean pines (Pinus spp.). Our results indicate that trnL is a very sensitive marker for delimiting species biodiversity. Furthermore, High Resolution Melting (HRM) analysis was exploited as a molecular fingerprint for fast and accurate discrimination of Pinus spp. DNA sequence variants. The trnL approach and the HRM analyses were extended to wood samples of two species (Pinus nigra and Pinus sylvestris) with excellent results, congruent to those obtained using leaf tissue. Both analyses demonstrate that hybrids from the P. brutia (maternal parent) × P. halepensis (paternal parent) cross, exhibit the P. halepensis profile, confirming paternal plastid inheritance in Group Halepensis pines. Our study indicates that a single one-step reaction method and DNA marker are sufficient for the identification of Mediterranean pines, their hybrids and the origin of pine wood. Furthermore, our results underline the potential for certain DNA regions to be used as novel biological information markers combined with existing morphological characters and suggest a relatively reliable and open taxonomic system that can link DNA variation to phenotype-based species or hybrid assignment status and direct taxa identification from recalcitrant tissues such as wood samples. PMID:23577179

  9. Characterization of Genetic Diversity of Bacillus anthracis in France by Using High-Resolution Melting Assays and Multilocus Variable-Number Tandem-Repeat Analysis ▿ †

    PubMed Central

    Derzelle, S.; Laroche, S.; Le Flèche, P.; Hauck, Y.; Thierry, S.; Vergnaud, G.; Madani, N.

    2011-01-01

    Using high-resolution melting (HRM) analysis, we developed a cost-effective method to genotype a set of 13 phylogenetically informative single-nucleotide polymorphisms (SNPs) within the genome of Bacillus anthracis. SNP discrimination assays were performed in monoplex or duplex and applied to 100 B. anthracis isolates collected in France from 1953 to 2009 and a few reference strains. HRM provided a reliable and cheap alternative to subtype B. anthracis into one of the 12 major sublineages or subgroups. All strains could be correctly positioned on the canonical SNP (canSNP) phylogenetic tree, except the divergent Pasteur vaccine strain ATCC 4229. We detected the cooccurrence of three canSNP subgroups in France. The dominant B.Br.CNEVA sublineage was found to be prevalent in the Alps, the Pyrenees, the Auvergne region, and the Saône-et-Loire department. Strains affiliated with the A.Br.008/009 subgroup were observed throughout most of the country. The minor A.Br.001/002 subgroup was restricted to northeastern France. Multiple-locus variable-number tandem-repeat analysis using 24 markers further resolved French strains into 60 unique profiles and identified some regional patterns. Diversity found within the A.Br.008/009 and B.Br.CNEVA subgroups suggests that these represent old, ecologically established clades in France. Phylogenetic relationships with strains from other parts of the world are discussed. PMID:21998431

  10. High-Throughput Screening for Spermatogenesis Candidate Genes in the AZFc Region of the Y Chromosome by Multiplex Real Time PCR Followed by High Resolution Melting Analysis

    PubMed Central

    Alechine, Evguenia; Corach, Daniel

    2014-01-01

    Microdeletions in the AZF region of the Y chromosome are among the most frequent genetic causes of male infertility, although the specific role of the genes located in this region is not fully understood. AZFa and AZFb deletions impair spermatogenesis since no spermatozoa are found in the testis. Deletions of the AZFc region, despite being the most frequent in azoospermic patients, do not correlate with spermatogenic failure. Therefore, the aim of this work was to develop a screening method to ascertain the presence of the main spermatogenesis candidate genes located in the AZFc region in the light of the identification of those responsible for spermatogenic failure. DAZ, CDY, BPY2, PRY, GOLGA2LY and CSGP4LY genes were selected on the basis of their location in the AZFc region, testis-only expression, and confirmed or predicted protein codification. AMEL and SRY were used as amplification controls. The identification of Real Time PCR products was performed by High Resolution Melting analysis with SYTO 9 as intercalating dye. The herein described method allows a rapid, simple, low-cost, high-throughput screening for deletions of the main AZFc genes in patients with spermatogenic failure. This provides a strategy that would accelerate the identification of spermatogenesis candidate genes in larger populations of patients with non-obstructive idiopathic azoospermia. PMID:24828879

  11. High-throughput screening for spermatogenesis candidate genes in the AZFc region of the Y chromosome by multiplex real time PCR followed by high resolution melting analysis.

    PubMed

    Alechine, Evguenia; Corach, Daniel

    2014-01-01

    Microdeletions in the AZF region of the Y chromosome are among the most frequent genetic causes of male infertility, although the specific role of the genes located in this region is not fully understood. AZFa and AZFb deletions impair spermatogenesis since no spermatozoa are found in the testis. Deletions of the AZFc region, despite being the most frequent in azoospermic patients, do not correlate with spermatogenic failure. Therefore, the aim of this work was to develop a screening method to ascertain the presence of the main spermatogenesis candidate genes located in the AZFc region in the light of the identification of those responsible for spermatogenic failure. DAZ, CDY, BPY2, PRY, GOLGA2LY and CSGP4LY genes were selected on the basis of their location in the AZFc region, testis-only expression, and confirmed or predicted protein codification. AMEL and SRY were used as amplification controls. The identification of Real Time PCR products was performed by High Resolution Melting analysis with SYTO 9 as intercalating dye. The herein described method allows a rapid, simple, low-cost, high-throughput screening for deletions of the main AZFc genes in patients with spermatogenic failure. This provides a strategy that would accelerate the identification of spermatogenesis candidate genes in larger populations of patients with non-obstructive idiopathic azoospermia.

  12. Rapid differentiation of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood by real-time PCR coupled to high resolution melting analysis.

    PubMed

    Albonico, Francesca; Loiacono, Monica; Gioia, Gloria; Genchi, Claudio; Genchi, Marco; Mortarino, Michele

    2014-02-24

    Dirofilaria immitis and D. repens are the principal causative agents of canine filariosis and, although the number of dogs subjected to specific prevention is increasing, the prevalence of these parasites remains high in many areas of the world. The discrimination between the two Dirofilaria species using the classical diagnostic methods can be difficult and may lead to misdiagnosis especially on samples from areas where both Dirofilaria are present. Over the last years, several molecular methods with higher sensitivity and specificity compared to classical microscopy and ELISA assays were designed. Nevertheless, a need for simple, rapid, and cost-effective molecular protocols to accurately discriminate between D. immitis and D. repens still remains. High resolution melting analysis coupled to real-time PCR (real-time PCR-HRMA) is a widely used technique to target sequence polymorphisms of the same gene in different species without the need to perform DNA sequencing or to use species-specific probes. In this work, a fast and cost-effective real-time PCR-HRMA protocol to detect and differentiate simultaneously and unequivocally D. immitis and D. repens microfilarial DNA extracted from peripheral dog blood samples is described. The present method is simpler to use than most other DNA-based methods and provides comparable discrimination between the two sibling species.

  13. Simultaneous detection of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus using real-time PCR and high resolution melting analysis.

    PubMed

    Komorowska, Beata; Fiore, Nicola; Zamorano, Alan; Li, Ruhui

    2014-08-01

    In this study, the real-time PCR assays were combined with high resolution melting (HRM) analysis for the simultaneous detection of Cherry necrotic rusty mottle virus (CNRMV) and Cherry green ring mottle virus (CGRMV) infection in sweet cherry trees. Detection of CNRMV and CGRMV was performed in a real-time PCR using a primer set for both of them or duplex real-time PCR that included one specific primer set for each virus. These two strategies allowed us to confirmed virus infection in all tested samples. In 17 field samples the technique revealed samples positive for CNRMV or CGRMV as well as positive for both viruses. In addition, the HRM analysis made it possible to differentiate clearly between CNRMV and CGRMV. Sequence variations among CNRMV and CGRMV isolates observed from the HRM peaks were confirmed by sequencing. To test the capability to use this method in field, forty one sweet cherry samples were examined by HRM analysis. The HRM data showed that seven samples were positive for CNRMV and three were infected with CGRMV. The results presented in this study indicated that real-time PCR followed by HRM analysis provides sensitive, automated and rapid tool to detect and differentiate between CNRMV and CGRMV isolates.

  14. Mutation Scanning in a Single and a Stacked Genetically Modified (GM) Event by Real-Time PCR and High Resolution Melting (HRM) Analysis

    PubMed Central

    Ben Ali, Sina-Elisabeth; Madi, Zita Erika; Hochegger, Rupert; Quist, David; Prewein, Bernhard; Haslberger, Alexander G.; Brandes, Christian

    2014-01-01

    Genetic mutations must be avoided during the production and use of seeds. In the European Union (EU), Directive 2001/18/EC requires any DNA construct introduced via transformation to be stable. Establishing genetic stability is critical for the approval of genetically modified organisms (GMOs). In this study, genetic stability of two GMOs was examined using high resolution melting (HRM) analysis and real-time polymerase chain reaction (PCR) employing Scorpion primers for amplification. The genetic variability of the transgenic insert and that of the flanking regions in a single oilseed rape variety (GT73) and a stacked maize (MON88017 × MON810) was studied. The GT73 and the 5' region of MON810 showed no instabilities in the examined regions. However; two out of 100 analyzed samples carried a heterozygous point mutation in the 3' region of MON810 in the stacked variety. These results were verified by direct sequencing of the amplified PCR products as well as by sequencing of cloned PCR fragments. The occurrence of the mutation suggests that the 5' region is more suitable than the 3' region for the quantification of MON810. The identification of the single nucleotide polymorphism (SNP) in a stacked event is in contrast to the results of earlier studies of the same MON810 region in a single event where no DNA polymorphism was found. PMID:25365178

  15. Molecular Identification of Mycobacterium avium subsp. silvaticum by Duplex High-Resolution Melt Analysis and Subspecies-Specific Real-Time PCR

    PubMed Central

    Csivincsik, Ágnes; Dán, Ádám

    2015-01-01

    Accurate identification of mycobacterial species and subspecies is essential to evaluate their significance and to perform epidemiological studies. The subspecies of Mycobacterium avium have different attributes but coincide in their zoonotic potential. Our knowledge about M. avium subsp. silvaticum is limited, since its identification is uncertain. Mycobacterium avium subsp. avium and M. avium subsp. silvaticum can be discriminated from each other based only on phenotypic characteristics, as they have almost identical genome sequences. Here we describe the development of a diagnostic method which enables the molecular identification of M. avium subsp. silvaticum and discrimination from M. avium subsp. avium based on genomic differences in a duplex high-resolution melt and M. avium subsp. silvaticum-specific mismatch real-time PCR. The developed assay was tested on reference strains and 199 field isolates, which were analyzed by phenotypic methods previously. This assay not only identified all 63 M. avium subsp. silvaticum and 138 M. avium subsp. avium strains correctly but also enabled the detection of mixed M. avium subsp. avium-M. avium subsp. silvaticum cultures. This is the first time that such a large panel of strains has been analyzed, and we also report the first isolation of M. avium subsp. silvaticum from red fox, red deer, wild boar, cattle, and badger. This assay is reliable, rapid, simple, inexpensive, and robust. It eliminates the long-existing problem of ambiguous phenotypic identification and opens up the possibility for detailed and comprehensive strain studies. PMID:25740770

  16. Assessment of the MAR regional climate model over the Antarctic Peninsula (1999 - 2009) through spaceborne enhanced spatial resolution melting maps and near-surface observations

    NASA Astrophysics Data System (ADS)

    Datta, R.; Tedesco, M.; Alexander, P. M.; Fettweis, X.; Steiner, N.; Gallee, H.

    2012-12-01

    We report results assessing the outputs of the regional climate model Modèle Atmosphérique Régionale (MAR) over the Antarctic peninsula for the period 1999 - 2009. Specifically, we compare maps of melt extent and duration generated by MAR with those obtained from the enhanced spatial resolution product (~ 5 km) distributed by the NASA Scatterometer Climate Record Pathfinder (SCP), at Brigham Young University (Utah, USA). Snowmelt is estimated from remote sensing observations using both a canonical threshold-based approach and a novel method based on wavelet methodology. MAR outputs are also evaluated against available surface observations (e.g., near-surface temperature, wind speed and direction, etc.). The additional effects of blowing snow upon the surface and energy balance can be uniquely explored by simulations in Antarctica (as compared to Greenland, for example). Because of this, as of the time of abstract submission, MAR is set up to run for a scenario with blowing snow as well as a scenario without blowing snow. Our final assessment will present the results of both, providing insight into the sensitivity of MAR outputs to the blowing snow model.

  17. Molecular identification of Mycobacterium avium subsp. silvaticum by duplex high-resolution melt analysis and subspecies-specific real-time PCR.

    PubMed

    Rónai, Zsuzsanna; Csivincsik, Ágnes; Dán, Ádám

    2015-05-01

    Accurate identification of mycobacterial species and subspecies is essential to evaluate their significance and to perform epidemiological studies. The subspecies of Mycobacterium avium have different attributes but coincide in their zoonotic potential. Our knowledge about M. avium subsp. silvaticum is limited, since its identification is uncertain. Mycobacterium avium subsp. avium and M. avium subsp. silvaticum can be discriminated from each other based only on phenotypic characteristics, as they have almost identical genome sequences. Here we describe the development of a diagnostic method which enables the molecular identification of M. avium subsp. silvaticum and discrimination from M. avium subsp. avium based on genomic differences in a duplex high-resolution melt and M. avium subsp. silvaticum-specific mismatch real-time PCR. The developed assay was tested on reference strains and 199 field isolates, which were analyzed by phenotypic methods previously. This assay not only identified all 63 M. avium subsp. silvaticum and 138 M. avium subsp. avium strains correctly but also enabled the detection of mixed M. avium subsp. avium-M. avium subsp. silvaticum cultures. This is the first time that such a large panel of strains has been analyzed, and we also report the first isolation of M. avium subsp. silvaticum from red fox, red deer, wild boar, cattle, and badger. This assay is reliable, rapid, simple, inexpensive, and robust. It eliminates the long-existing problem of ambiguous phenotypic identification and opens up the possibility for detailed and comprehensive strain studies.

  18. Rapid identification of dairy mesophilic and thermophilic sporeforming bacteria using DNA high resolution melt analysis of variable 16S rDNA regions.

    PubMed

    Chauhan, Kanika; Dhakal, Rajat; Seale, R Brent; Deeth, Hilton C; Pillidge, Christopher J; Powell, Ian B; Craven, Heather; Turner, Mark S

    2013-07-15

    Due to their ubiquity in the environment and ability to survive heating processes, sporeforming bacteria are commonly found in foods. This can lead to product spoilage if spores are present in sufficient numbers and where storage conditions favour spore germination and growth. A rapid method to identify the major aerobic sporeforming groups in dairy products, including Bacillus licheniformis group, Bacillus subtilis group, Bacillus pumilus group, Bacillus megaterium, Bacillus cereus group, Geobacillus species and Anoxybacillus flavithermus was devised. This method involves real-time PCR and high resolution melt analysis (HRMA) of V3 (~70 bp) and V6 (~100 bp) variable regions in the 16S rDNA. Comparisons of HRMA curves from 194 isolates of the above listed sporeforming bacteria obtained from dairy products which were identified using partial 16S rDNA sequencing, allowed the establishment of criteria for differentiating them from each other and several non-sporeforming bacteria found in samples. A blinded validation trial on 28 bacterial isolates demonstrated complete accuracy in unambiguous identification of the 7 different aerobic sporeformers. The reliability of HRMA method was also verified using boiled extractions of crude DNA, thereby shortening the time needed for identification. The HRMA method described in this study provides a new and rapid approach to identify the dominant mesophilic and thermophilic aerobic sporeforming bacteria found in a wide variety of dairy products.

  19. Rapid detection and identification of mucormycetes in bronchoalveolar lavage samples from immunocompromised patients with pulmonary infiltrates by use of high-resolution melt analysis.

    PubMed

    Lengerova, Martina; Racil, Zdenek; Hrncirova, Kristyna; Kocmanova, Iva; Volfova, Pavlina; Ricna, Dita; Bejdak, Petr; Moulis, Mojmir; Pavlovsky, Zdenek; Weinbergerova, Barbora; Toskova, Martina; Mayer, Jiri

    2014-08-01

    Rapid differential diagnostics of pulmonary infiltrates suspected of invasive fungal disease in an immunocompromised host and early initiation of effective antifungal therapy are crucial for patient outcomes. There are no serological tests available to detect mucormycetes; therefore, PCR-based methods are highly suitable. We validated our previously published PCR followed by high-resolution melt analysis (PCR/HRMA) to detect Rhizopus spp., Rhizomucor pusillus, Lichtheimia corymbifera, and Mucor spp. in bronchoalveolar lavage (BAL) samples from immunocompromised patients who were at risk of invasive fungal disease. All PCR/HRMA-positive samples were retested using novel real-time quantitative PCR (RQ PCR) assays specific to the species identified. In total, between January 2009 and December 2012 we analyzed 99 BAL samples from 86 patients with pulmonary abnormalities using PCR/HRMA. Ninety (91%) BAL samples were negative, and 9 (9%) samples were positive. The sensitivity and specificity of PCR/HRMA were 100% and 93%, respectively. By combining the positive results of PCR/HRMA with positive RQ PCR results, the specificity was raised to 98%. PCR/HRMA, due to its high negative predictive value (99%), represents a fast and reliable tool for routine BAL sample screening for the differential diagnosis of pulmonary infiltrates in immunocompromised patients for the four most clinically important mucormycetes.

  20. rpoB gene high-resolution melt curve analysis: a rapid approach for diagnosis and screening of drug resistance in tuberculous meningitis.

    PubMed

    Sharma, Kusum; Modi, Manish; Kaur, Harsimran; Sharma, Aman; Ray, Pallab; Varma, Subhash

    2015-10-01

    Timely and rapid diagnosis of multidrug resistance in tuberculous meningitis (TBM) is a challenge both for a microbiologist and neurologist. The present study was conducted to evaluate role of real-time polymerase chain reaction (PCR) using rpoB, IS6110, and MPB64 as targets in diagnosis of TBM in 110 patients and subsequent high-resolution melt (HRM) curve analysis of rpoB gene amplicons for screening of drug resistance. The sensitivity of smear, culture, and real-time PCR was 1.8%, 10.9%, and 83.63%, respectively. All 120 control patients showed negative results. With HRM rpoB analysis, rifampicin resistance was detected in 3 out of 110 cases of TBM (3.33%). Subsequently, results of HRM analysis were confirmed by rpoB gene sequencing, and mutations were observed at 516 (2 patients) and 531 (1 patient) codons, respectively. rpoB HRM analysis can be a promising tool for rapid diagnosis and screening of drug resistance in TBM patients in 90minutes.

  1. Rapid discrimination of MHC class I and killer cell lectin-like receptor allele variants by high-resolution melt analysis.

    PubMed

    Lundgren, Alyssa; Kim, Sharon; Stadnisky, Michael D; Brown, Michael G

    2012-08-01

    Ly49G and H-2 class I D(k) molecules are critical to natural killer cell-mediated viral control. To examine their contributions in greater depth, we established NK gene complex (NKC)/Ly49 congenic strains and a novel genetic model defined by MHC class I D(k) disparity in congenic and transgenic mouse strains. Generation and maintenance of Ly49 and H-2 class I select strains require efficient and reproducible genotyping assays for highly polygenic and polymorphic sequences. Thus, we coupled gene- and allele-specific PCR with high-resolution melt (HRM) analysis to discriminate Ly49g and H-2 class I D and K alleles in select strains and in the F(2) and backcross hybrid offspring of different genetic crosses. We show that HRM typing for these critical immune response genes is fast, accurate, and dependable. We further demonstrate that H-2 class I D HRM typing is competent to detect and quantify transgene copy numbers in different mice with distinct genetic backgrounds. Our findings substantiate the utility and practicality of HRM genotyping for highly related genes and alleles, even those belonging to clustered multigene families. Based on these findings, we envision that HRM is capable to interrogate and quantify gene- and allele-specific variations due to differential regulation of gene expression.

  2. Application of rock melting to construction of storage holes for nuclear waste

    SciTech Connect

    Neudecker, J.W. Jr.

    1988-12-31

    Rock melting technology can provide in-situ glass liners in nuclear waste package emplacement holes to reduce permeability and increase borehole stability. Reduction of permeability would reduce the time and probability of groundwater contacting the waste packages. Increasing the stability of the storage boreholes would enhance the retrievability of the nuclear waste packages. The rock melting hole forming technology has already been tested in volcanic tuff similar to the geology at the proposed nuclear waste repository at Yucca Mountain, Nevada. 6 refs., 5 figs., 2 tabs.

  3. Nickel and Cobalt Partitioning Between Spinel and Basaltic Melt: Applications to Planetary Basalt Suites

    NASA Technical Reports Server (NTRS)

    Righter, K.

    2002-01-01

    New experimental spinel/melt partition coefficients for Ni and Co have been measured in basalt samples with natural levels of Ni and Co, are lower than previous high doping experiments, and are applied to several planetary basalt suites. Additional information is contained in the original extended abstract.

  4. High spatial resolution geochemistry and textural characteristics of 'microtektite' glass spherules in proximal Cretaceous-Paleogene sections: Insights into glass alteration patterns and precursor melt lithologies

    NASA Astrophysics Data System (ADS)

    Belza, Joke; Goderis, Steven; Smit, Jan; Vanhaecke, Frank; Baert, Kitty; Terryn, Herman; Claeys, Philippe

    2015-03-01

    Using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), we have conducted spatially resolved trace element analysis on fresh, unaltered microtektite glasses linked to the Cretaceous-Paleogene (K-Pg) boundary Chicxulub crater and on their surrounding alteration phases. This unique approach offers the opportunity to study in situ and at high spatial resolution both the mixing of different target lithologies and the variation of the major and trace element budget during the alteration process. In addition, two-dimensional element distribution maps reveal important geochemical information beyond the capabilities of single spot laser drilling. Glasses from two localities in opposite quadrants from the source crater were studied. At the Beloc locality (Haiti), the glass population is dominated by the presence of yellow high-Ca glass and black andesitic glass formed by admixture of carbonate/dolomite/anhydrite platform lithologies with crystalline basement. These glasses alter according to the well-established hydration-palagonitization model postulated for mafic volcanic glasses. REEs become progressively leached from the glass to below the detection limit for the applied spot size, while immobile Zr, Hf, Nb, and Ta passively accumulate in the process exhibiting both inter-element ratios and absolute concentrations similar to those for the original glass. In contrast, The Arroyo El Mimbral locality (NE Mexico) is characterized by abundant green glass fragments high in Si, Al and alkalis, and low in Mg, Ca, Fe. Low Si black glass is less abundant though similar in composition to the black glass variety at Beloc. The alteration pattern of high-Si, Al green glass at the Mimbral locality is more complex, including numerous competing reaction processes (ion-exchange, hydration, dissolution, and secondary mineral precipitation) generally controlled by the pH and composition of the surrounding fluid. All green, high-Si, Al glasses are hydrated and

  5. Detection of sdhB Gene Mutations in SDHI-Resistant Isolates of Botrytis cinerea Using High Resolution Melting (HRM) Analysis

    PubMed Central

    Samaras, Anastasios; Madesis, Panagiotis; Karaoglanidis, George S.

    2016-01-01

    Botrytis cinerea, is a high risk pathogen for fungicide resistance development. Pathogen’ resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence of sdhB subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant’s DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdhB mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA–PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of several sdhB mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in

  6. Application of density functional theory calculations to the statistical mechanics of normal and anomalous melting

    NASA Astrophysics Data System (ADS)

    Rudin, Sven P.; Bock, Nicolas; Wallace, Duane C.

    2014-11-01

    Density functional theory (DFT) calculations reliably aid in understanding the relative stability of different crystal phases as functions of pressure and temperature. Our purpose here is to employ DFT to analyze the character of the melting process, with an emphasis on comparing normal and anomalous melting. The normal-anomalous distinction is the absence or presence, respectively, of a significant electronic structure change between crystal and liquid. We study the normal melters Na and Cu, which are metallic in both phases, and the anomalous melter Ga, which has a partially covalent crystal and a nearly free-electron liquid. We calculate free energies from lattice dynamics for the crystal and from vibration-transit (V-T) theory for the liquid, where the liquid formulation is similar to that of the crystal but has an additional term representing the diffusive transits. Internal energies U and entropies S calculated for both phases of Na and Cu were previously shown to be in good agreement with experiment; here we find the same agreement for Ga. The dominant theoretical terms in the melting Δ U and Δ S are the structural potential energy, the vibrational entropy, and the purely liquid transit terms in both U and S . The melting changes in structural energy and vibrational entropy are much larger in Ga than in Na and Cu. This behavior arises from the change in electronic structure in Ga, and is the identifying characteristic of anomalous melting. We interpret our DFT results in terms of the physical effects of the relatively few covalent bonds in the otherwise metallic Ga crystal.

  7. Solid-solid phase transformation via internal stress-induced virtual melting, significantly below the melting temperature. Application to HMX energetic crystal.

    PubMed

    Levitas, Valery I; Henson, Bryan F; Smilowitz, Laura B; Asay, Blaine W

    2006-05-25

    We theoretically predict a new phenomenon, namely, that a solid-solid phase transformation (PT) with a large transformation strain can occur via internal stress-induced virtual melting along the interface at temperatures significantly (more than 100 K) below the melting temperature. We show that the energy of elastic stresses, induced by transformation strain, increases the driving force for melting and reduces the melting temperature. Immediately after melting, stresses relax and the unstable melt solidifies. Fast solidification in a thin layer leads to nanoscale cracking which does not affect the thermodynamics or kinetics of the solid-solid transformation. Thus, virtual melting represents a new mechanism of solid-solid PT, stress relaxation, and loss of coherence at a moving solid-solid interface. It also removes the athermal interface friction and deletes the thermomechanical memory of preceding cycles of the direct-reverse transformation. It is also found that nonhydrostatic compressive internal stresses promote melting in contrast to hydrostatic pressure. Sixteen theoretical predictions are in qualitative and quantitative agreement with experiments conducted on the PTs in the energetic crystal HMX. In particular, (a) the energy of internal stresses is sufficient to reduce the melting temperature from 551 to 430 K for the delta phase during the beta --> delta PT and from 520 to 400 K for the beta phase during the delta --> beta PT; (b) predicted activation energies for direct and reverse PTs coincide with corresponding melting energies of the beta and delta phases and with the experimental values; (c) the temperature dependence of the rate constant is determined by the heat of fusion, for both direct and reverse PTs; results b and c are obtained both for overall kinetics and for interface propagation; (d) considerable nanocracking, homogeneously distributed in the transformed material, accompanies the PT, as predicted by theory; (e) the nanocracking does not

  8. High Resolution Reconstruction of the Ionosphere for SAR Applications

    NASA Astrophysics Data System (ADS)

    Minkwitz, David; Gerzen, Tatjana; Hoque, Mainul

    2014-05-01

    Caused by ionosphere's strong impact on radio signal propagation, high resolution and highly accurate reconstructions of the ionosphere's electron density distribution are demanded for a large number of applications, e.g. to contribute to the mitigation of ionospheric effects on Synthetic Aperture Radar (SAR) measurements. As a new generation of remote sensing satellites the TanDEM-L radar mission is planned to improve the understanding and modelling ability of global environmental processes and ecosystem change. TanDEM-L will operate in L-band with a wavelength of approximately 24 cm enabling a stronger penetration capability compared to X-band (3 cm) or C-band (5 cm). But accompanied by the lower frequency of the TanDEM-L signals the influence of the ionosphere will increase. In particular small scale irregularities of the ionosphere might lead to electron density variations within the synthetic aperture length of the TanDEM-L satellite and in turn might result into blurring and azimuth pixel shifts. Hence the quality of the radar image worsens if the ionospheric effects are not mitigated. The Helmholtz Alliance project "Remote Sensing and Earth System Dynamics" (EDA) aims in the preparation of the HGF centres and the science community for the utilisation and integration of the TanDEM-L products into the study of the Earth's system. One significant point thereby is to cope with the mentioned ionospheric effects. Therefore different strategies towards achieving this objective are pursued: the mitigation of the ionospheric effects based on the radar data itself, the mitigation based on external information like global Total Electron Content (TEC) maps or reconstructions of the ionosphere and the combination of external information and radar data. In this presentation we describe the geostatistical approach chosen to analyse the behaviour of the ionosphere and to provide a high resolution 3D electron density reconstruction. As first step the horizontal structure of

  9. A screening method for the detection of the 35S promoter and the nopaline synthase terminator in genetically modified organisms in a real-time multiplex polymerase chain reaction using high-resolution melting-curve analysis.

    PubMed

    Akiyama, Hiroshi; Nakamura, Fumi; Yamada, Chihiro; Nakamura, Kosuke; Nakajima, Osamu; Kawakami, Hiroshi; Harikai, Naoki; Furui, Satoshi; Kitta, Kazumi; Teshima, Reiko

    2009-11-01

    To screen for unauthorized genetically modified organisms (GMO) in the various crops, we developed a multiplex real-time polymerase chain reaction high-resolution melting-curve analysis method for the simultaneous qualitative detection of 35S promoter sequence of cauliflower mosaic virus (35SP) and the nopaline synthase terminator (NOST) in several crops. We selected suitable primer sets for the simultaneous detection of 35SP and NOST and designed the primer set for the detection of spiked ColE1 plasmid to evaluate the validity of the polymerase chain reaction (PCR) analyses. In addition, we optimized the multiplex PCR conditions using the designed primer sets and EvaGreen as an intercalating dye. The contamination of unauthorized GMO with single copy similar to NK603 maize can be detected as low as 0.1% in a maize sample. Furthermore, we showed that the present method would be applicable in identifying GMO in various crops and foods like authorized GM soybean, authorized GM potato, the biscuit which is contaminated with GM soybeans and the rice which is contaminated with unauthorized GM rice. We consider this method to be a simple and reliable assay for screening for unauthorized GMO in crops and the processing food products.

  10. Differentiation between pathogenic serotype 1 isolates of Marek's disease virus and the Rispens CVI988 vaccine in Australia using real-time PCR and high resolution melt curve analysis.

    PubMed

    Renz, K G; Cheetham, B F; Walkden-Brown, S W

    2013-01-01

    Two real-time PCR assays were developed which enable quantitation and differentiation between pathogenic Australian isolates of Marek's disease virus (MDV) serotype 1 and the serotype 1 vaccine strain Rispens CVI988. The assays are based on a DNA sequence variation in the meq gene between pathogenic and vaccinal MDV1 which has been confirmed by sequencing of 20 Australian field strains of MDV. Complete specificity has been demonstrated in samples containing pathogenic MDV (n=20), Rispens (3 commercial vaccine strains), or both. The limit of detection of both the Rispens-specific and the pathogenic MDV1-specific assays was 10 viral copies/reaction. The tests successfully differentiated and quantified MDV in mixtures of pathogenic and vaccinal Rispens virus. A high resolution melt curve analysis targeting the same SNP used for the real-time PCR assays was also developed which successfully detected sequence variation between Md5, six Australian MDV1 isolates and the three Rispens vaccines. However it was ineffective at differentiating mixtures of pathogenic and vaccinal MDV1. The real-time PCR assays have both diagnostic and epidemiological applications as they enable differentiation and quantitation of Rispens CVI988 and pathogenic MDV1 in co-infected chickens in Australia.

  11. Novel application of hot-melt extrusion for the preparation of monolithic matrices containing enteric-coated particles.

    PubMed

    Schilling, Sandra U; McGinity, James W

    2010-11-15

    The objective was to investigate a novel application of hot-melt extrusion for the preparation of multiparticulate matrices comprising delayed-release particles. Multiparticulates of different mechanical strengths (theophylline granules, wet-mass extruded/spheronized pellets and drug-layered microcrystalline cellulose spheres) were coated with Eudragit(®) L30D-55 and characterized regarding potency, moisture content, dissolution properties and tensile strength. The coated particles were incorporated into a water-soluble matrix using hot-melt extrusion. Six hydrophilic polymers including polyethylene glycols, poloxamers and polyethylene oxides were studied as the carrier material for the extrusion. Dissolution testing showed that the maintenance of the delayed-release properties of the incorporated particles was independent of the particle tensile strength, but influenced by the nature of the carrier polymer. High miscibility between the carrier and the coating polymer correlated with increased film permeability and higher drug release in acidic media. Of the materials tested, poloxamer 407 exhibited lower miscibility with the Eudragit(®) L polymer and matrices containing up to 40% enteric pellets were compliant with the USP dissolution requirements for delayed-release dosage forms. The potential advantages of hot-melt extrusion over direct compression for the processing of soft drug granules coated with Eudragit(®) L polymer were demonstrated.

  12. E-beam GIDC resolution enhancement technology in practical applications

    NASA Astrophysics Data System (ADS)

    Martens, S.; Butschke, J.; Galler, R.; Krüger, M.; Sailer, H.; Sülzle, M.

