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Sample records for amplicon quantification maq

  1. Amplicon sequencing for the quantification of spoilage microbiota in complex foods including bacterial spores.

    Science.gov (United States)

    de Boer, Paulo; Caspers, Martien; Sanders, Jan-Willem; Kemperman, Robèr; Wijman, Janneke; Lommerse, Gijs; Roeselers, Guus; Montijn, Roy; Abee, Tjakko; Kort, Remco

    2015-01-01

    Spoilage of food products is frequently caused by bacterial spores and lactic acid bacteria. Identification of these organisms by classic cultivation methods is limited by their ability to form colonies on nutrient agar plates. In this study, we adapted and optimized 16S rRNA amplicon sequencing for quantification of bacterial spores in a canned food matrix and for monitoring the outgrowth of spoilage microbiota in a ready-to-eat food matrix. The detection limit of bar-coded 16S rRNA amplicon sequencing was determined for the number of bacterial spores in a canned food matrix. Analysis of samples from a canned food matrix spiked with a mixture of equinumerous spores from the thermophiles, Geobacillus stearothermophilus and Geobacillus thermoglucosidans, and the mesophiles, Bacillus sporothermodurans, Bacillus cereus, and Bacillus subtilis, led to the detection of these spores with an average limit of 2 × 10(2) spores ml(-1). The data were normalized by setting the number of sequences resulting from DNA of an inactivated bacterial species, present in the matrix at the same concentration in all samples, to a fixed value for quantitative sample-to-sample comparisons. The 16S rRNA amplicon sequencing method was also employed to monitor population dynamics in a ready-to-eat rice meal, incubated over a period of 12 days at 7 °C. The most predominant outgrowth was observed by the genera Leuconostoc, Bacillus, and Paenibacillus. Analysis of meals pre-treated with weak acids showed inhibition of outgrowth of these three genera. The specificity of the amplicon synthesis was improved by the design of oligonucleotides that minimize the amplification of 16S rRNA genes from chloroplasts originating from plant-based material present in the food. This study shows that the composition of complex spoilage populations, including bacterial spores, can be monitored in complex food matrices by bar-coded amplicon sequencing in a quantitative manner. In order to allow sample

  2. Amplicon sequencing for the quantification of spoilage microbiota in complex foods including bacterial spores

    NARCIS (Netherlands)

    Boer, de P.; Caspers, M.; Sanders, J.W.; Kemperman, R.; Wijman, J.; Lommerse, G.; Roeselers, G.; Montijn, R.; Abee, T.; Kort, R.

    2015-01-01

    Background
    Spoilage of food products is frequently caused by bacterial spores and lactic acid bacteria. Identification of these organisms by classic cultivation methods is limited by their ability to form colonies on nutrient agar plates. In this study, we adapted and optimized 16S rRNA amplicon

  3. Mobile Air Quality Studies (MAQS-an international project

    Directory of Open Access Journals (Sweden)

    Sudik Claudia

    2010-04-01

    Full Text Available Abstract Due to an increasing awareness of the potential hazardousness of air pollutants, new laws, rules and guidelines have recently been implemented globally. In this respect, numerous studies have addressed traffic-related exposure to particulate matter using stationary technology so far. By contrast, only few studies used the advanced technology of mobile exposure analysis. The Mobile Air Quality Study (MAQS addresses the issue of air pollutant exposure by combining advanced high-granularity spatial-temporal analysis with vehicle-mounted, person-mounted and roadside sensors. The MAQS-platform will be used by international collaborators in order 1 to assess air pollutant exposure in relation to road structure, 2 to assess air pollutant exposure in relation to traffic density, 3 to assess air pollutant exposure in relation to weather conditions, 4 to compare exposure within vehicles between front and back seat (children positions, and 5 to evaluate "traffic zone"-exposure in relation to non-"traffic zone"-exposure. Primarily, the MAQS-platform will focus on particulate matter. With the establishment of advanced mobile analysis tools, it is planed to extend the analysis to other pollutants including NO2, SO2, nanoparticles and ozone.

  4. Maqâm-e Delkash: A Comparative Look at the Concept and Characteristics of Maqâm in Persian Dastgâhi Music

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    Ârash Mohâfez

    2015-12-01

    Full Text Available This is an English translation of an article originally published as “Maqâm-e Delkash: Negâhi tatbiqi be mafhum va khosusiyât-e maqâm dar musiqi-ye dastgâhi-ye Iran.” Faslnâme-ye Musiqi-ye Mâhur [Mahoor Music Quarterly] 53, 2011, 105–142. Draft translation: Leyla Rasuli, copy-editing: Kristen Wolf.

  5. Signifikansi Maqâsid al-Sharî‘ah sebagai Kerangka Berpikir Epistemik Ijtihad

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    Sanuri Sanuri

    2015-01-01

    Full Text Available This paper focuses on the importance of maqâsid al-sharî‘ah for the effectiveness of ijtihad in Islamic law. The emergence of controversies at the beginning of the tenth century AD on the issue of the closing of the gate of ijtihâd has resulted in the rigidity of Islamic law and its methodological framework. Along with these issues, some contemporary scholars on maqâsid agreed that sharî‘ah law (al-Qur’ân consists of partial (juz’îyât and universal values (kullîyyât that should be understood through a holistic approach in the frameworks of maqâsid. Shift in the meaning and orientation of maqâsid al-sharî‘ah in some contemporary Muslim scholars’ views, involving social sciences, philosophy of law, principles of morality, universality, social justice, human dignity, human rights, is a concrete manifestation of how Islamic law is able to provide answers to the current problems faced by the Muslim and non-Muslims community. This awareness has made contemporary Muslim thinkers strive to bring Islamic law into various achievements of progress in many aspects of life by emphasizing the importance of maqâsid al-sharî‘ah.

  6. Maqāmāt Dalam Manthiq Al-Thayr Al-Attār

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    Syamsun Ni`am

    2015-12-01

    Full Text Available Abstract : Attār is one to be accounted amongst the greatest sufi poets of Persia sufis in the history of Sufism who has notable contibution to the esoteric Islam (Sufism. He packed his mystical teachings in the form of poetry and verse, -to be more interactive and expressive- though his teachings are not different with his predecessors, particularly of those who inspired by the principle of mystical union; and Attār is among the Sufis who have managed to reach the highest peak in the mystical journey. According to Attār , there are seven valleys that must be passed by spiritual traveler (sālik in his mystical journey, as mentioned in Manthiq al-Thayr. In this work he use the language of poetry to reveal the process of a servant to God through the stages (maqāmāt, and Attār  gives the parable maqāmāt is like a bird -burung flying looking for the king. Attār call maqāmāt is the term valleys, of which there are seven valleys.Keywords : Maqāmāt, seven valleys, fanā’, divine loveAbstrak : Attar adalah salah satu sufi besar Persia yang pernah dikenal dalam sejarah tasawuf. Attār telah banyak memberikan kontribusi pemikirannya dalam khasanah keilmuan Islam esoteris (tasawuf. Ia mengemas ajaran tasawufnya dalam bentuk puisi dan syair, —sehingga tampak lebih menarik— meskipun ajaran-ajaran yang disampaikan kurang lebih sama dengan para sufi pendahulunya, terutama yang menganut paham mystical union; dan Attār adalah di antara sufi yang telah berhasil mencapai puncak tertinggi dalam perjalanan mistisnya itu. Menurut Attār, ada tujuh lembah yang harus dilalui sālik dalam menempuh perjalanan mistisnya, sebagaimana disebutkan dalam Manthiq al-Thayr. Dalam karyanya ini, Attār menggunakan bahasa puisi dan syair untuk mengungkap proses seorang hamba menuju Tuhan melalui tahapan-tahapan (Maqāmāt, dan Attār memberikan perumpamaan maqāmāt tersebut seperti burung-burung yang terbang mencari rajanya. Attār  menyebut maq

  7. Maqâsid al-Qur’ân dan Deradikalisasi Penafsiran dalam Konteks Keindonesiaan

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    Ulya Fikriyati

    2015-09-01

    Full Text Available This article deals with the viewpoint that the reading of the Qur’ân by a certain generation is subject to criticism by the following generation. The article seeks to offer, as an example, the deradicalization of interpreting the so-called ‘radical’ verses of the Qur’ân in Indonesian context. Islam in Indonesia always interact with various races, ethnicities, religions and beliefs, and therefore requires a type of exegesis different from other regions such as the Middle East. For the radicalization of interpretation, this article offers what is called the maqâsid al-Qur’ân as its parameter. The maqâsid al-Qur’ân consists of seven points: 1 Hifz al-dîn wa tatwîr wasâilih, 2 Hifz al-nafs wa tatwîruhâ, 3 Hifz al-‘aql wa tatwîruh, 4 Hifz al-mâl wa tanmîyat wasâilih, 5 Hifz al-‘ird wa tatwîr al-wasâil li al-husûl ‘alayh, 6 Tahqîq al-huqûq al-insânîyah wa mâ yandarij tahtahâ, 7 Hifz al-‘âlam wa ‘imâratuhâ. As spirit and parameter, the maqâsid al-Qur’ân necessitates the dialectics of dynamic interpretation without any judgment of infidelity or heresy. If a certain reading of the Qur’anic verses deviates from these seven maqâsid al-Qur’ân above, it deserves to be examined further, but not to be immediately suppressed.

  8. (SELAX strain) B2 amplicon

    African Journals Online (AJOL)

    USER

    2010-07-12

    Jul 12, 2010 ... Restriction enzyme digests of the clones suggested that the cloned portion of the B2 amplicon was 16 kb. Key words: B2 gene, A2B2 esterase, insecticide resistance. INTRODUCTION. Organophosphate resistance in Culex pipiens complex mosquitoes has been shown to be correlated with the presence of ...

  9. Removing Noise From Pyrosequenced Amplicons

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    Davenport Russell J

    2011-01-01

    Full Text Available Abstract Background In many environmental genomics applications a homologous region of DNA from a diverse sample is first amplified by PCR and then sequenced. The next generation sequencing technology, 454 pyrosequencing, has allowed much larger read numbers from PCR amplicons than ever before. This has revolutionised the study of microbial diversity as it is now possible to sequence a substantial fraction of the 16S rRNA genes in a community. However, there is a growing realisation that because of the large read numbers and the lack of consensus sequences it is vital to distinguish noise from true sequence diversity in this data. Otherwise this leads to inflated estimates of the number of types or operational taxonomic units (OTUs present. Three sources of error are important: sequencing error, PCR single base substitutions and PCR chimeras. We present AmpliconNoise, a development of the PyroNoise algorithm that is capable of separately removing 454 sequencing errors and PCR single base errors. We also introduce a novel chimera removal program, Perseus, that exploits the sequence abundances associated with pyrosequencing data. We use data sets where samples of known diversity have been amplified and sequenced to quantify the effect of each of the sources of error on OTU inflation and to validate these algorithms. Results AmpliconNoise outperforms alternative algorithms substantially reducing per base error rates for both the GS FLX and latest Titanium protocol. All three sources of error lead to inflation of diversity estimates. In particular, chimera formation has a hitherto unrealised importance which varies according to amplification protocol. We show that AmpliconNoise allows accurate estimates of OTU number. Just as importantly AmpliconNoise generates the right OTUs even at low sequence differences. We demonstrate that Perseus has very high sensitivity, able to find 99% of chimeras, which is critical when these are present at high

  10. Investigating Meat Milling Business in Yogyakarta: a Maqâshid al-Syarî‘ah Perspectives

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    Zein Muttaqin

    2017-12-01

    Full Text Available Investigating Meat Milling Business in Yogyakarta: A Maqâshid al-Syarî‘ah Perspectives. This article aims to investigate the meat milling business process in the light of maqâshid al-syarî‘ah perspectives. The ttopic arises due to some past cases related to the mixed process of meat milling of lawful and unlawful meat in the industry, while the demand on the meat milling business is pretty high according to the BAPPENAS Yogyakarta data. Lacking on supervision has threatened consumer protection and deviates from the sharia injunctions. Maqâshid al-syarî‘ah being consider as the basis of sharia compliances that serves to understand the purposes of sharia laws in the daily life in the form of prohibitions and injunctions. Qualitative method and content analysis is conducted to interpret 7 meats milling local shop owner responds regards the manifestation of maqâshid al-syarî‘ah in their business. It can be concluded that the implementation of maqâshid al-syarî‘ah of meat milling business in Yogyakarta on the level of input, process and output has been implemented accordingly. Although there are several cases of respondent that did not abide by the Islamic laws, but most of the businessman are. Halal logistic and supply chain is needed to become the insurance party of the legal aspect of business and also as the milestone of expanding the local meat milling to compete with the bigger competitor such as supermarket.

  11. Problematika Pendidikan Islam Perspektif Maqâṣid Sharîʻah

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    Rosidin Rosidin

    2016-11-01

    Full Text Available The article seeks to reveal problems faced by Islamic education from the perspective of Maqâṣid al-Sharîʻah, namely ideological (h}ifẓ al-dîn, practical (h}ifẓ al-nafs, academic (h}ifẓ al-‘aql, relationships or networks (h}ifẓ al-nasl, vocational (h}ifẓ al-mâl and quality (h}ifẓ al-‘irḍ aspects. In order to know the details of crucial problems of the Islamic education along with their alternative solutions, this paper suggests that the Islamic education should consider the insider’s and outsider’s perspective which has four types of roles as follow: a complete observer; b observer as participant; c participant as observer; d complete participant. This article proposes three important implications, are: first, the problems of the Islamic education can be categorized and mapped based on Maqâṣid al-Sharîʻah, in order to ease the analysis of such problems. Second, the analysis of the problems should be based on the insiders’ and outsiders’ point of views, so that it would be able to comprehensively detect the problems of Islamic education and show the inclusiveness of the Islamic education. Third, problem solving alternatives should be oriented at the level of theological (faith and religious [îmân], theoretical (philosophical and empirical [‘ilm], practical (learning and teaching [‘amal] and moral (ethics and aesthetics [akhlâq] realms.

  12. Al-‘Alāqah baina Ushūl al-Fiqh wa Maqāshidi al-Sharīah wa al-Da’wah ilā Ta’sīsi ‘Ilmi al-Maqāshid

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    Anggraini Binti Ramli

    2016-12-01

    Full Text Available The study of Maqāshid sharīa is an important point in the discussion of Islamic legal theory (ushūl al-fiqh. Serious debates began to emerge in the 19th century among Islamic jurists concerning the position of maqāshid sharīa. At least, there are three important debates in the history; first, whether maqāshid is part of the discussion ushūl al-fiqh; second, is maqāshid sharīa built upon a foundation of classical Islamic jurisprudence (fiqh; and third, whether the maqāshid sharīa study is able to become an independent science that is separated from the study of classical Islamic jurisprudence. This article tries to present a discussion of the three paradigms by employing a descriptive-analytic method. The results of this study uncover that the study of maqāshid sharīa is like two sides of one coin; theoretically it is a distinctive study from ushūl al-fiqh, but it cannot be separated from one another. Ushūl al-fiqh has become the foundation to find out more details about the study of maqashid sharia. The separation between classical Islamic jurisprudence (fiqh and maqāshid sharīa study conducted by Islamic jurists is a relative separation.

  13. Dispute management in Islamic financial services and products: A maqāṣid-based analysis

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    Umar A. Oseni

    2015-12-01

    Full Text Available The increasing expansion of the Islamic financial services industry beyond its original frontiers has not only come with success stories but has also been affected by the growing preference for litigation as the mode of dispute resolution. Exorbitant legal fees and cost of sustaining protracted litigation are two major challenges that require the attention of major stakeholders in the industry. This paper examines these challenges through a Maqāṣid al-Sharī‘ah focused prism considering the importance of the sustainable dispute management framework in Islamic financial services and products. While singling out the important higher objective (maqṣad of ḥifẓ al-māl, this study argues that preservation of wealth and financial resources requires effective means of resolving increasingly diverse disputes in the Islamic financial services industry. It is further argued that an effective dispute management framework will consider the original value proposition of Islamic financial intermediation which promotes maṣlaḥah (benefits and prevents mafsadah (hardship and ḍarar (financial harm. This makes a case for the affirmative relevance, potential adoption, and systemic modernisation of Islamic dispute management mechanisms such as ṣulḥ, taḥkīm, and muḥtasib in order to fulfil the overarching objective of protection and preservation of wealth and financial resources as one of the core objectives of Sharī‘ah.

  14. The Discourse of Medicine in the Čahār Maqāla (Four Discourses) of Nezami Aruzi of Samarghand.

    Science.gov (United States)

    Afshar, Ahmadreza

    2015-09-01

    Nezami Aruzi prepared Čahār Maqāla (Four Discourses) as a guide and admonishment for the rulers and kings. The fourth discourse of Čahār Maqāla with 12 anecdotes is devoted to the science of medicine and the characteristics of the physicians. The discourse presents the name of the eminent scientists, physicians, as well as Farsi and Arabic medical books that had professional acceptance in the medieval in Persia. The author has described how medicine was studied in the medieval in Persia and has presented notes on the physiology of the nervous system, pulse, uroscopy, fever, spiritual affairs and medical ethics. The current essay is a brief review of the medical subjects in Čahār Maqāla.

  15. MaqFACS (Macaque Facial Action Coding System can be used to document facial movements in Barbary macaques (Macaca sylvanus

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    Églantine Julle-Danière

    2015-09-01

    Full Text Available Human and non-human primates exhibit facial movements or displays to communicate with one another. The evolution of form and function of those displays could be better understood through multispecies comparisons. Anatomically based coding systems (Facial Action Coding Systems: FACS are developed to enable such comparisons because they are standardized and systematic and aid identification of homologous expressions underpinned by similar muscle contractions. To date, FACS has been developed for humans, and subsequently modified for chimpanzees, rhesus macaques, orangutans, hylobatids, dogs, and cats. Here, we wanted to test whether the MaqFACS system developed in rhesus macaques (Macaca mulatta could be used to code facial movements in Barbary macaques (M. sylvanus, a species phylogenetically close to the rhesus macaques. The findings show that the facial movement capacity of Barbary macaques can be reliably coded using the MaqFACS. We found differences in use and form of some movements, most likely due to specializations in the communicative repertoire of each species, rather than morphological differences.

  16. Freedom, Sufism, maqâm hurrîyah, hâl hurrîyah.

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    Ah. Haris Fakhrudi

    2015-09-01

    Full Text Available This paper will discuss the meaning of freedom in the discourse of Sufi thought, especially of Ibn ‘Arabî. This is based on the consideration that Sufism before Ibn ‘Arabî’s more focused on ritualistic orientation for students and only revealed variant of Sufi’s expressions, both on maqâmât and ahwâl. The presence of Ibn ‘Arabî, therefore, became the turning point in the discourse of Sufism by expressing his beliefs in the theoretical formulation. The doctrine of Sufism—which previously only implicitly contained in the words of the Sufi shaykh—in the hands of Ibn ‘Arabî flashed into an open, theoretical, and obvios and thus opened the door for anyone who has a high intelligence in reflecting at once and realizing the metaphysical theories through operational forms. Therefore, this article will discuss some of the key concepts in the thought of Ibn ‘Arabî including the meaning of freedom (al-hurrîyah in Sufism, maqâm hurrîyah, and hâl hurrîyah obtained by the Sufis during their spiritual journey.

  17. Cooperative Epigenetic Modulation by Cancer Amplicon Genes

    Science.gov (United States)

    Rui, Lixin; Tolga Emre, N. C.; Kruhlak, Michael J.; Chung, Hye-Jung; Steidl, Christian; Slack, Graham; Wright, George W.; Lenz, Georg; Ngo, Vu N.; Shaffer, Arthur L.; Xu, Weihong; Zhao, Hong; Yang, Yandan; Lamy, Laurence; Davis, R. Eric; Xiao, Wenming; Powell, John; Maloney, David; Thomas, Craig J.; Möller, Peter; Rosenwald, Andreas; Ott, German; Muller-Hermelink, Hans Konrad; Savage, Kerry; Connors, Joseph M.; Rimsza, Lisa M.; Campo, Elias; Jaffe, Elaine S.; Delabie, Jan; Smeland, Erlend B.; Weisenburger, Dennis D.; Chan, Wing C.; Gascoyne, Randy D.; Levens, David; Staudt, Louis M.

    2010-01-01

    Chromosome band 9p24 is frequently amplified in primary mediastinal B-cell lymphoma (PMBL) and Hodgkin lymphoma (HL). To identify oncogenes in this amplicon, we screened an RNA interference library targeting amplicon genes and thereby identified JAK2 and the histone demethylase JMJD2C as essential genes in these lymphomas. Inhibition of JAK2 and JMJD2C cooperated in killing these lymphomas by decreasing tyrosine 41 phosphorylation and increasing lysine 9 trimethylation of histone H3, promoting heterochromatin formation. MYC, a major target of JAK2-mediated histone phosphorylation, was silenced following JAK2 and JMJD2C inhibition, with a corresponding increase in repressive chromatin. Hence, JAK2 and JMJD2C cooperatively remodel the PMBL and HL epigenome, offering a mechanistic rationale for the development of JAK2 and JMJD2C inhibitors in these diseases. PMID:21156283

  18. Proposed Methodology for Application of Human-like gradual Multi-Agent Q-Learning (HuMAQ) for Multi-robot Exploration

    International Nuclear Information System (INIS)

    Ray, Dip Narayan; Majumder, Somajyoti

    2014-01-01

    Several attempts have been made by the researchers around the world to develop a number of autonomous exploration techniques for robots. But it has been always an important issue for developing the algorithm for unstructured and unknown environments. Human-like gradual Multi-agent Q-leaming (HuMAQ) is a technique developed for autonomous robotic exploration in unknown (and even unimaginable) environments. It has been successfully implemented in multi-agent single robotic system. HuMAQ uses the concept of Subsumption architecture, a well-known Behaviour-based architecture for prioritizing the agents of the multi-agent system and executes only the most common action out of all the different actions recommended by different agents. Instead of using new state-action table (Q-table) each time, HuMAQ uses the immediate past table for efficient and faster exploration. The proof of learning has also been established both theoretically and practically. HuMAQ has the potential to be used in different and difficult situations as well as applications. The same architecture has been modified to use for multi-robot exploration in an environment. Apart from all other existing agents used in the single robotic system, agents for inter-robot communication and coordination/ co-operation with the other similar robots have been introduced in the present research. Current work uses a series of indigenously developed identical autonomous robotic systems, communicating with each other through ZigBee protocol

  19. Maqâshid al-Qur’ân dalam Ayat Kebebasan Beragama Menurut Thahâ Jâbir al-‘Alwânî

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    Ah. Fawaid

    2017-12-01

    Full Text Available Maqâshid al-Qur’ân in the Verses of Religious Freedom in Interpretation of Thahâ Jâbir al-’Alwânî. This article is aimed at describing Thahâ Jâbir al-‘Alwânî’s interpretation on various verses dealing with the issues of religious freedom. Adapting an approach of maqâshid al-Qur’ân, the present paper comes to answer two main issues on the true of Alwânî’s maqâshid al-Qur’ân perception and the theoretical application of Alwânî’s maqâshid al-Qur’ân in interpreting verses of religious freedom. According to ‘Alwânî, it was concluded that there are three main segments that he called al-maqâshid al-Qur’âniyyah al-hâkimah: (1 al-tawhîd, (2 al-tazkiyah, and (3 al-‘umrân. ‘Alwânî stated that freedom of interfaith religion is important goal of sharia meaning. Freedom of interfaith religion, on the other hand, is one of the important embodiments in believing the God and tauhid. Seeing this pattern, the later purpose of the Quran is tazkiyah. This term is a value that enables people apply the message, fulfill the promise, and can perform the tasks of the caliphate. When such principles are implemented, something appears that Alwânî called ‘umrân as the next purpose of the Quran can be manifested well. ‘Umrân or ‘prosperity’ in which human being performs as a khalifah actually can create baldatun thayyibatun wa rabbun ghafûr as the real welfare.

  20. Optimal Activation of Isopsoralen To Prevent Amplicon Carryover

    OpenAIRE

    Fahle, Gary A.; Gill, Vee J.; Fischer, Steven H.

    1999-01-01

    We compared the efficiencies of activation of the photochemical isopsoralen compound 10 and its resulting amplicon neutralizations under conditions with a UV transilluminator box at room temperature (RT) and a HRI-300 UV photothermal reaction chamber at RT and at 5°C. Our data suggest that use of the HRI-300 reaction chamber at 5°C results in a statistically significantly higher degree of amplicon neutralization.

  1. Hak Kebebasan Berpendapat dalam Hubungannya dengan Pencemaran Nama Baik Menurut KUHP; Perspektif Teori Maqâṣid Sharî’ah

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    Moh. Faizur Rohman

    2017-11-01

    Full Text Available Human rights are often echoed by various parties are sometimes opposed to each other. Many journalists who exercised the right to freedom of expression expressed the opinion that the right has been protected by the 1945 Constitution article 28 E (3, precisely resulting in a criminal offense of defamation for those who do not like it. Defamation articles in the Criminal Code are often regarded as a powerful weapon against a criticism as a form of antipathy against criticism. Therefore, there should be efforts to classify the forms of the use of rights and forms of criminal acts. and will be seen from the standpoint of maqâṣid sharî’ah. Through approach statue approach, and case approach, obtained that the law has protected the rights of everyone in expressing opinions. Every person shall have the right and freedom to express opinions but such rights shall not infringe the rights of others. In maqâṣid is guaranteed and protected self-esteem, the soul for every human being is contained in the principle of ḥifḍu al-nafs wa al-‘ird. Therefore, in every opinion, communicate and socialize should pay attention to the rights of others is the dignity of one's dignity to create a balanced and harmonious life.

  2. Harmonising legality with morality in Islamic banking and finance: A quest for Maqāṣid al-Sharī‘ah paradigm

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    Luqman Zakariyah

    2015-12-01

    Full Text Available Scholars in Islamic Finance Industry (IFI have been calling for the integration of Islamic morality with legal theories in the industry. Among the reasons for this call is an unethical trend in product innovation. Implementing Islamic banking and financial practices would require adopting their undergirding Islamic legal and moral frameworks. Departing from these foundations of Islamic law could render the activities conducted under its name religiously unacceptable. Many approaches have been put forward to achieve this cause. One of the most complex yet subjective approaches is the quest for Maqāṣid al-Sharī‘ah. This paper critically examines the feasibility of harmonising morality with legality in Islamic finance. In doing so, it will reveal what constitutes morality and legality in Islamic legal theory, and critically examine the approaches of Muslim classical scholars in fusing the two elements together for the realisation and actualisation of the very objectives of Sharī‘ah. Questions of the relationship between morality and legality are raised, and samples of Islamic finance products are evaluated to expose their moral and legal dimensions. Lastly, the role of Maqāṣid al-Sharī‘ah in the process of harmonisation is discussed with some observations and reservations on the practicality of their implementation.

  3. High throughput 16S rRNA gene amplicon sequencing

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Larsen, Poul; Jørgensen, Mads Koustrup

    S rRNA gene amplicon sequencing has been developed over the past few years and is now ready to use for more comprehensive studies related to plant operation and optimization thanks to short analysis time, low cost, high throughput, and high taxonomic resolution. In this study we show how 16S r......RNA gene amplicon sequencing can be used to reveal factors of importance for the operation of full-scale nutrient removal plants related to settling problems and floc properties. Using optimized DNA extraction protocols, indexed primers and our in-house Illumina platform, we prepared multiple samples...... be correlated to the presence of the species that are regarded as “strong” and “weak” floc formers. In conclusion, 16S rRNA gene amplicon sequencing provides a high throughput approach for a rapid and cheap community profiling of activated sludge that in combination with multivariate statistics can be used...

  4. Innovación y ruptura en la métrica de la poesía en al-Maqāmāt al-Luzūmiyya de al-Saraqusṭī (s. VI/XII

    Directory of Open Access Journals (Sweden)

    Ferrando, Ignacio

    2016-06-01

    Full Text Available Al-Maqāmāt al-Luzūmiyya, a collection of 50 picaresque-like stories couched in rhymed prose by Abū l-Ṭāhir al-Saraqusṭī in al-Andalus in the VI/XIIth century, were considered by Western scholars, up until the eighties of XXth century, as highly rhetorical pieces with a negligible and irrelevant content. However, some contemporary scholars argued that they contain some degree of social and political criticism, as they constitute a fictional domain in which subversion and irony play a fundamental role. In this paper, we cast some light on the particular use of metrical patterns in Al-Maqāmāt al-Luzūmiyya, which should be considered another subversion field: a poetical rhythm that avoids the most usual Arabic metrical patterns, contributing to provide the maqāma a flavor of the countergenre.Al-Maqāmāt al-Luzūmiyya, la colección de 50 relatos de corte picaresco escritos en una exigente prosa rimada por Abū l-Ṭāhir al-Saraqusṭī en al-Andalus en el siglo VI/XII, han sido consideradas por los críticos occidentales como piezas de alta retórica y de virtuosismo lingüístico en las que el contenido es prácticamente irrelevante. Sin embargo, a partir de los años 80 del siglo pasado, algunos investigadores han tratado de incidir en el hecho de que esta obra contiene cierto grado de crítica social y política, puesto que constituye un espacio ficticio en el que la subversión y la ironía desempeñan un papel fundamental. En este trabajo vamos a tratar de arrojar luz sobre el uso particular de los esquemas métricos por parte de al-Saraqusṭī, tratando de mostrar que los metros constituyen otro más de los ámbitos de subversión tan caros al autor. De hecho, el uso de ritmos “invertidos” o contrarios a los más habituales de la poesía clásica contribuye a darle a la maqāma ese aire de anti género que la puebla.