    2013-09-01

    For nearly all relevant applications of e-beam lithography the resolution and pattern quality requirements are approaching or exceeding the limits of the available process. On one hand, for shrinking feature dimensions, the e-beam proximity effect and process effects such as photo acid diffusion limit the pattern contrast and process window. On the other hand, e-beam process related parasitic effects such as shot noise, fogging, developer loading, heating, charging, and inhomogeneous bake introduce some significant errors. Even though e-beam tool and process tool suppliers continue to implement new or improve current strategies to avoid or correct these effects, the amount of residual errors requires some reasonable e-beam process window, in particular for high end applications. For some patterns the undersize-overdose approach (SIZE) improves the pattern fidelity and process window. However, for patterns with high fill factors this approach increases the overall deposited electron dose, which due to the increased backscattering diminishes or even eliminates the advantages. The geometrically induced dose correction (GIDC) method overcomes this issue by combining the SIZE concept with a short range framing technique, which reduces the deposited dose in large filled pattern areas. This paper provides a comparison of the standard, SIZE, and GIDC correction approaches for 1D test patterns as well as production patterns. For a broad comparison, patterns were printed onto negative and positive chemically amplified resists and on wafer and mask substrates using a Vistec SB352HR variable shape e-beam writer. Both wafers were also etched. The outcome of the study is that the SIZE and GIDC approaches often outperform the standard proximity effect correction. For dense patterns, GIDC still provides a better pattern quality and process window, while the SIZE approach suffers from the increased overall deposited electron dose and clearly falls behind GIDC in terms of process

  13. Shallow subsurface applications of high-resolution seismic reflection

    NASA Astrophysics Data System (ADS)

    Steeples, Don

    2002-11-01

    Shallow seismic reflection surveys have been applied to a wide variety of problems. For example, in many geologic settings, variations and discontinuities on the surface of bedrock can influence the transport and eventual fate of contaminants introduced at or near the ground surface. Using seismic methods to determine the nature and location of anomalous bedrock can be an essential component of hydrologic characterization. Shallow seismic surveys can also be used to detect earthquake faults and to image underground voids. During the early 1980s, the advent of digital engineering seismographs designed for shallow, high-resolution surveying spurred significant improvements in engineering and environmental reflection seismology. Commonly, shallow seismic reflection methods are used in conjunction with other geophysical and geological methods, supported by a well-planned drilling-verification effort. To the extent that seismic reflection, refraction, and surface-wave methods can constrain shallow stratigraphy, geologic structure, engineering properties, and relative permeability, these methods are useful in civil-engineering applications and in characterizing environmental sites. Case histories from Kansas, California, and Texas illustrate how seismic reflection can be used to map bedrock beneath alluvium at hazardous waste sites, detect abandoned coal mines, follow the top of the saturated zone during an alluvial aquifer pumping test, and map shallow faults that serve as contaminant flowpaths.

  14. Application of spectroscopy and super-resolution microscopy: Excited state

    SciTech Connect

    Bhattacharjee, Ujjal

    2016-02-19

    Photophysics of inorganic materials and organic molecules in complex systems have been extensively studied with absorption and emission spectroscopy.1-4 Steady-state and time-resolved fluorescence studies are commonly carried out to characterize excited-state properties of fluorophores. Although steady-state fluorescence measurements are widely used for analytical applications, time-resolved fluorescence measurements provide more detailed information about excited-state properties and the environment in the vicinity of the fluorophore. Many photophysical processes, such as photoinduced electron transfer (PET), rotational reorientation, solvent relaxation, and energy transfer, occur on a nanosecond (10-9 s) timescale, thus affecting the lifetime of the fluorophores. Moreover, time-resolved microscopy methods, such as lifetimeimaging, combine the benefits of the microscopic measurement and information-rich, timeresolved data. Thus, time-resolved fluorescence spectroscopy combined with microscopy can be used to quantify these processes and to obtain a deeper understanding of the chemical surroundings of the fluorophore in a small area under investigation. This thesis discusses various photophysical and super-resolution microscopic studies of organic and inorganic materials, which have been outlined below.

  15. Application of mean-field model of polymer melt intercalation in organo-silicates for nanocomposites.

    PubMed

    Meneghetti, Paulo; Qutubuddin, Syed

    2005-08-15

    The mean-field, lattice-based model of polymer melt intercalation in organically-modified layered silicates (OLS) originally developed by Vaia and Giannelis was applied for different polymers such as poly(methyl methacrylate) (PMMA), polypropylene (PP), and poly(ethylene oxide) (PEO). The nature of each polymer controls significantly the intercalation of the system. The internal energy change caused by the interaction of polymer, surfactant and clay is the strongest factor in determining the equilibrium structure of the nanocomposite system.

  16. Gastrointestinal stromal tumors with KIT exon 9 mutations: Update on genotype-phenotype correlation and validation of a high-resolution melting assay for mutational testing.

    PubMed

    Künstlinger, Helen; Huss, Sebastian; Merkelbach-Bruse, Sabine; Binot, Elke; Kleine, Michaela Angelika; Loeser, Heike; Mittler, Jens; Hartmann, Wolfgang; Hohenberger, Peter; Reichardt, Peter; Büttner, Reinhard; Wardelmann, Eva; Schildhaus, Hans-Ulrich

    2013-11-01

    KIT exon 9 mutations in gastrointestinal stromal tumors (GISTs) are highly relevant and have direct therapeutic implications. In this context, we established and validated a fast and sensitive high-resolution melting assay. Analyzing 126 primary and 18 metastatic KIT exon 9-mutated cases from our registry, we demonstrate that the mutational spectrum of exon 9 is broader than previously thought and describe 3 novel mutations. Including these cases and the common p.A502_Y503dup mutation, we provide a comprehensive list of all known KIT exon 9 mutations according to the Human Genome Variation Society nomenclature. Two of the newly described mutations were associated with an aggressive phenotype and tumor progression while being treated with 400 mg imatinib, indicating that also GIST with rare exon 9 mutations could be treated with increased imatinib dosage. On the basis of >1500 GISTs from our registry, we have determined the frequency of KIT exon 9 mutations to be 9.2% among all GISTs and 22.5% among small-bowel cases. We describe for the first time that nearly 20% of exon 9-mutated GIST occur in the stomach or rectum. Furthermore, we provide first evidence that exon 9-mutated GISTs metastasize significantly more often to the peritoneum than to the liver. Performing extensive statistical analyses on data from our registry and from the literature, we demonstrate that KIT exon 9 mutations are neither associated with intermediate-risk/high-risk status nor overrepresented among metastatic lesions. Thus, we conclude that exon 9 mutations per se do not have prognostic relevance.

  17. Real-time PCR and high-resolution melt analysis for rapid detection of Mycobacterium leprae drug resistance mutations and strain types.

    PubMed

    Li, Wei; Matsuoka, Masanori; Kai, Masanori; Thapa, Pratibha; Khadge, Saraswoti; Hagge, Deanna A; Brennan, Patrick J; Vissa, Varalakshmi

    2012-03-01

    Drug resistance surveillance and strain typing of Mycobacterium leprae are necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR-high-resolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) of folP1, rpoB, and gyrA, targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included three folP1, two rpoB, and one gyrA mutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all the folP1 mutants representing two mutation types, including one not within the reference panel. The false positives (<5%) could be attributed to low DNA concentration or PCR inhibition. A second set of RT-PCR-HRM assays for identification of three previously reported single nucleotide polymorphisms (SNPs) that have been used for strain typing were developed and validated in 22 reference and 25 clinical strains. Real-time PCR-HRM is a sensitive, simple, rapid, and high-throughput tool for routine screening known DRDR mutants in new and relapsed cases, SNP typing, and detection of minor mutant alleles in the wild-type background at lower costs than current methods and with the potential for quality control in leprosy investigations.

  18. Evaluation of the Capacity of PCR and High-Resolution Melt Curve Analysis for Identification of Mixed Infection with Mycoplasma gallisepticum Strains.

    PubMed

    Ghorashi, Seyed A; Kanci, Anna; Noormohammadi, Amir H

    2015-01-01

    Pathogenicity and presentation of Mycoplasma gallisepticum (MG) infection may differ from one strain to another and this may have implications on control measures. Infection of individual birds with more than one MG strain has been reported. A PCR followed by high resolution melt (HRM) curve analysis has been developed in our laboratory and routinely used for detection and differentiation of MG strains. However the potential of this test for identification of MG strains in a mixed specimen has not been evaluated. In the present study, the capability of PCR-HRM curve analysis technique, targeting vlhA and pvpA genes was assessed for identification of individual MG strains in a mixed population. Different DNA ratios of two MG strains from 1 to 10(-4) ng were tested with some generated conventional and normalized curves distinct from those of individual strains alone. Using genotype confidence percentages (GCP) generated from HRM curve analysis, it was found that vlhA PCR-HRM was more consistent than pvpA PCR-HRM for the detection of MG ts-11 vaccine strain mixed with any of the MG strains 6/85, F, S6 or a field isolate. The potential of vlhA PCR-HRM to detect mixed MG strains in a specimen was found to be primarily dependent on quantity and proportion of the target DNAs in the mixture. This is the first study examining the capacity of PCR-HRM technique for identification of individual MG strains in a mixed strain population.

  19. Hypervariable pili and flagella genes provide suitable new targets for DNA high-resolution melt-based genotyping of dairy Geobacillus spp.

    PubMed

    Chauhan, Kanika; Seale, R Brent; Deeth, Hilton C; Turner, Mark S

    2014-10-01

    Although nonpathogenic in nature, spores of Geobacillus are able to attach to surfaces, germinate, and form biofilms, allowing rapid multiplication and persistence within milk powder processing plants, causing final product contamination, and eventually leading to a loss of revenue in terms of downgraded product quality. As a result, Geobacillus spp. have been found to be common contaminants of milk powder worldwide. Genotyping methods can help in gaining insight into the ecology and transmission of these thermophilic bacteria within and between dairy processing plants. The objective of this study was to use the assembled draft genomes of two Geobacillus spp. to identify and test new hypervariable genotyping targets for differentiating closely related dairy Geobacillus isolates. The two Geobacillus spp. strains obtained from high spore count powders were obtained in 2010 (isolate 7E) and in 1995 (isolate 126) and were previously shown to be of same genotype based on a variable number tandem repeat genotyping method. Significant nucleotide sequence variation was found in genes encoding pili and flagella, which were further investigated as suitable loci for a new high-resolution melt analysis (HRMA)-based genotyping method. Three genes encoding pulG (containing prepilin-type N-terminal cleavage domain), pilT (pili retraction protein), and fliW (flagellar assembly protein) were selected as targets for the new pili/flagella gene (PilFla) HRMA genotyping method. The three-gene-based PilFla-HRMA genotyping method differentiated 35 milk powder Geobacillus spp. isolates into 19 different genotype groups (D = 0.93), which compared favorably to the previous method (which used four variable number tandem repeat loci) that generated 16 different genotype groups (D = 0.90). In conclusion, through comparative genomics of two closely related dairy Geobacillus strains, we have identified new hypervariable regions that prove to be useful targets for highly discriminatory genotyping.

  20. Combining COLD-PCR and high-resolution melt analysis for rapid detection of low-level, rifampin-resistant mutations in Mycobacterium tuberculosis.

    PubMed

    Pang, Yu; Liu, Guan; Wang, Yufeng; Zheng, Suhua; Zhao, Yan-Lin

    2013-04-01

    Multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis) remains a serious threat to public health. Mutational analysis of the gene encoding the beta subunit of RNA polymerase (rpoB) is an established and widely used surrogate marker for multidrug-resistant tuberculosis (MDR-TB). The rpoB-based drug-resistant assay requires relatively less time to detect drug resistance in M. tuberculosis, yet it fails to detect low-level mutations in wild-type DNA. Here, we describe a low-level mutation detection method that combines co-amplification at lower denaturation temperature polymerase chain reaction (COLD-PCR) with high-resolution melting (HRM) analysis, aimed at detecting low-level, rifampin-resistant mutations in M. tuberculosis. Compared to conventional polymerase chain reaction (PCR), dilution experiments demonstrated a four- to eightfold improvement in selectivity using COLD-PCR/HRM to detect low-level, rifampin-resistant mutations. The mutation detection limit of conventional PCR/HRM was approximately 20%, whereas COLD-PCR/HRM had a mutation detection limit of 2.5%. Using traditional PCR/HRM and DNA sequencing, we found rpoB mutation in 110 rifampin-resistant isolates. The use of COLD-PCR/HRM allowed us to detect 10 low-level, rifampin-resistant mutations in 16 additional drug-resistant isolates. The sensitivity of COLD-PCR/HRM (95.2%) is significantly higher than that of PCR/HRM (87.3%). Our findings demonstrate that combined use of COLD-PCR with HRM can provide a sensitivity of at least 5% in detecting rpoB-mutated populations in a wild-type background, decreasing the delay in drug-resistant TB diagnosis and leading to faster, cheaper, more efficient, and more personalized antibiotic treatment, especially for low-level drug resistance mutations among the excess wild-type DNA.

  1. Bartonellae in domestic and stray cats from Israel: comparison of bacterial cultures and high-resolution melt real-time PCR as diagnostic methods.

    PubMed

    Gutiérrez, Ricardo; Morick, Danny; Gross, Ifat; Winkler, Ronen; Abdeen, Ziad; Harrus, Shimon

    2013-12-01

    To determine the occurrence of feline bartonellosis in Israel, blood samples were collected from 179 stray and 155 domestic cats from 18 cities or villages in central and northcentral Israel. Samples were screened for Bartonella infection by culture isolation and molecular detection using high-resolution melt (HRM) real-time PCR assay targeting the 16S-23S rRNA internal transcribed spacer (ITS). All positive samples were confirmed by two additional HRM real-time PCR assays targeting two fragments of the β-subunit of RNA polymerase (rpoB) and the 16S rRNA genes. The prevalence of Bartonella spp. infection in the general tested population was 25.1% (84/334). A higher prevalence was detected in the stray (30.7%; 55/179) than the domestic cats (18.7%; 29/155). Bartonella henselae, Bartonella clarridgeiae, and Bartonella koehlerae were highly prevalent in both cat populations, however their distribution among the two populations varied significantly (p=0.016). B. clarridgeiae and B. koehlerae were found to be more prevalent in stray than domestic cats, whereas B. henselae was evenly distributed. Co-infection with two or more different Bartonella spp. was determined in 2.1% (7) of the cats. The ITS HRM real-time PCR assay used in this study was shown to have a greater screening power than bacterial isolation, detecting 94.0% (79/84) compared to 35.7% (30/84), respectively, of all positive samples. The high prevalence of these zoonotic Bartonella species, coupled with the overpopulation of stray cats, and increased numbers of domestic cats in the major urban centers in Israel represent a significant threat for the public health in this country.

  2. Genotyping of present-day and historical Geobacillus species isolates from milk powders by high-resolution melt analysis of multiple variable-number tandem-repeat loci.

    PubMed

    Seale, R Brent; Dhakal, Rajat; Chauhan, Kanika; Craven, Heather M; Deeth, Hilton C; Pillidge, Christopher J; Powell, Ian B; Turner, Mark S

    2012-10-01

    Spores of thermophilic Geobacillus species are a common contaminant of milk powder worldwide due to their ability to form biofilms within processing plants. Genotyping methods can provide information regarding the source and monitoring of contamination. A new genotyping method was developed based on multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) in conjunction with high-resolution melt analysis (MLV-HRMA) and compared to the currently used method, randomized amplified polymorphic DNA PCR (RAPD-PCR). Four VNTR loci were identified and used to genotype 46 Geobacillus isolates obtained from retailed powder and samples from 2 different milk powder processing plants. These 46 isolates were differentiated into 16 different groups using MLV-HRMA (D = 0.89). In contrast, only 13 RAPD-PCR genotypes were identified among the 46 isolates (D = 0.79). This new method was then used to analyze 35 isolates obtained from powders with high spore counts (>10(4) spores · g(-1)) from a single processing plant together with 27 historical isolates obtained from powder samples processed in the same region of Australia 17 years ago. Results showed that three genotypes can coexist in a single processing run, while the same genotypes observed 17 years ago are present today. While certain genotypes could be responsible for powders with high spore counts, there was no correlation to specific genotypes being present in powder plants and retailed samples. In conclusion, the MLV-HRMA method is useful for genotyping Geobacillus spp. to provide insight into the prevalence and persistence of certain genotypes within milk powder processing plants.

  3. Comparison of high-resolution melting analysis, TaqMan Allelic discrimination assay, and sanger sequencing for Clopidogrel efficacy genotyping in routine molecular diagnostics.

    PubMed

    Zhang, Lina; Cui, Guanglin; Li, Zongzhe; Wang, Haoran; Ding, Hu; Wang, Dao Wen

    2013-09-01

    Clopidogrel, as a routine antiplatelet drug, is widely used in patients to reduce cardiovascular events following percutaneous coronary intervention. Because of genetic variation, patients undergoing percutaneous coronary intervention show differing responses to clopidogrel therapy. Recently, five single nucleotide polymorphisms (SNPs) within CYP2C19 (rs4244285, rs4986893, rs12248560), ABCB1 (rs1045642), and ITGB3 (rs5918) were identified that contribute prominently to variability in response to clopidogrel. Given that Sanger sequencing is labor intensive and time consuming, rapid genotyping methods for SNP detection are urgently required before clopidogrel therapy. Accordingly, we developed a high-resolution melting analysis (HRMA) and TaqMan allelic discrimination assay (TaqMan) to genotype those five SNPs, and compared these two assays with Sanger sequencing on accuracy of genotyping as well as operational characteristics. These two assays showed high accuracy (0.995, 95% CI 0.991 to 0.998 for HRMA; 0.997, 95% CI 0.994 to 0.999 for TaqMan, respectively), sensitivity (0.996, 95% CI 0.989 to 1.002 for HRMA; 0.998, 95% CI 0.993 to 1.002 for TaqMan, respectively), and specificity (0.995, 95% CI 0.991 to 0.999 for HRMA; 0.996, 95% CI 0.993 to 1.000 for TaqMan, respectively). Our study indicates that HRMA and TaqMan are easier to operate and obviously faster than Sanger sequencing. In conclusion, HRMA and TaqMan are rapid, convenient, and reliable assays for clopidogrel efficacy genotyping.

  4. Application of melt granulation technology to enhance tabletting properties of poorly compactible high-dose drugs.

    PubMed

    Lakshman, Jay P; Kowalski, James; Vasanthavada, Madhav; Tong, Wei-Qin; Joshi, Yatindra M; Serajuddin, Abu T M

    2011-04-01

    Using metformin HCl as the model drug and hydroxypropylcellulose (HPC) as the polymeric excipient, a melt granulation (MG) process that employs a twin-screw extruder has been developed to enhance compactibility of poorly compactible high-dose drug substances. A high (90%) drug-load tablet formulation, containing 1025 mg of active pharmaceutical ingredients and 109 mg of excipients, was produced. Drug-polymer-powder mixtures were melt granulated at a temperature above glass transition of HPC (130°C) but below melting point of metformin HCl (224°C). MG was compared with modified wet granulation (WG) and solvent granulation (SG) processes. Under identical compression force, the hardness of tablets produced was MG>SG>WG and the friability was MG

  5. Thermal Assessment of a Latent-Heat Energy Storage Module During Melting and Freezing for Solar Energy Applications

    NASA Astrophysics Data System (ADS)

    Ramos Archibold, Antonio

    Capital investment reduction, exergetic efficiency improvement and material compatibility issues have been identified as the primary techno-economic challenges associated, with the near-term development and deployment of thermal energy storage (TES) in commercial-scale concentrating solar power plants. Three TES techniques have gained attention in the solar energy research community as possible candidates to reduce the cost of solar-generated electricity, namely (1) sensible heat storage, (2) latent heat (tank filled with phase change materials (PCMs) or encapsulated PCMs packed in a vessel) and (3) thermochemical storage. Among these the PCM macro-encapsulation approach seems to be one of the most-promising methods because of its potential to develop more effective energy exchange, reduce the cost associated with the tank and increase the exergetic efficiency. However, the technological barriers to this approach arise from the encapsulation techniques used to create a durable capsule, as well as an assessment of the fundamental thermal energy transport mechanisms during the phase change. A comprehensive study of the energy exchange interactions and induced fluid flow during melting and solidification of a confined storage medium is reported in this investigation from a theoretical perspective. Emphasis has been placed on the thermal characterization of a single constituent storage module rather than an entire storage system, in order to, precisely capture the energy exchange contributions of all the fundamental heat transfer mechanisms during the phase change processes. Two-dimensional, axisymmetric, transient equations for mass, momentum and energy conservation have been solved numerically by the finite volume scheme. Initially, the interaction between conduction and natural convection energy transport modes, in the absence of thermal radiation, is investigated for solar power applications at temperatures (300--400°C). Later, participating thermal radiation

  6. 77 FR 70433 - Resolute Marine Energy, Inc.; Notice of Preliminary Permit Application Accepted for Filing and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-26

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Federal Energy Regulatory Commission Resolute Marine Energy, Inc.; Notice of Preliminary Permit Application Accepted for Filing and Soliciting Comments, Motions To Intervene, and Competing Applications On July 25, 2012, Resolute Marine Energy, Inc....

  7. Applications of web produced by hot air assisted melt differential electrospinning method

    NASA Astrophysics Data System (ADS)

    Bubakir, Mahmoud M.; Li, Haoyi; Wu, Weifeng; Li, Xiaohu; Ma, Shuai; Yang, Weimin

    2014-08-01

    Melt electrospinning, a technique that has gained increasing attention since it easily can generate continuous ultrafine fibers directly from polymer melts without the use of any solvent. Therefore, it is considered as a safe, cost effective, and environmental friendly technique. However, with all those great advantages, the technique still suffers some drawbacks such as: large fiber diameter and low throughput. The hot air assisted melt differential electrospinning (MDES) is a new technique invented by our research team that can solve or eliminate those drawbacks. The most important features of our used apparatus are: Needleless nozzle that could generate multiple Taylor cones around the bottom edge of the nozzle, which can result in a high throughput. The stretching force acting on the jets can be further strengthened by an air current provided by an air pressure gun. Interference between the high voltage supply and temperature sensors could be prevented through the grounding of the nozzle. The ultrafine pp webs produced using the same apparatus was in the micro/nano scale with a diameter of 600nm-6um and a smooth surface. Porosity of the webs ranges from 86.5%-99.4% when different collecting devices are used. The resultant ultrafine webs were applied in three areas: oil sorption, water treatment, and hydrophilic PP membrane. The results were very promising as for oil the sorption capacity was 129.0g/g; for water treatment, the rejection rate for 3um particles was 95%. And for the hydrophilic PP membrane, the water sorption capacity was 12.3 g/g.

  8. Application of mixtures of polymeric carriers for dissolution enhancement of fenofibrate using hot-melt extrusion.

    PubMed

    Kalivoda, Adela; Fischbach, Matthias; Kleinebudde, Peter

    2012-06-15

    Hot-melt extrusion was applied to improve dissolution behavior of poorly soluble model drug fenofibrate. Blends of polymers were used as carrier: copovidone (COP), polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer (PVCL-PVAc-PEG) and hypromellose 2910/5 (HPMC). The ratio of fenofibrate to COP remained constantly 1+3 (weighted parts) with varying amounts of PVCL-PVAc-PEG and HPMC. Solid state of fenofibrate was characterized by X-ray diffractometry and differential scanning calorimetry. Dissolution performance was compared to marketed formulations Lipidil and Lipidil-Ter. Stability studies were conducted at 25°C/60%rH. The dissolution rate from extrudates was significantly increased when compared to pure fenofibrate powder or physical mixture of the components. A supersaturation of 7.6-12.1 was reached with the pelletized extrudates. All extrudates were superior to marketed formulations. No recrystallization was observed after 26 weeks of storage for fenofibrate-COP extrudates 1+3 (weighted parts) with or without polymeric additives. Even so, both degree and duration of supersaturation decreased with increasing storage periods with the exception of fenofibrate-HPMC extrudates. Of particular interest is the finding that by adding polymers with differing release characteristics to the drug-carrier mixture, the dissolution performance of hot-melt extruded solid dosage forms can be readily adapted to meet specific requirements.

  9. Application of mixtures of polymeric carriers for dissolution enhancement of oxeglitazar using hot-melt extrusion.

    PubMed

    Kalivoda, Adela; Fischbach, Matthias; Kleinebudde, Peter

    2012-12-15

    Hot-melt extrusion was applied to improve the solubility of the poorly water-soluble drug oxeglitazar. Various polymers and their blends were used as carriers: copovidone (COP), polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer (PVCL-PVAc-PEG) and hypromellose 2910/5 (HPMC). After extrusion, the extrudate was pelletized. The physical state of the drug was assessed using X-ray diffraction and differential scanning calorimetry. The dissolution performance of the extrudates was compared to the physical mixture and pure oxeglitazar. The stability under long-term storage conditions (25 °C/60%rH) was investigated.The solubility of oxeglitazar was improved with all hot-melt extruded formulations: 26-66% of the drug was dissolved and a 1.9-5.0-fold supersaturation was reached with the pelletized extrudates. All extrudates which were assessed for their storage stability showed sufficient product stability. A super-additive effect of COP and HPMC as a polymeric blend was successfully demonstrated as a higher supersaturation and longer time of supersaturation were shown for the ternary blend. Through variations of the ratio COP:HPMC, it was shown that the shape of the dissolution curve is dominated by the polymer with the higher amount in the polymeric blend. If PVCL-PVAc-PEG is applied as single or additional carrier, the initial release rate is drastically reduced.

  10. Full Spatial Resolution Infrared Sounding Application in the Preconvection Environment

    NASA Astrophysics Data System (ADS)

    Liu, C.; Liu, G.; Lin, T.

    2013-12-01

    Advanced infrared (IR) sounders such as the Atmospheric Infrared Sounder (AIRS) and Infrared Atmospheric Sounding Interferometer (IASI) provide atmospheric temperature and moisture profiles with high vertical resolution and high accuracy in preconvection environments. The derived atmospheric stability indices such as convective available potential energy (CAPE) and lifted index (LI) from advanced IR soundings can provide critical information 1 ; 6 h before the development of severe convective storms. Three convective storms are selected for the evaluation of applying AIRS full spatial resolution soundings and the derived products on providing warning information in the preconvection environments. In the first case, the AIRS full spatial resolution soundings revealed local extremely high atmospheric instability 3 h ahead of the convection on the leading edge of a frontal system, while the second case demonstrates that the extremely high atmospheric instability is associated with the local development of severe thunderstorm in the following hours. The third case is a local severe storm that occurred on 7-8 August 2010 in Zhou Qu, China, which caused more than 1400 deaths and left another 300 or more people missing. The AIRS full spatial resolution LI product shows the atmospheric instability 3.5 h before the storm genesis. The CAPE and LI from AIRS full spatial resolution and operational AIRS/AMSU soundings along with Geostationary Operational Environmental Satellite (GOES) Sounder derived product image (DPI) products were analyzed and compared. Case studies show that full spatial resolution AIRS retrievals provide more useful warning information in the preconvection environments for determining favorable locations for convective initiation (CI) than do the coarser spatial resolution operational soundings and lower spectral resolution GOES Sounder retrievals. The retrieved soundings are also tested in a regional data assimilation WRF 3D-var system to evaluate the

  11. Ocean modeling at multiple resolutions for ISR applications

    NASA Astrophysics Data System (ADS)

    Cathcart, J. Michael; Teague, J. Ralph; Burdette, Ed; Kocher, Brian

    2011-05-01

    Recent research efforts at Georgia Tech have focused on the development of a multi-resolution ocean clutter model. This research was driven by the need to support both surveillance and search requirements set by several government customers. These requirements indicated a need to support target detection and tracking for both resolved and unresolved scenarios for targets located either above or on an ocean surface. As a result of this changing sensor resolution characteristic for the various acquisition scenarios, a need for accurate ocean surface models at different geometric resolutions arose. Georgia Tech met this need through development of a multi-resolution approach to modeling both the ocean surface and, subsequently, the ocean signature across the optical spectrum. This approach combined empirical overhead data with high resolution ocean surface models to construct a series of varying resolution ocean clutter models. This paper will describe the approach to utilizing and merging the various clutter models as well as the results of using these models in the target detection and tracking analysis. Remaining issues associated with this clutter model development will be identified and potential solutions discussed.

  12. Evaluation of extended-release applications for solid dispersion hot-melt fluid bed coatings utilizing hydrophobic coating agents.

    PubMed

    Kennedy, J P; Niebergall, P J

    1998-02-01

    A new hot-melt fluid bed coating method was evaluated for potential extended-release applications. Chlorpheniramine maleate (CPM) USP was chosen as a model drug. The assays for drug release and content uniformity were dictated by the USP Official Monograph for a Chlorpheniramine Maleate Extended-Release Capsule. The fluid bed chamber was charged with CPM-loaded nonpareils and hydrophobic coating agents in the solid state. The method consists of four processing stages: (a) warming, (b) preheating, (c) melting-spreading, and (d) cooling-congealing. Various hydrophobic coating agent candidates were evaluated for extended-release potential by a preliminary screen at a coating agent level of 1.5% (w/w). A beeswax coating agent was identified as the most promising candidate of the preliminary screen. After the level of beeswax was increased to 2.0%, the dissolution profile met all of the specifications of the USP Drug Release Test 1 for a CPM Extended-Release Capsule. The potency and content uniformity remained unchanged by the process. Dual coatings demonstrated a cumulative extension of release superior to the capability of a single coat. The new method is a viable alternative to hot-melt spray-coating methodologies. Organic solvents, spraying equipment, steam jackets, and/or heating tape are eliminated from the process. A reduction of equipment costs, setup time, and cleanup time may be realized. The method has demonstrated extended-release capabilities. No excessive attrition of potency or content uniformity has been noted. Additive, multiple coatings that have a cumulative effect on release retardation are feasible.

  13. Application of the Cold Crucible for Melting of UO{sub 2}/ZrO{sub 2} Mixtures

    SciTech Connect

    Hong, S.W.; Min, B.T.; Shin, Y.S.; Park, I.K.; Kim, J.H.; Song, J.H.; Kim, H.D.

    2002-07-01

    The melting and discharge technique of UO{sub 2}/ZrO{sub 2} mixtures using the cold crucible melting method that does not need a separate crucible such as tungsten one with high melting point is developed and applied to the KAERI FCI test called TROI. To discharge the melt from a cold crucible into a fuel-coolant interaction chamber after melting, a plug is specially designed using the concept for electro-magnetic field characteristics so as to as thin as possible the crust that is formed between the melt and plug. Its function keeps the melt in the crucible during melting period and provides the melt discharge path. About 8.5 kg melt is discharged from the cold crucible to the melt-water interaction chamber through the punched hole with 8 cm in diameter. The melt temperature is also measured and analyzed from observation of the melt surface. The power balance using the operating parameters such as current, voltage and coupling factor of R.F generator is analyzed. (authors)

  14. Application of melt ejection criterion in simulation of micromachining with laser

    NASA Astrophysics Data System (ADS)

    Semak, Vladimir V.; Schriempf, J. T.; Knorovsky, G. A.; MacCallum, D. O.

    2003-07-01

    The theoretical criterion defining the threshold pulse energy and beam intensity required for melt ejection is proposed. The results of numerical simulation present dependencies of the threshold pulse energy and beam intensity as functions of laser pulse duration and beam radius. The experimental verification of proposed criterion is described and the comparison of theoretical predictions and measurements is presented. The criterion is applied for simulation of laser drilling metal foil with thickness in the range 25 μm - 125 μm using laser beam with 12 μm beam radius and pulse durations 10 ns and 100 ns. The computational results are used to interpret the results of experimental study of laser drilling of 125 μm aluminum foil using a single mode beam of a XeCl laser performed at the Nederlands Centrum voor Laser Research (NCLR) and the University of Twente. Additional results on Nd:YAG spot welds in pure Ni are also presented.

  15. Application of tumbling melt granulation (TMG) method to prepare controlled-release fine granules.

    PubMed

    Maejima, T; Kubo, M; Osawa, T; Nakajima, K; Kobayashi, M

    1998-03-01

    The tumbling melt granulation (TMG) method was applied to prepare controlled-release fine granules of diltiazem hydrochloride (DH). The entire process, from the preparation of the cores by the adherence of DH to the sucrose crystal to the subsequent coating of the controlled-release layer, was performed without using any solvent. A mixture of meltable material, talc, and ethylcellulose was used for the controlled-release layer and controlled-release fine granules approximately 400 microns in diameter were obtained with excellent producibility. The dissolution rate of DH from these fine granules was similar to that of a once-a-day dosage form obtained in the market; further, the dependency of the dissolution profile on pH of the media was less. Thus, it was concluded that this TMG method was very useful for preparing not only controlled-release beads of granule size (usually 500 to 1400 microns) but also fine granules.

  16. Improvement of dissolution behavior for poorly water-soluble drug by application of cyclodextrin in extrusion process: comparison between melt extrusion and wet extrusion.

    PubMed

    Yano, Hideki; Kleinebudde, Peter

    2010-06-01

    The purpose of this study was to improve dissolution behavior of poorly water-soluble drugs by application of cyclodextrin in extrusion processes, which were melt extrusion process and wet extrusion process. Indomethacin (IM) was employed as a model drug. Extrudates containing IM and 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) in 1:1 w/w ratio were manufactured by both melt extrusion process and wet extrusion process. In vitro drug release properties of IM from extrudates and physiochemical properties of extrudates were investigated. The dissolution rates of IM from extrudates manufactured by melt extrusion and wet extrusion with HP-beta-CyD were significantly higher than that of the physical mixture of IM and HP-beta-CyD. In extrudate manufactured by melt extrusion, gamma-form of IM changed to amorphous completely during melt extrusion due to heating above melting point of IM. On the other hand, in extrudate manufactured by wet extrusion, gamma-form of IM changed to amorphous partially due to interaction between IM and HP-beta-CyD and mechanical agitating force during process. Application of HP-beta-CyD in extrusion process is useful for the enhancement of dissolution rate for poorly water-soluble drugs.

  17. Melt-processable hydrophobic acrylonitrile-based copolymer systems with adjustable elastic properties designed for biomedical applications.

    PubMed

    Cui, J; Trescher, K; Kratz, K; Jung, F; Hiebl, B; Lendlein, A

    2010-01-01

    Acrylonitrile-based polymer systems (PAN) are comprehensively explored as versatile biomaterials having various potential biomedical applications, such as membranes for extra corporal devices or matrixes for guided skin reconstruction. The surface properties (e.g. hydrophilicity or charges) of such materials can be tailored over a wide range by variation of molecular parameters such as different co-monomers or their sequence structure. Some of these materials show interesting biofunctionalities such as capability for selective cell cultivation. So far, the majority of AN-based copolymers, which were investigated in physiological environments, were processed from the solution (e.g. membranes), as these materials are thermo-sensitive and might degrade when heated. In this work we aimed at the synthesis of hydrophobic, melt-processable AN-based copolymers with adjustable elastic properties for preparation of model scaffolds with controlled pore geometry and size. For this purpose a series of copolymers from acrylonitrile and n-butyl acrylate (nBA) was synthesized via free radical copolymerisation technique. The content of nBA in the copolymer varied from 45 wt% to 70 wt%, which was confirmed by 1H-NMR spectroscopy. The glass transition temperatures (Tg) of the P(AN-co-nBA) copolymers determined by differential scanning calorimetry (DSC) decreased from 58 degrees C to 20 degrees C with increasing nBA-content, which was in excellent agreement with the prediction of the Gordon-Taylor equation based on the Tgs of the homopolymers. The Young's modulus obtained in tensile tests was found to decrease significantly with rising nBA-content from 1062 MPa to 1.2 MPa. All copolymers could be successfully processed from the melt with processing temperatures ranging from 50 degrees C to 170 degrees C, whereby thermally induced decomposition was only observed at temperatures higher than 320 degrees C in thermal gravimetric analysis (TGA). Finally, the melt processed P

  18. A new perturbation theory of solids and fluids and its applications to high-pressure melting problems

    SciTech Connect

    Ree, F.H.

    1990-05-01

    A statistical mechanical theory that can describe both solids and fluids in a self-consistent way is described. This theory utilizes a optimized reference potential whose repulsive range shrinks with density. A unique feature of the new theory is that solid- and fluid-phase thermodynamic properties are both computed within a single theoretical framework. Hence, it allows us to study melting phenomena in a self-consistent manner. For solids, the new theory treats both harmonic and anharmonic effects in thermodynamic properties on equal footing. Applications to several model and rare gas systems show that the new theory can accurately predict fluid, solid, and fluid-solid transition properties. Effective pair potentials inferred from the analysis of krypton and xenon isotherms contain short- and long-range modifications to the Aziz-Slaman pair potential. The long-range correction is repulsive and originates from the Axilrod-Teller three-body force, while the short-range correction probably originates from many-body forces. Using the computed melting curves of krypton and neon, we discuss the range of validity of the corresponding states principle for rare gas systems. 68 refs., 8 figs., 6 tabs.

  19. On the application and extension of Harten's high resolution scheme

    NASA Technical Reports Server (NTRS)

    Yee, H. C.; Warming, R. F.; Harten, A.

    1982-01-01

    Extensions of a second order high resolution explicit method for the numerical computation of weak solutions of one dimensonal hyperbolic conservation laws are discussed. The main objectives were (1) to examine the shock resoluton of Harten's method for a two dimensional shock reflection problem, (2) to study the use of a high resolution scheme as a post-processor to an approximate steady state solution, and (3) to construct an implicit in the delta-form using Harten's scheme for the explicit operator and a simplified iteration matrix for the implicit operator.

  20. Application of high-resolution depositional modeling to reservoir characterisation

    SciTech Connect

    Keyu, L.; Paterson, L.

    1995-08-01

    As hydrocarbon producing basins and fields become more intensely developed, conventional stratigraphic analysis methods can sometimes no longer meet the resolution required by petroleum geologists and engineers. High-resolution depositional modeling provides a quantitative alternative to the conventional approach of sequence stratigraphic analysis. A computer program, SEDPAK, which was designed primarily according to the basic premise of the sequence stratigraphic concept, was here used to conduct high-resolution simulations for two sedimentary sequences. One is a Pliocene to Recent shelf margin sediment sequence of the offshore Sydney Basin continental shelf, Australia; the other is the Early Cretaceous (Aptian) Windalia Sand reservoir of the Barrow Island Field, North West Shelf, Australia. In both instances, the simulations have well mimicked the variations of the sedimentary facies temporally and spatially in fine detail with each time step representing 10 ka and a vertical resolution of one meter. The reservoir heterogeneities and the observed cyclicity in the Windalia Sand were particularly well documented by the SEDPAK simulation using a locally derived sealevel curve. The simulation result suggested that high-frequency sealevel variations ?associated with Milankovitch cyclicity were probably the primary cause that controlled the reservoir heterogeneities of the Windalia Sand. This finding provides a working model for the Cretaceous coeval reservoirs in the North West Shelf, Australia.

  1. Application of Classification Models to Pharyngeal High-Resolution Manometry

    ERIC Educational Resources Information Center

    Mielens, Jason D.; Hoffman, Matthew R.; Ciucci, Michelle R.; McCulloch, Timothy M.; Jiang, Jack J.

    2012-01-01

    Purpose: The authors present 3 methods of performing pattern recognition on spatiotemporal plots produced by pharyngeal high-resolution manometry (HRM). Method: Classification models, including the artificial neural networks (ANNs) multilayer perceptron (MLP) and learning vector quantization (LVQ), as well as support vector machines (SVM), were…

  2. High resolution frequency analysis techniques with application to the redshift experiment

    NASA Technical Reports Server (NTRS)

    Decher, R.; Teuber, D.

    1975-01-01

    High resolution frequency analysis methods, with application to the gravitational probe redshift experiment, are discussed. For this experiment a resolution of .00001 Hz is required to measure a slowly varying, low frequency signal of approximately 1 Hz. Major building blocks include fast Fourier transform, discrete Fourier transform, Lagrange interpolation, golden section search, and adaptive matched filter technique. Accuracy, resolution, and computer effort of these methods are investigated, including test runs on an IBM 360/65 computer.

  3. On the accuracy of analytical models of impurity segregation during directional melt crystallization and their applicability for quantitative calculations

    NASA Astrophysics Data System (ADS)

    Voloshin, A. E.; Prostomolotov, A. I.; Verezub, N. A.

    2016-11-01

    The paper deals with the analysis of the accuracy of some one-dimensional (1D) analytical models of the axial distribution of impurities in the crystal grown from a melt. The models proposed by Burton-Prim-Slichter, Ostrogorsky-Muller and Garandet with co-authors are considered, these models are compared to the results of a two-dimensional (2D) numerical simulation. Stationary solutions as well as solutions for the initial transient regime obtained using these models are considered. The sources of errors are analyzed, a conclusion is made about the applicability of 1D analytical models for quantitative estimates of impurity incorporation into the crystal sample as well as for the solution of the inverse problems.

  4. Experiments on flow focusing in soluble porous media, with applications to melt extraction from the mantle

    SciTech Connect

    Kelemen, P.B.; Whitehead, J.A.; Aharonov, E.; Jordahl, K.A.

    1995-01-01

    We demonstrate finite strucutres formed as a consequence of the `reactive infiltration instability` in a series of laboratory and numerical experiments with growth of solution channels parallel to the fluid flow direction. Our experiments demonstrate channel growth in the presence of an initial solution front and without an initial solution front where there is a gradient in the solubility of the solid matrix. In the gradient case, diffuse flow is unstable everywhere, channels can form and grow at any point, and channels may extend over the length scale of the gradient. As a consequence of the gradient results, we suggest that the reactive infiltration instability is important in the Earth`s mantle, where partial melts in the mantle ascend adiabatically. This hypothesis represents an important alternative to mid-oceanic ridge basalts (MORB) extraction in fractures, since fractures may not form in weak, viscously deforming asthenospheric mantle. We also briefly consider the effects of crystallization, rather than dissolution reactions, on the morphology of porous flow via a second set of experiments where fluid becomes supersaturated in a solid phase. This process may produce a series of walled conduits, as in our experiments. Development of a low-porosity cap overlying high porosity conduits may create hydrostatic overpressure sufficient to cause fracture and magma transport to the surface in dikes.

  5. Dynamics in Polymer Melts and Nanocomposites

    NASA Astrophysics Data System (ADS)

    Schneider, Gerald

    Intense research has led to substantial progress in the field of polymer melts and nanocomposites, both regarding the fundamental understanding and the relationship to applications. From a fundamental point of view, knowing the microscopic single chain dynamics is important. It may even lead to optimized materials ranging from the classical car tire to battery or fuel cell applications. In polymer melts, different processes, such as diffusion, reptation, contour length fluctuations, etc. occur and determine the macroscopic results, e.g. obtained by rheology. In nanocomposites confinement effects and interactions of chains with surfaces play an important role. High resolution techniques, such as small-angle neutron scattering or neutron spin echo spectroscopy are suited to explore the structure and dynamics of chains. The presentation illuminates the fundamental relationship between the microscopic dynamics and the mesoscopic properties, exploiting different experimental techniques, such as dielectric spectroscopy, rheology, neutron scattering and neutron spin echo spectroscopy.

  6. An old model in a new perspective: The dynamic melting column and its application in intracontinental basalt petrogenesis

    NASA Astrophysics Data System (ADS)

    Taracsák, Zoltán; Molnár, Gábor; Harangi, Szabolcs

    2016-04-01

    Basaltic melt generation has been thought to be a simple process, involving distinct and well-defined melt batches generated in the asthenosphere or in the lithospheric mantle. However in the last decade, it became clear that complex processes including magma mixing from several sources may take place during magma generation, therefore single melting models might not be able to describe the melting process properly. We suggest that a trace element based melting column model assuming step by-step, dynamic melting caused by decompression would be a good estimation of how the mantle melts in intracontinental settings. This model makes it possible to assume a heterogeneous mantle source by adding melting cells with different composition and mineralogy, and can handle changes in bulk distribution coefficients caused by phase transitions for example the spinel-garnet transition in the upper mantle. If the uppermost part of the melting column reaches the stability of spinel, partitioning of rare earth elements (REEs) change drastically, as heavy REEs are compatible in the garnet stability field during melting while moderately incompatible in depths where spinel is stable. This way, trace element concentrations in primitive basalts can provide information on melting depth. This depth can be quantified with the step-by-step dynamic melting column model if the geotherm or the potential temperature is known. This way the depth of the spinel-garnet transition will be definable. We assume that melting beneath intracontinental volcanic fields is limited to the asthenosphere, and that the top of the melting column will represent the lithosphere-asthenosphere boundary (LAB). To test our model, we calculated lithospheric thicknesses for four Neogene-Quarternary basaltic volcanic fields from the Pannonian Basin, East Central Europe. Our results indicate slightly different LAB depths compared to previous, major element based calculations (Harangi et al., Int J Earth Sci, 2015).

  7. Recent advances in super-resolution fluorescence imaging and its applications in biology.

    PubMed

    Han, Rongcheng; Li, Zhenghong; Fan, Yanyan; Jiang, Yuqiang

    2013-12-20

    Fluorescence microscopy has become an essential tool for biological research because it can be minimally invasive, acquire data rapidly, and target molecules of interest with specific labeling strategies. However, the diffraction-limited spatial resolution, which is classically limited to about 200 nm in the lateral direction and about 500 nm in the axial direction, hampers its application to identify delicate details of subcellular structure. Extensive efforts have been made to break diffraction limit for obtaining high-resolution imaging of a biological specimen. Various methods capable of obtaining super-resolution images with a resolution of tens of nanometers are currently available. These super-resolution techniques can be generally divided into three primary classes: (1) patterned illumination-based super-resolution imaging, which employs spatially and temporally modulated illumination light to reconstruct sub-diffraction structures; (2) single-molecule localization-based super-resolution imaging, which localizes the profile center of each individual fluorophore at subdiffraction precision; (3) bleaching/blinking-based super-resolution imaging. These super-resolution techniques have been utilized in different biological fields and provide novel insights into several new aspects of life science. Given unique technical merits and commercial availability of super-resolution fluorescence microscope, increasing applications of this powerful technique in life science can be expected.

  8. Collimator application for microchannel plate image intensifier resolution improvement

    DOEpatents

    Thomas, Stanley W.

    1996-02-27

    A collimator is included in a microchannel plate image intensifier (MCPI). Collimators can be useful in improving resolution of MCPIs by eliminating the scattered electron problem and by limiting the transverse energy of electrons reaching the screen. Due to its optical absorption, a collimator will also increase the extinction ratio of an intensifier by approximately an order of magnitude. Additionally, the smooth surface of the collimator will permit a higher focusing field to be employed in the MCP-to-collimator region than is currently permitted in the MCP-to-screen region by the relatively rough and fragile aluminum layer covering the screen. Coating the MCP and collimator surfaces with aluminum oxide appears to permit additional significant increases in the field strength, resulting in better resolution.

  9. Collimator application for microchannel plate image intensifier resolution improvement

    DOEpatents

    Thomas, S.W.

    1996-02-27

    A collimator is included in a microchannel plate image intensifier (MCPI). Collimators can be useful in improving resolution of MCPIs by eliminating the scattered electron problem and by limiting the transverse energy of electrons reaching the screen. Due to its optical absorption, a collimator will also increase the extinction ratio of an intensifier by approximately an order of magnitude. Additionally, the smooth surface of the collimator will permit a higher focusing field to be employed in the MCP-to-collimator region than is currently permitted in the MCP-to-screen region by the relatively rough and fragile aluminum layer covering the screen. Coating the MCP and collimator surfaces with aluminum oxide appears to permit additional significant increases in the field strength, resulting in better resolution. 2 figs.

  10. Application of spectral phase shaping to high resolution CARS spectroscopy.

    PubMed

    Postma, S; van Rhijn, A C W; Korterik, J P; Gross, P; Herek, J L; Offerhaus, H L

    2008-05-26

    By spectral phase shaping of both the pump and probe pulses in coherent anti-Stokes Raman scattering (CARS) spectroscopy we demonstrate the extraction of the frequencies, bandwidths and relative cross sections of vibrational lines. We employ a tunable broadband Ti:Sapphire laser synchronized to a ps-Nd:YVO mode locked laser. A high resolution spectral phase shaper allows for spectroscopy with a precision better than 1 cm(-1) in the high frequency region around 3000 cm(-1). We also demonstrate how new spectral phase shaping strategies can amplify the resonant features of isolated vibrations to such an extent that spectroscopy and microscopy can be done at high resolution, on the integrated spectral response without the need for a spectrograph.

  11. Application and evaluation of high-resolution WRF-CMAQ with simple urban parameterization

    EPA Science Inventory

    The 2-way coupled WRF-CMAQ meteorology and air quality modeling system is evaluated for high-resolution applications by comparing to a regional air quality field study (Discover-AQ). The model was modified to better account for the effects of urban environments. High-resolution...

  12. Application and evaluation of high-resolution WRF-CMAQ with simple urban parameterization.

    EPA Science Inventory

    The 2-way coupled WRF-CMAQ meteorology and air quality modeling system is evaluated for high-resolution applications by comparing to a regional air quality field study (Discover-AQ). The model was modified to better account for the effects of urban environments. High-resolution...

  13. High Spatiotemporal-Resolution Magnetic Tweezers: Calibration and Applications for DNA Dynamics

    PubMed Central

    Dulin, David; Cui, Tao Ju; Cnossen, Jelmer; Docter, Margreet W.; Lipfert, Jan; Dekker, Nynke H.

    2015-01-01

    The observation of biological processes at the molecular scale in real time requires high spatial and temporal resolution. Magnetic tweezers are straightforward to implement, free of radiation or photodamage, and provide ample multiplexing capability, but their spatiotemporal resolution has lagged behind that of other single-molecule manipulation techniques, notably optical tweezers and AFM. Here, we present, to our knowledge, a new high-resolution magnetic tweezers apparatus. We systematically characterize the achievable spatiotemporal resolution for both incoherent and coherent light sources, different types and sizes of beads, and different types and lengths of tethered molecules. Using a bright coherent laser source for illumination and tracking at 6 kHz, we resolve 3 Å steps with a 1 s period for surface-melted beads and 5 Å steps with a 0.5 s period for double-stranded-dsDNA-tethered beads, in good agreement with a model of stochastic bead motion in the magnetic tweezers. We demonstrate how this instrument can be used to monitor the opening and closing of a DNA hairpin on millisecond timescales in real time, together with attendant changes in the hairpin dynamics upon the addition of deoxythymidine triphosphate. Our approach opens up the possibility of observing biological events at submillisecond timescales with subnanometer resolution using camera-based detection. PMID:26588570

  14. Field-calibrated model of melt, refreezing, and runoff for polar ice caps: Application to Devon Ice Cap

    NASA Astrophysics Data System (ADS)

    Morris, Richard M.; Mair, Douglas W. F.; Nienow, Peter W.; Bell, Christina; Burgess, David O.; Wright, Andrew P.

    2014-09-01

    Understanding the controls on the amount of surface meltwater that refreezes, rather than becoming runoff, over polar ice masses is necessary for modeling their surface mass balance and ultimately for predicting their future contributions to global sea level change. We present a modified version of a physically based model that includes an energy balance routine and explicit calculation of near-surface meltwater refreezing capacity, to simulate the evolution of near-surface density and temperature profiles across Devon Ice Cap in Arctic Canada. Uniquely, our model is initiated and calibrated using high spatial resolution measurements of snow and firn densities across almost the entire elevation range of the ice cap for the summer of 2004 and subsequently validated with the same type of measurements obtained during the very different meteorological conditions of summer 2006. The model captures the spatial variability across the transect in bulk snowpack properties although it slightly underestimates the flow of meltwater into the firn of previous years. The percentage of meltwater that becomes runoff is similar in both years; however, the spatial pattern of this melt-runoff relationship is different in the 2 years. The model is found to be insensitive to variation in the depth of impermeable layers within the firn but is very sensitive to variation in air temperature, since the refreezing capacity of firn decreases with increasing temperature. We highlight that the sensitivity of the ice cap's surface mass balance to air temperature is itself dependent on air temperature.

  15. High Resolution Atmospheric Modeling for Wind Energy Applications

    SciTech Connect

    Simpson, M; Bulaevskaya, V; Glascoe, L; Singer, M

    2010-03-18

    The ability of the WRF atmospheric model to forecast wind speed over the Nysted wind park was investigated as a function of time. It was found that in the time period we considered (August 1-19, 2008), the model is able to predict wind speeds reasonably accurately for 48 hours ahead, but that its forecast skill deteriorates rapidly after 48 hours. In addition, a preliminary analysis was carried out to investigate the impact of vertical grid resolution on the forecast skill. Our preliminary finding is that increasing vertical grid resolution does not have a significant impact on the forecast skill of the WRF model over Nysted wind park during the period we considered. Additional simulations during this period, as well as during other time periods, will be run in order to validate the results presented here. Wind speed is a difficult parameter to forecast due the interaction of large and small length scale forcing. To accurately forecast the wind speed at a given location, the model must correctly forecast the movement and strength of synoptic systems, as well as the local influence of topography / land use on the wind speed. For example, small deviations in the forecast track or strength of a large-scale low pressure system can result in significant forecast errors for local wind speeds. The purpose of this study is to provide a preliminary baseline of a high-resolution limited area model forecast performance against observations from the Nysted wind park. Validating the numerical weather prediction model performance for past forecasts will give a reasonable measure of expected forecast skill over the Nysted wind park. Also, since the Nysted Wind Park is over water and some distance from the influence of terrain, the impact of high vertical grid spacing for wind speed forecast skill will also be investigated.

  16. Application of the Plagioclase-Liquid Hygrometer to the Bishop Tuff: Consistency with Melt Inclusion H2O Contents

    NASA Astrophysics Data System (ADS)

    Jolles, J.; Lange, R. A.

    2015-12-01

    High-silica (74-77 wt% SiO2) rhyolites are the most evolved magmas on Earth and constitute some of the largest eruptions (1000s of km3). Of these, one classic example is the Bishop Tuff, a 760 ka eruption of >670 km3 of high-silica rhyolite erupted from Long Valley caldera, CA. Documenting dissolved H2O contents is crucial for understanding its origin and evolution. Analyses of water contents measured in quartz-hosted melt inclusions from the Bishop Tuff (Wallace et al. 1999; Anderson et al. 2000) show that the Early and Middle Bishop Tuff (Ig1Eb, Ig2Ea) have higher water contents (≤ 6.3 wt% H2O) than the Late Bishop Tuff (Ig2NWa; ≤ 5.2 wt%). Our work utilizes the revised plagioclase-liquid hygrometer (Waters & Lange, 2015), which is applicable to rhyolite, to evaluate internal consistency between Fe-Ti oxide temperatures, the plagioclase hygrometer, and melt inclusion H2O analyses. Two-oxide thermometry (Ghiorso & Evans, 2008), using all possible Fe-Ti oxide pairs (between 56 and 1500 pairs for individual samples), was carried out on 2-3 pumice clasts for each sampled eruptive unit. Resulting temperatures (°C ± 1σ) for individual clasts are: 705 ± 12, 728 ± 10 for unit Ig1Eb; 710 ± 12, 728 ± 11 for unit Ig2Ea; 752 ±10, 776 ± 8, 778 ± 7 for unit Ig2NWa; 791 ± 7, 795 ± 8 for unit Ig2Nb. The compositions of the most calcic plagioclase phenocrysts in the Early and Middle units are An17-19, whereas in the Late units they are An29-30. When the Fe-Ti oxide temperatures, whole rock analyses, and plagioclase compositions are incorporated into the plagioclase hygrometer, they give water contents at the onset of plagioclase crystallization of 6.6-6.9 wt% for the Early and Middle units and 4.8-4.9 wt % for the Late units. These results show internal consistency between melt inclusion analyses of water, Fe-Ti oxide thermometry, and the plagioclase-liquid hygrometer; they further support a temperature gradient across the Early, Middle, and Late Bishop Tuff units.

  17. High Spatial Resolution 40Ar/39Ar Geochronology of Impact Melt Breccias from Apollo 17 Boulders at Stations 2, 6, and 7

    NASA Astrophysics Data System (ADS)

    Mercer, C. M.; Hodges, K. V.; Jolliff, B. L.; Van Soest, M. C.; Wartho, J. A.; Weirich, J. R.

    2015-12-01

    Several boulders located at the bases of the North and South Massifs were among the primary field targets of the Apollo 17 mission to the Taurus-Littrow Valley on the Moon [1]. Some boulders are polylithologic, including Boulder 1 at Station 2 and the boulders at Stations 6 and 7. These boulders were the subjects of consortium studies [2, 3] that included 40Ar/39Ar geochronology to determine the ages of distinct lithologies within each boulder [e.g., 4-6]. We report new 40Ar/39Ar data for the impact melt breccias 72255, 76315, 77075, and 77135 obtained using the UV laser ablation microprobe (UVLAMP) methods of [7]. For 72255, we obtained a preliminary isochron date ca. 3814 Ma from 22 melt analyses, which is younger than published plateau dates (e.g., 3951-3835 Ma [4, 8]). Fifteen melt analyses of 76315 yield a preliminary isochron date ca. 3850 Ma, younger than the 3900 ± 16 Ma date reported by [8]. Melt analyses of 77075 yield preliminary dates between ca. 3797-3584 Ma, possibly reflecting partial loss of 40Ar. In this case, the oldest date may provide a minimum age for the formation of melt in 77075. Finally, the UVLAMP dates for the 77135 melt range from 3810-3361 Ma and corresponding Ca/K ratios range from ca. 100-6. Electron microprobe analyses of small (ca. 10s of microns wide) pockets of K-rich materials show that both K-rich glass and K-feldspar are present. The UVLAMP dates for 77135 likely reflect spatially variable 40Ar loss, consistent with published step heating results [e.g., 6]. References: [1] Schmitt (1973) Science, 182, 681-690. [2] Ryder (1993). Catalog of Apollo 17 Rocks: Volume 1 - Stations 2 and 3 (South Massif). LPI. [3] Ryder (1993). Catalog of Apollo 17 Rocks: Volume 4 - North Massif. LPI. [4] Leich et al. (1975) The Moon, 14, 407-444. [5] Cadogan & Turner (1976). LPSC, 7, 2267-2285. [6] Stettler et al. (1978). LPSC, 9, 1113-1115. [7] Mercer et al. (2015) Sci. Adv., 1, e1400050. [8] Dalrymple & Ryder (1996). JGR, 101, 26069-26084.

  18. Super-resolution imaging for cell biologists: concepts, applications, current challenges and developments.

    PubMed

    Fornasiero, Eugenio F; Opazo, Felipe

    2015-04-01

    The recent 2014 Nobel Prize in chemistry honored an era of discoveries and technical advancements in the field of super-resolution microscopy. However, the applications of diffraction-unlimited imaging in biology have a long road ahead and persistently engage scientists with new challenges. Some of the bottlenecks that restrain the dissemination of super-resolution techniques are tangible, and include the limited performance of affinity probes and the yet not capillary diffusion of imaging setups. Likewise, super-resolution microscopy has introduced new paradigms in the design of projects that require imaging with nanometer-resolution and in the interpretation of biological images. Besides structural or morphological characterization, super-resolution imaging is quickly expanding towards interaction mapping, multiple target detection and live imaging. Here we review the recent progress of biologists employing super-resolution imaging, some pitfalls, implications and new trends, with the purpose of animating the field and spurring future developments.

  19. Patch-Based Super-Resolution of MR Spectroscopic Images: Application to Multiple Sclerosis

    PubMed Central

    Jain, Saurabh; Sima, Diana M.; Sanaei Nezhad, Faezeh; Hangel, Gilbert; Bogner, Wolfgang; Williams, Stephen; Van Huffel, Sabine; Maes, Frederik; Smeets, Dirk

    2017-01-01

    Purpose: Magnetic resonance spectroscopic imaging (MRSI) provides complementary information to conventional magnetic resonance imaging. Acquiring high resolution MRSI is time consuming and requires complex reconstruction techniques. Methods: In this paper, a patch-based super-resolution method is presented to increase the spatial resolution of metabolite maps computed from MRSI. The proposed method uses high resolution anatomical MR images (T1-weighted and Fluid-attenuated inversion recovery) to regularize the super-resolution process. The accuracy of the method is validated against conventional interpolation techniques using a phantom, as well as simulated and in vivo acquired human brain images of multiple sclerosis subjects. Results: The method preserves tissue contrast and structural information, and matches well with the trend of acquired high resolution MRSI. Conclusions: These results suggest that the method has potential for clinically relevant neuroimaging applications. PMID:28197066

  20. A new method for simultaneous detection and discrimination of Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) using real time PCR with high resolution melting (HRM) analysis.

    PubMed

    Marin, M S; Quintana, S; Leunda, M R; Recavarren, M; Pagnuco, I; Späth, E; Pérez, S; Odeón, A

    2016-01-01

    Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are antigenically and genetically similar. The aim of this study was to develop a simple and reliable one-step real time PCR assay with high resolution melting (HRM) analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5. Optimization of assay conditions was performed with DNA from reference strains. Then, DNA from field isolates, clinical samples and tissue samples of experimentally infected animals were studied by real time PCR-HRM. An efficient amplification of real time PCR products was obtained, and a clear melting curve and appropriate melting peaks for both viruses were achieved in the HRM curve analysis for BoHV type identification. BoHV was identified in all of the isolates and clinical samples, and BoHV types were properly differentiated. Furthermore, viral DNA was detected in 12/18 and 7/18 samples from BoHV-1- and BoHV-5-infected calves, respectively. Real time PCR-HRM achieved a higher sensitivity compared with virus isolation or conventional PCR. In this study, HRM was used as a novel procedure. This method provides rapid, sensitive, specific and simultaneous detection of bovine alpha-herpesviruses DNA. Thus, this technique is an excellent tool for diagnosis, research and epidemiological studies of these viruses in cattle.

  1. Applications of High-Resolution Observations at Millimeter Wavelengths

    NASA Astrophysics Data System (ADS)

    Rosenfeld, Katherine

    Interferometric observations at millimeter wavelengths provide a precious, detailed view of certain astrophysical objects. This thesis is composed of studies that both rely on and enable this technique to study the structure of planet-forming disks and soon image the closest regions around super-massive black holes. Young stars form out of a cloud of gas and dust that, before its eventual dissipation, flattens to a disk. However the disk population is diverse and recent high-resolution images have revealed a wide variety of interesting features. To understand these observations we use detailed radiative transfer models to motivate various physical scenarios. First we identify a set of traits in the disk around V4046 Sgr that marks the coupled progression of the gas and dust distributions in the presence of at least one embedded companion. Next, we investigate how the vertical temperature structure of a disk can be spatially resolved and apply our framework to observations of the disk around HD163296. Lastly, we show how large-scale radial flows of gas may be observable and question how this phenomenon might be distinguished from other scenarios such as warps or outflows. The last chapter summarizes the APHIDS project which changes the sampling rate of data taken at the SMA so that it may be used for VLBI campaigns.

  2. Calculating Freshwater Input from Iceberg Melt in Greenlandic Fjords by Combining In Situ Observations of Iceberg Movement with High Resolution Satellite Imagery

    NASA Astrophysics Data System (ADS)

    Sulak, D. J.; Sutherland, D.; Stearns, L. A.; Hamilton, G. S.

    2015-12-01

    Understanding fjord circulation in Greenland's outlet glacial fjords is crucial to explaining recent temporal and spatial variability in glacier dynamics, as well as freshwater transport on the continental shelf. The fjords are commonly assumed to exhibit a plume driven circulation that draws in warmer and saltier Atlantic-origin water toward the glacier at depth. Freshwater input at glacier termini directly drives this circulation and significantly influences water column stratification, which indirectly feeds back on the plume driven circulation. Previous work has focused on freshwater inputs from surface runoff and submarine melting, but the contribution from iceberg melt, a potentially important freshwater source, has not been quantified. Here, we develop a new technique combining in situ observations of movement from iceberg-mounted GPS units with multispectral satellite imagery from Landsat 8. The combination of datasets allows us to examine the details of iceberg movement and quantify mean residence times in a given fjord. We then use common melt rate parameterizations to estimate freshwater input for a given iceberg, utilizing novel satellite-derived iceberg distributions to scale up to a fjord-wide freshwater contribution. We apply this technique to Rink Isbræ and Kangerlussuup Sermia in west Greenland, and Helheim Glacier in southeast Greenland. The analysis can be rapidly expanded to look at other systems as well as seasonal and interannual changes in how icebergs affect the circulation and stratification of Greenland's outlet glacial fjords. Ultimately, this work will lead to a more complete understanding of the wide range of factors that control the observed regional variability in Greenland's glaciers.

  3. A Survey on Large High-Resolution Display Technologies, Techniques, and Applications

    SciTech Connect

    Ni, Tao; Schmidt, Greg S.; Staadt, Oliver G.; Livingston, Mark A.; Ball, Robert; May, Richard A.

    2006-03-27

    Continued advances in display hardware, computing power, networking, and rendering algorithms have all converged to dramatically improve large high-resolution display capabilities. We present a survey on prior research with large high-resolution displays. In the hardware configurations section we examine systems including multi-monitor workstations, recon*gurable projector arrays, and others. Rendering and the data pipeline are addressed with an overview of current technologies. We discuss many applications for large high-resolution displays such as automotive design, scientific visualization, control centers, and others. Quantifying the effect of large high-resolution displays on human performance and other aspects is important as we look toward future advances in display technology and how it is applied in different situations. Interacting with these displays brings a different set of challenges for HCI professionals, so an overview of some of this work is provided. Finally, we present our view of the top ten greatest challenges in large high-resolution displays.

  4. [High-resolution distortion-product otoacoustic emissions: method and clinical applications].

    PubMed

    Janssen, T; Lodwig, A; Müller, J; Oswald, H

    2014-10-01

    Unlike pure tone thresholds that assess both peripheral and central sound processing, distortion-product otoacoustic emissions (DPOAEs) selectively mirror the functioning of the cochlear amplifier. High resolution DPOAEs are missing in the toolbox of routine audiometry due to the fact that high resolution DPOAE measurements are more time-consuming when compared to normal clinical DP grams with rough frequency resolution. Measurements of high resolution DPOAEs allow an early assessment of beginning sensory cell damage due to sound overexposure or administration of ototoxic drugs. When using a rough grid, sensory cell damage would be overlooked as in the early state damage only appears at some distinct cochlear sites. A review is given on the method and application of high resolution DPOAEs.

  5. Ultra high resolution stepper motors design, development, performance and application

    NASA Technical Reports Server (NTRS)

    Moll, H.; Roeckl, G.

    1979-01-01

    The design and development of stepper motors with steps in the 10 arc sec to 2 arc min range is described. Some of the problem areas, e.g. rotor suspension, tribology aspects and environmental conditions are covered. A summary of achieved test results and the employment in different mechanisms already developed and tested is presented to give some examples of the possible use of this interesting device. Adaptations to military and commercial requirements are proposed and show the wide range of possible applications.

  6. Microstructures and mechanical properties of Co-29Cr-6Mo alloy fabricated by selective laser melting process for dental applications.