  5. Swarm: robust and fast clustering method for amplicon-based studies

    Science.gov (United States)

    Rognes, Torbjørn; Quince, Christopher; de Vargas, Colomban; Dunthorn, Micah

    2014-01-01

    Popular de novo amplicon clustering methods suffer from two fundamental flaws: arbitrary global clustering thresholds, and input-order dependency induced by centroid selection. Swarm was developed to address these issues by first clustering nearly identical amplicons iteratively using a local threshold, and then by using clusters’ internal structure and amplicon abundances to refine its results. This fast, scalable, and input-order independent approach reduces the influence of clustering parameters and produces robust operational taxonomic units. PMID:25276506

  6. FlowClus: efficiently filtering and denoising pyrosequenced amplicons.

    Science.gov (United States)

    Gaspar, John M; Thomas, W Kelley

    2015-03-27

    Reducing the effects of sequencing errors and PCR artifacts has emerged as an essential component in amplicon-based metagenomic studies. Denoising algorithms have been designed that can reduce error rates in mock community data, but they change the sequence data in a manner that can be inconsistent with the process of removing errors in studies of real communities. In addition, they are limited by the size of the dataset and the sequencing technology used. FlowClus uses a systematic approach to filter and denoise reads efficiently. When denoising real datasets, FlowClus provides feedback about the process that can be used as the basis to adjust the parameters of the algorithm to suit the particular dataset. When used to analyze a mock community dataset, FlowClus produced a lower error rate compared to other denoising algorithms, while retaining significantly more sequence information. Among its other attributes, FlowClus can analyze longer reads being generated from all stages of 454 sequencing technology, as well as from Ion Torrent. It has processed a large dataset of 2.2 million GS-FLX Titanium reads in twelve hours; using its more efficient (but less precise) trie analysis option, this time was further reduced, to seven minutes. Many of the amplicon-based metagenomics datasets generated over the last several years have been processed through a denoising pipeline that likely caused deleterious effects on the raw data. By using FlowClus, one can avoid such negative outcomes while maintaining control over the filtering and denoising processes. Because of its efficiency, FlowClus can be used to re-analyze multiple large datasets together, thereby leading to more standardized conclusions. FlowClus is freely available on GitHub (jsh58/FlowClus); it is written in C and supported on Linux.

  7. Mobile air quality studies (MAQS in inner cities: particulate matter PM10 levels related to different vehicle driving modes and integration of data into a geographical information program

    Directory of Open Access Journals (Sweden)

    Uibel Stefanie

    2012-10-01

    Full Text Available Abstract Background Particulate matter (PM is assumed to exert a major burden on public health. Most studies that address levels of PM use stationary measure systems. By contrast, only few studies measure PM concentrations under mobile conditions to analyze individual exposure situations. Methods By combining spatial-temporal analysis with a novel vehicle-mounted sensor system, the present Mobile Air Quality Study (MAQS aimed to analyse effects of different driving conditions in a convertible vehicle. PM10 was continuously monitored in a convertible car, driven with roof open, roof closed, but windows open, or windows closed. Results PM10 values inside the car were nearly always higher with open roof than with roof and windows closed, whereas no difference was seen with open or closed windows. During the day PM10 values varied with high values before noon, and occasional high median values or standard deviation values due to individual factors. Vehicle speed in itself did not influence the mean value of PM10; however, at traffic speed (10 – 50 km/h the standard deviation was large. No systematic difference was seen between PM10 values in stationary and mobile cars, nor was any PM10 difference observed between driving within or outside an environmental (low emission zone. Conclusions The present study has shown the feasibility of mobile PM analysis in vehicles. Individual exposure of the occupants varies depending on factors like time of day as well as ventilation of the car; other specific factors are clearly identifiably and may relate to specific PM10 sources. This system may be used to monitor individual exposure ranges and provide recommendations for preventive measurements. Although differences in PM10 levels were found under certain ventilation conditions, these differences are likely not of concern for the safety and health of passengers.

  8. Deep amplicon sequencing reveals mixed phytoplasma infection within single grapevine plants

    DEFF Research Database (Denmark)

    Nicolaisen, Mogens; Contaldo, Nicoletta; Makarova, Olga

    2011-01-01

    The diversity of phytoplasmas within single plants has not yet been fully investigated. In this project, deep amplicon sequencing was used to generate 50,926 phytoplasma sequences from 11 phytoplasma-infected grapevine samples from a PCR amplicon in the 5' end of the 16S region. After clustering ...

  9. Multiplex PCR, amplicon size and hybridization efficiency on the NanoChip electronic microarray

    DEFF Research Database (Denmark)

    Børsting, Claus; Sanchez, Juan J; Morling, Niels

    2004-01-01

    . Hybridization to individual amplicons in multiplexes was less efficient suggesting that intramolecular and intermolecular interactions may block access to the target sequence on the NanoChip array. We observed a high risk of contamination with amplicons shorter than 60 bp and therefore, we recommend the use...

  10. Metatranscriptomics and Amplicon Sequencing Reveal Mutualisms in Seagrass Microbiomes

    Directory of Open Access Journals (Sweden)

    Byron C. Crump

    2018-03-01

    Full Text Available Terrestrial plants benefit from many well-understood mutualistic relationships with root- and leaf-associated microbiomes, but relatively little is known about these relationships for seagrass and other aquatic plants. We used 16S rRNA gene amplicon sequencing and metatranscriptomics to assess potential mutualisms between microorganisms and the seagrasses Zostera marina and Zostera japonica collected from mixed beds in Netarts Bay, OR, United States. The phylogenetic composition of leaf-, root-, and water column-associated bacterial communities were strikingly different, but these communities were not significantly different between plant species. Many taxa present on leaves were related to organisms capable of consuming the common plant metabolic waste product methanol, and of producing agarases, which can limit the growth of epiphytic algae. Taxa present on roots were related to organisms capable of oxidizing toxic sulfur compounds and of fixing nitrogen. Metatranscriptomic sequencing identified expression of genes involved in all of these microbial metabolic processes at levels greater than typical water column bacterioplankton, and also identified expression of genes involved in denitrification and in bacterial synthesis of the plant growth hormone indole-3-acetate. These results provide the first evidence using metatranscriptomics that seagrass microbiomes carry out a broad range of functions that may benefit their hosts, and imply that microbe–plant mutualisms support the health and growth of aquatic plants.

  11. Synthetic spike-in standards for high-throughput 16S rRNA gene amplicon sequencing.

    Science.gov (United States)

    Tourlousse, Dieter M; Yoshiike, Satowa; Ohashi, Akiko; Matsukura, Satoko; Noda, Naohiro; Sekiguchi, Yuji

    2017-02-28

    High-throughput sequencing of 16S rRNA gene amplicons (16S-seq) has become a widely deployed method for profiling complex microbial communities but technical pitfalls related to data reliability and quantification remain to be fully addressed. In this work, we have developed and implemented a set of synthetic 16S rRNA genes to serve as universal spike-in standards for 16S-seq experiments. The spike-ins represent full-length 16S rRNA genes containing artificial variable regions with negligible identity to known nucleotide sequences, permitting unambiguous identification of spike-in sequences in 16S-seq read data from any microbiome sample. Using defined mock communities and environmental microbiota, we characterized the performance of the spike-in standards and demonstrated their utility for evaluating data quality on a per-sample basis. Further, we showed that staggered spike-in mixtures added at the point of DNA extraction enable concurrent estimation of absolute microbial abundances suitable for comparative analysis. Results also underscored that template-specific Illumina sequencing artifacts may lead to biases in the perceived abundance of certain taxa. Taken together, the spike-in standards represent a novel bioanalytical tool that can substantially improve 16S-seq-based microbiome studies by enabling comprehensive quality control along with absolute quantification. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Use of AmpliWax to optimize amplicon sterilization by isopsoralen.

    OpenAIRE

    De la Viuda, M; Fille, M; Ruiz, J; Aslanzadeh, J

    1996-01-01

    The photochemical inactivation of amplicons by isopsoralen (IP-10) has been suggested as a possible means to prevent PCR carryover contamination. To evaluate the technique, serial dilutions of amplicons (10(11) to 10(3)) from the Borrelia burgdorferi OSP A gene were amplified in the presence of 0, 25, 50, and 100 micrograms of IP-10 per ml for 45 cycles. The PCR products were exposed to UV light for 15 min to activate IP-10 and sterilize the amplicons. One microliter of each sterilized sample...

  13. Characterization of Novel Genes With 8p11-12 Amplicon in Breast Cancer

    National Research Council Canada - National Science Library

    Yang, Zeng-Quan

    2004-01-01

    ...% of human breast cancer (HBC). Using chromosome comparative genomic hybridization (CGH) and array CGH, overlapping amplicons were found to be centered around chromosome 8p11-p12 in 3 breast cancer cell lines developed in Dr...

  14. JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms.

    Science.gov (United States)

    Petrillo, Mauro; Angers-Loustau, Alexandre; Henriksson, Peter; Bonfini, Laura; Patak, Alex; Kreysa, Joachim

    2015-01-01

    The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMO-Amplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website. Database URL: http://gmo-crl.jrc.ec.europa.eu/jrcgmoamplicons/. © The Author(s) 2015. Published by Oxford University Press.

  15. Efficient error correction for next-generation sequencing of viral amplicons

    Directory of Open Access Journals (Sweden)

    Skums Pavel

    2012-06-01

    Full Text Available Abstract Background Next-generation sequencing allows the analysis of an unprecedented number of viral sequence variants from infected patients, presenting a novel opportunity for understanding virus evolution, drug resistance and immune escape. However, sequencing in bulk is error prone. Thus, the generated data require error identification and correction. Most error-correction methods to date are not optimized for amplicon analysis and assume that the error rate is randomly distributed. Recent quality assessment of amplicon sequences obtained using 454-sequencing showed that the error rate is strongly linked to the presence and size of homopolymers, position in the sequence and length of the amplicon. All these parameters are strongly sequence specific and should be incorporated into the calibration of error-correction algorithms designed for amplicon sequencing. Results In this paper, we present two new efficient error correction algorithms optimized for viral amplicons: (i k-mer-based error correction (KEC and (ii empirical frequency threshold (ET. Both were compared to a previously published clustering algorithm (SHORAH, in order to evaluate their relative performance on 24 experimental datasets obtained by 454-sequencing of amplicons with known sequences. All three algorithms show similar accuracy in finding true haplotypes. However, KEC and ET were significantly more efficient than SHORAH in removing false haplotypes and estimating the frequency of true ones. Conclusions Both algorithms, KEC and ET, are highly suitable for rapid recovery of error-free haplotypes obtained by 454-sequencing of amplicons from heterogeneous viruses. The implementations of the algorithms and data sets used for their testing are available at: http://alan.cs.gsu.edu/NGS/?q=content/pyrosequencing-error-correction-algorithm

  16. Innovación y ruptura en la métrica de la poesía en al-Maqāmāt al-Luzūmiyya de al-Saraqusṭī (s. VI/XII)

    OpenAIRE

    Ferrando, Ignacio

    2016-01-01

    Al-Maqāmāt al-Luzūmiyya, la colección de 50 relatos de corte picaresco escritos en una exigente prosa rimada por Abū l-Ṭāhir al-Saraqusṭī en al-Andalus en el siglo VI/XII, han sido consideradas por los críticos occidentales como piezas de alta retórica y de virtuosismo lingüístico en las que el contenido es prácticamente irrelevante. Sin embargo, a partir de los años 80 del siglo pasado, algunos investigadores han tratado de incidir en el hecho de que esta obra contiene cierto grado de críti...

  17. Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis

    DEFF Research Database (Denmark)

    Lonowski, Lindsey A; Narimatsu, Yoshiki; Riaz, Anjum

    2017-01-01

    ). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second...... the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell...

  18. Use of AmpliWax to optimize amplicon sterilization by isopsoralen.

    Science.gov (United States)

    De la Viuda, M; Fille, M; Ruiz, J; Aslanzadeh, J

    1996-12-01

    The photochemical inactivation of amplicons by isopsoralen (IP-10) has been suggested as a possible means to prevent PCR carryover contamination. To evaluate the technique, serial dilutions of amplicons (10(11) to 10(3)) from the Borrelia burgdorferi OSP A gene were amplified in the presence of 0, 25, 50, and 100 micrograms of IP-10 per ml for 45 cycles. The PCR products were exposed to UV light for 15 min to activate IP-10 and sterilize the amplicons. One microliter of each sterilized sample was reamplified for an additional 45 cycles. The PCR products were then resolved in an agarose gel, blotted onto a nylon membrane, and probed with an alkaline phosphatase-conjugated chemiluminescent probe. Although IP-10 at concentrations of 50 and 100 micrograms/ml effectively sterilized up to 10(11) amplicons, the compound was inhibitory to PCR. IP-10 at a concentration of 25 micrograms/ml had slight inhibitory effect on PCR and did not completely sterilized all of the amplicons. Therefore, in subsequent experiments AmpliWax was substituted for mineral oil, and PCR was performed on 10(9) to 10(3) amplicons as described above. Following the amplification, the PCR tubes were cooled to solidify the AmpliWax and inoculated with various concentrations of IP-10. With this technique, PCR products produced from as many as 10(9) target amplicons were effectively sterilized with 200 micrograms of IP-10 per ml. Similarly, the addition of IP-10 (50 micrograms/ml) before and after PCR was evaluated for the detection of B. burgdorferi in 62 ticks from a region of Southern Connecticut where the organism is highly endemic. PCR performed in the presence of 50 micrograms of IP-10 per ml detected B. burgdorferi-specific DNA in 17 of 62 ticks (27%) following gel electrophoresis and in 34 of 62 ticks (55%) following Southern blot hybridization of the PCR products. In contrast, post-PCR addition of IP-10 detected borrelia-specific DNA in 31 of 62 ticks (50%) following gel electrophoresis and in

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

    DEFF Research Database (Denmark)

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

    2018-01-01

    of libraries exists. We assessed eight methods of quantification of libraries by quantifying 54 amplicon, six capture, and six shotgun fragment libraries. Chemically synthesized double-stranded DNA was also quantified. Light spectrophotometry, i.e. NanoDrop, was found to give the highest concentration......Quantification of massively parallel sequencing libraries is important for acquisition of monoclonal beads or clusters prior to clonal amplification and to avoid large variations in library coverage when multiple samples are included in one sequencing analysis. No gold standard for quantification...

  20. Overexpressed Genes/ESTs and Characterization of Distinct Amplicons on 17823 in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ayse E. Erson

    2001-01-01

    Full Text Available 17823 is a frequent site of gene amplification in breast cancer. Several lines of evidence suggest the presence of multiple amplicons on 17823. To characterize distinct amplicons on 17823 and localize putative oncogenes, we screened genes and expressed sequence tags (ESTs in existing physical and radiation hybrid maps for amplification and overexpression in breast cancer cell lines by semiquantitative duplex PCR, semiquantitative duplex RT-PCR, Southern blot, Northern blot analyses. We identified two distinct amplicons on 17823, one including TBX2 and another proximal region including RPS6KB1 (PS6K and MUL. In addition to these previously reported overexpressed genes, we also identified amplification and overexpression of additional uncharacterized genes and ESTs, some of which suggest potential oncogenic activity. In conclusion, we have further defined two distinct regions of gene amplification and overexpression on 17823 with identification of new potential oncogene candidates. Based on the amplification and overexpression patterns of known and as of yet unrecognized genes on 17823, it is likely that some of these genes mapping to the discrete amplicons function as oncogenes and contribute to tumor progression in breast cancer cells.

  1. Global Perspectives on Activated Sludge Community Composition analyzed using 16S rRNA amplicon sequencing

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Saunders, Aaron Marc; Albertsen, Mads

    Activated sludge is the most commonly applied bioprocess throughout the world for wastewater treatment. Microorganisms are key to the process, yet our knowledge of their identity and function is still limited. High-througput16S rRNA amplicon sequencing can reliably characterize microbial...

  2. Improved sensitivity of circulating tumor DNA measurement using short PCR amplicons

    DEFF Research Database (Denmark)

    Andersen, Rikke Fredslund; Spindler, Karen-Lise Garm; Brandslund, Ivan

    2015-01-01

    , however, presents a number of challenges that require attention. The amount of DNA is low and highly fragmented and analyses need to be optimized accordingly. KRAS ARMS-qPCR assays with amplicon lengths of 120 and 85 base pairs, respectively, were compared using positive control material (PCR fragments...

  3. Characterization of the 17p amplicon in human sarcomas: microsatellite marker analysis

    NARCIS (Netherlands)

    Wolf, M.; Tarkkanen, M.; Hulsebos, T.; Larramendy, M. L.; Forus, A.; Myklebost, O.; Aaltonen, L. A.; Elomaa, I.; Knuutila, S.

    1999-01-01

    The structure of the 17p amplicon from 9 human sarcoma specimens evaluated by comparative genomic hybridization (CGH) has been studied by analyzing 28 microsatellite markers by PCR. Eleven sarcoma specimens showing no DNA copy number increases at 17p by CGH were analyzed as control samples. Five

  4. The influence of amplicon length on real-time PCR results

    Directory of Open Access Journals (Sweden)

    Debode, F.

    2017-01-01

    Full Text Available Description of the subject. This paper discusses the influence of amplicon length on real-time PCR results. Objectives. The aim of the experiments was to show that amplicon size has an influence on detection. Method. Tests were performed on genomic and plasmid DNA. Double-dye probes and SYBR® Green were used for detection by real-time PCR. Primers were selected in order to produce fragments with increasing sizes. Experiments dealt with two targets: an endogenous target for soybean (part of the lectin gene and a transgenic target (junction P35S-CTP of the MON40-3-2 soybean. Results. The results show that the kinetics of amplification curves evolve as a function of amplicon length, and smaller amplicons yield a higher level of fluorescence for the plateau phase. DNA degradation within the sample as well as the principles of fluorescence acquisition as a function of the chemistry used can also be factors. Conclusions. It was experimentally shown that the observed effect is linked to the suboptimal elongation temperature used in real-time PCR. Detection using SYBR® Green is less impacted as the loss of efficiency is partially compensated by the greater integration of SYBR® Green molecules in the larger fragments.

  5. Polyadenylated Sequencing Primers Enable Complete Readability of PCR Amplicons Analyzed by Dideoxynucleotide Sequencing

    Directory of Open Access Journals (Sweden)

    Martin Beránek

    2012-01-01

    Full Text Available Dideoxynucleotide DNA sequencing is one of the principal procedures in molecular biology. Loss of an initial part of nucleotides behind the 3' end of the sequencing primer limits the readability of sequenced amplicons. We present a method which extends the readability by using sequencing primers modified by polyadenylated tails attached to their 5' ends. Performing a polymerase chain reaction, we amplified eight amplicons of six human genes (AMELX, APOE, HFE, MBL2, SERPINA1 and TGFB1 ranging from 106 bp to 680 bp. Polyadenylation of the sequencing primers minimized the loss of bases in all amplicons. Complete sequences of shorter products (AMELX 106 bp, SERPINA1 121 bp, HFE 208 bp, APOE 244 bp, MBL2 317 bp were obtained. In addition, in the case of TGFB1 products (366 bp, 432 bp, and 680 bp, respectively, the lengths of sequencing readings were significantly longer if adenylated primers were used. Thus, single strand dideoxynucleotide sequencing with adenylated primers enables complete or near complete readability of short PCR amplicons.

  6. Microbial community composition and diversity via 16S rRNA gene amplicons: evaluating the illumina platform.

    Directory of Open Access Journals (Sweden)

    Lucas Sinclair

    Full Text Available As new sequencing technologies become cheaper and older ones disappear, laboratories switch vendors and platforms. Validating the new setups is a crucial part of conducting rigorous scientific research. Here we report on the reliability and biases of performing bacterial 16S rRNA gene amplicon paired-end sequencing on the MiSeq Illumina platform. We designed a protocol using 50 barcode pairs to run samples in parallel and coded a pipeline to process the data. Sequencing the same sediment sample in 248 replicates as well as 70 samples from alkaline soda lakes, we evaluated the performance of the method with regards to estimates of alpha and beta diversity. Using different purification and DNA quantification procedures we always found up to 5-fold differences in the yield of sequences between individually barcodes samples. Using either a one-step or a two-step PCR preparation resulted in significantly different estimates in both alpha and beta diversity. Comparing with a previous method based on 454 pyrosequencing, we found that our Illumina protocol performed in a similar manner - with the exception for evenness estimates where correspondence between the methods was low. We further quantified the data loss at every processing step eventually accumulating to 50% of the raw reads. When evaluating different OTU clustering methods, we observed a stark contrast between the results of QIIME with default settings and the more recent UPARSE algorithm when it comes to the number of OTUs generated. Still, overall trends in alpha and beta diversity corresponded highly using both clustering methods. Our procedure performed well considering the precisions of alpha and beta diversity estimates, with insignificant effects of individual barcodes. Comparative analyses suggest that 454 and Illumina sequence data can be combined if the same PCR protocol and bioinformatic workflows are used for describing patterns in richness, beta-diversity and taxonomic

  7. Bacterial and viral identification and differentiation by amplicon sequencing on the MinION nanopore sequencer.

    Science.gov (United States)

    Kilianski, Andy; Haas, Jamie L; Corriveau, Elizabeth J; Liem, Alvin T; Willis, Kristen L; Kadavy, Dana R; Rosenzweig, C Nicole; Minot, Samuel S

    2015-01-01

    The MinION™ nanopore sequencer was recently released to a community of alpha-testers for evaluation using a variety of sequencing applications. Recent reports have tested the ability of the MinION™ to act as a whole genome sequencer and have demonstrated that nanopore sequencing has tremendous potential utility. However, the current nanopore technology still has limitations with respect to error-rate, and this is problematic when attempting to assemble whole genomes without secondary rounds of sequencing to correct errors. In this study, we tested the ability of the MinION™ nanopore sequencer to accurately identify and differentiate bacterial and viral samples via directed sequencing of characteristic genes shared broadly across a target clade. Using a 6 hour sequencing run time, sufficient data were generated to identify an E. coli sample down to the species level from 16S rDNA amplicons. Three poxviruses (cowpox, vaccinia-MVA, and vaccinia-Lister) were identified and differentiated down to the strain level, despite over 98% identity between the vaccinia strains. The ability to differentiate strains by amplicon sequencing on the MinION™ was accomplished despite an observed per-base error rate of approximately 30%. While nanopore sequencing, using the MinION™ platform from Oxford Nanopore in particular, continues to mature into a commercially available technology, practical uses are sought for the current versions of the technology. This study offers evidence of the utility of amplicon sequencing by demonstrating that the current versions of MinION™ technology can accurately identify and differentiate both viral and bacterial species present within biological samples via amplicon sequencing.

  8. Pregnancy-associated glycoprotein (PAG) family: transcripts and gene amplicons in camelids.

    Science.gov (United States)

    Majewska, Marta; Panasiewicz, Grzegorz; Klisch, Karl; Olivera, Louis V M; Mamani, Javier M; Abd-Elnaeim, Mahmoud M; Szafranska, Bozena

    2009-07-01

    In this study, the placental localization of PAG-like transcripts and genomic existence of PAG-like amplicons in new-world (Lp, Lama pacos, alpaca) and old-world camelids (Cb, Camelus bactrianus, bactrian; Cd, Camelus dromedarius; dromedary) are reported for the first time. Sections of Lp (150-347 days post coitum), Cd (43-90 cm crown-rump length) and Cb (term) placentas were used for heterologous (ht; cross-species) autoradiographic in situ hybridization (aISH) with single-stranded diagnostic (antisense) or control (sense) [alpha-(35)S]dATP-labeled 323 nt porcine PAG8 (pPAG8) cDNA probes produced by asymmetric PCRs. The aISH with antisense (35)S-pPAG8 probe identified camelid PAG-like (LpPAG, CbPAG and CdPAG) mRNA expression restricted to chorionic epithelium cells within placentas of camelids. In addition, genomic DNA (gDNA), isolated from placental sections were used as templates for camelid PAG-like gene amplicon production by PCR. Specificity of the obtained multiple camelid gDNA PAG-like amplicons was confirmed by double ht-Southern hybridizations with [alpha-(32)P]dATP-labeled 611 bp pPAG5 and pPAG10 double-stranded cDNA probes. The double ht-Southern hybridizations of camelid gDNA amplicons (with pPAG5 and -10 probes) allowed the identification of length-polymorphism of LpPAG, CbPAG and CdPAG genes, coding catalytically active and potentially inactive forms. Such an application of porcine PAG probes may be advantageous for future identification of still undiscovered PAG-like families in other eutherian species.

  9. Analysing 454 amplicon resequencing experiments using the modular and database oriented Variant Identification Pipeline

    Directory of Open Access Journals (Sweden)

    Deforce Dieter

    2010-05-01

    Full Text Available Abstract Background Next-generation amplicon sequencing enables high-throughput genetic diagnostics, sequencing multiple genes in several patients together in one sequencing run. Currently, no open-source out-of-the-box software solution exists that reliably reports detected genetic variations and that can be used to improve future sequencing effectiveness by analyzing the PCR reactions. Results We developed an integrated database oriented software pipeline for analysis of 454/Roche GS-FLX amplicon resequencing experiments using Perl and a relational database. The pipeline enables variation detection, variation detection validation, and advanced data analysis, which provides information that can be used to optimize PCR efficiency using traditional means. The modular approach enables customization of the pipeline where needed and allows researchers to adopt their analysis pipeline to their experiments. Clear documentation and training data is available to test and validate the pipeline prior to using it on real sequencing data. Conclusions We designed an open-source database oriented pipeline that enables advanced analysis of 454/Roche GS-FLX amplicon resequencing experiments using SQL-statements. This modular database approach allows easy coupling with other pipeline modules such as variant interpretation or a LIMS system. There is also a set of standard reporting scripts available.

  10. Simultaneous analysis of multiple PCR amplicons enhances capillary SSCP discrimination of MHC alleles.

    Science.gov (United States)

    Alcaide, Miguel; López, Lidia; Tanferna, Alessandro; Blas, Julio; Sergio, Fabrizio; Hiraldo, Fernando

    2010-04-01

    Major histocompatibility complex (MHC) genotyping still remains one of the most challenging issues for evolutionary ecologists. To date, none of the proposed methods have proven to be perfect, and all provide both important pros and cons. Although denaturing capillary electrophoresis has become a popular alternative, allele identification commonly relies upon conformational polymorphisms of two single-stranded DNA molecules at the most. Using the MHC class II (beta chain, exon 2) of the black kite (Aves: Accipitridae) as our model system, we show that the simultaneous analysis of overlapping PCR amplicons from the same target region substantially enhances allele discrimination. To cover this aim, we designed a multiplex PCR capable to generate four differentially sized and labeled amplicons from the same allele. Informative peaks to assist allele calling then fourfold those generated by the analysis of single PCR amplicons. Our approach proved successful to differentiate all the alleles (N=13) isolated from eight unrelated birds at a single optimal run temperature and electrophoretic conditions. In particular, we emphasize that this approach may constitute a straightforward and cost-effective alternative for the genotyping of single or duplicated MHC genes displaying low to moderate sets of divergent alleles.

  11. Comparison of somatic mutation calling methods in amplicon and whole exome sequence data.

    Science.gov (United States)

    Xu, Huilei; DiCarlo, John; Satya, Ravi Vijaya; Peng, Quan; Wang, Yexun

    2014-03-28

    High-throughput sequencing is rapidly becoming common practice in clinical diagnosis and cancer research. Many algorithms have been developed for somatic single nucleotide variant (SNV) detection in matched tumor-normal DNA sequencing. Although numerous studies have compared the performance of various algorithms on exome data, there has not yet been a systematic evaluation using PCR-enriched amplicon data with a range of variant allele fractions. The recently developed gold standard variant set for the reference individual NA12878 by the NIST-led "Genome in a Bottle" Consortium (NIST-GIAB) provides a good resource to evaluate admixtures with various SNV fractions. Using the NIST-GIAB gold standard, we compared the performance of five popular somatic SNV calling algorithms (GATK UnifiedGenotyper followed by simple subtraction, MuTect, Strelka, SomaticSniper and VarScan2) for matched tumor-normal amplicon and exome sequencing data. We demonstrated that the five commonly used somatic SNV calling methods are applicable to both targeted amplicon and exome sequencing data. However, the sensitivities of these methods vary based on the allelic fraction of the mutation in the tumor sample. Our analysis can assist researchers in choosing a somatic SNV calling method suitable for their specific needs.

  12. Superposition Quantification

    Science.gov (United States)

    Chang, Li-Na; Luo, Shun-Long; Sun, Yuan

    2017-11-01

    The principle of superposition is universal and lies at the heart of quantum theory. Although ever since the inception of quantum mechanics a century ago, superposition has occupied a central and pivotal place, rigorous and systematic studies of the quantification issue have attracted significant interests only in recent years, and many related problems remain to be investigated. In this work we introduce a figure of merit which quantifies superposition from an intuitive and direct perspective, investigate its fundamental properties, connect it to some coherence measures, illustrate it through several examples, and apply it to analyze wave-particle duality. Supported by Science Challenge Project under Grant No. TZ2016002, Laboratory of Computational Physics, Institute of Applied Physics and Computational Mathematics, Beijing, Key Laboratory of Random Complex Structures and Data Science, Chinese Academy of Sciences, Grant under No. 2008DP173182

  13. Sequence-specific validation of LAMP amplicons in real-time optomagnetic detection of Dengue serotype 2 synthetic DNA

    DEFF Research Database (Denmark)

    Minero, Gabriel Khose Antonio; Nogueira, Catarina; Rizzi, Giovanni

    2017-01-01

    magnetic nanoparticles attached to biotinylated LAMP amplicons. We demonstrate detection of sub-femtomolar Dengue DNA target concentrations in the ideal contamination-free lab environment within 20 min. The second detection approach is based on sequence-specific binding of functionalised magnetic...... nanoparticles to loops of LAMP amplicons. Melting studies reveal that true positive and spurious amplicons have different melting points and this allows us to discriminate between them. This is found to be in a good agreement with subsequent studies on real-time sequence-specific discrimination of LAMP...... amplicons. The specific binding causes clustering of magnetic nanoparticles via binding to multiple sites (loops) emerging in the elongation phase of LAMP. Formation of nanoclusters is monitored via the depletion of the optomagnetic signal due to free nanoparticles. After sequence-specific validation, we...

  14. Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing

    Directory of Open Access Journals (Sweden)

    Lank Simon M

    2012-08-01

    Full Text Available Abstract Background High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. Results We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. Conclusions The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons.

  15. Mining environmental high-throughput sequence data sets to identify divergent amplicon clusters for phylogenetic reconstruction and morphotype visualization.