    PubMed

    Takaichi, Atsushi; Suyalatu; Nakamoto, Takayuki; Joko, Natsuka; Nomura, Naoyuki; Tsutsumi, Yusuke; Migita, Satoshi; Doi, Hisashi; Kurosu, Shingo; Chiba, Akihiko; Wakabayashi, Noriyuki; Igarashi, Yoshimasa; Hanawa, Takao

    2013-05-01

    The selective laser melting (SLM) process was applied to a Co-29Cr-6Mo alloy, and its microstructure, mechanical properties, and metal elution were investigated to determine whether the fabrication process is suitable for dental applications. The microstructure was evaluated using scanning electron microscopy with energy-dispersed X-ray spectroscopy (SEM-EDS), X-ray diffractometry (XRD), and electron back-scattered diffraction pattern analysis. The mechanical properties were evaluated using a tensile test. Dense builds were obtained when the input energy of the laser scan was higher than 400 J mm⁻³, whereas porous builds were formed when the input energy was lower than 150 J mm⁻³. The microstructure obtained was unique with fine cellular dendrites in the elongated grains parallel to the building direction. The γ phase was dominant in the build and its preferential <001> orientation was confirmed along the building direction, which was clearly observed for the builds fabricated at lower input energy. Although the mechanical anisotropy was confirmed in the SLM builds due to the unique microstructure, the yield strength, UTS, and elongation were higher than those of the as-cast alloy and satisfied the type 5 criteria in ISO22764. Metal elution from the SLM build was smaller than that of the as-cast alloy, and thus, the SLM process for the Co-29Cr-6Mo alloy is a promising candidate for fabricating dental devices.

  7. Effects of milling media on the fabrication of melt-derived bioactive glass powder for biomaterial application

    NASA Astrophysics Data System (ADS)

    Ibrahim, Nurul Farhana; Mohamad, Hasmaliza; Noor, Siti Noor Fazliah Mohd

    2016-12-01

    The present work aims to study the effects of using different milling media on bioactive glass produced through melt-derived method for biomaterial application. The bioactive glass powder based on SiO2-CaO-Na2O-P2O5 system was fabricated using two different types of milling media which are tungsten carbide (WC) and zirconia (ZrO2) balls. However, in this work, no P2O5 was added in the new composition. XRF analysis indicated that tungsten trioxide (WO3) was observed in glass powder milled using WC balls whereas ZrO2 was observed in glass powder milled using ZrO2 balls. Amorphous structure was detected with no crystalline peak observed through XRD analysis for both glass powders. FTIR analysis confirmed the formation of silica network with the existence of functional groups Si-O-Si (bend), Si-O-Si (tetrahedral) and Si-O-Si (stretch) for both glass powders. The results revealed that there was no significant effect of milling media on amorphous silica network glass structure which shows that WC and zirconia can be used as milling media for bioactive glass fabrication without any contamination. Therefore, the fabricated BG can be tested safely for bioactivity assessment in biological fluids environment.

  8. [Spatial resolution standardization of payload on board of remote sensing satellite based on application requirements].

    PubMed

    Wei, Xiang-qin; Gu, Xing-fa; Yu, Tao; Meng, Qing-yan; Li, Bin; Guo, Hong

    2012-03-01

    Remote sensing application requirements are the starting point for design of payload on board earth observation satellite. The generalization, standardization and serialization of payload are the future development trend for payload design. In the present paper, based on the analysis of remote sensing application requirements, the spatial resolution standardization of satellite remote sensing payload, which is the main concerned indicator, was investigated. The design standards of national payload spatial resolution of earth observation satellite are presented, which are important to the promotion of satellite payload production and saving in design cost.

  9. Vitis vinifera L. Single-Nucleotide Polymorphism Detection with High-Resolution Melting Analysis Based on the UDP-Glucose:Flavonoid 3-O-Glucosyltransferase Gene.

    PubMed

    Pereira, Leonor; Martins-Lopes, Paula

    2015-10-21

    Vitis vinifera L. is a species with a large number of varieties, which differ in terms of anthocyanin content. The genes involved in the anthocyanin biosynthesis pathway have a direct effect in the anthocyanin profile of each variety, being potentially interesting for varietal identification. The current study aimed at the design of an assay suitable for the discrimination of the largest number of grapevine varieties. Two genes of the anthocyanin pathway, chalcone isomerase (CHI) and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT), were sequenced in 22 grapevine varieties. The CHI gene presented 5 SNPs within the sequence. A total of 58 SNPs and 1 INDEL were found among the UFGT gene, allowing the discrimination of 18 different genotypes within the 22 grapevine varieties. A HRM assay designed for UFGT, containing 704 bp, produced differentiated melting curves for each of the 18 haplotypes. The developed HRM assay is efficient in grapevine varietal discrimination.

  10. Experimental investigation of the electrical behavior of olivine during partial melting under pressure and application to the lunar mantle

    NASA Astrophysics Data System (ADS)

    Pommier, A.; Leinenweber, K.; Tasaka, M.

    2015-09-01

    Electrical conductivity measurements were performed during melting experiments of olivine compacts (dry and hydrous Fo77 and Fo90) at 4 and 6 GPa in order to investigate melt transport properties and quantify the effect of partial melting on electrical properties. Experiments were performed in the multi-anvil apparatus and electrical measurements were conducted using the impedance spectroscopy technique with the two-electrode method. Changes in impedance spectra were used to identify the transition from an electrical response controlled by the solid matrix to an electrical response controlled by the melt phase. This transition occurs slightly above the solidus temperature and lasts until Tsolidus + 75 °C (±25). At higher temperature, a significant increase in conductivity (corresponding to an increase in conductivity values by a factor ranging from ∼30 to 100) is observed, consistent with the transition from a tube-dominated network to a structure in which melt films and pools become prominent features. This increase in conductivity corresponds to an abrupt jump for all dry samples and to a smoother increase for the hydrous sample. It is followed by a plateau at higher temperature, suggesting that the electrical response of the investigated samples lacks sensitivity to temperature at an advanced stage of partial melting. Electron microprobe analyses on quenched products indicated an increase in Mg# (molar Mg / (Mg +Fe)) of olivine during experiments (∼77-93 in the quenched samples with an initial Fo77 composition and ∼92-97 in the quenched samples with an initial Fo90 composition) due to the partitioning of iron to the melt phase. Assuming a respective melt fraction of 0.10 and 0.20 before and after the phase of significant increase in conductivity, in agreement with previous electrical and permeability studies, our results can be reproduced satisfactorily by two-phase electrical models (the Hashin and Shtrikman bounds and the modified brick layer model

  11. Thermal Modeling of Permafrost Melt by Overlying Lava Flows with Applications to Flow-associated Outflow Channel Volumes in the Cerberus Plains, Mars

    NASA Technical Reports Server (NTRS)

    Chase, Z. A. J.; Sakimoto, S. E. H.

    2003-01-01

    The Cerberus region of Mars has numerous geologically recent fluvial and volcanic features superimposed spatially, with some of them using the same flow channels and apparent vent structures. Lava-water interaction landforms such as psuedocraters suggest some interaction of emplacing lava flows with underlying ground ice or water. This study investigates a related interaction type a region where the emplaced lava might have melted underlying ice in the regolith, as there are small outflow channel networks emerging from the flank flows of a lava shield over a portion of the Eastern Cerberus Rupes. Specifically, we use high-resolution Mars Orbiter Laser Altimeter (MOLA) topography to constrain channel and flow dimensions, and thus estimate the thermal pulse from the emplaced lava into the substrate and the resulting melting durations and refreezing intervals. These preliminary thermal models indicate that the observed flows could easily create thermal pulse(s) sufficient to melt enough ground ice to fill the observed fluvial small outflow channels. Depending on flow eruption timing and hydraulic recharge times, this system could easily have produced multiple thermal pulses and fluvial releases. This specific case suggests that regional small water releases from similar cases may be more common than suspected, and that there is a possibility for future fluvial releases if ground ices are currently present and future volcanic eruptions in this young region are possible.

  12. Effect of surface conditioning on the flowability of Ti6Al7Nb powder for selective laser melting applications

    NASA Astrophysics Data System (ADS)

    Marcu, T.; Todea, M.; Gligor, I.; Berce, P.; Popa, C.

    2012-01-01

    Porous metallic implants have been developed to provide a good implant-tissue mechanical attachment and an as close as possible elastic modulus to that of human bone, by the means of a proper porosity. A viable manufacturing technology for personalized implants is rapid prototyping (RP), which employs fine feeding powders which usually display a poor flow behaviour. In the present work, the required flowability of Ti6Al7Nb powder aimed for selective laser melting (SLM) applications was improved by applying a simple heat treatment in air, at temperatures higher than 500 °C. As flowability depends on the powder surface condition, the surface chemical composition before and after heat treatment was characterized by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) analysis. The results indicated that the amount of basic and acidic -OH groups is almost the same for powders treated at 400 °C, which do not flow, and powders treated at 500 °C, which display flowability. An increase of the Al2O3 amount in the TiO2-based surface layer on the powder particles with the treatment temperature was noticed. Based on the results, the temperature of 500 °C was selected for the further research. The conditioned powder displayed a very good technological behaviour, being successfully used for obtaining specimens with both cellular and fully dense structures through SLM. The microstructure of the manufactured samples, characterized by optical microscopy and scanning electron microscopy (SEM), has shown mainly α‧ martensite, with columnar grains.

  13. Regional Studies and Applications with a Variable Resolution Stretched Grid Data Assimilation System

    NASA Technical Reports Server (NTRS)

    Fox-Rabinovitz, Michael; Dee, Dick; Takacs, Lawrence

    1999-01-01

    The variable resolution stretched grid (SG) version of the Goddard Earth Observing System (GEOS) Data Assimilation System (DAS) incorporating the GEOS SG-GCM, is used for regional analysis, forecast, and climate applications. The region of interest with enhanced horizontal resolution, mostly used in experiments, is a rectangle over the U.S. The SG-DAS is capable of reproducing regional mesoscale fields, patterns and diagnostics that are not produced by the medium uniform resolution run with the same amount of grid points as for the SG. The SG-DAS regional analyses and diagnostics are used for: validation of regional climate simulation experiments produced with the SG-GCM for the U.S. 1988 summer drought; and are planned to be used for atmospheric chemistry transport experiments. Also, a case study is conducted on a super-typhoon development in December 1997. The SG-DAS appears to be a viable candidate for a variety of regional studies and applications.

  14. Architecture and applications of a high resolution gated SPAD image sensor

    PubMed Central

    Burri, Samuel; Maruyama, Yuki; Michalet, Xavier; Regazzoni, Francesco; Bruschini, Claudio; Charbon, Edoardo

    2014-01-01

    We present the architecture and three applications of the largest resolution image sensor based on single-photon avalanche diodes (SPADs) published to date. The sensor, fabricated in a high-voltage CMOS process, has a resolution of 512 × 128 pixels and a pitch of 24 μm. The fill-factor of 5% can be increased to 30% with the use of microlenses. For precise control of the exposure and for time-resolved imaging, we use fast global gating signals to define exposure windows as small as 4 ns. The uniformity of the gate edges location is ∼140 ps (FWHM) over the whole array, while in-pixel digital counting enables frame rates as high as 156 kfps. Currently, our camera is used as a highly sensitive sensor with high temporal resolution, for applications ranging from fluorescence lifetime measurements to fluorescence correlation spectroscopy and generation of true random numbers. PMID:25090572

  15. Mechanisms, Capabilities, and Applications of High-Resolution Electrohydrodynamic Jet Printing.

    PubMed

    Onses, M Serdar; Sutanto, Erick; Ferreira, Placid M; Alleyne, Andrew G; Rogers, John A

    2015-09-09

    This review gives an overview of techniques used for high-resolution jet printing that rely on electrohydrodynamically induced flows. Such methods enable the direct, additive patterning of materials with a resolution that can extend below 100 nm to provide unique opportunities not only in scientific studies but also in a range of applications that includes printed electronics, tissue engineering, and photonic and plasmonic devices. Following a brief historical perspective, this review presents descriptions of the underlying processes involved in the formation of liquid cones and jets to establish critical factors in the printing process. Different printing systems that share similar principles are then described, along with key advances that have been made in the last decade. Capabilities in terms of printable materials and levels of resolution are reviewed, with a strong emphasis on areas of potential application.

  16. High-resolution particle analysis--its application to platelet counting and suggestions for further application in blood cell analysis.

    PubMed

    Haynes, J L

    1980-01-01

    The characteristics of an instrument for high-resolution particle analysis in flow are discussed. It employs a combination of hydrodynamic focusing, fluid resistors, and electronic techniques to achieve precision and ease of use heretofore unobtainable in a moderate-cost clinical instrument. Its application to whole blood platelet counting is discussed, and suggestions are made for its possible application to a wide variety of blood cell measurements.

  17. Multi-storm, multi-catchment investigation of rainfall spatial resolution requirements for urban hydrological applications

    NASA Astrophysics Data System (ADS)

    Ochoa Rodriguez, Susana; ten Veldhuis, Marie-Claire; Bruni, Guendalina; Gires, Auguste; van Assel, Johan; Wang, Lipen; Reinoso-Rodinel, Ricardo; Ichiba, Abdellah; Kroll, Stefan; Schertzer, Daniel; Onof, Christian; Willems, Patrick

    2014-05-01

    Rainfall estimates of the highest possible resolution are required for urban hydrological applications, given the small size and fast response which characterise urban catchments. While significant progress has been made over the last few decades in high resolution measurement of rainfall at urban scales and in the modelling of urban runoff processes, a number of questions as to the actual resolution requirements for input data and models remain to be answered. With the aim of answering some of these questions, this work investigates the impact of rainfall estimates of different spatial resolutions and structures on the hydraulic outputs of models of several urban catchments with different characteristics. For this purpose multiple storm events, including convective and stratiform ones, measured by a polarimetric X-band radar located in Cabauw (NL) were selected for analysis. The original radar estimates, at 100 m and 1 min resolutions, were aggregated to coarser spatial resolutions of up to 1000 m. These estimates were then applied to the high-resolution semi distributed hydraulic models of four urban catchments of similar size (approx. 7 km2), but different morphological and land use characteristics; these are: the Herent catchment (Belgium), the Cranbrook catchment (UK), the Morée Sausset catchment (France) and the Kralingen District of Rotterdam (The Netherlands). When doing so, methodologies for standardising rainfall inputs and making results comparable were implemented. Moreover, the results were analysed considering different points at each catchment, while also taking into account the particular storm and catchment characteristics. The results obtained for the storms used in this study show that flat and less compact catchments (e.g. polder areas) may be more sensitive to the spatial resolution of rainfall estimates, as compared to catchments with higher slopes and compactness, which in general show little sensitivity to changes in spatial resolution

  18. Rutile solubility in hydrous rhyolite melts at 750-900 °C and 2 kbar, with application to titanium-in-quartz (TitaniQ) thermobarometry

    NASA Astrophysics Data System (ADS)

    Kularatne, Kanchana; Audétat, Andreas

    2014-01-01

    The solubility of rutile in water-saturated haplogranite melts with molar Al/(Na + K)-ratios ranging from 0.84 to 1.25 was determined at 750-900 °C and 2 kbar in cold-seal pressure vessel experiments. Due to the low diffusivity of Ti at these conditions a new method was developed to determine TiO2 solubility. In this method, glasses with TiO2 gradients were used as starting material, and after the experiments the TiO2 content was measured at the contact between rutile-bearing and rutile-free glass. The glasses were either directly equilibrated at the desired P-T conditions (i.e., crystallization experiments), or first treated at 50-150 °C lower temperatures and then subjected to the desired conditions (i.e., dissolution experiments). The results obtained in crystallization and dissolution experiments agree well with each other, suggesting that equilibrium was attained. Rutile solubility in peralkaline melts strongly increases with temperature and the amount of excess alkalies according to the relation: log TiO2 (wt%)=(1.8∗ΔANK-0.53)∗10,000/T-(12.8∗ΔANK-4.3) in which ΔANK is the deviation of the molar Al/(Na + K)-ratio from unity and T is given in Kelvin. Excess alumina does not seem to promote TiO2 solubility. For natural melt compositions it was found that if Ca, Mg and Fe are assumed to be 10 times less effective in promoting TiO2 solubility than excess Na and K, good fits to previous solubility data in silicic (⩾70 wt% SiO2) melts at upper crustal pressures (⩽10 kbar) are obtained. Application of this extended TiO2 solubility model to natural melt inclusions in quartz phenocrysts from five silicic volcanic systems returns TiO2 activities that are similar to those calculated with a previous experiment-based model and to those calculated from pairs of coexisting Fe-Ti-oxides, but are up to 2.9 times higher than those calculated with MELTS and rhyolite-MELTS. Pressures calculated from Ti concentrations in the host quartz using the most recent

  19. Modeling of hot fragment conductive ignition of solid propellants with applications to melting and evaporation of solids

    SciTech Connect

    Kuo, K.K.; Hsieh, W.H.; Hsieh, K.C. ); Miller, M.S. )

    1988-08-01

    A comprehensive theoretical model has been formulated for studying the degree of vulnerability of various solid propellants being heated by hot spall fragments. The model simulates the hot fragment conductive ignition (HFCI) processes caused by direct contact of hot inert particles with solid propellant samples. The model describes the heat transfer and displacement of the hot particle, the generation of the melt (or foam) layer caused by the liquefaction, pyrolysis, and decomposition of the propellant, the regression of the propellant as well as the timw variation of its temperature distributions. To validate partially the theoretical model in the absence of the necessary chemical kinetic data, an ice melting and evaporation experiment was designed and conducted. These experiments provide features of the conductive heating, melting and evaporating processes. Calculated results compare well with experimental data in temperature-time traces, spall particle sinking velocity, and displacement.

  20. Phase-equilibrium geobarometers for silicic rocks based on rhyolite-MELTS. Part 2: application to Taupo Volcanic Zone rhyolites

    NASA Astrophysics Data System (ADS)

    Bégué, Florence; Gualda, Guilherme A. R.; Ghiorso, Mark S.; Pamukcu, Ayla S.; Kennedy, Ben M.; Gravley, Darren M.; Deering, Chad D.; Chambefort, Isabelle

    2014-11-01

    Constraining the pressure of crystallisation of large silicic magma bodies gives important insight into the depth and vertical extent of magmatic plumbing systems; however, it is notably difficult to constrain pressure at the level of detail necessary to understand shallow magmatic systems. In this study, we use the recently developed rhyolite-MELTS geobarometer to constrain the crystallisation pressures of rhyolites from the Taupo Volcanic Zone (TVZ). As sanidine is absent from the studied deposits, we calculate the pressures at which quartz and feldspar are found to be in equilibrium with melt now preserved as glass (the quartz +1 feldspar constraint of Gualda and Ghiorso, Contrib Mineral Petrol 168:1033. doi:10.1007/s00410-014-1033-3. 2014). We use glass compositions (matrix glass and melt inclusions) from seven eruptive deposits dated between ~320 and 0.7 ka from four distinct calderas in the central TVZ, and we discuss advantages and limitations of the rhyolite-MELTS geobarometer in comparison with other geobarometers applied to the same eruptive deposits. Overall, there is good agreement with other pressure estimates from the literature (amphibole geobarometry and H2O-CO2 solubility models). One of the main advantages of this new geobarometer is that it can be applied to both matrix glass and melt inclusions—regardless of volatile saturation. The examples presented also emphasise the utility of this method to filter out spurious glass compositions. Pressure estimates obtained with the new rhyolite-MELTS geobarometer range between ~250 to ~50 MPa, with a large majority at ~100 MPa. These results confirm that the TVZ hosts some of the shallowest rhyolitic magma bodies on the planet, resulting from the extensional tectonic regime and thinning of the crust. Distinct populations with different equilibration pressures are also recognised, which is consistent with the idea that multiple batches of eruptible magma can be present in the crust at the same time and

  1. Common Variants in 6 Lipid-Related Genes Discovered by High-Resolution DNA Melting Analysis and Their Association with Plasma Lipids.

    PubMed

    Carlquist, John F; McKinney, Jason T; Horne, Benjamin D; Camp, Nicola J; Cannon-Albright, Lisa; Muhlestein, Joseph B; Hopkins, Paul; Clarke, Jessica L; Mower, Chrissa P; Park, James J; Nicholas, Zachary P; Huntinghouse, John A; Anderson, Jeffrey L

    2011-07-10

    BACKGROUND: Total cholesterol was among the earliest identified risk factors for coronary heart disease (CHD). We sought to identify genetic variants in six genes associated with lipid metabolism and estimate their respective contribution to risk for CHD. METHODS: For 6 lipid-associated genes (LCAT, CETP, LIPC, LPL, SCARB1, and ApoF) we scanned exons, 5' and 3' untranslated regions, and donor and acceptor splice sites for variants using Hi-Res Melting® curve analysis (HRMCA) with confirmation by cycle sequencing. Healthy subjects were used for SNP discovery (n=64), haplotype determination/tagging SNP discovery (n=339), and lipid association testing (n=786). RESULTS: In 17,840 bases of interrogated sequence, 90 variant SNPs were identified; 19 (21.1%) previously unreported. Thirty-four variants (37.8%) were exonic(16 non-synonymous), 28 (31.1%) in intron-exon boundaries, and 28 (31.1%) in the 5' and 3' untranslated regions. Compared to cycle sequencing, HRMCA had sensitivity of 99.4% and specificity of 97.7%. Tagging SNPs (n=38) explained >90% of the variation in the 6 genes and identified linkage disequilibrium (LD) groups. Significant beneficial lipid profiles were observed for CETP LD group 2, LIPC LD groups 1 and 7, and SCARB1 LD groups 1, 3 and 4. Risk profiles worsened for CETP LD group 3, LPL LD group 4. CONCLUSIONS: These findings demonstrate the feasibility, sensitivity, and specificity of HRMCA for SNP discovery. Variants identified in these genes may be used to predict lipid-associated risk and reclassification of clinical CHD risk.

  2. Additive Manufacturing by selective laser melting the realizer desktop machine and its application for the dental industry

    NASA Astrophysics Data System (ADS)

    Gebhardt, Andreas; Schmidt, Frank-Michael; Hötter, Jan-Steffen; Sokalla, Wolfgang; Sokalla, Patrick

    Additive Manufacturing of metal parts by Selective Laser Melting has become a powerful tool for the direct manufacturing of complex parts mainly for the aerospace and medical industry. With the introduction of its desktop machine, Realizer targeted the dental market. The contribution describes the special features of the machine, discusses details of the process and shows manufacturing results focused on metal dental devices.

  3. Combined application of extrusion-spheronization and hot-melt coating technologies for improving moisture-proofing of herbal extracts.

    PubMed

    Chen, Hao; Shi, Shuai; Liu, Aina; Tang, Xing

    2010-05-01

    The aim of this research was to investigate the moisture-proofing effect and its mechanism for herbal extracts using extrusion-spheronization combined with hot-melt coating. Guizhi Fuling (GF) compound herbal extract with high hygroscopicity was used as a model drug. In the process of extrusion-spheronization, pellets containing 100% GF were prepared, and then coated with hot-melt coating material using a traditional coating pan. The moisture sorption data for GF were determined by storage at a series of different relative humidities. When the pellets were coated with a 96:4 mixture of stearic acid and polyethylene glycol 6000, the cumulative drug release was over 90% at 45 min while the moisture content was 4.9% at 75% RH within 10 days. These pellets have better moisture-proofing than those coated with Opadry AMB at the same coating level due to a different moisture sorption mechanism. The moisture sorption behavior of the hot-melt coating can be attributed to water vapor diffusion via a porous matrix system, while the Opadry AMB coating system involved a swelling controlled system. The Higuchi model was the best fit for the moisture sorption of the hot-melt coating in all formulations whereas the Opadry AMB coating fitted the Nuttanan model.

  4. Application of temporal moments and other signal processing algorithms to analysis of ultrasonic signals through melting wax

    SciTech Connect

    Lau, Sarah J.; Moore, David G.; Stair, Sarah L.; Nelson, Ciji L.

    2016-01-01

    Ultrasonic analysis is being explored as a way to capture events during melting of highly dispersive wax. Typical events include temperature changes in the material, phase transition of the material, surface flows and reformations, and void filling as the material melts. Melt tests are performed with wax to evaluate the usefulness of different signal processing algorithms in capturing event data. Several algorithm paths are being pursued. The first looks at changes in the velocity of the signal through the material. This is only appropriate when the changes from one ultrasonic signal to the next can be represented by a linear relationship, which is not always the case. The second tracks changes in the frequency content of the signal. The third algorithm tracks changes in the temporal moments of a signal over a full test. This method does not require that the changes in the signal be represented by a linear relationship, but attaching changes in the temporal moments to physical events can be difficult. This study describes the algorithm paths applied to experimental data from ultrasonic signals as wax melts and explores different ways to display the results.

  5. Application of temporal moments and other signal processing algorithms to analysis of ultrasonic signals through melting wax

    DOE PAGES

    Lau, Sarah J.; Moore, David G.; Stair, Sarah L.; ...

    2016-01-01

    Ultrasonic analysis is being explored as a way to capture events during melting of highly dispersive wax. Typical events include temperature changes in the material, phase transition of the material, surface flows and reformations, and void filling as the material melts. Melt tests are performed with wax to evaluate the usefulness of different signal processing algorithms in capturing event data. Several algorithm paths are being pursued. The first looks at changes in the velocity of the signal through the material. This is only appropriate when the changes from one ultrasonic signal to the next can be represented by a linearmore » relationship, which is not always the case. The second tracks changes in the frequency content of the signal. The third algorithm tracks changes in the temporal moments of a signal over a full test. This method does not require that the changes in the signal be represented by a linear relationship, but attaching changes in the temporal moments to physical events can be difficult. This study describes the algorithm paths applied to experimental data from ultrasonic signals as wax melts and explores different ways to display the results.« less

  6. A New High-Resolution Direction Finding Architecture Using Photonics and Neural Network Signal Processing for Miniature Air Vehicle Applications

    DTIC Science & Technology

    2015-09-01

    RESOLUTION DIRECTION FINDING ARCHITECTURE USING PHOTONICS AND NEURAL NETWORK SIGNAL PROCESSING FOR MINIATURE AIR VEHICLE APPLICATIONS by Robert...RESOLUTION DIRECTION FINDING ARCHITECTURE USING PHOTONICS AND NEURAL NETWORK SIGNAL PROCESSING FOR MINIATURE AIR VEHICLE APPLICATIONS 5. FUNDING...unlimited 12b. DISTRIBUTION CODE 13. ABSTRACT (maximum 200 words) This paper investigates the design of an interferometric direction finding receiver

  7. An application of electrothermal feedback for high resolution cryogenic particle detection

    SciTech Connect

    Irwin, K.D.

    1995-04-10

    A novel type of superconducting transition edge sensor is proposed. In this sensor, the temperature of a superconducting film is held constant by feeding back to its position on the resistive transition edge. Energy deposited in the film is measured by a reduction in the feedback Joule heating. This mode of operation should lead to substantial improvements in resolution, linearity, dynamic range, and count rate. Fundamental resolution limits are below {Delta}{ital E}={radical}{ital kT}{sup 2}{ital C}, which is sometimes incorrectly referred to as the thermodynamic limit. This performance is better than any existing technology operating at the same temperature, count rate, and absorber heat capacity. Applications include high resolution x-ray spectrometry, dark matter searches, and neutrino detection.

  8. Thermodynamics of Oligonucleotide Duplex Melting

    NASA Astrophysics Data System (ADS)

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-05-01

    Melting temperatures of oligonucleotides are useful for a number of molecular biology applications, such as the polymerase chain reaction (PCR). Although melting temperatures are often calculated with simplistic empirical equations, application of thermodynamics provides more accurate melting temperatures and an opportunity for students to apply rigorous thermodynamic analysis to an important biochemical problem. Because the stacking of base pairs on top of one another is a significant factor in the energetics of oligonucleotide melting, several investigators have applied van't Hoff analysis to melting temperature data using a nearest-neighbor model and have obtained entropies and enthalpies for the stacking of bases. The present article explains how the equilibrium constant for the dissociation of strands from double-stranded oligonucleotides can be expressed in terms of the total strand concentration and thus how the total strand concentration influences the melting temperature. It also presents a simplified analysis based on the entropies and enthalpies of stacking that is manually tractable so that students can work examples to help them understand the thermodynamics of oligonucleotide melting.

  9. Multi-focus, high resolution inspection system for extended range applications

    NASA Astrophysics Data System (ADS)

    Harding, Kevin

    2016-05-01

    Visual inspection of parts or structures for defects typically requires good spatial resolution to see the defects, but may also require a large focus range. But to obtain the best resolution from an imaging system, it needs to have a low f-number which limits the usable depth of field. Methods to use autofocus or focus stacking provides more range at high resolution, but often at the expense of computation time, loss of a real time image and uncertainty in scale changes. This paper describes an approach to quickly move through a range of focus positions without the need to move optics mechanically in a manner that is highly repeatable, maintains high resolution and provides the potential for a live image directly viewable by an inspector, even at microscope level magnifications. This paper will present the approach we investigated and discuss the pros and cons for a range of applications from large structures to small feature inspection. The paper will present examples of what resolution was achieved and how the multiple images might also be used to determine other parameters such as pose of a test surface.

  10. Resolution in QCM sensors for the viscosity and density of liquids: application to lead acid batteries.

    PubMed

    Cao-Paz, Ana María; Rodríguez-Pardo, Loreto; Fariña, José; Marcos-Acevedo, Jorge

    2012-01-01

    In battery applications, particularly in automobiles, submarines and remote communications, the state of charge (SoC) is needed in order to manage batteries efficiently. The most widely used physical parameter for this is electrolyte density. However, there is greater dependency between electrolyte viscosity and SoC than that seen for density and SoC. This paper presents a Quartz Crystal Microbalance (QCM) sensor for electrolyte density-viscosity product measurements in lead acid batteries. The sensor is calibrated in H(2)SO(4) solutions in the battery electrolyte range to obtain sensitivity, noise and resolution. Also, real-time tests of charge and discharge are conducted placing the quartz crystal inside the battery. At the same time, the present theoretical "resolution limit" to measure the square root of the density-viscosity product [Formula: see text] of a liquid medium or best resolution achievable with a QCM oscillator is determined. Findings show that the resolution limit only depends on the characteristics of the liquid to be studied and not on frequency. The QCM resolution limit for [Formula: see text] measurements worsens when the density-viscosity product of the liquid is increased, but it cannot be improved by elevating the work frequency.

  11. Multiplex real-time PCR and high-resolution melting analysis for detection of white spot syndrome virus, yellow-head virus, and Penaeus monodon densovirus in penaeid shrimp.

    PubMed

    Panichareon, Benjaporn; Khawsak, Paisarn; Deesukon, Warin; Sukhumsirichart, Wasana

    2011-12-01

    A multiplex real-time PCR and high-resolution melting (HRM) analysis was developed to detect simultaneously three of the major viruses of penaeid shrimp including white spot syndrome virus (WSSV), yellow-head virus (YHV), and Penaeus monodon densovirus (PmDNV). Plasmids containing DNA/cDNA fragments of WSSV and YHV, and genomic DNAs of PmDNV and normal shrimp were used to test sensitivity of the procedure. Without the need of any probe, the products were identified by HRM analysis after real-time PCR amplification using three sets of viral specific primers. The results showed DNA melting curves that were specific for individual virus. No positive result was detected with nucleic acids from shrimp, Penaeus monodon nucleopolyhedrovirus (PemoNPV), Penaeus stylirostris densovirus (PstDNV), or Taura syndrome virus (TSV). The detection limit for PmDNV, YHV and WSSV DNAs were 40fg, 50fg, and 500fg, respectively, which was 10 times more sensitive than multiplex real-time PCR analyzed by agarose gel electrophoresis. In viral nucleic acid mixtures, HRM analysis clearly identified each virus in dual and triple infection. To test the capability to use this method in field, forty-one of field samples were examined by HRM analysis in comparison with agarose gel electrophoresis. For HRM analysis, 11 (26.83%), 9 (21.95%), and 4 (9.76%) were infected with WSSV, PmDNV, and YHV, respectively. Agarose gel electrophoresis detected lesser number of PmDNV infection which may due to the limit of sensitivity. No multiple infection was found in these samples. This method provides a rapid, sensitive, specific, and simultaneous detection of three major viruses making it as a useful tool for diagnosis and epidemiological studies of these viruses in shrimp and carriers.

  12. A mitochondrial species identification assay for Australian blacktip sharks (Carcharhinus tilstoni, C. limbatus and C. amblyrhynchoides) using real-time PCR and high-resolution melt analysis.

    PubMed

    Morgan, Jess A T; Welch, David J; Harry, Alistair V; Street, Raewyn; Broderick, Damien; Ovenden, Jennifer R

    2011-09-01

    Tropical Australian shark fisheries target two morphologically indistinguishable blacktip sharks, the Australian blacktip (Carcharhinus tilstoni) and the common blacktip (C. limbatus). Their relative contributions to northern and eastern Australian coastal fisheries are unclear because of species identification difficulties. The two species differ in their number of precaudal vertebrae, which is difficult and time consuming to obtain in the field. But, the two species can be distinguished genetically with diagnostic mutations in their mitochondrial DNA ND4 gene. A third closely related sister species, the graceful shark C. amblyrhynchoides, can also be distinguished by species-specific mutations in this gene. DNA sequencing is an effective diagnostic tool, but is relatively expensive and time consuming. In contrast, real-time high-resolution melt (HRM) PCR assays are rapid and relatively inexpensive. These assays amplify regions of DNA with species-specific genetic mutations that result in PCR products with unique melt profiles. A real-time HRM PCR species-diagnostic assay (RT-HRM-PCR) has been developed based on the mtDNA ND4 gene for rapid typing of C. tilstoni, C. limbatus and C. amblyrhynchoides. The assay was developed using ND4 sequences from 66 C. tilstoni, 33. C. limbatus and five C. amblyrhynchoides collected from Indonesia and Australian states and territories; Western Australia, the Northern Territory, Queensland and New South Wales. The assay was shown to be 100% accurate on 160 unknown blacktip shark tissue samples by full mtDNA ND4 sequencing.

  13. Comparison of COBAS 4800 KRAS, TaqMan PCR and high resolution melting PCR assays for the detection of KRAS somatic mutations in formalin-fixed paraffin embedded colorectal carcinomas.

    PubMed

    Harlé, Alexandre; Busser, Benoit; Rouyer, Marie; Harter, Valentin; Genin, Pascal; Leroux, Agnès; Merlin, Jean-Louis

    2013-03-01

    Many studies documented the influence of KRAS mutation status on the response of patients with metastatic colorectal cancer (mCRC) to anti-EGFR monoclonal antibodies. The COBAS 4800 KRAS is an assay using real time PCR and TaqMelt technology, CE-IVD validated, for the detection of 19 KRAS somatic mutations in exons 2 and 3. We compared COBAS with previously validated PCR TaqMan and High Resolution Melting (HRM) assays on 156 formalin-fixed paraffin embedded (FFPE) specimens of colorectal carcinoma. DNA extraction procedures, using the Qiagen QiAMP kit and the Roche COBAS DNA kit, were also compared. Of the 156 samples, 132 were interpretable using COBAS and TaqMan and 92 using COBAS and HRM. No statistically significant difference was found between COBAS/TaqMan and COBAS/HRM (k = 0.937; p < 0.001 - four discordant cases were found, mostly concerning codon 61 mutations and k = 0.891; p < 0.001 - five discordant cases were found, three regarding codon 61 and two on codon 12/13, respectively). No difference was found between the two DNA extraction methods (t = 1.7185; dol = 39; α = 5 %). The three assays were found suitable to detect accurately KRAS mutations in colon FFPE specimens. COBAS and TaqMan were found to be more robust than HRM, as they yielded fewer non-interpretable results. DNA extraction kits were found to provide equivalent results. The present study shows that pre-screening using COBAS with further TaqMan mutation characterization constitutes an easy and reliable approach for routine diagnostic purposes.

  14. Potential Offshore Wind Energy Applications for Enhanced Resolution Scatterometer Products (Invited)

    NASA Astrophysics Data System (ADS)

    Plagge, A. M.; Epps, B.

    2013-12-01

    The multi-decadal record of ocean surface vector winds provided by scatterometer measurements is a valuable resource that has been underutilized by the wind energy sector. Previously, these data were not considered applicable for offshore wind energy analysis; chiefly, the sensors' low resolution limited their desirability. Now, however, enhanced products provide high quality wind vectors at resolutions between 3 and 5km. Potential energy applications currently under investigation include (1) validation of existing commercial wind resource assessment models, (2) investigations of interactions between large existing wind farms and the atmospheric boundary layer including attempts to identify wakes, and (3) an extension of previous studies comparing SAR and scatterometer wind fields with regard to specific wind energy concerns, including wind spectra and Weibull parameters.

  15. A transportable high-resolution gamma-ray spectrometer and analysis system applicable to mobile, autonomous or unattended applications

    SciTech Connect

    Buckley, W.M.; Neufeld, K.W.

    1995-07-01

    The Safeguards Technology Program at the Lawrence Livermore National Laboratory is developing systems based on a compact electro-mechanically cooled high-purity germanium (HPGe) detector. This detector system broadens the practicality of performing high- resolution gamma-ray spectrometry in the field. Utilizing portable computers, multi-channel analyzers and software these systems greatly improve the ease of performing mobile high-resolution gamma-ray spectrometry. Using industrial computers, we can construct systems that will run autonomously for extended periods of time without operator input or maintenance. These systems can start or make decisions based on sensor inputs rather than operator interactions. Such systems can provide greater capability for wider domain of safeguards, treaty verification application, and other unattended, autonomous or in-situ applications.

  16. Fault rheology beyond frictional melting

    PubMed Central

    Lavallée, Yan; Hirose, Takehiro; Kendrick, Jackie E.; Hess, Kai-Uwe; Dingwell, Donald B.

    2015-01-01

    During earthquakes, comminution and frictional heating both contribute to the dissipation of stored energy. With sufficient dissipative heating, melting processes can ensue, yielding the production of frictional melts or “pseudotachylytes.” It is commonly assumed that the Newtonian viscosities of such melts control subsequent fault slip resistance. Rock melts, however, are viscoelastic bodies, and, at high strain rates, they exhibit evidence of a glass transition. Here, we present the results of high-velocity friction experiments on a well-characterized melt that demonstrate how slip in melt-bearing faults can be governed by brittle fragmentation phenomena encountered at the glass transition. Slip analysis using models that incorporate viscoelastic responses indicates that even in the presence of melt, slip persists in the solid state until sufficient heat is generated to reduce the viscosity and allow remobilization in the liquid state. Where a rock is present next to the melt, we note that wear of the crystalline wall rock by liquid fragmentation and agglutination also contributes to the brittle component of these experimentally generated pseudotachylytes. We conclude that in the case of pseudotachylyte generation during an earthquake, slip even beyond the onset of frictional melting is not controlled merely by viscosity but rather by an interplay of viscoelastic forces around the glass transition, which involves a response in the brittle/solid regime of these rock melts. We warn of the inadequacy of simple Newtonian viscous analyses and call for the application of more realistic rheological interpretation of pseudotachylyte-bearing fault systems in the evaluation and prediction of their slip dynamics. PMID:26124123

  17. Fault rheology beyond frictional melting.

    PubMed

    Lavallée, Yan; Hirose, Takehiro; Kendrick, Jackie E; Hess, Kai-Uwe; Dingwell, Donald B

    2015-07-28

    During earthquakes, comminution and frictional heating both contribute to the dissipation of stored energy. With sufficient dissipative heating, melting processes can ensue, yielding the production of frictional melts or "pseudotachylytes." It is commonly assumed that the Newtonian viscosities of such melts control subsequent fault slip resistance. Rock melts, however, are viscoelastic bodies, and, at high strain rates, they exhibit evidence of a glass transition. Here, we present the results of high-velocity friction experiments on a well-characterized melt that demonstrate how slip in melt-bearing faults can be governed by brittle fragmentation phenomena encountered at the glass transition. Slip analysis using models that incorporate viscoelastic responses indicates that even in the presence of melt, slip persists in the solid state until sufficient heat is generated to reduce the viscosity and allow remobilization in the liquid state. Where a rock is present next to the melt, we note that wear of the crystalline wall rock by liquid fragmentation and agglutination also contributes to the brittle component of these experimentally generated pseudotachylytes. We conclude that in the case of pseudotachylyte generation during an earthquake, slip even beyond the onset of frictional melting is not controlled merely by viscosity but rather by an interplay of viscoelastic forces around the glass transition, which involves a response in the brittle/solid regime of these rock melts. We warn of the inadequacy of simple Newtonian viscous analyses and call for the application of more realistic rheological interpretation of pseudotachylyte-bearing fault systems in the evaluation and prediction of their slip dynamics.

  18. Resonant absorption induced fast melting studied with mid-IR QCLs

    NASA Astrophysics Data System (ADS)

    Lu, Jie; Lv, Yankun; Ji, Youxin; Tang, Xiaoliang; Qi, Zeming; Li, Liangbin

    2017-02-01

    We demonstrate the use of a pump-probe setup based on two mid-infrared quantum cascade lasers (QCLs) to investigate the melting and crystallization of materials through resonant absorption. A combination of pump and probe beams fulfills the two-color synchronous detection. Furthermore, narrow linewidth advances the accuracy of measurements and the character of broad tuning range of QCLs enables wide applications in various sample and multiple structures. 1-Eicosene was selected as a simple model system to verify the feasibility of this method. A pulsed QCL was tuned to the absorption peak of CH2 bending vibration at 1467 cm-1 to resonantly heat the sample. The other QCL in continuous mode was tuned to 1643 cm-1 corresponding the C=C stretching vibration to follow the fast melting dynamics. By monitoring the transmission intensity variation of pump and probe beams during pump-probe experiments, the resonant absorption induced fast melting and re-crystallization of 1-Eicosene can be studied. Results show that the thermal effect and melting behaviors strongly depend on the pump wavelength (resonant or non-resonant) and energy, as well as the pump time. The realization and detection of melting and recrystallization can be performed in tens of milliseconds, which improves the time resolution of melting process study based on general mid-infrared spectrum by orders of magnitude. The availability of resonant heating and detections based on mid-infrared QCLs is expected to enable new applications in melting study.

  19. An update on clinical applications of magnetic nanoparticles for increasing the resolution of magnetic resonance imaging.

    PubMed

    Zeinali Sehrig, Fatemeh; Majidi, Sima; Asvadi, Sahar; Hsanzadeh, Arash; Rasta, Seyed Hossein; Emamverdy, Masumeh; Akbarzadeh, Jamshid; Jahangiri, Sahar; Farahkhiz, Shahrzad; Akbarzadeh, Abolfazl

    2016-11-01

    Today, technologies based on magnetic nanoparticles (MNPs) are regularly applied to biological systems with diagnostic or therapeutic aims. Nanoparticles made of the elements iron (Fe), gadolinium (Gd) or manganese (Mn) are generally used in many diagnostic applications performed under magnetic resonance imaging (MRI). Similar to molecular-based contrast agents, nanoparticles can be used to increase the resolution of imaging while offering well biocompatibility, poisonousness and biodistribution. Application of MNPs enhanced MRI sensitivity due to the accumulation of iron in the liver caused by discriminating action of the hepatobiliary system. The aim of this study is about the use, properties and advantages of MNPs in MRI.

  20. Improved resolution and sensitivity on the ANSTO microprobe and it’s application to μ-PIXE

    NASA Astrophysics Data System (ADS)

    Siegele, R.; Kachenko, A. G.; Ionescu, M.; Cohen, D. D.

    2009-06-01

    We report on the improved spatial resolution of the ANSTO heavy ion microprobe, achieved through the use of a higher brightness ion source for hydrogen. The improved resolution will be demonstrated for applications of μ-PIXE. With the higher brightness source, a 3 μm resolution was achieved for μ-PIXE elemental analysis. This is illustrated in high resolution images of nickel (Ni)-hyperaccumulating Hybanthus floribundus subsp. floribundus leaf tissues, where individual cells were clearly visible in the acquired elemental images. The higher resolution images illustrated that Ni was localised in epidermal cell walls.

  1. Application of spatially resolved high resolution crystal spectrometry to inertial confinement fusion plasmas

    SciTech Connect

    Hill, K. W.; Bitter, M.; Delgado-Aparacio, L.; Pablant, N. A.; Beiersdorfer, P.; Schneider, M.; Widmann, K.; Sanchez del Rio, M.; Zhang, L.

    2012-10-15

    High resolution ({lambda}/{Delta}{lambda}{approx} 10 000) 1D imaging x-ray spectroscopy using a spherically bent crystal and a 2D hybrid pixel array detector is used world wide for Doppler measurements of ion-temperature and plasma flow-velocity profiles in magnetic confinement fusion plasmas. Meter sized plasmas are diagnosed with cm spatial resolution and 10 ms time resolution. This concept can also be used as a diagnostic of small sources, such as inertial confinement fusion plasmas and targets on x-ray light source beam lines, with spatial resolution of micrometers, as demonstrated by laboratory experiments using a 250-{mu}m {sup 55}Fe source, and by ray-tracing calculations. Throughput calculations agree with measurements, and predict detector counts in the range 10{sup -8}-10{sup -6} times source x-rays, depending on crystal reflectivity and spectrometer geometry. Results of the lab demonstrations, application of the technique to the National Ignition Facility (NIF), and predictions of performance on NIF will be presented.

  2. Imaging live cells at high spatiotemporal resolution for lab-on-a-chip applications.

    PubMed

    Chin, Lip Ket; Lee, Chau-Hwang; Chen, Bi-Chang

    2016-05-24

    Conventional optical imaging techniques are limited by the diffraction limit and difficult-to-image biomolecular and sub-cellular processes in living specimens. Novel optical imaging techniques are constantly evolving with the desire to innovate an imaging tool that is capable of seeing sub-cellular processes in a biological system, especially in three dimensions (3D) over time, i.e. 4D imaging. For fluorescence imaging on live cells, the trade-offs among imaging depth, spatial resolution, temporal resolution and photo-damage are constrained based on the limited photons of the emitters. The fundamental solution to solve this dilemma is to enlarge the photon bank such as the development of photostable and bright fluorophores, leading to the innovation in optical imaging techniques such as super-resolution microscopy and light sheet microscopy. With the synergy of microfluidic technology that is capable of manipulating biological cells and controlling their microenvironments to mimic in vivo physiological environments, studies of sub-cellular processes in various biological systems can be simplified and investigated systematically. In this review, we provide an overview of current state-of-the-art super-resolution and 3D live cell imaging techniques and their lab-on-a-chip applications, and finally discuss future research trends in new and breakthrough research areas of live specimen 4D imaging in controlled 3D microenvironments.

  3. On the application of ENO scheme with subcell resolution to conservation laws with stiff source terms

    NASA Technical Reports Server (NTRS)

    Chang, Shih-Hung

    1991-01-01

    Two approaches are used to extend the essentially non-oscillatory (ENO) schemes to treat conservation laws with stiff source terms. One approach is the application of the Strang time-splitting method. Here the basic ENO scheme and the Harten modification using subcell resolution (SR), ENO/SR scheme, are extended this way. The other approach is a direct method and a modification of the ENO/SR. Here the technique of ENO reconstruction with subcell resolution is used to locate the discontinuity within a cell and the time evolution is then accomplished by solving the differential equation along characteristics locally and advancing in the characteristic direction. This scheme is denoted ENO/SRCD (subcell resolution - characteristic direction). All the schemes are tested on the equation of LeVeque and Yee (NASA-TM-100075, 1988) modeling reacting flow problems. Numerical results show that these schemes handle this intriguing model problem very well, especially with ENO/SRCD which produces perfect resolution at the discontinuity.

  4. Fluorescence in situ hybridization applications for super-resolution 3D structured illumination microscopy.

    PubMed

    Markaki, Yolanda; Smeets, Daniel; Cremer, Marion; Schermelleh, Lothar

    2013-01-01

    Fluorescence in situ hybridization on three-dimensionally preserved cells (3D-FISH) is an efficient tool to analyze the subcellular localization and spatial arrangement of targeted DNA sequences and RNA transcripts at the single cell level. 3D reconstructions from serial optical sections obtained by confocal laser scanning microscopy (CLSM) have long been considered the gold standard for 3D-FISH analyses. Recent super-resolution techniques circumvent the diffraction-limit of optical resolution and have defined a new state-of-the-art in bioimaging. Three-dimensional structured illumination microscopy (3D-SIM) represents one of these technologies. Notably, 3D-SIM renders an eightfold improved volumetric resolution over conventional imaging, and allows the simultaneous visualization of differently labeled target structures. These features make this approach highly attractive for the analysis of spatial relations and substructures of nuclear targets that escape detection by conventional light microscopy. Here, we focus on the application of 3D-SIM for the visualization of subnuclear 3D-FISH preparations. In comparison with conventional fluorescence microscopy, the quality of 3D-SIM data is dependent to a much greater extent on the optimal sample preparation, labeling and acquisition conditions. We describe typical problems encountered with super-resolution imaging of in situ hybridizations in mammalian tissue culture cells and provide optimized DNA-/(RNA)-FISH protocols including combinations with immunofluorescence staining (Immuno-FISH) and DNA replication labeling using click chemistry.

  5. High-resolution solid-state NMR study of the effect of composition on network connectivity and structural disorder in multi-component glasses in the diopside and jadeite join: Implications for structure of andesitic melts

    NASA Astrophysics Data System (ADS)

    Park, Sun Young; Lee, Sung Keun

    2014-12-01

    The structural evolution of andesitic melts with varying compositions remains one of the unsolved questions in high-temperature geochemistry and petrology. In this article, we report the structural details of model andesitic glasses [CaO-MgO-Na2O-Al2O3-SiO2 (CMNAS)] in the diopside (CaMgSi2O6) and jadeite (NaAlSi2O6) join using high-resolution, multi-nuclear, solid-state nuclear magnetic resonance (NMR). The 27Al NMR spectra of CMNAS glasses confirm that [4]Al is dominant. While a minor fraction of [5]Al is observed, its presence is only prevalent in the glasses with higher Ca-Mg content. Topological disorder in the glass network also tends to increase with Ca-Mg content as evidenced by the increase in the quadrupolar coupling constant (Cq) of [4]Al for glasses with increasing diopside contents (XDiopside). Despite the complex nature of the glasses studied here (with five oxide components), the 17O 3QMAS NMR spectra resolve diverse bridging oxygens (BOs) and non-bridging oxygens (NBOs), from which the degree of Al avoidance among framework cations (Si and Al) and preferential proximity among non-network cations (Ca2+, Mg2+, and Na+) and each oxygen site can be estimated: presence of Al-O-Al in jadeite glass implies a violation of the Al-avoidance rule in the glasses and the decrease in the fraction of NBOs with increasing XDiopside is consistent with a decrease in their viscosity. Analysis of the peak position of {Ca, Mg}-mixed NBOs, along with the absence of Na-NBO peak, and the peak shape of Si-O-Al reveals preferential partitioning of Ca2+ and Mg2 into NBOs and the proximity of Na+ to BOs. The fraction of highly coordinated Al has been linked to thermodynamic and transport properties of the melts. Considering all the experimental Al coordination environments available in the literature, together with the current experimental studies, we attempt to establish the relationship between the fractions of highly coordinated Al and composition, particularly average

  6. Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region.

    PubMed

    Jeffery, Nathan; Gasser, Robin B; Steer, Penelope A; Noormohammadi, Amir H

    2007-08-01

    Mycoplasma synoviae is an economically important pathogen of poultry worldwide, causing respiratory infection and synovitis in chickens and turkeys. Identification of M. synoviae isolates is of critical importance, particularly in countries in which poultry flocks are vaccinated with the live attenuated M. synoviae strain MS-H. Using oligonucleotide primers complementary to the single-copy conserved 5' end of the variable lipoprotein and haemagglutinin gene (vlhA), amplicons of approximately 400 bp were generated from 35 different M. synoviae strains/isolates from chickens and subjected to mutation scanning analysis. Analysis of the amplicons by single-strand conformation polymorphism (SSCP) revealed 10 distinct profiles (A-J). Sequencing of the amplicons representing these profiles revealed that each profile related to a unique sequence, some differing from each other by only one base-pair substitution. Comparative high-resolution melting (HRM) curve analysis of the amplicons using SYTO 9 green fluorescent dye also displayed profiles which were concordant with the same 10 SSCP profiles (A-J) and their sequences. For both mutation detection methods, the Australian M. synoviae strains represented one of the A, B, C or D profiles, while the USA strains represented one of the E, F, G, H, I or J profiles. The results presented in this study show that the PCR-based SSCP or HRM curve analyses of vlhA provide high-resolution mutation detection tools for the detection and identification of M. synoviae strains. In particular, the HRM curve analysis is a rapid and effective technique which can be performed in a single test tube in less than 2 h.

  7. A Compact "Water Window" Microscope with 60 nm Spatial Resolution for Applications in Biology and Nanotechnology.

    PubMed

    Wachulak, Przemyslaw; Torrisi, Alfio; Nawaz, Muhammad F; Bartnik, Andrzej; Adjei, Daniel; Vondrová, Šárka; Turňová, Jana; Jančarek, Alexandr; Limpouch, Jiří; Vrbová, Miroslava; Fiedorowicz, Henryk

    2015-10-01

    Short illumination wavelength allows an extension of the diffraction limit toward nanometer scale; thus, improving spatial resolution in optical systems. Soft X-ray (SXR) radiation, from "water window" spectral range, λ=2.3-4.4 nm wavelength, which is particularly suitable for biological imaging due to natural optical contrast provides better spatial resolution than one obtained with visible light microscopes. The high contrast in the "water window" is obtained because of selective radiation absorption by carbon and water, which are constituents of the biological samples. The development of SXR microscopes permits the visualization of features on the nanometer scale, but often with a tradeoff, which can be seen between the exposure time and the size and complexity of the microscopes. Thus, herein, we present a desk-top system, which overcomes the already mentioned limitations and is capable of resolving 60 nm features with very short exposure time. Even though the system is in its initial stage of development, we present different applications of the system for biology and nanotechnology. Construction of the microscope with recently acquired images of various samples will be presented and discussed. Such a high resolution imaging system represents an interesting solution for biomedical, material science, and nanotechnology applications.

  8. Comparison of high-resolution melting analysis with direct sequencing for the detection of recurrent mutations in DNA methyltransferase 3A and isocitrate dehydrogenase 1 and 2 genes in acute myeloid leukemia patients.

    PubMed

    Gorniak, Patryk; Ejduk, Anna; Borg, Katarzyna; Makuch-Lasica, Hanna; Nowak, Grazyna; Lech-Maranda, Ewa; Prochorec-Sobieszek, Monika; Warzocha, Krzysztof; Juszczynski, Przemyslaw

    2016-02-01

    Acute myeloid leukemia (AML) cells harbor frequent mutations in genes responsible for epigenetic modifications. Increasing evidence of clinical role of DNMT3A and IDH1/2 mutations highlights the need for a robust and inexpensive test to identify these mutations in routine diagnostic work-up. Herein, we compared routinely used direct sequencing method with high-resolution melting (HRM) assay for screening DNMT3A and IDH1/2 mutations in patients with AML. We show very high concordance between HRM and Sanger sequencing (100% samples for IDH2-R140 and DNMT3-R882 mutations, 99% samples for IDH1-R132 and IDH2-R172 mutations). HRM method reported no false-negative results, suggesting that it can be used for mutations screening. Moreover, HRM displayed much higher sensitivity in comparison with DNA sequencing in all assessed loci. With Sanger sequencing, robust calls were observed when the sample contained 50% of mutant DNA in the background of wild-type DNA. In marked contrast, the detection limit of HRM improved down to 10% of mutated DNA. Given the ubiquitous presence of wild-type DNA background in bone marrow aspirates and clonal variations regarding mutant allele burden, these results favor HRM as a sensitive, specific, labor-, and cost-effective tool for screening and detection of mutations in IDH1/2 and DNMT3A genes in patients with AML.

  9. Development of a novel real-time PCR assay with high-resolution melt analysis to detect and differentiate OXA-48-Like β-lactamases in carbapenem-resistant Enterobacteriaceae.

    PubMed

    Hemarajata, Peera; Yang, Shangxin; Hindler, Janet A; Humphries, Romney M

    2015-09-01

    The rapid global spread of carbapenem-resistant Enterobacteriaceae (CRE) poses an urgent threat to public health. More than 250 class D β-lactamases (OXAs) have been described in recent years, with variations in hydrolytic activity for β-lactams. The plasmid-borne OXA-48 β-lactamase and its variants are identified only sporadically in the United States but are common in Europe. Recognition of these OXA-48-like carbapenemases is vital in order to control their dissemination. We developed a real-time PCR assay based on high-resolution melt analysis, using bla OXA-48-like-specific primers coupled with an unlabeled 3'-phosphorylated oligonucleotide probe (LunaProbe) homologous to OXA-48-like carbapenemase genes. The assay was validated using genomic DNA from 48 clinical isolates carrying a variety of carbapenemase genes, including bla KPC, bla SME, bla IMP, bla NDM-1, bla VIM, bla OXA-48, bla OXA-162, bla OXA-181, bla OXA-204, bla OXA-244, bla OXA-245, and bla OXA-232. Our assay identified the presence of bla OXA-48-like β-lactamase genes and clearly distinguished between bla OXA-48 and its variants in control strains, including between bla OXA-181 and bla OXA-232, which differ by only a single base pair in the assay target region. This approach has potential for use in epidemiological investigations and continuous surveillance to help control the spread of CRE strains producing OXA-48-like enzymes.

  10. Identification of bacterial pathogens in ascitic fluids from patients with suspected spontaneous bacterial peritonitis by use of broad-range PCR (16S PCR) coupled with high-resolution melt analysis.

    PubMed

    Hardick, Justin; Won, Helen; Jeng, Kevin; Hsieh, Yu-Hsiang; Gaydos, Charlotte A; Rothman, Richard E; Yang, Samuel

    2012-07-01

    Spontaneous bacterial peritonitis (SBP) can be a severe complication occurring in patients with cirrhosis and ascites, with associated mortality often as high as 40%. Traditional diagnostics for SBP rely on culture techniques for proper diagnosis, although recent reports suggest that the presence of bacterial DNA in peritoneal fluid in patients with cirrhosis and ascites is an indicator of SBP. A previously published broad-range PCR (16S PCR) coupled with high-resolution melt analysis (HRMA) was compared with standard culture techniques for diagnosis of SBP in 106 peritoneal fluid samples from patients with suspected SBP. The sensitivity and specificity for 16S PCR for detecting eubacterial DNA compared with those of standard culture techniques were 100% (17/17) and 91.5% (85/89), respectively. Overall, HRMA concordance with species identification was 70.6% (12/17), although the 5 samples that were discordant at the species level were SBP resulting from a polymicrobial infection, and species-level identification for polymicrobial infections is outside the capability of HRMA. Both the broad-range 16S PCR and HRMA analysis provide useful diagnostic adjunctive assays for clinicians in detecting and identifying pathogens responsible for SBP.

  11. Volcanic degassing of bromine and iodine: experimental fluid/melt partitioning data and applications to stratospheric chemistry

    NASA Astrophysics Data System (ADS)

    Bureau, Hélène; Keppler, Hans; Métrich, Nicole

    2000-11-01

    In order to understand the degassing behavior of heavy halogens in volcanic processes, we experimentally studied the distribution of Cl, Br, and I between albite melt and hydrous fluids containing 0.01-2 wt% of NaCl, NaBr, or NaI, respectively. All experiments were carried out at 2 kbar and 900°C in rapid-quench cold-seal autoclaves with a run duration of 7 days. The major element compositions and Cl contents of the glassy run products were measured by electron microprobe. Bromine and iodine were measured by proton-induced X-ray emission. Fluid compositions were obtained by mass balance. All halogens investigated were found to partition strongly into the fluid phase. Over the range of concentrations studied, the halogen contents in the melt are proportional to the concentrations in the fluid. The fluid/melt partition coefficients, Df/m, are 8.1±0.2 for Cl, 17.5±0.6 for Br, and 104±7 for I. The logarithm of Df/m is linearly correlated with the ionic radius of the halogenide ion. On the basis of our experimental data, we estimate the amount of bromine injected into the stratosphere by major volcanic explosions. For the 1991 Mount Pinatubo eruption, we obtain Br yields of 11-25 kt as minimum estimates. These numbers are comparable to the total annual influx of bromine into the stratosphere from all other natural and anthropogenic sources (about 100 kt/year). Since bromine is much more efficient in destroying stratospheric ozone than chlorine, it could at least be partially responsible for the massive ozone depletion observed after the 1991 Mount Pinatubo eruption.

  12. Accuracy and Resolution of Kinect Depth Data for Indoor Mapping Applications

    PubMed Central

    Khoshelham, Kourosh; Elberink, Sander Oude

    2012-01-01

    Consumer-grade range cameras such as the Kinect sensor have the potential to be used in mapping applications where accuracy requirements are less strict. To realize this potential insight into the geometric quality of the data acquired by the sensor is essential. In this paper we discuss the calibration of the Kinect sensor, and provide an analysis of the accuracy and resolution of its depth data. Based on a mathematical model of depth measurement from disparity a theoretical error analysis is presented, which provides an insight into the factors influencing the accuracy of the data. Experimental results show that the random error of depth measurement increases with increasing distance to the sensor, and ranges from a few millimeters up to about 4 cm at the maximum range of the sensor. The quality of the data is also found to be influenced by the low resolution of the depth measurements. PMID:22438718

  13. Quantifying melting and mobilistaion of interstitial melts in crystal mushes

    NASA Astrophysics Data System (ADS)

    Veksler, Ilya; Dobson, Katherine; Hess, Kai-Uwe; Ertel-Ingrisch, Werner; Humphreys, Madeleine

    2015-04-01

    The deformation of crystals mushes and separation of melts and crystals in is critical to understanding the development of physical and chemical heterogeneity in magma chambers and has been invoked as an eruption trigger mechanism. Here we investigate the behaviour of the melt in the well characterised, classic crystal mush system of the Skaergaard intrusion by combining experimental petrology and the non-destructive 3D imaging methods. Starting materials for partial melting experiments were four samples from the upper Middle Zone of the Layered Series. Cylinders, 15 mm in diameter and 20 mm in length, were drilled out of the rock samples, placed in alumina crucibles and held for 5 days in electric furnaces at atmospheric pressure and 1050-1100 °C. Redox conditions set by the CO-CO2 gas mixture were kept close to those of the FMQ buffer. We then use spatially registered 3D x-ray computed tomography images, collected before and after the experiment, to determine the volume and distribution of the crystal framework and interstitial phases, and the volume, distribution and connectivity the interstitial phases that undergo melting and extraction while at elevated temperature. Image analysis has allowed us to quantify these physical changes with high spatial resolution. Our work is a first step towards quantitative understanding of the melt mobilisation and migration processes operating in notionally locked crystal rich magmatic systems.

  14. High Resolution Applications of Laser-Induced Breakdown Spectroscopy for Environmental and Forensic Applications

    SciTech Connect

    Martin, Madhavi Z; Labbe, Nicole; Andre, Nicolas O; Harris, Ronny D; Ebinger, Michael H; Wullschleger, Stan D; Vass, Arpad Alexander

    2007-01-01

    Laser-induced breakdown spectroscopy (LIBS) has been used in the elemental analysis for a variety of environmental samples and as a proof of concept for a host of forensic applications. In the first application, LIBS was used for the rapid detection of carbon from a number of different soil types. In this application, a major breakthrough was achieved by using a multivariate analytical approach that has brought us closer towards a "universal calibration curve". In a second application, it has been demonstrated that LIBS in combination with multivariate analysis can be employed to analyze the chemical composition of annual tree growth rings and correlate them to external parameters such as changes in climate, forest fires, and disturbances involving human activity. The objectives of using this technology in fire scar determinations are: 1) To determine the characteristic spectra of wood exposed to forest fires and 2) To examine the viability of this technique for detecting fire occurrences in stems that did not develop fire scars. These examples demonstrate that LIBS-based techniques are inherently well suited for diverse environmental applications. LIBS was also applied to a variety of proof of concept forensic applications such as the analysis of cremains (human cremation remains) and elemental composition analysis of prosthetic implants.

  15. High resolution applications of laser-induced breakdown spectroscopy for environmental and forensic applications

    NASA Astrophysics Data System (ADS)

    Martin, Madhavi Z.; Labbé, Nicole; André, Nicolas; Harris, Ronny; Ebinger, Michael; Wullschleger, Stan D.; Vass, Arpad A.

    2007-12-01

    Laser-induced breakdown spectroscopy (LIBS) has been used in the elemental analysis for a variety of environmental samples and as a proof of concept for a host of forensic applications. In the first application, LIBS was used for the rapid detection of carbon from a number of different soil types. In this application, a major breakthrough was achieved by using a multivariate analytical approach that has brought us closer towards a "universal calibration curve". In a second application, it has been demonstrated that LIBS in combination with multivariate analysis can be employed to analyze the chemical composition of annual tree growth rings and correlate them to external parameters such as changes in climate, forest fires, and disturbances involving human activity. The objectives of using this technology in fire scar determinations are: 1) To determine the characteristic spectra of wood exposed to forest fires and 2) To examine the viability of this technique for detecting fire occurrences in stems that did not develop fire scars. These examples demonstrate that LIBS-based techniques are inherently well suited for diverse environmental applications. LIBS was also applied to a variety of proof of concept forensic applications such as the analysis of cremains (human cremation remains) and elemental composition analysis of prosthetic implants.

  16. A Solvent-Free Surface Suspension Melt Technique for Making Biodegradable PCL Membrane Scaffolds for Tissue Engineering Applications.

    PubMed

    Suntornnond, Ratima; An, Jia; Tijore, Ajay; Leong, Kah Fai; Chua, Chee Kai; Tan, Lay Poh

    2016-03-21

    In tissue engineering, there is limited availability of a simple, fast and solvent-free process for fabricating micro-porous thin membrane scaffolds. This paper presents the first report of a novel surface suspension melt technique to fabricate a micro-porous thin membrane scaffolds without using any organic solvent. Briefly, a layer of polycaprolactone (PCL) particles is directly spread on top of water in the form of a suspension. After that, with the use of heat, the powder layer is transformed into a melted layer, and following cooling, a thin membrane is obtained. Two different sizes of PCL powder particles (100 µm and 500 µm) are used. Results show that membranes made from 100 µm powders have lower thickness, smaller pore size, smoother surface, higher value of stiffness but lower ultimate tensile load compared to membranes made from 500 µm powder. C2C12 cell culture results indicate that the membrane supports cell growth and differentiation. Thus, this novel membrane generation method holds great promise for tissue engineering.

  17. Application of ICH Q9 Quality Risk Management Tools for Advanced Development of Hot Melt Coated Multiparticulate Systems.

    PubMed

    Stocker, Elena; Becker, Karin; Hate, Siddhi; Hohl, Roland; Schiemenz, Wolfgang; Sacher, Stephan; Zimmer, Andreas; Salar-Behzadi, Sharareh

    2017-01-01

    This study aimed to apply quality risk management based on the The International Conference on Harmonisation guideline Q9 for the early development stage of hot melt coated multiparticulate systems for oral administration. N-acetylcysteine crystals were coated with a formulation composing tripalmitin and polysorbate 65. The critical quality attributes (CQAs) were initially prioritized using failure mode and effects analysis. The CQAs of the coated material were defined as particle size, taste-masking efficiency, and immediate release profile. The hot melt coated process was characterized via a flowchart, based on the identified potential critical process parameters (CPPs) and their impact on the CQAs. These CPPs were prioritized using a process failure mode, effects, and criticality analysis and their critical impact on the CQAs was experimentally confirmed using a statistical design of experiments. Spray rate, atomization air pressure, and air flow rate were identified as CPPs. Coating amount and content of polysorbate 65 in the coating formulation were identified as critical material attributes. A hazard and critical control points analysis was applied to define control strategies at the critical process points. A fault tree analysis evaluated causes for potential process failures. We successfully demonstrated that a standardized quality risk management approach optimizes the product development sustainability and supports the regulatory aspects.