    Science.gov (United States)

    Gimmler, Anna; Stoeck, Thorsten

    2015-08-01

    Environmental high-throughput sequencing (envHTS) is a very powerful tool, which in protistan ecology is predominantly used for the exploration of diversity and its geographic and local patterns. We here used a pyrosequenced V4-SSU rDNA data set from a solar saltern pond as test case to exploit such massive protistan amplicon data sets beyond this descriptive purpose. Therefore, we combined a Swarm-based blastn network including 11 579 ciliate V4 amplicons to identify divergent amplicon clusters with targeted polymerase chain reaction (PCR) primer design for full-length small subunit of the ribosomal DNA retrieval and probe design for fluorescence in situ hybridization (FISH). This powerful strategy allows to benefit from envHTS data sets to (i) reveal the phylogenetic position of the taxon behind divergent amplicons; (ii) improve phylogenetic resolution and evolutionary history of specific taxon groups; (iii) solidly assess an amplicons (species') degree of similarity to its closest described relative; (iv) visualize the morphotype behind a divergent amplicons cluster; (v) rapidly FISH screen many environmental samples for geographic/habitat distribution and abundances of the respective organism and (vi) to monitor the success of enrichment strategies in live samples for cultivation and isolation of the respective organisms. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  16. Amplicon-based metagenomics identified candidate organisms in soils that caused yield decline in strawberry

    OpenAIRE

    Xiangming Xu; Thomas Passey; Feng Wei; Robert Saville; Richard J. Harrison

    2015-01-01

    A phenomenon of yield decline due to weak plant growth in strawberry was recently observed in non-chemo-fumigated soils, which was not associated with the soil fungal pathogen Verticillium dahliae, the main target of fumigation. Amplicon-based metagenomics was used to profile soil microbiota in order to identify microbial organisms that may have caused the yield decline. A total of 36 soil samples were obtained in 2013 and 2014 from four sites for metagenomic studies; two of the four sites ha...

  17. Analysis and Visualization Tool for Targeted Amplicon Bisulfite Sequencing on Ion Torrent Sequencers.

    Directory of Open Access Journals (Sweden)

    Stephan Pabinger

    Full Text Available Targeted sequencing of PCR amplicons generated from bisulfite deaminated DNA is a flexible, cost-effective way to study methylation of a sample at single CpG resolution and perform subsequent multi-target, multi-sample comparisons. Currently, no platform specific protocol, support, or analysis solution is provided to perform targeted bisulfite sequencing on a Personal Genome Machine (PGM. Here, we present a novel tool, called TABSAT, for analyzing targeted bisulfite sequencing data generated on Ion Torrent sequencers. The workflow starts with raw sequencing data, performs quality assessment, and uses a tailored version of Bismark to map the reads to a reference genome. The pipeline visualizes results as lollipop plots and is able to deduce specific methylation-patterns present in a sample. The obtained profiles are then summarized and compared between samples. In order to assess the performance of the targeted bisulfite sequencing workflow, 48 samples were used to generate 53 different Bisulfite-Sequencing PCR amplicons from each sample, resulting in 2,544 amplicon targets. We obtained a mean coverage of 282X using 1,196,822 aligned reads. Next, we compared the sequencing results of these targets to the methylation level of the corresponding sites on an Illumina 450k methylation chip. The calculated average Pearson correlation coefficient of 0.91 confirms the sequencing results with one of the industry-leading CpG methylation platforms and shows that targeted amplicon bisulfite sequencing provides an accurate and cost-efficient method for DNA methylation studies, e.g., to provide platform-independent confirmation of Illumina Infinium 450k methylation data. TABSAT offers a novel way to analyze data generated by Ion Torrent instruments and can also be used with data from the Illumina MiSeq platform. It can be easily accessed via the Platomics platform, which offers a web-based graphical user interface along with sample and parameter storage

  18. Amplicon-based metagenomics identified candidate organisms in soils that caused yield decline in strawberry.

    Science.gov (United States)

    Xu, Xiangming; Passey, Thomas; Wei, Feng; Saville, Robert; Harrison, Richard J

    2015-01-01

    A phenomenon of yield decline due to weak plant growth in strawberry was recently observed in non-chemo-fumigated soils, which was not associated with the soil fungal pathogen Verticillium dahliae, the main target of fumigation. Amplicon-based metagenomics was used to profile soil microbiota in order to identify microbial organisms that may have caused the yield decline. A total of 36 soil samples were obtained in 2013 and 2014 from four sites for metagenomic studies; two of the four sites had a yield-decline problem, the other two did not. More than 2000 fungal or bacterial operational taxonomy units (OTUs) were found in these samples. Relative abundance of individual OTUs was statistically compared for differences between samples from sites with or without yield decline. A total of 721 individual comparisons were statistically significant - involving 366 unique bacterial and 44 unique fungal OTUs. Based on further selection criteria, we focused on 34 bacterial and 17 fungal OTUs and found that yield decline resulted probably from one or more of the following four factors: (1) low abundance of Bacillus and Pseudomonas populations, which are well known for their ability of supressing pathogen development and/or promoting plant growth; (2) lack of the nematophagous fungus (Paecilomyces species); (3) a high level of two non-specific fungal root rot pathogens; and (4) wet soil conditions. This study demonstrated the usefulness of an amplicon-based metagenomics approach to profile soil microbiota and to detect differential abundance in microbes.

  19. A priori Considerations When Conducting High-Throughput Amplicon-Based Sequence Analysis

    Directory of Open Access Journals (Sweden)

    Aditi Sengupta

    2016-03-01

    Full Text Available Amplicon-based sequencing strategies that include 16S rRNA and functional genes, alongside “meta-omics” analyses of communities of microorganisms, have allowed researchers to pose questions and find answers to “who” is present in the environment and “what” they are doing. Next-generation sequencing approaches that aid microbial ecology studies of agricultural systems are fast gaining popularity among agronomy, crop, soil, and environmental science researchers. Given the rapid development of these high-throughput sequencing techniques, researchers with no prior experience will desire information about the best practices that can be used before actually starting high-throughput amplicon-based sequence analyses. We have outlined items that need to be carefully considered in experimental design, sampling, basic bioinformatics, sequencing of mock communities and negative controls, acquisition of metadata, and in standardization of reaction conditions as per experimental requirements. Not all considerations mentioned here may pertain to a particular study. The overall goal is to inform researchers about considerations that must be taken into account when conducting high-throughput microbial DNA sequencing and sequences analysis.

  20. Multiplex amplicon sequencing for microbe identification in community-based culture collections.

    Science.gov (United States)

    Armanhi, Jaderson Silveira Leite; de Souza, Rafael Soares Correa; de Araújo, Laura Migliorini; Okura, Vagner Katsumi; Mieczkowski, Piotr; Imperial, Juan; Arruda, Paulo

    2016-07-12

    Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC.

  1. Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform.

    Science.gov (United States)

    Schirmer, Melanie; Ijaz, Umer Z; D'Amore, Rosalinda; Hall, Neil; Sloan, William T; Quince, Christopher

    2015-03-31

    With read lengths of currently up to 2 × 300 bp, high throughput and low sequencing costs Illumina's MiSeq is becoming one of the most utilized sequencing platforms worldwide. The platform is manageable and affordable even for smaller labs. This enables quick turnaround on a broad range of applications such as targeted gene sequencing, metagenomics, small genome sequencing and clinical molecular diagnostics. However, Illumina error profiles are still poorly understood and programs are therefore not designed for the idiosyncrasies of Illumina data. A better knowledge of the error patterns is essential for sequence analysis and vital if we are to draw valid conclusions. Studying true genetic variation in a population sample is fundamental for understanding diseases, evolution and origin. We conducted a large study on the error patterns for the MiSeq based on 16S rRNA amplicon sequencing data. We tested state-of-the-art library preparation methods for amplicon sequencing and showed that the library preparation method and the choice of primers are the most significant sources of bias and cause distinct error patterns. Furthermore we tested the efficiency of various error correction strategies and identified quality trimming (Sickle) combined with error correction (BayesHammer) followed by read overlapping (PANDAseq) as the most successful approach, reducing substitution error rates on average by 93%. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. CASPER: context-aware scheme for paired-end reads from high-throughput amplicon sequencing.

    Science.gov (United States)

    Kwon, Sunyoung; Lee, Byunghan; Yoon, Sungroh

    2014-01-01

    Merging the forward and reverse reads from paired-end sequencing is a critical task that can significantly improve the performance of downstream tasks, such as genome assembly and mapping, by providing them with virtually elongated reads. However, due to the inherent limitations of most paired-end sequencers, the chance of observing erroneous bases grows rapidly as the end of a read is approached, which becomes a critical hurdle for accurately merging paired-end reads. Although there exist several sophisticated approaches to this problem, their performance in terms of quality of merging often remains unsatisfactory. To address this issue, here we present a context-aware scheme for paired-end reads (CASPER): a computational method to rapidly and robustly merge overlapping paired-end reads. Being particularly well suited to amplicon sequencing applications, CASPER is thoroughly tested with both simulated and real high-throughput amplicon sequencing data. According to our experimental results, CASPER significantly outperforms existing state-of-the art paired-end merging tools in terms of accuracy and robustness. CASPER also exploits the parallelism in the task of paired-end merging and effectively speeds up by multithreading. CASPER is freely available for academic use at http://best.snu.ac.kr/casper.

  3. Direct recovery of infectious Pestivirus from a full-length RT-PCR amplicon

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, Ilona; Hoffmann, Bernd

    2008-01-01

    This study describes the use of a novel and rapid long reverse transcription (RT)-PCR for the generation of infectious full-length cDNA of pestiviruses. To produce rescued viruses, full-length RT-PCR amplicons of 12.3 kb, including a T7-promotor, were transcribed directly in vitro, and the result......This study describes the use of a novel and rapid long reverse transcription (RT)-PCR for the generation of infectious full-length cDNA of pestiviruses. To produce rescued viruses, full-length RT-PCR amplicons of 12.3 kb, including a T7-promotor, were transcribed directly in vitro......, and the resulting RNA transcripts were electroporated into ovine cells. Infectious virus was obtained after one cell culture passage. The rescued viruses had a phenotype similar to the parental Border Disease virus strain. Therefore, direct generation of infectious pestiviruses from full-length RT-PCR cDNA products...

  4. Photochemical immobilization of anthraquinone conjugated oligonucleotides and PCR amplicons on solid surfaces

    DEFF Research Database (Denmark)

    Koch, T.; Jacobsen, N.; Fensholdt, J.

    2000-01-01

    Ligand immobilization on solid surfaces is an essential step in fields such as diagnostics, bio sensor manufacturing, and new material sciences in general. In this paper a photochemical approach based on anthraquinone as the chromophore is presented. Photochemical procedures offer special...... advantages as they are able to generate highly reactive species in an orientation specific manner. As presented here, anthraquinone (AQ) mediated covalent DNA immobilization appears to be superior to currently known procedures. A synthetic procedure providing AQ-phosphoramidites is presented. These reagents...... facilitate AQ conjugation during routine DNA synthesis, thus enabling the AQ-oligonucleotides to be immobilized in a very convenient and efficient manner. AQ-conjugated PCR primers can be used directly in PCR. When the PCR is performed in solution, the amplicons can be immobilized after the PCR. Moreover...

  5. Swarm v2: highly-scalable and high-resolution amplicon clustering.

    Science.gov (United States)

    Mahé, Frédéric; Rognes, Torbjørn; Quince, Christopher; de Vargas, Colomban; Dunthorn, Micah

    2015-01-01

    Previously we presented Swarm v1, a novel and open source amplicon clustering program that produced fine-scale molecular operational taxonomic units (OTUs), free of arbitrary global clustering thresholds and input-order dependency. Swarm v1 worked with an initial phase that used iterative single-linkage with a local clustering threshold (d), followed by a phase that used the internal abundance structures of clusters to break chained OTUs. Here we present Swarm v2, which has two important novel features: (1) a new algorithm for d = 1 that allows the computation time of the program to scale linearly with increasing amounts of data; and (2) the new fastidious option that reduces under-grouping by grafting low abundant OTUs (e.g., singletons and doubletons) onto larger ones. Swarm v2 also directly integrates the clustering and breaking phases, dereplicates sequencing reads with d = 0, outputs OTU representatives in fasta format, and plots individual OTUs as two-dimensional networks.

  6. Nitrogenase gene amplicons from global marine surface waters are dominated by genes of non-cyanobacteria

    DEFF Research Database (Denmark)

    Farnelid, Hanna; Andersson, Anders F.; Bertilsson, Stefan

    2011-01-01

    by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least......Cyanobacteria are thought to be the main N2-fixing organisms (diazotrophs) in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our...... of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related...

  7. Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology.

    Science.gov (United States)

    Sie, Daoud; Snijders, Peter J F; Meijer, Gerrit A; Doeleman, Marije W; van Moorsel, Marinda I H; van Essen, Hendrik F; Eijk, Paul P; Grünberg, Katrien; van Grieken, Nicole C T; Thunnissen, Erik; Verheul, Henk M; Smit, Egbert F; Ylstra, Bauke; Heideman, Daniëlle A M

    2014-10-01

    Next generation DNA sequencing (NGS) holds promise for diagnostic applications, yet implementation in routine molecular pathology practice requires performance evaluation on DNA derived from routine formalin-fixed paraffin-embedded (FFPE) tissue specimens. The current study presents a comprehensive analysis of TruSeq Amplicon Cancer Panel-based NGS using a MiSeq Personal sequencer (TSACP-MiSeq-NGS) for somatic mutation profiling. TSACP-MiSeq-NGS (testing 212 hotspot mutation amplicons of 48 genes) and a data analysis pipeline were evaluated in a retrospective learning/test set approach (n = 58/n = 45 FFPE-tumor DNA samples) against 'gold standard' high-resolution-melting (HRM)-sequencing for the genes KRAS, EGFR, BRAF and PIK3CA. Next, the performance of the validated test algorithm was assessed in an independent, prospective cohort of FFPE-tumor DNA samples (n = 75). In the learning set, a number of minimum parameter settings was defined to decide whether a FFPE-DNA sample is qualified for TSACP-MiSeq-NGS and for calling mutations. The resulting test algorithm revealed 82% (37/45) compliance to the quality criteria and 95% (35/37) concordant assay findings for KRAS, EGFR, BRAF and PIK3CA with HRM-sequencing (kappa = 0.92; 95% CI = 0.81-1.03) in the test set. Subsequent application of the validated test algorithm to the prospective cohort yielded a success rate of 84% (63/75), and a high concordance with HRM-sequencing (95% (60/63); kappa = 0.92; 95% CI = 0.84-1.01). TSACP-MiSeq-NGS detected 77 mutations in 29 additional genes. TSACP-MiSeq-NGS is suitable for diagnostic gene mutation profiling in oncopathology.