  18. Clinopyroxene megacrysts from Enmelen melanephelinitic volcanoes (Chukchi Peninsula, Russia): application to composition and evolution of mantle melts

    NASA Astrophysics Data System (ADS)

    Akinin, Vyacheslav V.; Sobolev, Alexander V.; Ntaflos, Theodoros; Richter, Wolfram.

    2005-08-01

    Clinopyroxene megacrysts from young melanephelinitic lavas were divided into Cr-rich and Cr-poor suites. Sr, Nd, and Pb isotopic ratios of leached megacrysts and host lava are indistinguishable from each other and indicate a depleted source. Host lavas do not display chemical evidence for significant fractional crystallization, which is required to explain the compositional range of the megacrysts. This rules out a simple cognate genetic relationship between the two, and strictly defines megacrysts as xenocrysts. The well-defined correlations of trace elements with the Mg-numbers in the megacrysts are interpreted as the result of extensive fractional/equilibrium crystallization of magma over a large temperature range at near isobaric condition in the upper mantle. Trace element variations in megacrysts are consistent with fractional crystallization of clinopyroxene alone for the Cr-rich suite, and clinopyroxene + garnet for the Cr-poor suite from at least two bathes of related melts. Megacrysts parent magma might represent mantle melts, which were never erupted in their initial composition.

  19. Soft X-ray microscope with nanometer spatial resolution and its applications

    NASA Astrophysics Data System (ADS)

    Wachulak, P. W.; Torrisi, A.; Bartnik, A.; Wegrzynski, L.; Fok, T.; Patron, Z.; Fiedorowicz, H.

    2016-12-01

    A compact size microscope based on nitrogen double stream gas puff target soft X-ray source, which emits radiation in water-window spectral range at the wavelength of λ = 2.88 nm is presented. The microscope employs ellipsoidal grazing incidence condenser mirror for sample illumination and silicon nitride Fresnel zone plate objective for object magnification and imaging. The microscope is capable of capturing water-window images of objects with 60 nm spatial resolution and exposure time as low as a few seconds. Details about the microscopy system as well as some examples of different applications from various fields of science, are presented and discussed.

  20. The resolution capability of an irregularly sampled dataset: With application to Geosat altimeter data

    NASA Technical Reports Server (NTRS)

    Chelton, Dudley B.; Schlax, Michael G.

    1994-01-01

    A formalism is presented for determining the wavenumber-frequency transfer function associated with an irregularly sampled multidimensional dataset. This transfer function reveals the filtering characteristics and aliasing patterns inherent in the sample design. In combination with information about the spectral characteristics of the signal, the transfer function can be used to quantify the spatial and temporal resolution capability of the dataset. Application of the method to idealized Geosat altimeter data (i.e., neglecting measurement errors and data dropouts) concludes that the Geosat orbit configuration is capable of resolving scales of about 3 deg in latitude and longitude by about 30 days.

  1. Applications of high resolution ICP-AES in the nuclear industry

    SciTech Connect

    Johnson, S.G.; Giglio, J.J.; Goodall, P.S.; Cummings, D.G.

    1998-07-01

    Application of high resolution ICP-AES to selected problems of importance in the nuclear industry is a growing field. The advantages in sample preparation time, waste minimization and equipment cost are considerable. Two examples of these advantages are presented in this paper, burnup analysis of spent fuel and analysis of major uranium isotopes. The determination of burnup, an indicator of fuel cycle efficiency, has been accomplished by the determination of {sup 139}La by high resolution inductively coupled plasma atomic emission spectroscopy (HR-ICP-AES). Solutions of digested samples of reactor fuel rods were introduced into a shielded glovebox housing an inductively coupled plasma (ICP) and the resulting atomic emission transmitted to a high resolution spectrometer by a 31 meter fiber optic bundle. Total and isotopic U determination by thermal ionization mass spectrometry (TIMS) is presented to allow for the calculation of burnup for the samples. This method of burnup determination reduces the time, material, sample handling and waste generated associated with typical burnup determinations which require separation of lanthanum from the other fission products with high specific activities. Work concerning an alternative burnup indicator, {sup 236}U, is also presented for comparison. The determination of {sup 235}U:{sup 238}U isotope ratios in U-Zr fuel alloys is also presented to demonstrate the versatility of HR-ICP-AES.

  2. Ground-based high resolution Fourier transform spectrometer and its application in Beijing

    NASA Astrophysics Data System (ADS)

    Fan, Dongdong

    2013-10-01

    The B3M-FTS instrument, inherited from ACE-FTS and PARIS, is built by Canadian ABB and Beijing Vision Sky Aerospace Co., Ltd. The B3M is a complete stand-alone spectrometer designed to operate from the ground in moderate environment. It can acquire atmospheric spectra with the Sun as back illumination. This instrument is an adapted version of the classical Michelson interferometer using an optimized optical layout, and it is a high-resolution infrared Fourier transform spectrometer operating in the 750 to 4100cm-1 spectral range. In this paper, the instrument concept of a compact, portable, high-resolution Fourier transform spectrometer is introduced. Some test results of the instrument such as ILS and SNR are presented, and the spectral resolution of 0.028cm-1 @ 750cm-1 and SNR over 100:1 are achieved. Sample atmospheric absorption spectra and corresponding retrieval results measured by the FTS are given. The B3M-FTS, with its high performance, provides the capability to monitor the atmospheric composition changes by measuring the atmospheric absorption spectra of solar radiance. Lots of measurements have been acquired at the Olympics atmospheric observation super-station. Up to now, the VMRs of near 10 trace gases have been retrieved. The success of atmospheric composition profile retrieval using the FTS measurements makes the further application of FTS type payload possible in China.

  3. Theory of a continuous stripe melting transition in a two-dimensional metal: a possible application to cuprate superconductors.

    PubMed

    Mross, David F; Senthil, T

    2012-06-29

    We construct a theory of continuous stripe melting quantum phase transitions in two-dimensional metals and the associated Fermi surface reconstruction. Such phase transitions are strongly coupled but yet theoretically tractable in situations where the stripe ordering is destroyed by proliferating doubled dislocations of the charge stripe order. The resulting non-Landau quantum critical point has strong stripe fluctuations which we show decouple dynamically from the Fermi surface even though static stripe ordering reconstructs the Fermi surface. We discuss connections to various stripe phenomena in the cuprates. We point out several puzzling aspects of old experimental results [G. Aeppli et al., Science 278, 1432 (1997)] on singular stripe fluctuations in the cuprates, and provide a possible explanation within our theory. These results may thus have been the first observation of non-Landau quantum criticality in an experiment.

  4. Melt-spun shaped fibers with enhanced surface effects: fiber fabrication, characterization and application to woven scaffolds.

    PubMed

    Park, S J; Lee, B-K; Na, M H; Kim, D S

    2013-08-01

    Scaffolds with a high surface-area-to-volume ratio (SA:V) are advantageous with regard to the attachment and proliferation of cells in the field of tissue engineering. This paper reports on the development of novel melt-spun fibers with a high SA:V, which enhanced the surface effects of a fiber-based scaffold while maintaining its mechanical strength. The cross-section of the fibers was altered to a non-circular shape, producing a higher SA:V for a similar cross-sectional area. To obtain fibers with non-circular cross-sectional shape, or shaped fibers, three different types of metal spinnerets were fabricated for the melt-spinning process, each with circular, triangular or cruciform capillaries, using deep X-ray lithography followed by nickel electroforming. Using these spinnerets, circular and shaped fibers were manufactured with biodegradable polyester, polycaprolactone. The SA:V increase in the shaped fibers was experimentally investigated under different processing conditions. Tensile tests on the fibers and indentation tests on the woven fiber scaffolds were performed. The tested fibers and scaffolds exhibited similar mechanical characteristics, due to the similar cross-sectional area of the fibers. The degradation of the shaped fibers was notably faster than that of circular fibers, because of the enlarged surface area of the shaped fibers. The woven scaffolds composed of the shaped fibers significantly increased the proliferation of human osteosarcoma MG63 cells. This approach to increase the SA:V in shaped fibers could be useful for the fabrication of programmable, biodegradable fiber-based scaffolds in tissue engineering.

  5. Processing of large YBa2 Cu3Ox domains for levitation applications by a Nd1+x Ba2-x Cu3 Oy-Seeded melt-growth technique

    NASA Astrophysics Data System (ADS)

    Todt, V. R.; Sengupta, S.; Shi, Donglu; Sahm, P. R.; McGinn, P. J.; Poeppel, R. B.; Hull, J. R.

    1994-11-01

    YBa2Cu3Ox domains for levitation applications have been produced by a seeding technology that includes Nd1+x Ba2-x Cu3Oy seeds and melt-processing technologies such as conventional melt-textured growth, melt-texturing with PtO2 and Y2BaCuO5 additions, and the new solid-liquid-melt-growth technology. Large domains (˜20 mm) with high levitation forces (F1 up to 8.2 N) have been produced. The reproducibility of the results is good, and the capability of producing a large number of pellets in a single batch indicates good potential for the production of large amounts of this material.

  6. Viking High-Resolution Topography and Mars '01 Site Selection: Application to the White Rock Area

    NASA Astrophysics Data System (ADS)

    Tanaka, K. L.; Kirk, Randolph L.; Mackinnon, D. J.; Howington-Kraus, E.

    1999-06-01

    Definition of the local topography of the Mars '01 Lander site is crucial for assessment of lander safety and rover trafficability. According to Golombek et al., steep surface slopes may (1) cause retro-rockets to be fired too early or late for a safe landing, (2) the landing site slope needs to be < 1deg to ensure lander stability, and (3) a nearly level site is better for power generation of both the lander and the rover and for rover trafficability. Presently available datasets are largely inadequate to determine surface slope at scales pertinent to landing-site issues. Ideally, a topographic model of the entire landing site at meter-scale resolution would permit the best assessment of the pertinent topographic issues. MOLA data, while providing highly accurate vertical measurements, are inadequate to address slopes along paths of less than several hundred meters, because of along-track data spacings of hundreds of meters and horizontal errors in positioning of 500 to 2000 m. The capability to produce stereotopography from MOC image pairs is not yet in hand, nor can we necessarily expect a suitable number of stereo image pairs to be acquired. However, for a limited number of sites, high-resolution Viking stereo imaging is available at tens of meters horizontal resolution, capable of covering landing-ellipse sized areas. Although we would not necessarily suggest that the chosen Mars '01 Lander site should be located where good Viking stereotopography is available, an assessment of typical surface slopes at these scales for a range of surface types may be quite valuable in landing-site selection. Thus this study has a two-fold application: (1) to support the proposal of White Rock as a candidate Mars '01 Lander site, and (2) to evaluate how Viking high resolution stereotopography may be of value in the overall Mars '01 Lander site selection process.

  7. Enhancing resolution properties of array antennas via field extrapolation: application to MIMO systems

    NASA Astrophysics Data System (ADS)

    Reggiannini, Ruggero

    2015-12-01

    This paper is concerned with spatial properties of linear arrays of antennas spaced less than half wavelength. Possible applications are in multiple-input multiple-output (MIMO) wireless links for the purpose of increasing the spatial multiplexing gain in a scattering environment, as well as in other areas such as sonar and radar. With reference to a receiving array, we show that knowledge of the received field can be extrapolated beyond the actual array size by exploiting the finiteness of the interval of real directions from which the field components impinge on the array. This property permits to increase the performance of the array in terms of angular resolution. A simple signal processing technique is proposed allowing formation of a set of beams capable to cover uniformly the entire horizon with an angular resolution better than that achievable by a classical uniform-weighing half-wavelength-spaced linear array. Results are also applicable to active arrays. As the above approach leads to arrays operating in super-directive regime, we discuss all related critical aspects, such as sensitivity to external and internal noises and to array imperfections, and bandwidth, so as to identify the basic design criteria ensuring the array feasibility.

  8. High-resolution infrared detector and its electronic unit for space application

    NASA Astrophysics Data System (ADS)

    Meftah, M.; Montmessin, F.; Korablev, O.; Trokhimovsky, A.; Poiet, G.; Bel, J.-B.

    2015-05-01

    High-resolution infrared detector is used extensively for military and civilian purposes. Military applications include target acquisition, surveillance, night vision, and tracking. Civilian applications include, among others, scientific observations. For our space systems, we want to use the products developed by SOFRADIR Company. Thus, we have developed a space electronic unit that is used to control the high-resolution SCORPIO-MW infrared detector, which has a format of 640×512 pixels with 15μm×15μm pixel pitch. The detector within microelectronics based on infrared mid-wave (MW) complementary metal oxide semiconductors (CMOS) uses a micro-cooler in order to keep its temperature around 100 K. The standard wavelength range (3 to 5μm) is adapted to the 2.2 to 4.3μm wavelength range thanks to adaptation of the optical interface of the detector and with an antireflection coating. With our electronic system, we can acquire 3 images per second. To increase the signal to noise ratio, we have the opportunity to make a summation of 15 frames per image. Through this article, we will describe the space electronic system that we have developed in order to achieve space observations (e.g. Atmospheric Chemistry Suite package for ExoMars Trace Gas Orbiter).

  9. Application of multivariate curve resolution alternating least squares (MCR-ALS) to remote sensing hyperspectral imaging.

    PubMed

    Zhang, Xin; Tauler, Romà

    2013-01-31

    The application of the MCR-ALS method is demonstrated on two simulated remote sensing spectroscopic images and on one experimental reference remote sensing spectroscopic image obtained by the Airborn Visible/Infrared Imaging Spectrometer (AVIRIS). By application of MCR-ALS, the spectra signatures of the pure constituents present in the image and their concentration distribution at a pixel level are estimated. Results obtained by MCR-ALS are compared to those obtained by other methods frequently used in the remote sensing spectroscopic imaging field like VCA and MVSA. In the case of the analysis of the experimental data set, the resolved pure spectra signatures were compared to reference spectra from USGS library for their identification. In all cases, results were also evaluated for the presence of rotational ambiguities using the MCR-BANDS method. The obtained results confirmed that the MCR-ALS method can be successfully used for remote sensing hyperspectral image resolution purposes. However, the amount of rotation ambiguity still present in the solutions obtained by this and other resolution methods (like VCA or MVSA) can still be large and it should be evaluated with care, trying to reduce its effects by selecting the more appropriate constraints. Only in this way it is possible to increase the reliability of the solutions provided by these methods and decrease the uncertainties associated to their use.

  10. Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations

    PubMed Central

    2014-01-01

    Background The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma. Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response. Therefore, the precise identification of the underlying somatic mutations is essential. Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations. Methods Samples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples. DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction. BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC). All mutations were independently reassessed by Sanger sequencing. Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively). Results There was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%). All mutations down to 6.6% allele frequency could be detected with 100% specificity. In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity. The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib. NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity. IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM. Conclusion Therefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients. PMID:24410877

  11. Comparison of multiple genes and 16S-23S rRNA intergenic space region for their capacity in high resolution melt curve analysis to differentiate Mycoplasma gallisepticum vaccine strain ts-11 from field strains.

    PubMed

    Ghorashi, Seyed A; Bradbury, Janet M; Ferguson-Noel, Naola M; Noormohammadi, Amir H

    2013-12-27

    Mycoplasma gallisepticum (MG) is an important avian pathogen causing significant economic losses in the global poultry industry. In an attempt to compare and evaluate existing genotyping methods for differentiation of MG strains/isolates, high resolution melt (HRM) curve analysis was applied to 5 different PCR methods targeting vlhA, pvpA, gapA, mgc2 genes and 16S-23S rRNA intergenic space region (IGSR). To assess the discriminatory power of PCR-HRM of examined genes and IGSR, MG strains ts-11, F, 6/85 and S6, and, initially, 8 field isolates were tested. All MG strains/isolates were differentiated using PCR-HRM curve analysis and genotype confidence percentage (GCP) values of vlhA and pvpA genes, while only 0, 3 and 4 out of 12 MG strains/isolates were differentiated using gapA, mgc2 genes and IGSR, respectively. The HRM curve analysis of vlhA and pvpA genes was found to be highly correlated with the genetic diversity of the targeted genes confirmed by sequence analysis of amplicons generated from MG strains. The potential of the vlhA and pvpA genes was also demonstrated for genotyping of 12 additional MG strains from Europe and the USA. Results from this study provide a direct comparison between genes previously used in sequencing-based genotyping methods for MG strain identification and highlight the usefulness of vlhA and pvpA HRM curve analyses as rapid and reliable tools specially for diagnosis and differentiation of MG strains used here.

  12. Selective comprehensive multidimensional separation for resolution enhancement in high performance liquid chromatography. Part II: applications.

    PubMed

    Groskreutz, Stephen R; Swenson, Michael M; Secor, Laura B; Stoll, Dwight R

    2012-03-09

    In this second paper of a two-part series, we demonstrate the utility of an approach to enhancing the resolution of select portions of conventional 1D-LC separations, which we refer to as selective comprehensive two-dimensional HPLC (sLC × LC), in three quite different example applications. In the first paper of the series we described the principles of this approach, which breaks the long-standing link in online multi-dimensional chromatography between the timescales of sampling the first dimension (¹D) separation and the separation of fractions of ¹D effluent in the second dimension. In the first example, the power of the sLC × LC approach to significantly reduce the analysis time and method development effort is demonstrated by selectively enhancing the resolution of critical pairs of peaks that are unresolved by a one-dimensional separation (1D-LC) alone. Transfer and subsequent ²D separations of multiple fractions of a particular ¹D peak produces a two-dimensional chromatogram that reveals the coordinates of the peaks in the 2D separation space. The added time dimension of sLC × LC chromatograms also facilitates the application of sophisticated chemometric curve resolution algorithms to further resolve peaks that are otherwise chromatographically unresolved. This is demonstrated in this work by the targeted analysis of phenytoin in urban wastewater effluent using UV diode array detection. Quantitation by both standard addition and external calibration methods yielded results that were not statistically different from 2D-LC/MS/MS analysis of the same samples. Next, we demonstrate the utility of sLC × LC for reducing ion suppression due to matrix effects in electrospray ionization mass spectrometry through the analysis of cocaine in urban wastewater effluent. Finally, we explore the flexibility of the approach in its application to two select regions of a single ¹D separation of triclosan and cocaine. The diversity of these applications demonstrates the

  13. Applications of High Resolution Laser: Induced Breakdown Spectroscopy for Environmental and Biological Samples

    NASA Astrophysics Data System (ADS)

    Martin, Madhavi Z.; Labbe, Nicole; Wagner, Rebekah J.

    This chapter details the application of LIBS in a number of environmental areas of research such as carbon sequestration and climate change. LIBS has also been shown to be useful in other high resolution environmental applications for example, elemental mapping and detection of metals in plant materials. LIBS has also been used in phytoremediation applications. Other biological research involves a detailed understanding of wood chemistry response to precipitation variations and also to forest fires. A cross-section of Mountain pine (pinceae Pinus pungen Lamb.) was scanned using a translational stage to determine the differences in the chemical features both before and after a fire event. Consequently, by monitoring the elemental composition pattern of a tree and by looking for abrupt changes, one can reconstruct the disturbance history of a tree and a forest. Lastly we have shown that multivariate analysis of the LIBS data is necessary to standardize the analysis and correlate to other standard laboratory techniques. LIBS along with multivariate statistical analysis makes it a very powerful technology that can be transferred from laboratory to field applications with ease.

  14. Applications of High Resolution Laser Induced Breakdown Spectroscopy for Environmental and Biological Samples

    SciTech Connect

    Martin, Madhavi Z; Labbe, Nicole; Wagner, Rebekah J.

    2013-01-01

    This chapter details the application of LIBS in a number of environmental areas of research such as carbon sequestration and climate change. LIBS has also been shown to be useful in other high resolution environmental applications for example, elemental mapping and detection of metals in plant materials. LIBS has also been used in phytoremediation applications. Other biological research involves a detailed understanding of wood chemistry response to precipitation variations and also to forest fires. A cross-section of Mountain pine (pinceae Pinus pungen Lamb.) was scanned using a translational stage to determine the differences in the chemical features both before and after a fire event. Consequently, by monitoring the elemental composition pattern of a tree and by looking for abrupt changes, one can reconstruct the disturbance history of a tree and a forest. Lastly we have shown that multivariate analysis of the LIBS data is necessary to standardize the analysis and correlate to other standard laboratory techniques. LIBS along with multivariate statistical analysis makes it a very powerful technology that can be transferred from laboratory to field applications with ease.

  15. Development of a high resolution voxelised head phantom for medical physics applications.

    PubMed

    Giacometti, V; Guatelli, S; Bazalova-Carter, M; Rosenfeld, A B; Schulte, R W

    2017-01-01

    Computational anthropomorphic phantoms have become an important investigation tool for medical imaging and dosimetry for radiotherapy and radiation protection. The development of computational phantoms with realistic anatomical features contribute significantly to the development of novel methods in medical physics. For many applications, it is desirable that such computational phantoms have a real-world physical counterpart in order to verify the obtained results. In this work, we report the development of a voxelised phantom, the HIGH_RES_HEAD, modelling a paediatric head based on the commercial phantom 715-HN (CIRS). HIGH_RES_HEAD is unique for its anatomical details and high spatial resolution (0.18×0.18mm(2) pixel size). The development of such a phantom was required to investigate the performance of a new proton computed tomography (pCT) system, in terms of detector technology and image reconstruction algorithms. The HIGH_RES_HEAD was used in an ad-hoc Geant4 simulation modelling the pCT system. The simulation application was previously validated with respect to experimental results. When compared to a standard spatial resolution voxelised phantom of the same paediatric head, it was shown that in pCT reconstruction studies, the use of the HIGH_RES_HEAD translates into a reduction from 2% to 0.7% of the average relative stopping power difference between experimental and simulated results thus improving the overall quality of the head phantom simulation. The HIGH_RES_HEAD can also be used for other medical physics applications such as treatment planning studies. A second version of the voxelised phantom was created that contains a prototypic base of skull tumour and surrounding organs at risk.

  16. Application of polyelectrolyte theories for analysis of DNA melting in the presence of Na+ and Mg2+ ions.

    PubMed Central

    Korolev, N; Lyubartsev, A P; Nordenskiöld, L

    1998-01-01

    Numerical calculations, using Poisson-Boltzmann (PB) and counterion condensation (CC) polyelectrolyte theories, of the electrostatic free energy difference, DeltaGel, between single-stranded (coil) and double-helical DNA have been performed for solutions of NaDNA + NaCl with and without added MgCl2. Calculations have been made for conditions relevant to systems where experimental values of helix coil transition temperature (Tm) and other thermodynamic quantities have been measured. Comparison with experimental data has been possible by invoking values of Tm for solutions containing NaCl salt only. Resulting theoretical values of enthalpy, entropy, and heat capacity (for NaCl salt-containing solutions) and of Tm as a function of NaCl concentration in NaCl + MgCl2 solutions have thus been obtained. Qualitative and, to a large extent, quantitative reproduction of the experimental Tm, DeltaHm, DeltaSm, and DeltaCp values have been found from the results of polyelectrolyte theories. However, the quantitative resemblance of experimental data is considerably better for PB theory as compared to the CC model. Furthermore, some rather implausible qualitative conclusions are obtained within the CC results for DNA melting in NaCl + MgCl2 solutions. Our results argue in favor of the Poisson-Boltzmann theory, as compared to the counterion condensation theory. PMID:9826624

  17. Surface chemistry of Ti6Al4V components fabricated using selective laser melting for biomedical applications.

    PubMed

    Vaithilingam, Jayasheelan; Prina, Elisabetta; Goodridge, Ruth D; Hague, Richard J M; Edmondson, Steve; Rose, Felicity R A J; Christie, Steven D R

    2016-10-01

    Selective laser melting (SLM) has previously been shown to be a viable method for fabricating biomedical implants; however, the surface chemistry of SLM fabricated parts is poorly understood. In this study, X-ray photoelectron spectroscopy (XPS) was used to determine the surface chemistries of (a) SLM as-fabricated (SLM-AF) Ti6Al4V and (b) SLM fabricated and mechanically polished (SLM-MP) Ti6Al4V samples and compared with (c) traditionally manufactured (forged) and mechanically polished Ti6Al4V samples. The SLM-AF surface was observed to be porous with an average surface roughness (Ra) of 17.6±3.7μm. The surface chemistry of the SLM-AF was significantly different to the FGD-MP surface with respect to elemental distribution and their existence on the outermost surface. Sintered particles on the SLM-AF surface were observed to affect depth profiling of the sample due to a shadowing effect during argon ion sputtering. Surface heterogeneity was observed for all three surfaces; however, vanadium was witnessed only on the mechanically polished (SLM-MP and FGD-MP) surfaces. The direct and indirect 3T3 cell cytotoxicity studies revealed that the cells were viable on the SLM fabricated Ti6Al4V parts. The varied surface chemistry of the SLM-AF and SLM-MP did not influence the cell behaviour.

  18. Thermodynamics of gas-metal-slag equilibria for applications in in situ and ex situ vitrification melts

    SciTech Connect

    Miller, R.L.; Reimann, G.A.

    1993-05-01

    An equilibrium thermodynamic model for melting mixed waste was evaluated using the STEPSOL computer code. STEPSOL uses free energy minimization techniques to predict equilibrium composition from input species and user selected species in the output. The model assumes equilibrium between gas, slag, and metallic phases. Input for the model was developed using compositional data from Pit 9 of the Subsurface Disposal Area at the Idaho National Engineering Laboratory. Thermodynamic data were primarily from compilations published by the US Government. The results of model evaluation indicate that the amount of plutonium chloride or plutonium oxyhydroxide that would be evaporated into the vapor phase would be minor. Relatively more uranium chloride and uranium oxyhydroxide would be vaporized. However, a hazards analysis was not part of the present task. Minor amounts of plutonium and uranium would be reduced to the metallic state, but these amounts should alloy with the iron-chromium-nickel metallic phase. The vast majority of the plutonium and uranium are in the slag phase as oxides. Results of the calculations show that silica and silicates dominate the products and that the system is very reducing. The major gases are carbon monoxide and hydrogen, with lesser amounts of carbon dioxide and water. High vapor pressure metals are considered but were not analyzed using STEPSOL. STEPSOL does not make predictions of distribution of species between phases.

  19. Stabilizing Crystal Oscillators With Melting Metals

    NASA Technical Reports Server (NTRS)

    Stephens, J. B.; Miller, C. G.

    1984-01-01

    Heat of fusion provides extended period of constant temperature and frequency. Crystal surrounded by metal in spherical container. As outside temperature rises to melting point of metal, metal starts to liquefy; but temperature stays at melting point until no solid metal remains. Potential terrestrial applications include low-power environmental telemetering transmitters and instrumentation transmitters for industrial processes.

  20. Application and correlation of nano resolution microscopy techniques to viral protein localization

    NASA Astrophysics Data System (ADS)

    Hodges, Jeffery Allen

    This dissertation is primarily focused on the application of super-resolution microscopy techniques to localization of viral proteins within envelope viruses. Advances in optical super-resolution microscopy techniques have enabled scientists to observe phenomena much smaller than the Abbe diffraction limit by stochastically limiting the number of molecules excited at a given instance and localizing their positions one at a time. Additionally, methods such as Atomic Force Microscopy (AFM) allow scientists to measure the topological features and material properties of samples through contact with a force probe. This dissertation describes the application of these two techniques to virology in order to localize internal viral proteins of enveloped virions, and measure their effect on the elastic properties of the virion. By utilizing super-resolution microscopy techniques such as Fluorescent Photo-Activated Localization Microscopy (fPALM) on virions, which have had their surface glycoproteins labeled with a photo-switchable label, the viral envelope may be accurately recovered. This dissertation describes the development and application of this technique as it applies to envelope recovery of Vesicular Stomatitis Virus (VSV) and Human Immunodeficiency Virus-1 (HIV-1). By fluorescently labeling proteins, which are internal to each of these viruses, I have been able to localize a variety of viral proteins within their recovered envelopes. This is done without significant damage to the virion, making this method a highly effective in vivo technique. In the case of VSV, an asymmetric localization along the central axis towards the blunt 5' end was found to exist for both the polymerase and phosphoproteins. These have been determined to occupy a region in the central cavity of ˜57 +/- 12 nm on the 5' end. This inhomogeneity of the underlying proteins such an asymmetry would predict that the Young's modulus would vary along the central axis of the virion. This dissertation

  1. Development and Applications of a New, High-Resolution, Operational MISR Aerosol Product

    NASA Astrophysics Data System (ADS)

    Garay, M. J.; Diner, D. J.; Kalashnikova, O.

    2014-12-01

    Since early 2000, the Multi-angle Imaging SpectroRadiometer (MISR) instrument on NASA's Terra satellite has been providing aerosol optical depth (AOD) and particle property retrievals at 17.6 km spatial resolution. Capitalizing on the capabilities provided by multi-angle viewing, the operational MISR algorithm performs well, with about 75% of MISR AOD retrievals falling within 0.05 or 20% × AOD of the paired validation data from the ground-based Aerosol Robotic Network (AERONET), and is able to distinguish aerosol particles by size and sphericity, over both land and water. These attributes enable a variety of applications, including aerosol transport model validation and global air quality assessment. Motivated by the adverse impacts of aerosols on human health at the local level, and taking advantage of computational speed advances that have occurred since the launch of Terra, we have implemented an operational MISR aerosol product with 4.4 km spatial resolution that maintains, and sometimes improves upon, the quality of the 17.6 km resolution product. We will describe the performance of this product relative to the heritage 17.6 km product, the global AERONET validation network, and high spatial density AERONET-DRAGON sites. Other changes that simplify product content, and make working with the data much easier for users, will also be discussed. Examples of how the new product demonstrates finer spatial variability of aerosol fields than previously retrieved, and ways this new dataset can be used for studies of local aerosol effects, will be shown.

  2. Flood and Landslide Applications of High Time Resolution Satellite Rain Products

    NASA Technical Reports Server (NTRS)

    Adler, Robert F.; Hong, Yang; Huffman, George J.

    2006-01-01

    Experimental, potentially real-time systems to detect floods and landslides related to heavy rain events are described. A key basis for these applications is high time resolution satellite rainfall analyses. Rainfall is the primary cause for devastating floods across the world. However, in many countries, satellite-based precipitation estimation may be the best source of rainfall data due to insufficient ground networks and absence of data sharing along many trans-boundary river basins. Remotely sensed precipitation from the NASA's TRMM Multi-satellite Precipitation Analysis (TMPA) operational system (near real-time precipitation at a spatial-temporal resolution of 3 hours and 0.25deg x 0.25deg) is used to monitor extreme precipitation events. Then these data are ingested into a macro-scale hydrological model which is parameterized using spatially distributed elevation, soil and land cover datasets available globally from satellite remote sensing. Preliminary flood results appear reasonable in terms of location and frequency of events, with implementation on a quasi-global basis underway. With the availability of satellite rainfall analyses at fine time resolution, it has also become possible to assess landslide risk on a near-global basis. Early results show that landslide occurrence is closely associated with the spatial patterns and temporal distribution of TRMM rainfall characteristics. Particularly, the number of landslides triggered by rainfall is related to rainfall climatology, antecedent rainfall accumulation, and intensity-duration of rainstorms. For the purpose of prediction, an empirical TMPA-based rainfall intensity-duration threshold is developed and shown to have skill in determining potential areas of landslides. These experimental findings, in combination with landslide surface susceptibility information based on satellite-based land surface information, form a starting point towards a potential operational landslide monitoring/warning system

  3. Methods for Melting Temperature Calculation

    NASA Astrophysics Data System (ADS)

    Hong, Qi-Jun

    Melting temperature calculation has important applications in the theoretical study of phase diagrams and computational materials screenings. In this thesis, we present two new methods, i.e., the improved Widom's particle insertion method and the small-cell coexistence method, which we developed in order to capture melting temperatures both accurately and quickly. We propose a scheme that drastically improves the efficiency of Widom's particle insertion method by efficiently sampling cavities while calculating the integrals providing the chemical potentials of a physical system. This idea enables us to calculate chemical potentials of liquids directly from first-principles without the help of any reference system, which is necessary in the commonly used thermodynamic integration method. As an example, we apply our scheme, combined with the density functional formalism, to the calculation of the chemical potential of liquid copper. The calculated chemical potential is further used to locate the melting temperature. The calculated results closely agree with experiments. We propose the small-cell coexistence method based on the statistical analysis of small-size coexistence MD simulations. It eliminates the risk of a metastable superheated solid in the fast-heating method, while also significantly reducing the computer cost relative to the traditional large-scale coexistence method. Using empirical potentials, we validate the method and systematically study the finite-size effect on the calculated melting points. The method converges to the exact result in the limit of a large system size. An accuracy within 100 K in melting temperature is usually achieved when the simulation contains more than 100 atoms. DFT examples of Tantalum, high-pressure Sodium, and ionic material NaCl are shown to demonstrate the accuracy and flexibility of the method in its practical applications. The method serves as a promising approach for large-scale automated material screening in which

  4. Rate-gyro-integral constraint for ambiguity resolution in GNSS attitude determination applications.

    PubMed

    Zhu, Jiancheng; Li, Tao; Wang, Jinling; Hu, Xiaoping; Wu, Meiping

    2013-06-21

    In the field of Global Navigation Satellite System (GNSS) attitude determination, the constraints usually play a critical role in resolving the unknown ambiguities quickly and correctly. Many constraints such as the baseline length, the geometry of multi-baselines and the horizontal attitude angles have been used extensively to improve the performance of ambiguity resolution. In the GNSS/Inertial Navigation System (INS) integrated attitude determination systems using low grade Inertial Measurement Unit (IMU), the initial heading parameters of the vehicle are usually worked out by the GNSS subsystem instead of by the IMU sensors independently. However, when a rotation occurs, the angle at which vehicle has turned within a short time span can be measured accurately by the IMU. This measurement will be treated as a constraint, namely the rate-gyro-integral constraint, which can aid the GNSS ambiguity resolution. We will use this constraint to filter the candidates in the ambiguity search stage. The ambiguity search space shrinks significantly with this constraint imposed during the rotation, thus it is helpful to speeding up the initialization of attitude parameters under dynamic circumstances. This paper will only study the applications of this new constraint to land vehicles. The impacts of measurement errors on the effect of this new constraint will be assessed for different grades of IMU and current average precision level of GNSS receivers. Simulations and experiments in urban areas have demonstrated the validity and efficacy of the new constraint in aiding GNSS attitude determinations.

  5. The online application of binding condition B in native and non-native pronoun resolution

    PubMed Central

    Patterson, Clare; Trompelt, Helena; Felser, Claudia

    2014-01-01

    Previous research has shown that anaphor resolution in a non-native language may be more vulnerable to interference from structurally inappropriate antecedents compared to native anaphor resolution. To test whether previous findings on reflexive anaphors generalize to non-reflexive pronouns, we carried out an eye-movement monitoring study investigating the application of binding condition B during native and non-native sentence processing. In two online reading experiments we examined when during processing local and/or non-local antecedents for pronouns were considered in different types of syntactic environment. Our results demonstrate that both native English speakers and native German-speaking learners of English showed online sensitivity to binding condition B in that they did not consider syntactically inappropriate antecedents. For pronouns thought to be exempt from condition B (so-called “short-distance pronouns”), the native readers showed a weak preference for the local antecedent during processing. The non-native readers, on the other hand, showed a preference for the matrix subject even where local coreference was permitted, and despite demonstrating awareness of short-distance pronouns' referential ambiguity in a complementary offline task. This indicates that non-native comprehenders are less sensitive during processing to structural cues that render pronouns exempt from condition B, and prefer to link a pronoun to a salient subject antecedent instead. PMID:24611060

  6. The online application of binding condition B in native and non-native pronoun resolution.

    PubMed

    Patterson, Clare; Trompelt, Helena; Felser, Claudia

    2014-01-01

    Previous research has shown that anaphor resolution in a non-native language may be more vulnerable to interference from structurally inappropriate antecedents compared to native anaphor resolution. To test whether previous findings on reflexive anaphors generalize to non-reflexive pronouns, we carried out an eye-movement monitoring study investigating the application of binding condition B during native and non-native sentence processing. In two online reading experiments we examined when during processing local and/or non-local antecedents for pronouns were considered in different types of syntactic environment. Our results demonstrate that both native English speakers and native German-speaking learners of English showed online sensitivity to binding condition B in that they did not consider syntactically inappropriate antecedents. For pronouns thought to be exempt from condition B (so-called "short-distance pronouns"), the native readers showed a weak preference for the local antecedent during processing. The non-native readers, on the other hand, showed a preference for the matrix subject even where local coreference was permitted, and despite demonstrating awareness of short-distance pronouns' referential ambiguity in a complementary offline task. This indicates that non-native comprehenders are less sensitive during processing to structural cues that render pronouns exempt from condition B, and prefer to link a pronoun to a salient subject antecedent instead.

  7. High-Resolution Microscopy-Coil MR Imaging of Skin Tumors: Techniques and Novel Clinical Applications.

    PubMed

    Budak, Matthew J; Weir-McCall, Jonathan R; Yeap, Phey M; White, Richard D; Waugh, Shelley A; Sudarshan, Thiru A P; Zealley, Ian A

    2015-01-01

    High-resolution magnetic resonance (MR) imaging performed with a microscopy coil is a robust radiologic tool for the evaluation of skin lesions. Microscopy-coil MR imaging uses a small surface coil and a 1.5-T or higher MR imaging system. Simple T1- and T2-weighted imaging protocols can be implemented to yield high-quality, high-spatial-resolution images that provide an excellent depiction of dermal anatomy. The primary application of microscopy-coil MR imaging is to delineate the deep margins of skin tumors, thereby providing a preoperative road map for dermatologic surgeons. This information is particularly useful for surgeons who perform Mohs micrographic surgery and in cases of nasofacial neoplasms, where the underlying anatomy is complex. Basal cell carcinoma is the most common nonmelanocytic skin tumor and has a predilection to manifest on the face, where it can be challenging to achieve complete surgical excision while preserving the cosmetic dignity of the patient. Microscopy-coil MR imaging provides dermatologic surgeons with valuable preoperative anatomic information that is not available at conventional clinical examination.

  8. CALIOP high resolution IRF modeling based on in-situ observations and application to AOD retrieval.

    NASA Astrophysics Data System (ADS)

    Guilmo, A.; Reagan, J. A.

    2014-12-01

    The CALIOP Lidar system onboard the CALIPSO satellite delivers daily atmospheric cloud and aerosol data that is widely used throughout the Atmospheric Science community.However, the lidar atmospheric returns suffer a number of modifications before downlinking which result in narrow-bandwidth signals. Additionally, the 532nm channel photomultiplier tube (PMT) detector introduces an artifact described in previous literature as "after-pulsing" or "tail effect" that has a direct impact on the received energy measurement.We present here a technique that allows for the full characterization of the detection channel's impulse response function (IRF) and the recovery of high-resolution signals. Emphasis is laid on the tail part of the 532nm channel's IRF caused by the PMT. The technique involves the use of multiple surface return (SR) signals to derive a super-resolution model of the IRF. We then demonstrate the use of this model for ocean SR data fitting and the application to aerosol optical depth (AOD) retrieval and to the 1064nm channel calibration.

  9. Multi-resolution correlative focused ion beam scanning electron microscopy: applications to cell biology.

    PubMed

    Narayan, Kedar; Danielson, Cindy M; Lagarec, Ken; Lowekamp, Bradley C; Coffman, Phil; Laquerre, Alexandre; Phaneuf, Michael W; Hope, Thomas J; Subramaniam, Sriram

    2014-03-01

    Efficient correlative imaging of small targets within large fields is a central problem in cell biology. Here, we demonstrate a series of technical advances in focused ion beam scanning electron microscopy (FIB-SEM) to address this issue. We report increases in the speed, robustness and automation of the process, and achieve consistent z slice thickness of ∼3 nm. We introduce "keyframe imaging" as a new approach to simultaneously image large fields of view and obtain high-resolution 3D images of targeted sub-volumes. We demonstrate application of these advances to image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently labeled cell membranes, proteins and HIV cores, we first produce a "target map" of an HIV infected cell by fluorescence microscopy. We then generate a correlated 3D EM volume of the entire cell as well as high-resolution 3D images of individual HIV cores, achieving correlative imaging across a volume scale of 10(9) in a single automated experimental run.

  10. Rate-Gyro-Integral Constraint for Ambiguity Resolution in GNSS Attitude Determination Applications

    PubMed Central

    Zhu, Jiancheng; Li, Tao; Wang, Jinling; Hu, Xiaoping; Wu, Meiping

    2013-01-01

    In the field of Global Navigation Satellite System (GNSS) attitude determination, the constraints usually play a critical role in resolving the unknown ambiguities quickly and correctly. Many constraints such as the baseline length, the geometry of multi-baselines and the horizontal attitude angles have been used extensively to improve the performance of ambiguity resolution. In the GNSS/Inertial Navigation System (INS) integrated attitude determination systems using low grade Inertial Measurement Unit (IMU), the initial heading parameters of the vehicle are usually worked out by the GNSS subsystem instead of by the IMU sensors independently. However, when a rotation occurs, the angle at which vehicle has turned within a short time span can be measured accurately by the IMU. This measurement will be treated as a constraint, namely the rate-gyro-integral constraint, which can aid the GNSS ambiguity resolution. We will use this constraint to filter the candidates in the ambiguity search stage. The ambiguity search space shrinks significantly with this constraint imposed during the rotation, thus it is helpful to speeding up the initialization of attitude parameters under dynamic circumstances. This paper will only study the applications of this new constraint to land vehicles. The impacts of measurement errors on the effect of this new constraint will be assessed for different grades of IMU and current average precision level of GNSS receivers. Simulations and experiments in urban areas have demonstrated the validity and efficacy of the new constraint in aiding GNSS attitude determinations. PMID:23793044

  11. Applications of high-resolution 1H solid-state NMR.

    PubMed

    Brown, Steven P

    2012-02-01

    This article reviews the large increase in applications of high-resolution (1)H magic-angle spinning (MAS) solid-state NMR, in particular two-dimensional heteronuclear and homonuclear (double-quantum and spin-diffusion NOESY-like exchange) experiments, in the last five years. These applications benefit from faster MAS frequencies (up to 80 kHz), higher magnetic fields (up to 1 GHz) and pulse sequence developments (e.g., homonuclear decoupling sequences applicable under moderate and fast MAS). (1)H solid-state NMR techniques are shown to provide unique structural insight for a diverse range of systems including pharmaceuticals, self-assembled supramolecular structures and silica-based inorganic-organic materials, such as microporous and mesoporous materials and heterogeneous organometallic catalysts, for which single-crystal diffraction structures cannot be obtained. The power of NMR crystallography approaches that combine experiment with first-principles calculations of NMR parameters (notably using the GIPAW approach) are demonstrated, e.g., to yield quantitative insight into hydrogen-bonding and aromatic CH-π interactions, as well as to generate trial three-dimensional packing arrangements. It is shown how temperature-dependent changes in the (1)H chemical shift, linewidth and DQ-filtered signal intensity can be analysed to determine the thermodynamics and kinetics of molecular level processes, such as the making and breaking of hydrogen bonds, with particular application to proton-conducting materials. Other applications to polymers and biopolymers, inorganic compounds and bioinorganic systems, paramagnetic compounds and proteins are presented. The potential of new technological advances such as DNP methods and new microcoil designs is described.

  12. Application of the low-finesse γ -ray frequency comb for high-resolution spectroscopy

    NASA Astrophysics Data System (ADS)

    Shakhmuratov, R. N.; Vagizov, F. G.; Scully, Marlan O.; Kocharovskaya, Olga

    2016-10-01

    High-finesse frequency combs (HFC) with large ratio of the frequency spacing to the width of the spectral components have demonstrated remarkable results in many applications such as precision spectroscopy and metrology. We found that low-finesse frequency combs having very small ratio of the frequency spacing to the width of the spectral components are more sensitive to the exact resonance with absorber than HFC. Our method is based on time domain measurements reviling oscillations of the radiation intensity after passing through an optically thick absorber. Fourier analysis of the oscillations allows to reconstruct the spectral content of the comb. If the central component of the incident comb is in exact resonance with the single line absorber, the contribution of the first sideband frequency to oscillations is exactly zero. We demonstrated this technique with γ -photon absorption by Mössbauer nuclei providing the spectral resolution beyond the natural broadening.

  13. Design of an eight-element refractive lens for high resolution imaging applications

    NASA Astrophysics Data System (ADS)

    Rao, D. V. B.

    1987-06-01

    An 8 element refractive lens of 324.4 mm EFL and f/4.5 was designed for the spectral band 520nm to 590nm. The FOV of the lens was +- 5 degrees. The computed MTF of this lens system was better than 0.7 for spatial frequencies up to 60 1p/mm, as against the diffraction limited MTF of 0.81. A similar lens was designed for the spectral band 770nm to 860nm. These two lenses will be configured alongwith a panchromatic catadioptric lens of 900 mm EFL for high resolution spacecraft remote sensing applications. The design details of these lenses were presented in this paper.

  14. Experiments and Monte Carlo modeling of a higher resolution Cadmium Zinc Telluride detector for safeguards applications

    NASA Astrophysics Data System (ADS)

    Borella, Alessandro

    2016-09-01

    The Belgian Nuclear Research Centre is engaged in R&D activity in the field of Non Destructive Analysis on nuclear materials, with focus on spent fuel characterization. A 500 mm3 Cadmium Zinc Telluride (CZT) with enhanced resolution was recently purchased. With a full width at half maximum of 1.3% at 662 keV, the detector is very promising in view of its use for applications such as determination of uranium enrichment and plutonium isotopic composition, as well as measurement on spent fuel. In this paper, I report about the work done with such a detector in terms of its characterization. The detector energy calibration, peak shape and efficiency were determined from experimental data. The data included measurements with calibrated sources, both in a bare and in a shielded environment. In addition, Monte Carlo calculations with the MCNPX code were carried out and benchmarked with experiments.

  15. High resolution remote sensing image processing technology and its application to uranium geology

    NASA Astrophysics Data System (ADS)

    Zhang, Jie-lin

    2008-12-01

    Hyperspectral and high spatial resolution remote sensing technology take important role in uranium geological application, data mining and knowledge discovery methods are key to character spectral and spatial information of uranium mineralization factors. Based on curvelet transform algorithm, this paper developed the image fusion technology of hyperspectral (Hyperion) and high spatial data (SPOT5), and results demonstrated that fusion image had advantage in denoising, enhancing and information identification. Used discrete wavelet transform, the spectral parameters of uranium mineralization factors were acquired, the spectral identification pedigrees of typical quadrivalence and hexavalence uranium minerals were established. Furthermore, utilizing hyperspectral remote sensing observation technology, this paper developed hyperspectral logging of drill cores and trench, it can quickly processed lots of geological and spectral information, and the relationship between radioactive intensity and abnormal spectral characteristics of Fe3+ was established. All those provided remote sensing technical bases to uranium geology, and the better results have been achieved in Taoshan uranium deposits in south China.

  16. New fluorescence imaging probe with high spatial resolution for in vivo applications.

    PubMed

    Bonnans, V; Gharbi, T; Pieralli, C; Wacogne, B; Humbert, Ph

    2004-01-01

    A new fiberized fluorescence imaging probe is presented. This device can potentially be used for a wide range of biological or medical applications. By exploiting the chromatic aberrations of gradient index lenses, the excitation blue or near-UV excitation light is focused on the sample surface, while the red fluorescence signal is efficiently launched back to collecting fibers. The excitation fiber is single mode at the working wavelength so that a resolution of 5 microm is obtained over a scanning area of several square millimeters. Experimental fluorescence images are presented. They concern either self-fabricated fluorescent microsamples or views of leaves that constitute an example of biological tissues analysis. The probe can also be adapted for spectroscopic investigations.

  17. A hybrid particle-continuum resolution method and its application to a homopolymer solution

    NASA Astrophysics Data System (ADS)

    Qi, S.; Behringer, H.; Raasch, T.; Schmid, F.

    2016-10-01

    We discuss in detail a recently proposed hybrid particle-continuum scheme for complex fluids and evaluate it at the example of a confined homopolymer solution in slit geometry. The hybrid scheme treats polymer chains near the impenetrable walls as particles keeping the configuration details, and chains in the bulk region as continuous density fields. Polymers can switch resolutions on the fly, controlled by an inhomogeneous tuning function. By properly choosing the tuning function, the representation of the system can be adjusted to reach an optimal balance between physical accuracy and computational efficiency. The hybrid simulation reproduces the results of a reference particle simulation and is significantly faster (about a factor of 3.5 in our application example).

  18. Valence state partitioning of V between pyroxene-melt: Effects of pyroxene and melt composition, and direct determination of V valence states by XANES. Application to Martian basalt QUE 94201 composition

    SciTech Connect

    Karner, J.M.; Papike, J.J.; Sutton, S.R.; Shearer, C.K.; Burger, P.; McKay, G.; Le, L.

    2009-01-13

    Experiments on a Martian basalt composition show that D{sub V} augite/melt is greater than D{sub V} pigeonite/melt in samples equilibrated under the same fO{sub 2} conditions. This increase is due to the increased availability of elements for coupled substitution with the V{sup 3+} or V{sup 4+} ions, namely Al and Na. for this bulk composition, both Al and Na are higher in concentration in augite compared with pigeonite; therefore more V can enter augite than pigeonite. Direct valence state determination by XANES shows that the V{sup 3+} and V{sup 4+} are the main V species in the melt at fO{sub 2} conditions of IW-1 to IW+3.5, whereas pyroxene grains at IW-1, IW, and IW+1 contain mostly V{sup 3+}. This confirms the idea that V{sup 3+} is more compatible in pyroxene than V{sup 4+}. The Xanes data also indicates that a small percentage of V{sup 2+} may exist in melt and pyroxene at IW-1. The similar valence of V in glass and pyroxene at IW-1 suggests that V{sup 2+} and V{sup 3+} may have similar compatibilities in pyroxene.

  19. MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay

    PubMed Central

    Nagy, Alexander; Černíková, Lenka; Vitásková, Eliška; Křivda, Vlastimil; Dán, Ádám; Dirbáková, Zuzana; Jiřincová, Helena; Procházka, Bohumír; Sedlák, Kamil; Havlíčková, Martina

    2016-01-01

    In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence. PMID:27031831

  20. MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay.

    PubMed

    Nagy, Alexander; Černíková, Lenka; Vitásková, Eliška; Křivda, Vlastimil; Dán, Ádám; Dirbáková, Zuzana; Jiřincová, Helena; Procházka, Bohumír; Sedlák, Kamil; Havlíčková, Martina

    2016-01-01

    In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.

  1. Multiple High Resolution Climate Scenarios over North America for Application to Water Resources (Invited)

    NASA Astrophysics Data System (ADS)

    Mearns, L. O.

    2009-12-01

    The North American Regional Climate Change Assessment Program (NARCCAP) is an international program that is serving the climate scenario needs of the United States, Canada, and northern Mexico. We are systematically investigating the uncertainties in regional scale projections of future climate and producing high resolution climate change scenarios using multiple regional climate models(RCMs)and multiple global model responses to a future emission scenario, by nesting the RCMs within atmosphere ocean general circulation models (AOGCMs) forced with the A2 SRES scenario, over a domain covering the conterminous US, northern Mexico, and most of Canada. The project also includes a validation component through nesting the participating RCMs within NCEP reanalyses. The basic spatial resolution of the RCM simulations is 50 km. This program includes RCMs that participated in the European PRUDENCE program (HadRM3 and RegCM), the Canadian regional climate model (CRCM) as well as the NCEP regional spectral model (RSM), the NCAR/PSU MM5, and NCAR WRF. Candidate AOGCMs include the Hadley Centre HadCM3, NCAR CCSM, the Canadian CGCM3 and the GFDL model. The resulting climate model runs form the basis for multiple high resolution climate scenarios that can be used in climate change impacts assessments over North America. High resolution (50 km) global time-slice experiments based on the GFDL atmospheric model and the NCAR atmospheric model (CAM3) have also been produced and will be compared with the simulations of the regional models. There also will be opportunities for double nesting over key regions through which additional modelers in the regional modeling community will be able to participate in NARCCAP. Additional key science issues are being investigated such as the importance of compatible physics in the nested and nesting models. Measures of uncertainty across the multiple runs are being developed by geophysical statisticians. In this overview talk, results from Phase II

  2. Influences of composition, melt viscosity and crystallization on the color strength and stability of multioxide glass frit/zircon-vanadium pigment systems for ceramic whitewares coatings applications

    NASA Astrophysics Data System (ADS)

    Earl, David Alonzo

    Color control is becoming increasingly important in the industrial processing of ceramics coatings. Multi-oxide glass frits are the predominant materials in ceramic whitewares coatings, and zircon doped pigments are the most commonly used colorants. The primary objective of this research was to determine if glass frits could be formulated to improve the fired color strength and high-temperature stability of ceramic coatings colored with zircon-vanadium (Zr-V) blue pigments. The results would also be applicable to other ceramic pigments that utilize the same zircon structure to incorporate colorant metal ions. A secondary goal was to relate the frit oxide composition, pigment content, firing temperature, melt viscosity and microstructural development to the fired color. A ceramic tile process was applied to fabricate sample coatings for the study. A coating's color was quantified and related to human perception with CIE L*, a* and b* values and pigment absorption factors (K/S), calculated based on spectral reflectance data. The research was successful in quantifying the influence of individual glass frit oxides on the fired color strength and high-temperature stability of the coatings. Opaque and transparent glossy frit compositions which yield excellent color strength and stability were formulated. Mathematical models for predicting a coating's color strength and stability given the frit oxide composition, Zr-V pigment loading and peak firing temperature were derived. Frit oxides of ZrO2, SrO, ZnO, Al2O3, Na 2O and K2O were found to have a significant influence on crystallization, pigment dissolution and color development. The properties, sizes, morphologies and quantities of crystalline phases that precipitated in the coatings during firing were related to the color. A technique for producing uniquely light yet high chroma colors through control of zircon precipitate particle size was demonstrated. In addition, a statistical model was developed for calculating the

  3. Application of the CloudSat and NEXRAD Radars Toward Improvements in High Resolution Operational Forecasts

    NASA Technical Reports Server (NTRS)

    Molthan, A. L.; Haynes, J. A.; Case, J. L.; Jedlovec, G. L.; Lapenta, W. M.

    2008-01-01

    As computational power increases, operational forecast models are performing simulations with higher spatial resolution allowing for the transition from sub-grid scale cloud parameterizations to an explicit forecast of cloud characteristics and precipitation through the use of single- or multi-moment bulk water microphysics schemes. investments in space-borne and terrestrial remote sensing have developed the NASA CloudSat Cloud Profiling Radar and the NOAA National Weather Service NEXRAD system, each providing observations related to the bulk properties of clouds and precipitation through measurements of reflectivity. CloudSat and NEXRAD system radars observed light to moderate snowfall in association with a cold-season, midlatitude cyclone traversing the Central United States in February 2007. These systems are responsible for widespread cloud cover and various types of precipitation, are of economic consequence, and pose a challenge to operational forecasters. This event is simulated with the Weather Research and Forecast (WRF) Model, utilizing the NASA Goddard Cumulus Ensemble microphysics scheme. Comparisons are made between WRF-simulated and observed reflectivity available from the CloudSat and NEXRAD systems. The application of CloudSat reflectivity is made possible through the QuickBeam radiative transfer model, with cautious application applied in light of single scattering characteristics and spherical target assumptions. Significant differences are noted within modeled and observed cloud profiles, based upon simulated reflectivity, and modifications to the single-moment scheme are tested through a supplemental WRF forecast that incorporates a temperature dependent snow crystal size distribution.

  4. Variable high-resolution color CCD camera system with online capability for professional photo studio application

    NASA Astrophysics Data System (ADS)

    Breitfelder, Stefan; Reichel, Frank R.; Gaertner, Ernst; Hacker, Erich J.; Cappellaro, Markus; Rudolf, Peter; Voelk, Ute

    1998-04-01

    Digital cameras are of increasing significance for professional applications in photo studios where fashion, portrait, product and catalog photographs or advertising photos of high quality have to be taken. The eyelike is a digital camera system which has been developed for such applications. It is capable of working online with high frame rates and images of full sensor size and it provides a resolution that can be varied between 2048 by 2048 and 6144 by 6144 pixel at a RGB color depth of 12 Bit per channel with an also variable exposure time of 1/60s to 1s. With an exposure time of 100 ms digitization takes approx. 2 seconds for an image of 2048 by 2048 pixels (12 Mbyte), 8 seconds for the image of 4096 by 4096 pixels (48 Mbyte) and 40 seconds for the image of 6144 by 6144 pixels (108 MByte). The eyelike can be used in various configurations. Used as a camera body most commercial lenses can be connected to the camera via existing lens adaptors. On the other hand the eyelike can be used as a back to most commercial 4' by 5' view cameras. This paper describes the eyelike camera concept with the essential system components. The article finishes with a description of the software, which is needed to bring the high quality of the camera to the user.

  5. Sierra Nevada snow melt from SMS-2

    NASA Technical Reports Server (NTRS)

    Breaker, L. C.; Mcmillan, M. C.

    1975-01-01

    A film loop from SMS-2 imagery shows snow melt over the Sierra Nevadas from May 10 to July 8, 1975. The sequence indicates a successful application of geostationary satellite data for monitoring dynamic hydrologic conditions.

  6. Application of a high-resolution x-ray fluorescence analyzer.

    SciTech Connect

    Adams, B. W.; Attenkofer, K.; Experimental Facilities Division

    2006-01-01

    We have developed a high resolution x-ray fluorescence analyzer based on the principle of active optics. It combines a resolution of ca. 5 eV with a tunability over several keV and a wide solid-angle coverage (ca. 2 by 5 degrees). To date, this analyzer has been used in near-edge spectroscopy of gallium in GaAs, and for the detection and chemical speciation of trace amounts platinum in soot from an diesel engine. The latter application illustrates the use of the analyzer to enhance the signal-to-background ratio in trace-element x-ray fluorescence analysis. The analyzer is shown schematically in Fig. 1. In it, a strip of silicon is bent by an axial force from two pushers at its ends, and eight correctors act from above to bring the shape of the bent crystal to approximate a logarithmic spiral. A more detailed description of the device, its underlying theory, and adjustment procedures may be found elsewhere. The sample consisted of soot collected from the exhaust of a diesel engine burning a fuel with a platinum-based additive that was tested for the purpose of facilitating clean combustion. The concentration of platinum in the soot was about 100 ppm, and the chemical speciation (oxidation state, dispersed or in the form of nanoparticles, etc.) was unknown. A small speck of this soot containing 10{sup 12} to 10{sup 13} atoms was placed into the x-ray beam of the 11-ID-D station of the APS. The incident photon energy was scanned over the Pt L{sub 3} edge, and the Pt L{sub {alpha}1} fluorescence was detected using two silicon drift detectors (Vortex), one directly and one with the analyzer. The purpose of the analyzer in this application was to enhance the energy resolution by a factor of about 50 (250 eV for the drift detector, 5 eV for the analyzer), and thus reduce the background of elastically or Compton-scattered photons, while keeping the fluorescent line. Whereas the detector with the analyzer recorded a clear signature of platinum in the form of an absorption

  7. Surface melting on ice shelves and icebergs

    NASA Astrophysics Data System (ADS)

    Sergienko, Olga V.

    Disintegration of Larsen Ice Shelf A and B, in 1995 and 2002, respectively, were preceded by two decades of extended summer melt seasons and by surface melt-water accumulation in ponds, surface crevasses and depressions produced by the elastic flexure of the ice. The extraordinary rapidity of ice-shelf fragmentation into large iceberg plumes following the appearance of surface melt water implies that the mechanical effects of surface melt water accumulation may represent an unforeseen process allowing abrupt, large-scale change of Antarctica's ice mass. The present study of surface melting and subsequent movement of melt water, both vertically (i.e., downward percolation into underlying firn) and horizontally (e.g., into crevasses and surface depressions created by ice-shelf flexure in response to both side boundary conditions and the melt-water load itself), is motivated by the need to further describe the energy, mass and momentum balances associated with ice shelves and their surrogates-large tabular icebergs-in the face of unprecedented changes in surface mass balance. The goal of this dissertation is to examine both the thermodynamic and mechanical aspects of surface melting on ice shelves and icebergs subject to sudden changes in climate conditions (e.g ., global warming). Thermodynamic aspects of the study include the development and application of surface energy balance models capable of describing the process of surface melting and subsequent vertical movement of melt water through a porous firn. Mechanical aspects of this study include the analysis of vertical melt-water flow, and more particularly, the elastic flexure response of the ice shelf or iceberg to the melt-water loads. Work presented here involves three methodologies, numerical modeling, field observation, and mathematical analysis (e.g., development of analytic solutions to simple, idealized ice-shelf flexure problems).

  8. A sub-millimeter resolution detector module for small-animal PET applications

    NASA Astrophysics Data System (ADS)

    Sacco, I.; Dohle, R.; Fischer, P.; Gola, A.; Piemonte, C.; Ritzert, M.

    2017-01-01

    We present a gamma detection module optimized for very high resolution PET applications, able to resolve arrays of scintillating crystals with sub-millimeter pitch. The detector is composed of a single ceramic substrate (LTCC): it hosts four flip-chip mounted PETA5 ASICs on the bottom side and an array of SiPM sensors on the top surface, fabricated in HD-RGB technology by FBK. Each chip has 36 channels, for a maximum of 144 readout channels on a sensitive area of about 32 mm × 32 mm. The module is MR-compatible. The thermal decoupling of the readout electronics from the photon sensors is obtained with an efficient internal liquid channel, integrated within the ceramic substrate. Two modules have been designed, based on different SiPM topologies: • Light spreader-based: an array of 12 × 12 SiPMs, with an overall pitch of 2.5 mm, is coupled with a scintillators array using a 1 mm thick glass plate. The light from one crystal is spread over a group of SiPMs, which are read out in parallel using PETA5 internal neighbor logic. • Interpolating SiPM-based: ISiPMs are intrinsic position-sensitive sensors. The photon diodes in the array are connected to one of the four available outputs so that the center of gravity of any bunch of detected photons can be reconstructed using a proper weight function of the read out amplitudes. An array of ISiPMs, each 7.5 mm× 5 mm sized, is directly coupled with the scintillating crystals. Both modules can clearly resolve LYSO arrays with a pitch of only 0.833 mm. The detector can be adjusted for clinical PET, where it has already shown ToF resolution of about 230 ps CRT at FWHM. The module designs, their features and results are described.

  9. Applications and Advancements in the use of High-Resolution Microendoscopy for Detection of Gastrointestinal Neoplasia

    PubMed Central

    Louie, Justin S.; Richards-Kortum, Rebecca; Anandasabapathy, Sharmila

    2014-01-01

    The high-resolution microendoscope (HRME) is a novel imaging modality that allows real-time epithelial imaging at subcellular resolution. Used in concert with any standard endoscope, this portable, low cost, ‘optical biopsy’ technology has the ability to provide images of cellular morphology during a procedure. This technology has been the subject of a number of studies investigating its use in screening and surveillance of a range of gastrointestinal neoplasia, including esophageal adenocarcinoma(EAC), esophageal squamous cell cancer(ESCC), colorectal neoplasia(CRC) and anal neoplasia. These studies have shown that HRME is a modality that consistently provides high specificity, negative predictive value, and accuracy across different diseases. In addition, they have illustrated that HRME users can be relatively easily trained in a short period of time and that users have demonstrated solid inter-rater reliability. These features make HRME a potential complement to high definition white light imaging, narrow band imaging and other ‘red flag technologies’ in facilitating real-time clinical diagnosis, endoscopic therapy and margin determination. Further clinical validation is needed to determine whether this translates to reduced procedure times, pathology costs, and follow up procedures. Finally, the HRME has a relatively simple design compared to other similar technologies, making it portable, simple to maintain, and low cost. This may allow the HRME device to function in both advanced care settings as well as in places with less resources and specialized support systems. As a whole, the HRME device has shown good performance along with low-cost and portable construction, and its application in different conditions and settings has been promising. PMID:25108219

  10. Effect of melt composition on basalt and peridotite interaction: laboratory dissolution experiments with applications to mineral compositional variations in mantle xenoliths from the North China Craton

    NASA Astrophysics Data System (ADS)

    Wang, Chunguang; Liang, Yan; Xu, Wenliang; Dygert, Nick

    2013-11-01

    Interaction between basaltic melts and peridotites has played an important role in modifying the lithospheric and asthenospheric mantle during magma genesis in a number of tectonic settings. Compositions of basaltic melts vary considerably and may play an important role in controlling the kinetics of melt-peridotite interaction. To better understand the effect of melt composition on melt-peridotite interaction, we conducted spinel lherzolite dissolution experiments at 2 GPa and 1,425 °C using the dissolution couple method. The reacting melts include a basaltic andesite, a ferro-basalt, and an alkali basalt. Dissolution of lherzolite in the basaltic andesite and the ferro-basalt produced harzburgite-lherzolite sequences with a thin orthopyroxenite layer at the melt-harzburgite interface, whereas dissolution of lherzolite in the alkali basalt produced a dunite-harzburgite-lherzolite sequence. Systematic variations in mineral compositions across the lithological units are observed. These mineral compositional variations are attributed to grain-scale processes that involve dissolution, precipitation, and reprecipitation and depend strongly on reacting melt composition. Comparison of mineral compositional variations across the dissolution couples with those observed in mantle xenoliths from the North China Craton (NCC) helps to assess the spatial and temporal variations in the extent of siliceous melt and peridotite interaction in modifying the lithospheric mantle beneath the NCC. We found that such melt-rock interaction mainly took place in Early Cretaceous, and is responsible for the enrichment of pyroxene in the lithospheric mantle. Spatially, siliceous melt-peridotite interaction took place in the ancient orogens with thickened lower crust.

  11. UT-CT: A National Resource for Applications of High-Resolution X-ray Computed Tomography in the Geological Sciences

    NASA Astrophysics Data System (ADS)

    Carlson, W. D.; Ketcham, R. A.; Rowe, T. B.