  8. Amplicon Sequencing Reveals Microbiological Signatures in Spent Nuclear Fuel Storage Basins

    Directory of Open Access Journals (Sweden)

    Christopher E. Bagwell

    2018-03-01

    Full Text Available Water quality is an important determinant for the structural integrity of alloy cladded fuels and assemblies during long-term wet storage. Detailed characterization of a water filled storage basin for spent nuclear reactor fuel was performed following the formation and proliferation of an amorphous white flocculent. White precipitant was sampled throughout the storage basin for chemical and spectroscopic characterization, and environmental DNA was extracted for 454 pyrosequencing of bacterial 16S rRNA gene diversity. Accordingly, spectroscopic analyses indicated the precipitant to be primarily amorphous to crystalline aluminum (oxy hydroxides with minor associated elemental components including Fe, Si, Ti, and U. High levels of organic carbon were co-localized with the precipitant relative to bulk dissolved organic concentrations. Bacterial densities were highly variable between sampling locations and with depth within the water filled storage basin; cell numbers ranged from 4 × 103to 4 × 104 cells/mL. Bacterial diversity that was physically associated with the aluminum (oxy hydroxide complexes exceeded an estimated 4,000 OTUs/amplicon library (3% cutoff and the majority of sequences were aligned to the families Burkholderiaceae (23%, Nitrospiraceae (23%, Hyphomicrobiaceae (17%, and Comamonadaceae (6%. We surmise that episodic changes in the physical and chemical properties of the basin contribute to the polymerization of aluminum (oxy hydroxides, which in turn can chemisorb nutrients, carbon ligands and bacterial cells from the surrounding bulk aqueous phase. As such, these precipitants should establish favorable microhabitats for bacterial colonization and growth. Comparative analyses of 16S rRNA gene amplicon libraries across a selection of natural and engineered aquatic ecosystems were performed and microbial community and taxonomic signatures unique to the spent nuclear fuel (SNF storage basin environment were revealed. These insights

  9. Similar diversity of Alphaproteobacteria and nitrogenase gene amplicons on two related Sphagnum mosses

    Directory of Open Access Journals (Sweden)

    Anastasia eBragina

    2012-01-01

    Full Text Available Sphagnum mosses represent a main component in ombrotrophic wetlands. They harbor a specific and diverse microbial community with essential functions for the host. To understand extend and degree of host specificity, Sphagnum fallax and S. angustifolium, two phylogenetically closely related species, which show distinct habitat preference with respect to the nutrient level, were analyzed by a multifaceted approach. Microbial fingerprints obtained by PCR-SSCP (single-strand conformation polymorphism using universal, group-specific and functional primers were highly similar. Similarity was confirmed for colonization patterns obtained by fluorescence in situ hybridization (FISH coupled with confocal laser scanning microscopy (CLSM: Alphaproteobacteria were the main colonizers inside the hyaline cells of Sphagnum leaves. A deeper survey of Alphaproteobacteria by 16S rRNA gene amplicon sequencing reveals a high diversity with Acidocella, Acidisphaera, Rhodopila and Phenylobacterium as major genera for both mosses. Pathogen defense and nitrogen fixation are important functions of Sphagnum-associated bacteria, which are fulfilled by microbial communities of both Sphagna in a similar way. NifH libraries of Sphagnum-associated microbial communities were characterized by high diversity and abundance of Alphaproteobacteria but contained also diverse amplicons of other taxa, e.g. Cyanobacteria, Geobacter and Spirochaeta. Statistically significant differences between the microbial communities of both Sphagnum species could not be discovered in any of the experimental approach. Our results show that the same close relationship, which exists between the physical, morphological and chemical characteristics of Sphagnum mosses and the ecology and function of bog ecosystems, also connects moss plantlets with their associated bacterial communities.

  10. Sequence-specific validation of LAMP amplicons in real-time optomagnetic detection of Dengue serotype 2 synthetic DNA.

    Science.gov (United States)

    Minero, Gabriel Antonio S; Nogueira, Catarina; Rizzi, Giovanni; Tian, Bo; Fock, Jeppe; Donolato, Marco; Strömberg, Mattias; Hansen, Mikkel F

    2017-09-08

    We report on an optomagnetic technique optimised for real-time molecular detection of Dengue fever virus under ideal as well as non-ideal laboratory conditions using two different detection approaches. The first approach is based on the detection of the hydrodynamic volume of streptavidin coated magnetic nanoparticles attached to biotinylated LAMP amplicons. We demonstrate detection of sub-femtomolar Dengue DNA target concentrations in the ideal contamination-free lab environment within 20 min. The second detection approach is based on sequence-specific binding of functionalised magnetic nanoparticles to loops of LAMP amplicons. Melting studies reveal that true positive and spurious amplicons have different melting points and this allows us to discriminate between them. This is found to be in a good agreement with subsequent studies on real-time sequence-specific discrimination of LAMP amplicons. The specific binding causes clustering of magnetic nanoparticles via binding to multiple sites (loops) emerging in the elongation phase of LAMP. Formation of nanoclusters is monitored via the depletion of the optomagnetic signal due to free nanoparticles. After sequence-specific validation, we claim detection of down to 100 fM of Dengue target after 20 min of LAMP with a contamination background.

  11. Function of the EGR-1/TIS8 radiation inducible promoter in a minimal HSV-1 amplicon system

    International Nuclear Information System (INIS)

    Spear, M.A.; Sakamoto, K.M.; Herrlinger, U.; Pechan, P.; Breakefield, X.O.

    1997-01-01

    Purpose: To evaluate function of the EGR-1/TIS8 promoter region in minimal HSV-1 amplicon system in order to determine the feasibility of using the system to regulate vector replication with radiation. Materials and Methods: A 600-base pair 5' upstream region of the EGR-1 promoter linked to chloramphenicol acetyltransferase (CAT) was recombined into a minimal HSV-1 amplicon vector system (pONEC). pONEC or a control plasmid was transfected into U87 glioma cells using the Lipofectamine method. Thirty-six hours later one aliquot of cells from each transfection was irradiated to a dose of 20 Gy and another identical aliquot served as a control. CAT activity was assayed 8 hours after irradiation. Results: pONEC transfected cells irradiated with 20 Gy demonstrated 2.0 fold increase in CAT activity compared to non-irradiated cells. Cells transfected with control plasmid showed no change in CAT activity. Unirradiated pONEC cells had CAT activity 1.3 times those transfected with control plasmid. Conclusion: We have previously created HSV-1 gene therapy amplicon vector systems which allow virus-amplicon interdependent replication, with the intent of regulating replication. The above data demonstrates that a minimal amplicon system will allow radiation dependent regulation by the EGR-1 promoter, thus indicating the possibility of using this system to regulate onsite, spatially and temporally regulated vector production. Baseline CAT activity was higher and relative induction lower than other reported expression constructs, which raises concern for the ability of the system to produce a differential in transcription levels sufficient for this purpose. This is possibly the result of residual promoter/enhancer elements remaining in the HSV-1 sequences. We are attempting to create constructs lacking these elements. Addition of secondary promoter sequences may also be of use. We are also currently evaluating the efficacy of the putative IEX-1 radiation inducible promoter region in

  12. Herpes simplex virus type 1-based amplicon vectors for fundamental research in neurosciences and gene therapy of neurological diseases.

    Science.gov (United States)

    Jerusalinsky, Diana; Baez, María Verónica; Epstein, Alberto Luis

    2012-01-01

    Somatic manipulation of the nervous system without the involvement of the germinal line appears as a powerful counterpart of the transgenic strategy. The use of viral vectors to produce specific, transient and localized knockout, knockdown, ectopic expression or overexpression of a gene, leads to the possibility of analyzing both in vitro and in vivo molecular basis of neural function. In this approach, viral particles engineered to carry transgenic sequences are delivered into discrete brain regions, to transduce cells that will express the transgenic products. Amplicons are replication-incompetent helper-dependent vectors derived from herpes simplex virus type 1 (HSV-1), with several advantages that potentiate their use in neurosciences: (1) minimal toxicity: amplicons do not encode any virus proteins, are neither toxic for the infected cells nor pathogenic for the inoculated animals and elicit low levels of adaptive immune responses; (2) extensive transgene capacity to carry up to 150-kb of foreign DNA; i.e., entire genes with regulatory sequences could be delivered; (3) widespread cellular tropism: amplicons can experimentally infect several cell types including glial cells, though naturally the virus infects mainly neurons and epithelial cells; (4) since the viral genome does not integrate into cellular chromosomes there is low probability to induce insertional mutagenesis. Recent investigations on gene transfer into the brain using these vectors, have focused on gene therapy of inherited genetic diseases affecting the nervous system, such as ataxias, or on neurodegenerative disorders using experimental models of Parkinson's or Alzheimer's disease. Another group of studies used amplicons to investigate complex neural functions such as neuroplasticity, anxiety, learning and memory. In this short review, we summarize recent data supporting the potential of HSV-1 based amplicon vector model for gene delivery and modulation of gene expression in primary cultures

  13. 16S rRNA Amplicon Sequencing for Epidemiological Surveys of Bacteria in Wildlife.

    Science.gov (United States)

    Galan, Maxime; Razzauti, Maria; Bard, Emilie; Bernard, Maria; Brouat, Carine; Charbonnel, Nathalie; Dehne-Garcia, Alexandre; Loiseau, Anne; Tatard, Caroline; Tamisier, Lucie; Vayssier-Taussat, Muriel; Vignes, Helene; Cosson, Jean-François

    2016-01-01

    The human impact on natural habitats is increasing the complexity of human-wildlife interactions and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential for recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of the large numbers of samples needed to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to mitigate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a comprehensive trimming strategy for managing these biases. We demonstrated the application of this strategy using 711 commensal rodents, including 208 Mus musculus domesticus , 189 Rattus rattus , 93 Mastomys natalensis , and 221 Mastomys erythroleucus , collected from 24 villages in Senegal. Seven major genera of pathogenic bacteria were detected in their spleens: Borrelia , Bartonella , Mycoplasma , Ehrlichia , Rickettsia , Streptobacillus , and Orientia . Mycoplasma , Ehrlichia , Rickettsia , Streptobacillus , and Orientia have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90% of individuals per site, depending on the bacterial taxon, rodent species, and site considered, and 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of

  14. Differential amplicons (ΔAmp)-a new molecular method to assess RNA integrity.

    Science.gov (United States)

    Björkman, J; Švec, D; Lott, E; Kubista, M; Sjöback, R

    2016-01-01

    Integrity of the mRNA in clinical samples has major impact on the quality of measured expression levels. This is independent of the measurement technique being next generation sequencing (NGS), Quantitative real-time PCR (qPCR) or microarray profiling. If mRNA is highly degraded or damaged, measured data will be very unreliable and the whole study is likely a waste of time and money. It is therefore common strategy to test the quality of RNA in samples before conducting large and costly studies. Most methods today to assess the quality of RNA are ignorant to the nature of the RNA and, therefore, reflect the integrity of ribosomal RNA, which is the dominant species, rather than of mRNAs, microRNAs and long non-coding RNAs, which usually are the species of interest. Here, we present a novel molecular approach to assess the quality of the targeted RNA species by measuring the differential amplification (ΔAmp) of an Endogenous RNase Resistant (ERR) marker relative to a reference gene, optionally combined with the measurement of two amplicons of different lengths. The combination reveals any mRNA degradation caused by ribonucleases as well as physical, chemical or UV damage. ΔAmp has superior sensitivity to common microfluidic electrophoretic methods, senses the integrity of the actual targeted RNA species, and allows for a smoother and more cost efficient workflow.

  15. Differential amplicons (ΔAmp—a new molecular method to assess RNA integrity

    Directory of Open Access Journals (Sweden)

    J. Björkman

    2016-01-01

    Full Text Available Integrity of the mRNA in clinical samples has major impact on the quality of measured expression levels. This is independent of the measurement technique being next generation sequencing (NGS, Quantitative real-time PCR (qPCR or microarray profiling. If mRNA is highly degraded or damaged, measured data will be very unreliable and the whole study is likely a waste of time and money. It is therefore common strategy to test the quality of RNA in samples before conducting large and costly studies. Most methods today to assess the quality of RNA are ignorant to the nature of the RNA and, therefore, reflect the integrity of ribosomal RNA, which is the dominant species, rather than of mRNAs, microRNAs and long non-coding RNAs, which usually are the species of interest. Here, we present a novel molecular approach to assess the quality of the targeted RNA species by measuring the differential amplification (ΔAmp of an Endogenous RNase Resistant (ERR marker relative to a reference gene, optionally combined with the measurement of two amplicons of different lengths. The combination reveals any mRNA degradation caused by ribonucleases as well as physical, chemical or UV damage. ΔAmp has superior sensitivity to common microfluidic electrophoretic methods, senses the integrity of the actual targeted RNA species, and allows for a smoother and more cost efficient workflow.

  16. Hi-Plex for Simple, Accurate, and Cost-Effective Amplicon-based Targeted DNA Sequencing.

    Science.gov (United States)

    Pope, Bernard J; Hammet, Fleur; Nguyen-Dumont, Tu; Park, Daniel J

    2018-01-01

    Hi-Plex is a suite of methods to enable simple, accurate, and cost-effective highly multiplex PCR-based targeted sequencing (Nguyen-Dumont et al., Biotechniques 58:33-36, 2015). At its core is the principle of using gene-specific primers (GSPs) to "seed" (or target) the reaction and universal primers to "drive" the majority of the reaction. In this manner, effects on amplification efficiencies across the target amplicons can, to a large extent, be restricted to early seeding cycles. Product sizes are defined within a relatively narrow range to enable high-specificity size selection, replication uniformity across target sites (including in the context of fragmented input DNA such as that derived from fixed tumor specimens (Nguyen-Dumont et al., Biotechniques 55:69-74, 2013; Nguyen-Dumont et al., Anal Biochem 470:48-51, 2015), and application of high-specificity genetic variant calling algorithms (Pope et al., Source Code Biol Med 9:3, 2014; Park et al., BMC Bioinformatics 17:165, 2016). Hi-Plex offers a streamlined workflow that is suitable for testing large numbers of specimens without the need for automation.

  17. Extracellular DNA amplicon sequencing reveals high levels of benthic eukaryotic diversity in the central Red Sea

    KAUST Repository

    Pearman, John K.

    2015-11-01

    The present study aims to characterize the benthic eukaryotic biodiversity patterns at a coarse taxonomic level in three areas of the central Red Sea (a lagoon, an offshore area in Thuwal and a shallow coastal area near Jeddah) based on extracellular DNA. High-throughput amplicon sequencing targeting the V9 region of the 18S rRNA gene was undertaken for 32 sediment samples. High levels of alpha-diversity were detected with 16,089 operational taxonomic units (OTUs) being identified. The majority of the OTUs were assigned to Metazoa (29.2%), Alveolata (22.4%) and Stramenopiles (17.8%). Stramenopiles (Diatomea) and Alveolata (Ciliophora) were frequent in a lagoon and in shallower coastal stations, whereas metazoans (Arthropoda: Maxillopoda) were dominant in deeper offshore stations. Only 24.6% of total OTUs were shared among all areas. Beta-diversity was generally lower between the lagoon and Jeddah (nearshore) than between either of those and the offshore area, suggesting a nearshore–offshore biodiversity gradient. The current approach allowed for a broad-range of benthic eukaryotic biodiversity to be analysed with significantly less labour than would be required by other traditional taxonomic approaches. Our findings suggest that next generation sequencing techniques have the potential to provide a fast and standardised screening of benthic biodiversity at large spatial and temporal scales.