    2002-12-01

    An NSF-sponsored (EAR-IF) shared multi-user facility dedicated to research applications of high-resolution X-ray computed tomography (CT) in the geological sciences has been in operation since 1997 at the University of Texas at Austin. The centerpiece of the facility is an industrial CT scanner custom-designed for geological applications. Because the instrument can optimize trade-offs among penetrating ability, spatial resolution, density discrimination, imaging modes, and scan times, it can image a very broad range of geological specimens and materials, and thus offers significant advantages over medical scanners and desktop microtomographs. Two tungsten-target X-ray sources (200-kV microfocal and 420-kV) and three X-ray detectors (image-intensifier, high-sensitivity cadmium tungstate linear array, and high-resolution gadolinium-oxysulfide radiographic line scanner) can be used in various combinations to meet specific imaging goals. Further flexibility is provided by multiple imaging modes: second-generation (translate-rotate), third-generation (rotate-only; centered and variably offset), and cone-beam (volume CT). The instrument can accommodate specimens as small as about 1 mm on a side, and as large as 0.5 m in diameter and 1.5 m tall. Applications in petrology and structural geology include measuring crystal sizes and locations to identify mechanisms governing the kinetics of metamorphic reactions; visualizing relationships between alteration zones and abundant macrodiamonds in Siberian eclogites to elucidate metasomatic processes in the mantle; characterizing morphologies of spiral inclusion trails in garnet to test hypotheses of porphyroblast rotation during growth; measuring vesicle size distributions in basaltic flows for determination of elevation at the time of eruption to constrain timing and rates of continental uplift; analysis of the geometry, connectivity, and tortuosity of migmatite leucosomes to define the topology of melt flow paths, for numerical

  12. A MELCOR Application to Two Light Water Reactor Nuclear Power Plant Core Melt Scenarios with Assumed Cavity Flooding Action

    SciTech Connect

    Martin-Fuertes, Francisco; Martin-Valdepenas, Juan Manuel; Mira, Jose; Sanchez, Maria Jesus

    2003-10-15

    The MELCOR 1.8.4 code Bottom Head package has been applied to simulate two reactor cavity flooding scenarios for when the corium material relocates to the lower-plenum region in postulated severe accidents. The applications were preceded by a review of two main physical models, which highly impacted the results. A model comparison to available bibliography models was done, which allowed some code modifications on selected default assumptions to be undertaken. First, the corium convective heat transfer to the wall when it becomes liquid was modified, and second, the default nucleate boiling regime curve in a submerged hemisphere was replaced by a new curve (and, to a much lesser extent, the critical heat flux curve was slightly varied).The applications were devoted to two prototypical light water reactor nuclear power plants, a 2700-MW(thermal) pressurized water reactor (PWR) and a 1381-MW(thermal) boiling water reactor (BWR). The main conclusions of the cavity flooding simulations were that the PWR lower-head survivability is extended although it is clearly not guaranteed, while in the BWR sequence the corium seems to be successfully arrested in the lower plenum.Three applications of the CFX 4.4 computational fluid dynamics code were carried out in the context of the BWR scenario to support the first modification of the aforementioned two scenarios for MELCOR.Finally, in the same BWR context, a statistic predictor of selected output parameters as a function of input parameters is presented, which provides reasonable results when compared to MELCOR full calculations in much shorter CPU processing times.

  13. Cloud screening and melt water detection over melting sea ice using AATSR/SLSTR

    NASA Astrophysics Data System (ADS)

    Istomina, Larysa; Heygster, Georg

    2014-05-01

    With the onset of melt in the Arctic Ocean, the fraction of melt water on sea ice, the melt pond fraction, increases. The consequences are: the reduced albedo of sea ice, increased transmittance of sea ice and affected heat balance of the system with more heat passing through the ice into the ocean, which facilitates further melting. The onset of melt, duration of melt season and melt pond fraction are good indicators of the climate state of the Arctic and its change. In the absence of reliable sea ice thickness retrievals in summer, melt pond fraction retrieval from satellite is in demand as input for GCM as an indicator of melt state of the sea ice. The retrieval of melt pond fraction with a moderate resolution radiometer as AATSR is, however, a non-trivial task due to a variety of subpixel surface types with very different optical properties, which give non-unique combinations if mixed. In this work this has been solved by employing additional information on the surface and air temperature of the pixel. In the current work, a concept of melt pond detection on sea ice is presented. The basis of the retrieval is the sensitivity of AATSR reflectance channels 550nm and 860nm to the amount of melt water on sea ice. The retrieval features extensive usage of a database of in situ surface albedo spectra. A tree of decisions is employed to select the feasible family of in situ spectra for the retrieval, depending on the melt stage of the surface. Reanalysis air temperature at the surface and brightness temperature measured by the satellite sensor are analyzed in order to evaluate the melting status of the surface. Case studies for FYI and MYI show plausible retrieved melt pond fractions, characteristic for both of the ice types. The developed retrieval can be used to process the historical AATSR (2002-2012) dataset, as well as for the SLSTR sensor onboard the future Sentinel-3 mission (scheduled for launch in 2015), to keep the continuity and obtain longer time sequence

  14. A high brightness laser-cooled atomic beam for application in high resolution FIB

    NASA Astrophysics Data System (ADS)

    Wouters, Steinar; Geer, Bas; Haaf, Gijs; Jansen, Bart; Mutsaers, Peter

    2013-05-01

    A new type of high-brightness ion source is under development which employs transverse laser cooling and compression of a thermal atomic rubidium beam, followed by in-field photo-ionization. When attached to a focusing column, this Focused Ion Beam (FIB) has the advantage of supplying a higher current in a smaller spot compared to conventional LMIS-based FIBs, thus increasing both the resolution and the speed of the FIB. Furthermore, different types of ion species can be used, broadening the range of applications of the FIB. Simulations using a 10 cm long laser cooling and compression stage and a realistic ionization and acceleration structure, predict an achievable brightness for 87Rb+ of order 107 A/m2 sr eV at an energy spread of less than 1 eV and a current of tens of pA. This would lead to a spot size below 5 nm. Simulations and modeling on the ionization process have led to a better understanding of stochastic heating. Experimental realization of the compact ion source has recently started with the development of an efficient high-flux atom source and a 2D laser cooler and compressor. Progress on simulations and experimental results will be reported.

  15. High-resolution permanent photoresist laminate TMMF for sealed microfluidic structures in biological applications

    NASA Astrophysics Data System (ADS)

    Wangler, N.; Gutzweiler, L.; Kalkandjiev, K.; Müller, C.; Mayenfels, F.; Reinecke, H.; Zengerle, R.; Paust, N.

    2011-09-01

    We demonstrate the use of photosensitive epoxy laminate TMMF S2045 for the fabrication and sealing of tapered microfluidic channels. The 45 µm thick resist enables the fabrication of shallow sealed cavities featuring extreme aspect ratios of less than 1:40 (h = 45 µm, w = 2000 µm). It also provides high resolution and enables minimum feature sizes of 10 µm. For the fabrication of free-standing structures, an aspect ratio of up to 7:1 was achieved. The dry-film photoresist can be applied easily by lamination onto structured substrates. The total thickness variation of the resist across a 100 mm wafer was determined to be less than ±0.6 µm. Process parameters for the fabrication and sealing of various micro-channels are discussed and optimized in this paper. The main focus was to minimize thermal impact during lamination, soft-bake, exposure and post-exposure bake, which could lead to lid sagging or channel clogging due to liquefaction of uncured resist. We tested TMMF according to ISO 10995-5 and found it to be non-cytotoxic, enabling its use for biological applications. Swelling of less than 5% for incubation of the dry-film resist in several biologically relevant solvents, buffers and cleaning solutions was observed.

  16. Nanotexturing of surfaces to reduce melting point.

    SciTech Connect

    Garcia, Ernest J.; Zubia, David; Mireles, Jose; Marquez, Noel; Quinones, Stella

    2011-11-01

    This investigation examined the use of nano-patterned structures on Silicon-on-Insulator (SOI) material to reduce the bulk material melting point (1414 C). It has been found that sharp-tipped and other similar structures have a propensity to move to the lower energy states of spherical structures and as a result exhibit lower melting points than the bulk material. Such a reduction of the melting point would offer a number of interesting opportunities for bonding in microsystems packaging applications. Nano patterning process capabilities were developed to create the required structures for the investigation. One of the technical challenges of the project was understanding and creating the specialized conditions required to observe the melting and reshaping phenomena. Through systematic experimentation and review of the literature these conditions were determined and used to conduct phase change experiments. Melting temperatures as low as 1030 C were observed.

  17. Terrestrial Analogues for Lunar Impact Melt Flows

    NASA Technical Reports Server (NTRS)

    Neish, C. D.; Hamilton, C. W.; Hughes, S. S.; Nawotniak, S. Kobs; Garry, W. B.; Skok, J. R.; Elphic, R. C.; Schaefer, E.; Carter, L. M.; Bandfield, J. L.; Osinski, G. R.; Lim, D.; Heldmann, J. L.

    2016-01-01

    Lunar impact melt deposits have unique physical properties. They have among the highest observed radar returns at S-Band (12.6 cm wavelength), implying that they are rough at the decimeter scale. However, they are also observed in high-resolution optical imagery to be quite smooth at the meter scale. These characteristics distinguish them from well-studied terrestrial analogues, such as Hawaiian pahoehoe and ?a ?a lava flows. The morphology of impact melt deposits can be related to their emplacement conditions, so understanding the origin of these unique surface properties will help to inform us as to the circumstances under which they were formed. In this work, we seek to find a terrestrial analogue for well-preserved lunar impact melt flows by examining fresh lava flows on Earth. We compare the radar return and high-resolution topographic variations of impact melt flows to terrestrial lava flows with a range of surface textures. The lava flows examined in this work range from smooth Hawaiian pahoehoe to transitional basaltic flows at Craters of the Moon (COTM) National Monument and Preserve in Idaho to rubbly and spiny pahoehoe-like flows at the recent eruption at Holuhraun in Iceland. The physical properties of lunar impact melt flows appear to differ from those of all the terrestrial lava flows studied in this work. This may be due to (a) differences in post-emplacement modification processes or (b) fundamental differences in the surface texture of the melt flows due to the melts' unique emplacement and/or cooling environment. Information about the surface properties of lunar impact melt deposits will be critical for future landed missions that wish to sample these materials.

  18. Terrestrial analogues for lunar impact melt flows

    NASA Astrophysics Data System (ADS)

    Neish, C. D.; Hamilton, C. W.; Hughes, S. S.; Nawotniak, S. Kobs; Garry, W. B.; Skok, J. R.; Elphic, R. C.; Schaefer, E.; Carter, L. M.; Bandfield, J. L.; Osinski, G. R.; Lim, D.; Heldmann, J. L.

    2017-01-01

    Lunar impact melt deposits have unique physical properties. They have among the highest observed radar returns at S-Band (12.6 cm wavelength), implying that they are rough at the decimeter scale. However, they are also observed in high-resolution optical imagery to be quite smooth at the meter scale. These characteristics distinguish them from well-studied terrestrial analogues, such as Hawaiian pāhoehoe and ´a´ā lava flows. The morphology of impact melt deposits can be related to their emplacement conditions, so understanding the origin of these unique surface properties will help to inform us as to the circumstances under which they were formed. In this work, we seek to find a terrestrial analogue for well-preserved lunar impact melt flows by examining fresh lava flows on Earth. We compare the radar return and high-resolution topographic variations of impact melt flows to terrestrial lava flows with a range of surface textures. The lava flows examined in this work range from smooth Hawaiian pāhoehoe to transitional basaltic flows at Craters of the Moon (COTM) National Monument and Preserve in Idaho to rubbly and spiny pāhoehoe-like flows at the recent eruption at Holuhraun in Iceland. The physical properties of lunar impact melt flows appear to differ from those of all the terrestrial lava flows studied in this work. This may be due to (a) differences in post-emplacement modification processes or (b) fundamental differences in the surface texture of the melt flows due to the melts' unique emplacement and/or cooling environment. Information about the surface properties of lunar impact melt deposits will be critical for future landed missions that wish to sample these materials.

  19. Melt containment member

    SciTech Connect

    Rieken, Joel R.; Heidloff, Andrew J.

    2014-09-09

    A tubular melt containment member for transient containment of molten metals and alloys, especially reactive metals and alloys, includes a melt-contacting layer or region that comprises an oxygen-deficient rare earth oxide material that is less reactive as compared to the counterpart stoichiometric rare earth oxide. The oxygen-deficient (sub-stoichiometric) rare earth oxide can comprise oxygen-deficient yttria represented by Y.sub.2O.sub.3-x wherein x is from 0.01 to 0.1. Use of the oxygen-deficient rare earth oxide as the melt-contacting layer or region material reduces reaction with the melt for a given melt temperature and melt contact time.

  20. A Honeycomb-Structured Ti-6Al-4V Oil-Gas Separation Rotor Additively Manufactured by Selective Electron Beam Melting for Aero-engine Applications

    NASA Astrophysics Data System (ADS)

    Tang, H. P.; Wang, Q. B.; Yang, G. Y.; Gu, J.; Liu, N.; Jia, L.; Qian, M.

    2016-03-01

    Oil -gas separation is a key process in an aero-engine lubrication system. This study reports an innovative development in oil -gas separation. A honeycomb-structured rotor with hexagonal cone-shaped pore channels has been designed, additively manufactured from Ti-6Al-4V using selective electron beam melting (SEBM) and assessed for oil -gas separation for aero-engine application. The Ti-6Al-4V honeycomb structure showed a high compressive strength of 110 MPa compared to less than 20 MPa for metal foam structures. The oil -gas separation efficiency of the honeycomb-structured separation rotor achieved 99.8% at the rotation speed of 6000 rpm with much lower ventilation resistance (17.3 kPa) than that of the separator rotor constructed using a Ni-Cr alloy foam structure (23.5 kPa). The honeycomb-structured Ti-6Al-4V separator rotor produced by SEBM provides a promising solution to more efficient oil -gas separation in the aero-engine lubrication system.

  1. Investigation on the microstructure, mechanical property and corrosion behavior of the selective laser melted CoCrW alloy for dental application.

    PubMed

    Lu, Yanjin; Wu, Songquan; Gan, Yiliang; Li, Junlei; Zhao, Chaoqian; Zhuo, Dongxian; Lin, Jinxin

    2015-04-01

    In this study, an experimental investigation on fabricating Ni-free CoCrW alloys by selective laser melting (SLM) for dental application was conducted in terms of microstructure, hardness, mechanical property, electrochemical behavior, and metal release; and line and island scanning strategy were applied to determine whether these strategies are able to obtain expected CoCrW parts. The XRD revealed that the γ-phase and ε-phase coexisted in the as-SLM CoCrW alloys; The OM and SEM images showed that the microstructure of CoCrW alloys appeared square-like pattern with the fine cellular dendrites at the borders; tensile test suggested that the difference of mechanical properties of line- and island-formed specimens was very small; whilst the outcomes from the electrochemical and metal release tests indicated that the island-formed alloys showed slightly better corrosion resistance than line-formed ones in PBS and Hanks solutions. Considering that the mechanical properties and corrosion resistance of line-formed and island-formed specimens meet the standards of ISO 22674:2006 and EN ISO 10271, CoCrW dental alloys can be successfully fabricated by line and island scanning strategies in the SLM process.

  2. Selective Laser Melting: a regular unit cell approach for the manufacture of porous, titanium, bone in-growth constructs, suitable for orthopedic applications.

    PubMed

    Mullen, Lewis; Stamp, Robin C; Brooks, Wesley K; Jones, Eric; Sutcliffe, Christopher J

    2009-05-01

    In this study, a novel porous titanium structure for the purpose of bone in-growth has been designed, manufactured and evaluated. The structure was produced by Selective Laser Melting (SLM); a rapid manufacturing process capable of producing highly intricate, functionally graded parts. The technique described utilizes an approach based on a defined regular unit cell to design and produce structures with a large range of both physical and mechanical properties. These properties can be tailored to suit specific requirements; in particular, functionally graded structures with bone in-growth surfaces exhibiting properties comparable to those of human bone have been manufactured. The structures were manufactured and characterized by unit cell size, strand diameter, porosity, and compression strength. They exhibited a porosity (10-95%) dependant compression strength (0.5-350 Mpa) comparable to the typical naturally occurring range. It is also demonstrated that optimized structures have been produced that possesses ideal qualities for bone in-growth applications and that these structures can be applied in the production of orthopedic devices.

  3. Novel melt-processable poly(ether ether ketone)(PEEK)/inorganic fullerene-like WS(2) nanoparticles for critical applications.

    PubMed

    Naffakh, Mohammed; Díez-Pascual, Ana M; Marco, Carlos; Gómez, Marián A; Jiménez, Ignacio

    2010-09-09

    The combination of high-performance thermoplastic poly(ether ether ketone) (PEEK) with inorganic fullerene-like tungsten disulfide (IF-WS(2)) nanoparticles offers an attractive way to combine the merits of organic and inorganic materials into novel polymer nanocomposite materials. Here, we report the processing of novel PEEK/IF-WS(2) nanocomposites, which overcome the nanoparticle agglomerate formation and provide PEEK-particle interactions. The IF-WS(2) nanoparticles do not require exfoliation or modification, making it possible to obtain stronger, lighter materials without the complexity and processing cost associated with these treatments. The nanocomposites were fabricated by melt blending, after a predispersion step based on ball milling and mechanical treatments in organic solvent, which leads to the dispersion of individually IF-WS(2) nanoparticles in the PEEK matrix as confirmed by scanning electron microscopy. In order to determine the performance of the PEEK/IF-WS(2) nanocomposites for potential critical applications, particularly for the aircraft industry, we have extensively investigated these materials with a wide range of structural, thermal, and mechanical techniques using time-resolved synchrotron X-ray diffraction, thermogravimetric analysis, differential scanning calorimetry, dynamic-mechanical analysis, and tensile and impact tests as well as thermal measurements. Modulus, tensile strengh, thermal stability, and thermal conductivity of PEEK exhibited remarkable improvement with the addition of IF-WS(2).

  4. Application of melt extrusion in the development of a physically and chemically stable high-energy amorphous solid dispersion of a poorly water-soluble drug.

    PubMed

    Lakshman, Jay P; Cao, Yu; Kowalski, James; Serajuddin, Abu T M

    2008-01-01

    Formulation of active pharmaceutical ingredients (API) in high-energy amorphous forms is a common strategy to enhance solubility, dissolution rate and, consequently, oral bioavailability of poorly water-soluble drugs. Amorphous APIs are, however, susceptible to recrystallization and, therefore, there is a need to physically stabilize them as solid dispersions in polymeric carriers. Hot melt extrusion has in recent years gained wide acceptance as a method of choice for the preparation of solid dispersions. There is a potential that the API, the polymer or both may degrade if excessively high temperature is needed in the melt extrusion process, especially when the melting point of the API is high. This report details a novel method where the API was first converted to an amorphous form by solvent evaporation and then melt-extruded with a suitable polymer at a drug load of at least 20% w/w. By this means, melt extrusion could be performed much below the melting temperature of the drug substance. Since the glass transition temperature of the amorphous drug was lower than that of the polymer used, the drug substance itself served as the plasticizer for the polymer. The addition of surfactants in the matrix enhanced dispersion and subsequent dissolution of the drug in aqueous media. The amorphous melt extrusion formulations showed higher bioavailability than formulations containing the crystalline API. There was no conversion of amorphous solid to its crystalline form during accelerated stability testing of dosage forms.

  5. A partial melting study of an ordinary (H) chondrite composition with application to the unique achondrite Graves Nunataks 06128 and 06129

    NASA Astrophysics Data System (ADS)

    Usui, Tomohiro; Jones, John H.; Mittlefehldt, David W.

    2015-04-01

    Melting experiments of a synthesized, alkali-bearing, H-chondrite composition were conducted at ambient pressure with three distinct oxygen fugacity conditions (IW-1, IW, and IW+2). Oxygen fugacity conditions significantly influence the compositions of partial melts. Partial melts at IW-1 are distinctly enriched in SiO2 relative to those of IW and IW+2 melts. The silica-enriched, reduced (IW-1) melts are characterized by high alkali contents and have silica-oversaturated compositions. In contrast, the silica-depleted, oxidized (≥IW) melts, which are also enriched in alkali contents, have distinctly silica-undersaturated compositions. These experimental results suggest that alkali-rich, felsic, asteroidal crusts as represented by paired achondrites Graves Nunataks 06128 and 06129 should originate from a low-degree, relatively reduced partial melt from a parent body having near-chondritic compositions. Based on recent chronological constraints and numerical considerations as well as our experimental results, we propose that such felsic magmatism should have occurred in a parent body that is smaller in size and commenced accreting later than those highly differentiated asteroids having basaltic crusts and metallic cores.

  6. A multi-resolution method for climate system modeling: application of Spherical Centroidal A multi-resolution method for climate system modeling: Application of Spherical Centroidal Voroni Tessellations

    SciTech Connect

    Ringler, Todd D; Gunzburger, Max; Ju, Lili

    2008-01-01

    During the next decade and beyond, climate system models will be challenged to resolve scales and processes that are far beyond their current scope. Each climate system component has its prototypical example of an unresolved process that may strongly influence the global climate system, ranging from eddy activity within ocean models, to ice streams within ice sheet models, to surface hydrological processes within land system models, to cloud processes within atmosphere models. These new demands will almost certainly result in the develop of multi-resolution schemes that are able, at least regional to faithfully simulate these fine-scale processes. Spherical Centroidal Voronoi Tessellations (SCVTs) offer one potential path toward the development of robust, multi-resolution climate system component models, SCVTs allow for the generation of high quality Voronoi diagrams and Delaunay triangulations through the use of an intuitive, user-defined density function, each of the examples provided, this method results in high-quality meshes where the quality measures are guaranteed to improve as the number of nodes is increased. Real-world examples are developed for the Greenland ice sheet and the North Atlantic ocean. Idealized examples are developed for ocean-ice shelf interaction and for regional atmospheric modeling. In addition to defining, developing and exhibiting SCVTs, we pair this mesh generation technique with a previously developed finite-volume method. Our numerical example is based on the nonlinear shallow-water equations spanning the entire surface of the sphere. This example is used to elucidate both the potential benefits of this multi-resolution method and the challenges ahead.

  7. Exact feature extraction using finite rate of innovation principles with an application to image super-resolution.

    PubMed

    Baboulaz, Loïc; Dragotti, Pier Luigi

    2009-02-01

    The accurate registration of multiview images is of central importance in many advanced image processing applications. Image super-resolution, for example, is a typical application where the quality of the super-resolved image is degrading as registration errors increase. Popular registration methods are often based on features extracted from the acquired images. The accuracy of the registration is in this case directly related to the number of extracted features and to the precision at which the features are located: images are best registered when many features are found with a good precision. However, in low-resolution images, only a few features can be extracted and often with a poor precision. By taking a sampling perspective, we propose in this paper new methods for extracting features in low-resolution images in order to develop efficient registration techniques. We consider, in particular, the sampling theory of signals with finite rate of innovation and show that some features of interest for registration can be retrieved perfectly in this framework, thus allowing an exact registration. We also demonstrate through simulations that the sampling model which enables the use of finite rate of innovation principles is well suited for modeling the acquisition of images by a camera. Simulations of image registration and image super-resolution of artificially sampled images are first presented, analyzed and compared to traditional techniques. We finally present favorable experimental results of super-resolution of real images acquired by a digital camera available on the market.

  8. Magnetic Biocomposites for Remote Melting.

    PubMed

    Zhou, Mengbo; Liebert, Tim; Müller, Robert; Dellith, Andrea; Gräfe, Christine; Clement, Joachim H; Heinze, Thomas

    2015-08-10

    A new approach toward the fabrication of biocompatible composites suitable for remote melting is presented. It is shown that magnetite nanoparticles (MNP) can be embedded into a matrix of biocompatible thermoplastic dextran esters. For that purpose, fatty acid esters of dextran with adjustable melting points in the range of 30-140 °C were synthesized. Esterification of the polysaccharide by activation of the acid as iminium chlorides guaranteed mild reaction conditions leading to high quality products as confirmed by FTIR- and NMR spectroscopy as well as by gel permeation chromatography (GPC). A method for the preparation of magnetically responsive bionanocomposites was developed consisting of combined dissolution/suspension of the dextran ester and hydrophobized MNPs in an organic solvent followed by homogenization with ultrasonication, casting of the solution, drying and melting of the composite for a defined shaping. This process leads to a uniform distribution of MNPs in nanocomposite as revealed by scanning electron microscope. Samples of different geometries were exposed to high frequency alternating magnetic field. It could be shown that defined remote melting of such biocompatible nanocomposites is possible for the first time. This may lead to a new class of magnetic remote control systems, which are suitable for controlled release applications or self-healing materials.

  9. Application of high resolution images from unmanned aerial vehicles for hydrology and rangeland science

    NASA Astrophysics Data System (ADS)

    Rango, A.; Vivoni, E. R.; Anderson, C. A.; Perini, N. A.; Saripalli, S.; Laliberte, A.

    2012-12-01

    A common problem in many natural resource disciplines is the lack of high-enough spatial resolution images that can be used for monitoring and modeling purposes. Advances have been made in the utilization of Unmanned Aerial Vehicles (UAVs) in hydrology and rangeland science. By utilizing low flight altitudes and velocities, UAVs are able to produce high resolution (5 cm) images as well as stereo coverage (with 75% forward overlap and 40% sidelap) to extract digital elevation models (DEM). Another advantage of flying at low altitude is that the potential problems of atmospheric haze obscuration are eliminated. Both small fixed-wing and rotary-wing aircraft have been used in our experiments over two rangeland areas in the Jornada Experimental Range in southern New Mexico and the Santa Rita Experimental Range in southern Arizona. The fixed-wing UAV has a digital camera in the wing and six-band multispectral camera in the nose, while the rotary-wing UAV carries a digital camera as payload. Because we have been acquiring imagery for several years, there are now > 31,000 photos at one of the study sites, and 177 mosaics over rangeland areas have been constructed. Using the DEM obtained from the imagery we have determined the actual catchment areas of three watersheds and compared these to previous estimates. At one site, the UAV-derived watershed area is 4.67 ha which is 22% smaller compared to a manual survey using a GPS unit obtained several years ago. This difference can be significant in constructing a watershed model of the site. From a vegetation species classification, we also determined that two of the shrub types in this small watershed(mesquite and creosote with 6.47 % and 5.82% cover, respectively) grow in similar locations(flat upland areas with deep soils), whereas the most predominant shrub(mariola with 11.9% cover) inhabits hillslopes near stream channels(with steep shallow soils). The positioning of these individual shrubs throughout the catchment using

  10. Measurement and Analysis of Porosity in Al-10Si-1Mg Components Additively Manufactured by Selective Laser Melting

    SciTech Connect

    Rao, Suraj; Cunningham, Ross; Ozturk, Tugce; Rollett, Anthony D.

    2016-10-18

    Aluminum alloys are candidate materials for weight critical applications because of their excellent strength and stiffness to weight ratio. However, defects such as voids decrease the strength and fatigue life of these alloys, which can limit the application of Selective Laser Melting. In this study, the average volume fraction, average size, and size distribution of pores in Al10-Si-1Mg samples built using Selective Laser Melting have been characterized. Synchrotron high energy X-rays were used to perform computed tomography on volumes of order one cubic millimeter with a resolution of approximately 1.5 μm. Substantial variations in the pore size distributions were found as a function of process conditions. Even under conditions that ensured that all locations were melted at least once, a significant number density was found of pores above 5 μm in diameter.

  11. Spatial resolution in CBCT machines for dental/maxillofacial applications-what do we know today?

    PubMed

    Brüllmann, D; Schulze, R K W

    2015-01-01

    Spatial resolution is one of the most important parameters objectively defining image quality, particularly in dental imaging, where fine details often have to be depicted. Here, we review the current status on assessment parameters for spatial resolution and on published data regarding spatial resolution in CBCT images. The current concepts of visual [line-pair (lp) measurements] and automated [modulation transfer function (MTF)] assessment of spatial resolution in CBCT images are summarized and reviewed. Published measurement data on spatial resolution in CBCT are evaluated and analysed. Effective (i.e. actual) spatial resolution available in CBCT images is being influenced by the two-dimensional detector, the three-dimensional reconstruction process, patient movement during the scan and various other parameters. In the literature, the values range between 0.6 and 2.8 lp mm(-1) (visual assessment; median, 1.7 lp mm(-1)) vs MTF (range, 0.5-2.3 cycles per mm; median, 2.1 lp mm(-1)). Spatial resolution of CBCT images is approximately one order of magnitude lower than that of intraoral radiographs. Considering movement, scatter effects and other influences in real-world scans of living patients, a realistic spatial resolution of just above 1 lp mm(-1) could be expected.

  12. Application of model output statistics to the GEM-AQ high resolution air quality forecast

    NASA Astrophysics Data System (ADS)

    Struzewska, J.; Kaminski, J. W.; Jefimow, M.

    2016-11-01

    The aim of the presented work was to analyse the impact of data stratification on the efficiency of the Model Output Statistics (MOS) methodology as applied to a high-resolution deterministic air quality forecast carried out with the GEM-AQ model. The following parameters forecasted by the GEM-AQ model were selected as predictors for the MOS equation: pollutant concentration, air temperature in the lowest model layer, wind speed in the lowest model layer, temperature inversion and the precipitation rate. A representative 2-year series were used to construct regression functions. Data series were divided into two subsets. Approximately 75% of the data (first 3 weeks of each month) were used to estimate the regression function parameters. Remaining 25% (last week of each month) were used to test the method (control period). The subsequent 12 months were used for method verification (verification period). A linear model fitted the function based on forecasted parameters to the observations. We have assumed four different temperature-based data stratification methods (for each method, separate equations were constructed). For PM10 and PM2.5, SO2 and NO2 the best correction results were obtained with the application of temperature thresholds in the cold season and seasonal distribution combined with temperature thresholds in the warm season. For the PM10, PM2.5 and SO2 the best results were obtained using a combination of two stratification methods separately for cold and warm seasons. For CO, the systematic bias of the forecasted concentrations was partly corrected. For ozone more sophisticated methods of data stratification did not bring a significant improvement.

  13. Analyzing Protein Clusters on the Plasma Membrane: Application of Spatial Statistical Analysis Methods on Super-Resolution Microscopy Images.

    PubMed

    Paparelli, Laura; Corthout, Nikky; Pavie, Benjamin; Annaert, Wim; Munck, Sebastian

    2016-01-01

    The spatial distribution of proteins within the cell affects their capability to interact with other molecules and directly influences cellular processes and signaling. At the plasma membrane, multiple factors drive protein compartmentalization into specialized functional domains, leading to the formation of clusters in which intermolecule interactions are facilitated. Therefore, quantifying protein distributions is a necessity for understanding their regulation and function. The recent advent of super-resolution microscopy has opened up the possibility of imaging protein distributions at the nanometer scale. In parallel, new spatial analysis methods have been developed to quantify distribution patterns in super-resolution images. In this chapter, we provide an overview of super-resolution microscopy and summarize the factors influencing protein arrangements on the plasma membrane. Finally, we highlight methods for analyzing clusterization of plasma membrane proteins, including examples of their applications.

  14. Melt inclusions: Chapter 6

    USGS Publications Warehouse

    ,; Lowenstern, J. B.

    2014-01-01

    Melt inclusions are small droplets of silicate melt that are trapped in minerals during their growth in a magma. Once formed, they commonly retain much of their initial composition (with some exceptions) unless they are re-opened at some later stage. Melt inclusions thus offer several key advantages over whole rock samples: (i) they record pristine concentrations of volatiles and metals that are usually lost during magma solidification and degassing, (ii) they are snapshots in time whereas whole rocks are the time-integrated end products, thus allowing a more detailed, time-resolved view into magmatic processes (iii) they are largely unaffected by subsolidus alteration. Due to these characteristics, melt inclusions are an ideal tool to study the evolution of mineralized magma systems. This chapter first discusses general aspects of melt inclusions formation and methods for their investigation, before reviewing studies performed on mineralized magma systems.

  15. Congruent Melting Kinetics: Constraints on Chondrule Formation

    NASA Technical Reports Server (NTRS)

    Greenwood, James P.; Hess, Paul C.

    1995-01-01

    The processes and mechanisms of melting and their applications to chondrule formation are discussed A model for the kinetics of congruent melting is developed and used to place constraints on the duration and maximum temperature experienced by the interiors of relict-bearing chondrules. Specifically, chondrules containing relict forsteritic olivine or enstatitic pyroxene cannot have been heated in excess of 1901 C or 1577 C, respectively, for more than a few seconds.

  16. Ultra high resolution molecular beam cars spectroscopy with application to planetary atmospheric molecules

    NASA Technical Reports Server (NTRS)

    Byer, R. L.

    1982-01-01

    The measurement of high resolution pulsed and continuous wave (CW) coherent anti-Stokes Raman spectroscopy (CARS) measurements in pulsed and steady state supersonic expansions were demonstrated. Pulsed molecular beam sources were characterized, and saturation of a Raman transition and, for the first time, the Raman spectrum of a complex molecular cluster were observed. The observation of CW CARS spectra in a molecular expansion and the effects of transit time broadening is described. Supersonic expansion is established as a viable technique for high resolution Raman spectroscopy of cold molecules with resolutions of 100 MH2.

  17. Nanoparticle-induced unusual melting and solidification behaviours of metals.

    PubMed

    Ma, Chao; Chen, Lianyi; Cao, Chezheng; Li, Xiaochun

    2017-01-18

    Effective control of melting and solidification behaviours of materials is significant for numerous applications. It has been a long-standing challenge to increase the melted zone (MZ) depth while shrinking the heat-affected zone (HAZ) size during local melting and solidification of materials. In this paper, nanoparticle-induced unusual melting and solidification behaviours of metals are reported that effectively solve this long-time dilemma. By introduction of Al2O3 nanoparticles, the MZ depth of Ni is increased by 68%, while the corresponding HAZ size is decreased by 67% in laser melting at a pulse energy of 0.18 mJ. The addition of SiC nanoparticles shows similar results. The discovery of the unusual melting and solidification of materials that contain nanoparticles will not only have impacts on existing melting and solidification manufacturing processes, such as laser welding and additive manufacturing, but also on other applications such as pharmaceutical processing and energy storage.