  18. Functional Overexpression of Vomeronasal Receptors Using a Herpes Simplex Virus Type 1 (HSV-1-Derived Amplicon.

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    Benjamin Stein

    Full Text Available In mice, social behaviors such as mating and aggression are mediated by pheromones and related chemosignals. The vomeronasal organ (VNO detects olfactory information from other individuals by sensory neurons tuned to respond to specific chemical cues. Receptors expressed by vomeronasal neurons are implicated in selective detection of these cues. Nearly 400 receptor genes have been identified in the mouse VNO, but the tuning properties of individual receptors remain poorly understood, in part due to the lack of a robust heterologous expression system. Here we develop a herpes virus-based amplicon delivery system to overexpress three types of vomeronasal receptor genes and to characterize cell responses to their proposed ligands. Through Ca2+ imaging in native VNO cells we show that virus-induced overexpression of V1rj2, V2r1b or Fpr3 caused a pronounced increase of responsivity to sulfated steroids, MHC-binding peptide or the synthetic hexapeptide W-peptide, respectively. Other related ligands were not recognized by infected individual neurons, indicating a high degree of selectivity by the overexpressed receptor. Removal of G-protein signaling eliminates Ca2+ responses, indicating that the endogenous second messenger system is essential for observing receptor activation. Our results provide a novel expression system for vomeronasal receptors that should be useful for understanding the molecular logic of VNO ligand detection. Functional expression of vomeronasal receptors and their deorphanization provides an essential requirement for deciphering the neural mechanisms controlling behavior.

  19. Swarm v2: highly-scalable and high-resolution amplicon clustering

    Directory of Open Access Journals (Sweden)

    Frédéric Mahé

    2015-12-01

    Full Text Available Previously we presented Swarm v1, a novel and open source amplicon clustering program that produced fine-scale molecular operational taxonomic units (OTUs, free of arbitrary global clustering thresholds and input-order dependency. Swarm v1 worked with an initial phase that used iterative single-linkage with a local clustering threshold (d, followed by a phase that used the internal abundance structures of clusters to break chained OTUs. Here we present Swarm v2, which has two important novel features: (1 a new algorithm for d = 1 that allows the computation time of the program to scale linearly with increasing amounts of data; and (2 the new fastidious option that reduces under-grouping by grafting low abundant OTUs (e.g., singletons and doubletons onto larger ones. Swarm v2 also directly integrates the clustering and breaking phases, dereplicates sequencing reads with d = 0, outputs OTU representatives in fasta format, and plots individual OTUs as two-dimensional networks.

  20. Shedding light on the microbial community of the macropod foregut using 454-amplicon pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Lisa-Maree Gulino

    Full Text Available Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering. Thirty-two OTUs were identified as 'shared' OTUS (i.e. present in all samples belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales. These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry.

  1. High-Throughput Analysis of DNA Break-Induced Chromosome Rearrangements by Amplicon Sequencing.

    Science.gov (United States)

    Brown, Alexander J; Al-Soodani, Aneesa T; Saul, Miles; Her, Stephanie; Garcia, Juan C; Ramsden, Dale A; Her, Chengtao; Roberts, Steven A

    2018-01-01

    The mechanistic understanding of how DNA double-strand breaks (DSB) are repaired is rapidly advancing in part due to the advent of inducible site-specific break model systems as well as the employment of next-generation sequencing (NGS) technologies to sequence repair junctions at high depth. Unfortunately, the sheer volume of data produced by these methods makes it difficult to analyze the structure of repair junctions manually or with other general-purpose software. Here, we describe methods to produce amplicon libraries of DSB repair junctions for sequencing, to map the sequencing reads, and then to use a robust, custom python script, Hi-FiBR, to analyze the sequence structure of mapped reads. The Hi-FiBR analysis processes large data sets quickly and provides information such as number and type of repair events, size of deletion, size of insertion and inserted sequence, microhomology usage, and whether mismatches are due to sequencing error or biological effect. The analysis also corrects for common alignment errors generated by sequencing read mapping tools, allowing high-throughput analysis of DSB break repair fidelity to be accurately conducted regardless of which suite of NGS analysis software is available. © 2018 Elsevier Inc. All rights reserved.

  2. Species tree estimation of North American chorus frogs (Hylidae: Pseudacris) with parallel tagged amplicon sequencing.

    Science.gov (United States)

    Barrow, Lisa N; Ralicki, Hannah F; Emme, Sandra A; Lemmon, Emily Moriarty

    2014-06-01

    The field of phylogenetics is changing rapidly with the application of high-throughput sequencing to non-model organisms. Cost-effective use of this technology for phylogenetic studies, which often include a relatively small portion of the genome but several taxa, requires strategies for genome partitioning and sequencing multiple individuals in parallel. In this study we estimated a multilocus phylogeny for the North American chorus frog genus Pseudacris using anonymous nuclear loci that were recently developed using a reduced representation library approach. We sequenced 27 nuclear loci and three mitochondrial loci for 44 individuals on 1/3 of an Illumina MiSeq run, obtaining 96.5% of the targeted amplicons at less than 20% of the cost of traditional Sanger sequencing. We found heterogeneity among gene trees, although four major clades (Trilling Frog, Fat Frog, crucifer, and West Coast) were consistently supported, and we resolved the relationships among these clades for the first time with strong support. We also found discordance between the mitochondrial and nuclear datasets that we attribute to mitochondrial introgression and a possible selective sweep. Bayesian concordance analysis in BUCKy and species tree analysis in (*)BEAST produced largely similar topologies, although we identify taxa that require additional investigation in order to clarify taxonomic and geographic range boundaries. Overall, we demonstrate the utility of a reduced representation library approach for marker development and parallel tagged sequencing on an Illumina MiSeq for phylogenetic studies of non-model organisms. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Comparative analyses of amplicon migration behavior in differing denaturing gradient gel electrophoresis (DGGE) systems

    Science.gov (United States)

    Thornhill, D. J.; Kemp, D. W.; Sampayo, E. M.; Schmidt, G. W.

    2010-03-01

    Denaturing gradient gel electrophoresis (DGGE) is commonly utilized to identify and quantify microbial diversity, but the conditions required for different electrophoretic systems to yield equivalent results and optimal resolution have not been assessed. Herein, the influence of different DGGE system configuration parameters on microbial diversity estimates was tested using Symbiodinium, a group of marine eukaryotic microbes that are important constituents of coral reef ecosystems. To accomplish this, bacterial clone libraries were constructed and sequenced from cultured isolates of Symbiodinium for the ribosomal DNA internal transcribed spacer 2 (ITS2) region. From these, 15 clones were subjected to PCR with a GC clamped primer set for DGGE analyses. Migration behaviors of the resulting amplicons were analyzed using a range of conditions, including variation in the composition of the denaturing gradient, electrophoresis time, and applied voltage. All tests were conducted in parallel on two commercial DGGE systems, a C.B.S. Scientific DGGE-2001, and the Bio-Rad DCode system. In this context, identical nucleotide fragments exhibited differing migration behaviors depending on the model of apparatus utilized, with fragments denaturing at a lower gradient concentration and applied voltage on the Bio-Rad DCode system than on the C.B.S. Scientific DGGE-2001 system. Although equivalent PCR-DGGE profiles could be achieved with both brands of DGGE system, the composition of the denaturing gradient and application of electrophoresis time × voltage must be appropriately optimized to achieve congruent results across platforms.

  4. Fluorescent duplex allele-specific PCR and amplicon melting for rapid homogeneous mtDNA haplogroup H screening and sensitive mixture detection.

    Directory of Open Access Journals (Sweden)

    Harald Niederstätter

    Full Text Available BACKGROUND: For large scale studies aiming at a better understanding of mitochondrial DNA (mtDNA, sequence variation in particular mt haplogroups (hgs and population structure, reliable low-cost high-throughput genotyping assays are needed. Furthermore, methods facilitating sensitive mixture detection and relative quantification of allele proportions are indispensable for the study of heteroplasmy, mitochondrial sequence evolution, and mitochondrial disorders. Here the properties of a homogeneous competitive duplex allele specific PCR (ARMS assay were scrutinized in the light of these requirements. METHODOLOGY/PRINCIPAL FINDINGS: A duplex ARMS assay amplifying either the ancestral mtDNA 2706G allele (non-hg H samples or the derived 7028C allele (hg H samples in the presence of SYBR Green fluorescent reporter dye was developed and characterized. Product detection, allele calling, and hg inference were based on the amplicon-characteristic melting-point temperatures obtained with on-line post-PCR fluorescent dissociation curve analysis (DCA. The analytical window of the assay covered at least 5 orders of magnitude of template DNA input with a detection limit in the low picogram range of genomic DNA. A set of forensically relevant test specimens was analyzed successfully. The presence of mtDNA mixtures was detected over a broad range of input DNA amounts and mixture ratios, and the estimation of allele proportions in samples with known total mtDNA content was feasible with limitations. A qualified DNA analyst successfully analyzed approximately 2,200 DNA extracts within three regular working days, without using robotic lab-equipment. By performing the amplification on-line, the assay also facilitated absolute mtDNA quantification. CONCLUSIONS: Although this assay was developed just for a particular purpose, the approach is general in that it is potentially suitable in a broad variety of assay-layouts for many other applications, including the

  5. Sensitive multiplex RNA quantification using capillary electrophoresis-based single-strand conformation polymorphism.

    Science.gov (United States)

    Shin, Gi Won; Hwang, Hee Sung; Nam, Hong Gil; Oh, Mi-Hwa; Jung, Gyoo Yeol

    2010-05-01

    Quantification of RNA provides information crucial for various biological studies, including analysis of mRNA expression and that of microRNAs. Reverse transcription (RT) coupled with real-time polymerase chain reaction (PCR) is known to be the most accurate method for quantifying nucleic acids, and thus represents the state-of-the-art for RNA quantification. However, the use of real-time PCR for RNA quantification is limited to a single target per analytical run because of reductions in quantification power and limitations of fluorescence dyes associated with multiplex applications. Here, we report a novel multiplex RNA quantification method that uses capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) coupled with modified RT and asymmetric PCR. The reverse transcripts of seven in vitro transcribed RNAs were modified with common sequence tags and amplified by asymmetric PCR using primers specific to the common tags. The resulting amplicons were separated and quantified by CE-SSCP. A series of experiments using different amounts of RNA demonstrated that the assay had a limit of detection of 2 amol and a dynamic range of approximately 10(5). These results clearly indicate the potential of this method to provide robust and precise multiplex RNA quantification.

  6. DNA Methylation Analysis by Bisulfite Conversion Coupled to Double Multiplexed Amplicon-Based Next-Generation Sequencing (NGS).

    Science.gov (United States)

    Bashtrykov, Pavel; Jeltsch, Albert

    2018-01-01

    Methylation of cytosine bases in DNA is one of the main epigenetic signals regulating gene expression and chromatin structure. The distribution of DNA methylation in the genome has a cell-type-specific pattern and can be modulated by internal or external stimuli. One of the most powerful approaches to investigate DNA methylation patterns is bisulfite conversion of the DNA followed by DNA sequencing, which allows to determine methylation patterns at a single-cytosine resolution. Here, we present a protocol for bisulfite DNA methylation analysis of targeted genomic regions using amplicon-based next-generation sequencing (NGS) on an Illumina sequencing system. We use a PCR-free library generation approach and implement a nested strategy for double molecular barcoding of samples (combining indexing of adapters and in-line barcoding of individual amplicons) which allows highly multiplexed sequencing. Also, we discuss the main limitations of this technology in particular in relation to clonal DNA amplification and other PCR artifacts.

  7. Network analysis of the microorganism in 25 Danish wastewater treatment plants over 7 years using high-throughput amplicon sequencing

    DEFF Research Database (Denmark)

    Albertsen, Mads; Larsen, Poul; Saunders, Aaron Marc

    Wastewater treatment is the world’s largest biotechnological processes and a perfect model system for microbial ecology as the habitat is well defined and replicated all over the world. Extensive investigations on Danish wastewater treatment plants using fluorescent in situ hybridization have...... the existing knowledge on core genera with high-throughput amplicon sequencing, plant design and process data in order to identify interactions and community shaping factors. We investigated 25 Danish full-scale wastewater treatment plants with nutrient removal over a period of 7 years with two to four samples...... a year, totaling over 400 samples. All samples were subjected to 16S rDNA amplicon sequencing using V13 primers on the Illumina MiSeq platform (2x300bp) to a depth of at least 20.000 quality filtered reads per sample. The OTUs were assigned taxonomy based on a manually curated version of the greengenes...

  8. Competitive amplification of differentially melting amplicons (CADMA improves KRAS hotspot mutation testing in colorectal cancer

    Directory of Open Access Journals (Sweden)

    Kristensen Lasse

    2012-11-01

    Full Text Available Abstract Background Cancer is an extremely heterogeneous group of diseases traditionally categorized according to tissue of origin. However, even among patients with the same cancer subtype the cellular alterations at the molecular level are often very different. Several new therapies targeting specific molecular changes found in individual patients have initiated the era of personalized therapy and significantly improved patient care. In metastatic colorectal cancer (mCRC a selected group of patients with wild-type KRAS respond to antibodies against the epidermal growth factor receptor (EGFR. Testing for KRAS mutations is now required prior to anti-EGFR treatment, however, less sensitive methods based on conventional PCR regularly fail to detect KRAS mutations in clinical samples. Methods We have developed sensitive and specific assays for detection of the seven most common KRAS mutations based on a novel methodology named Competitive Amplification of Differentially Melting Amplicons (CADMA. The clinical applicability of these assays was assessed by analyzing 100 colorectal cancer samples, for which KRAS mutation status has been evaluated by the commercially available TheraScreen® KRAS mutation kit. Results The CADMA assays were sensitive to at least 0.5% mutant alleles in a wild-type background when using 50 nanograms of DNA in the reactions. Consensus between CADMA and the TheraScreen kit was observed in 96% of the colorectal cancer samples. In cases where disagreement was observed the CADMA result could be confirmed by a previously published assay based on TaqMan probes and by fast COLD-PCR followed by Sanger sequencing. Conclusions The high analytical sensitivity and specificity of CADMA may increase diagnostic sensitivity and specificity of KRAS mutation testing in mCRC patients.

  9. Evaluating the accuracy of amplicon-based microbiome computational pipelines on simulated human gut microbial communities.

    Science.gov (United States)

    Golob, Jonathan L; Margolis, Elisa; Hoffman, Noah G; Fredricks, David N

    2017-05-30

    Microbiome studies commonly use 16S rRNA gene amplicon sequencing to characterize microbial communities. Errors introduced at multiple steps in this process can affect the interpretation of the data. Here we evaluate the accuracy of operational taxonomic unit (OTU) generation, taxonomic classification, alpha- and beta-diversity measures for different settings in QIIME, MOTHUR and a pplacer-based classification pipeline, using a novel software package: DECARD. In-silico we generated 100 synthetic bacterial communities approximating human stool microbiomes to be used as a gold-standard for evaluating the colligative performance of microbiome analysis software. Our synthetic data closely matched the composition and complexity of actual healthy human stool microbiomes. Genus-level taxonomic classification was correctly done for only 50.4-74.8% of the source organisms. Miscall rates varied from 11.9 to 23.5%. Species-level classification was less successful, (6.9-18.9% correct); miscall rates were comparable to those of genus-level targets (12.5-26.2%). The degree of miscall varied by clade of organism, pipeline and specific settings used. OTU generation accuracy varied by strategy (closed, de novo or subsampling), reference database, algorithm and software implementation. Shannon diversity estimation accuracy correlated generally with OTU-generation accuracy. Beta-diversity estimates with Double Principle Coordinate Analysis (DPCoA) were more robust against errors introduced in processing than Weighted UniFrac. The settings suggested in the tutorials were among the worst performing in all outcomes tested. Even when using the same classification pipeline, the specific OTU-generation strategy, reference database and downstream analysis methods selection can have a dramatic effect on the accuracy of taxonomic classification, and alpha- and beta-diversity estimation. Even minor changes in settings adversely affected the accuracy of the results, bringing them far from the

  10. De novo origin of VCY2 from autosome to Y-transposed amplicon.

    Directory of Open Access Journals (Sweden)

    Peng-Rong Cao

    Full Text Available The formation of new genes is a primary driving force of evolution in all organisms. The de novo evolution of new genes from non-protein-coding genomic regions is emerging as an important additional mechanism for novel gene creation. Y chromosomes underlie sex determination in mammals and contain genes that are required for male-specific functions. In this study, a search was undertaken for Y chromosome de novo genes derived from non-protein-coding sequences. The Y chromosome orphan gene variable charge, Y-linked (VCY2, is an autosome-derived gene that has sequence similarity to large autosomal fragments but lacks an autosomal protein-coding homolog. VCY2 locates in the amplicon containing long DNA fragments that were transposed from autosomes to the Y chromosome before the ape-monkey split. We confirmed that VCY2 cannot be encoded by autosomes due to the presence of multiple disablers that disrupt the open reading frame, such as the absence of start or stop codons and the presence of premature stop codons. Similar observations have been made for homologs in the autosomes of the chimpanzee, gorilla, rhesus macaque, baboon and out-group marmoset, which suggests that there was a non-protein-coding ancestral VCY2 that was common to apes and monkeys that predated the transposition event. Furthermore, while protein-coding orthologs are absent, a putative non-protein-coding VCY2 with conserved disablers was identified in the rhesus macaque Y chromosome male-specific region. This finding implies that VCY2 might have not acquired its protein-coding ability before the ape-monkey split. VCY2 encodes a testis-specific expressed protein and is involved in the pathologic process of male infertility, and the acquisition of this gene might improve male fertility. This is the first evidence that de novo genes can be generated from transposed autosomal non-protein-coding segments, and this evidence provides novel insights into the evolutionary history of the Y

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

    Science.gov (United States)

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

    2016-03-01

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

  12. Unraveling Core Functional Microbiota in Traditional Solid-State Fermentation by High-Throughput Amplicons and Metatranscriptomics Sequencing

    Directory of Open Access Journals (Sweden)

    Zhewei Song

    2017-07-01

    Full Text Available Fermentation microbiota is specific microorganisms that generate different types of metabolites in many productions. In traditional solid-state fermentation, the structural composition and functional capacity of the core microbiota determine the quality and quantity of products. As a typical example of food fermentation, Chinese Maotai-flavor liquor production involves a complex of various microorganisms and a wide variety of metabolites. However, the microbial succession and functional shift of the core microbiota in this traditional food fermentation remain unclear. Here, high-throughput amplicons (16S rRNA gene amplicon sequencing and internal transcribed space amplicon sequencing and metatranscriptomics sequencing technologies were combined to reveal the structure and function of the core microbiota in Chinese soy sauce aroma type liquor production. In addition, ultra-performance liquid chromatography and headspace-solid phase microextraction-gas chromatography-mass spectrometry were employed to provide qualitative and quantitative analysis of the major flavor metabolites. A total of 10 fungal and 11 bacterial genera were identified as the core microbiota. In addition, metatranscriptomic analysis revealed pyruvate metabolism in yeasts (genera Pichia, Schizosaccharomyces, Saccharomyces, and Zygosaccharomyces and lactic acid bacteria (genus Lactobacillus classified into two stages in the production of flavor components. Stage I involved high-level alcohol (ethanol production, with the genus Schizosaccharomyces serving as the core functional microorganism. Stage II involved high-level acid (lactic acid and acetic acid production, with the genus Lactobacillus serving as the core functional microorganism. The functional shift from the genus Schizosaccharomyces to the genus Lactobacillus drives flavor component conversion from alcohol (ethanol to acid (lactic acid and acetic acid in Chinese Maotai-flavor liquor production. Our findings provide

  13. Obtaining representative community profiles of anaerobic digesters through optimisation of 16S rRNA amplicon sequencing protocols

    DEFF Research Database (Denmark)

    Kirkegaard, Rasmus Hansen; McIlroy, Simon Jon; Karst, Søren Michael

    RNA gene amplicon sequencing is rapid, cheap, high throughput, and has high taxonomic resolution. However, biases are introduced in multiple steps of this approach, including non-representative DNA extraction and uneven taxonomic coverage of selected PCR primers, potentially giving a skewed view...... parameters involving harsher lysis conditions than recommended for the commercial kit, and appeared to be similar for both the mesophilic and thermophilic reactor biomass samples. The community profiling was found to be greatly influenced by the selected PCR primers, and showed that in silico designed...

  14. Next-generation sequencing of multiple individuals per barcoded library by deconvolution of sequenced amplicons using endonuclease fragment analysis.

    Science.gov (United States)

    Andersen, Jeppe D; Pereira, Vania; Pietroni, Carlotta; Mikkelsen, Martin; Johansen, Peter; Børsting, Claus; Morling, Niels

    2014-08-01

    The simultaneous sequencing of samples from multiple individuals increases the efficiency of next-generation sequencing (NGS) while also reducing costs. Here we describe a novel and simple approach for sequencing DNA from multiple individuals per barcode. Our strategy relies on the endonuclease digestion of PCR amplicons prior to library preparation, creating a specific fragment pattern for each individual that can be resolved after sequencing. By using both barcodes and restriction fragment patterns, we demonstrate the ability to sequence the human melanocortin 1 receptor (MC1R) genes from 72 individuals using only 24 barcoded libraries.

  15. Analysis of intra-host genetic diversity of Prunus necrotic ringspot virus (PNRSV using amplicon next generation sequencing.

    Directory of Open Access Journals (Sweden)

    Wycliff M Kinoti

    Full Text Available PCR amplicon next generation sequencing (NGS analysis offers a broadly applicable and targeted approach to detect populations of both high- or low-frequency virus variants in one or more plant samples. In this study, amplicon NGS was used to explore the diversity of the tripartite genome virus, Prunus necrotic ringspot virus (PNRSV from 53 PNRSV-infected trees using amplicons from conserved gene regions of each of PNRSV RNA1, RNA2 and RNA3. Sequencing of the amplicons from 53 PNRSV-infected trees revealed differing levels of polymorphism across the three different components of the PNRSV genome with a total number of 5040, 2083 and 5486 sequence variants observed for RNA1, RNA2 and RNA3 respectively. The RNA2 had the lowest diversity of sequences compared to RNA1 and RNA3, reflecting the lack of flexibility tolerated by the replicase gene that is encoded by this RNA component. Distinct PNRSV phylo-groups, consisting of closely related clusters of sequence variants, were observed in each of PNRSV RNA1, RNA2 and RNA3. Most plant samples had a single phylo-group for each RNA component. Haplotype network analysis showed that smaller clusters of PNRSV sequence variants were genetically connected to the largest sequence variant cluster within a phylo-group of each RNA component. Some plant samples had sequence variants occurring in multiple PNRSV phylo-groups in at least one of each RNA and these phylo-groups formed distinct clades that represent PNRSV genetic strains. Variants within the same phylo-group of each Prunus plant sample had ≥97% similarity and phylo-groups within a Prunus plant sample and between samples had less ≤97% similarity. Based on the analysis of diversity, a definition of a PNRSV genetic strain was proposed. The proposed definition was applied to determine the number of PNRSV genetic strains in each of the plant samples and the complexity in defining genetic strains in multipartite genome viruses was explored.

  16. Introduction to uncertainty quantification

    CERN Document Server

    Sullivan, T J

    2015-01-01

    Uncertainty quantification is a topic of increasing practical importance at the intersection of applied mathematics, statistics, computation, and numerous application areas in science and engineering. This text provides a framework in which the main objectives of the field of uncertainty quantification are defined, and an overview of the range of mathematical methods by which they can be achieved. Complete with exercises throughout, the book will equip readers with both theoretical understanding and practical experience of the key mathematical and algorithmic tools underlying the treatment of uncertainty in modern applied mathematics. Students and readers alike are encouraged to apply the mathematical methods discussed in this book to their own favourite problems to understand their strengths and weaknesses, also making the text suitable as a self-study. This text is designed as an introduction to uncertainty quantification for senior undergraduate and graduate students with a mathematical or statistical back...

  17. Assessment of DNA degradation induced by thermal and UV radiation processing: implications for quantification of genetically modified organisms.

    Science.gov (United States)

    Ballari, Rajashekhar V; Martin, Asha

    2013-12-01

    DNA quality is an important parameter for the detection and quantification of genetically modified organisms (GMO's) using the polymerase chain reaction (PCR). Food processing leads to degradation of DNA, which may impair GMO detection and quantification. This study evaluated the effect of various processing treatments such as heating, baking, microwaving, autoclaving and ultraviolet (UV) irradiation on the relative transgenic content of MON 810 maize using pRSETMON-02, a dual target plasmid as a model system. Amongst all the processing treatments examined, autoclaving and UV irradiation resulted in the least recovery of the transgenic (CaMV 35S promoter) and taxon-specific (zein) target DNA sequences. Although a profound impact on DNA degradation was seen during the processing, DNA could still be reliably quantified by Real-time PCR. The measured mean DNA copy number ratios of the processed samples were in agreement with the expected values. Our study confirms the premise that the final analytical value assigned to a particular sample is independent of the degree of DNA degradation since the transgenic and the taxon-specific target sequences possessing approximately similar lengths degrade in parallel. The results of our study demonstrate that food processing does not alter the relative quantification of the transgenic content provided the quantitative assays target shorter amplicons and the difference in the amplicon size between the transgenic and taxon-specific genes is minimal. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. |SE|S|AM|E| Barcode: NGS-oriented software for amplicon characterization--application to species and environmental barcoding.

    Science.gov (United States)

    Piry, S; Guivier, E; Realini, A; Martin, J-F

    2012-11-01

    Progress in NGS technologies has opened up new opportunities for characterizing biodiversity, both for individual specimen identification and for environmental barcoding. Although the amount of data available to biologist is increasing, user-friendly tools to facilitate data analysis have yet to be developed. Our aim, with |SE|S|AM|E| Barcode, is to provide such support through a unified platform. The sequences are analysed through a pipeline that (i) processes NGS amplicon runs, filtering markers and samples, (ii) builds reference libraries and finally (iii) identifies (barcodes) the sequences in each amplicon from the reference library. We use a simulated data set for specimen identification and a recently published data set for environmental barcoding to validate the method. The results obtained are consistent with the expected characterizations (in silico and previously published, respectively). |SE|S|AM|E| Barcode and its documentation are freely available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported Licence for Windows and Linux from http://www1.montpellier.inra.fr/CBGP/NGS/. © 2012 Blackwell Publishing Ltd.

  19. Improved Efficiency and Reliability of NGS Amplicon Sequencing Data Analysis for Genetic Diagnostic Procedures Using AGSA Software

    Directory of Open Access Journals (Sweden)

    Axel Poulet

    2016-01-01

    Full Text Available Screening for BRCA mutations in women with familial risk of breast or ovarian cancer is an ideal situation for high-throughput sequencing, providing large amounts of low cost data. However, 454, Roche, and Ion Torrent, Thermo Fisher, technologies produce homopolymer-associated indel errors, complicating their use in routine diagnostics. We developed software, named AGSA, which helps to detect false positive mutations in homopolymeric sequences. Seventy-two familial breast cancer cases were analysed in parallel by amplicon 454 pyrosequencing and Sanger dideoxy sequencing for genetic variations of the BRCA genes. All 565 variants detected by dideoxy sequencing were also detected by pyrosequencing. Furthermore, pyrosequencing detected 42 variants that were missed with Sanger technique. Six amplicons contained homopolymer tracts in the coding sequence that were systematically misread by the software supplied by Roche. Read data plotted as histograms by AGSA software aided the analysis considerably and allowed validation of the majority of homopolymers. As an optimisation, additional 250 patients were analysed using microfluidic amplification of regions of interest (Access Array Fluidigm of the BRCA genes, followed by 454 sequencing and AGSA analysis. AGSA complements a complete line of high-throughput diagnostic sequence analysis, reducing time and costs while increasing reliability, notably for homopolymer tracts.

  20. A Systematic Assessment of Accuracy in Detecting Somatic Mosaic Variants by Deep Amplicon Sequencing: Application to NF2 Gene.

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    Elisa Contini

    Full Text Available The accurate detection of low-allelic variants is still challenging, particularly for the identification of somatic mosaicism, where matched control sample is not available. High throughput sequencing, by the simultaneous and independent analysis of thousands of different DNA fragments, might overcome many of the limits of traditional methods, greatly increasing the sensitivity. However, it is necessary to take into account the high number of false positives that may arise due to the lack of matched control samples. Here, we applied deep amplicon sequencing to the analysis of samples with known genotype and variant allele fraction (VAF followed by a tailored statistical analysis. This method allowed to define a minimum value of VAF for detecting mosaic variants with high accuracy. Then, we exploited the estimated VAF to select candidate alterations in NF2 gene in 34 samples with unknown genotype (30 blood and 4 tumor DNAs, demonstrating the suitability of our method. The strategy we propose optimizes the use of deep amplicon sequencing for the identification of low abundance variants. Moreover, our method can be applied to different high throughput sequencing approaches to estimate the background noise and define the accuracy of the experimental design.

  1. A Portable Automatic Endpoint Detection System for Amplicons of Loop Mediated Isothermal Amplification on Microfluidic Compact Disk Platform

    Directory of Open Access Journals (Sweden)

    Shah Mukim Uddin

    2015-03-01

    Full Text Available In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD. The sensitivity test has been performed with detection limit up to 2.5 × 10−3 ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment.

  2. Disease quantification in dermatology

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  3. Multitracer Positron Emission Tomographic Imaging of Exogenous Gene Expression Mediated by a Universal Herpes Simplex Virus 1 Amplicon Vector

    Directory of Open Access Journals (Sweden)

    Christiane Kummer

    2007-05-01

    Full Text Available To develop efficient and safe gene therapy approaches, the herpes simplex virus type 1 thymidine kinase gene (HSV-1-tk has been shown to function as a marker gene for the direct noninvasive in vivo localization of thymidine kinase (TK expression by positron emission tomography (PET using radiolabeled nucleoside analogues as specific TK substrates. Moreover, the gene encoding dopamine type 2 receptor (d2r could be used as a PET marker gene using specific radiolabeled receptor binding compounds. Here we describe the quantitative colocalization of d2r and HSV-1-tk gene expression mediated from a universal HSV-1 amplicon vector in a subcutaneous human Gli36dEGFR glioma model by PET. The HSV-1 amplicon vector was constructed using a bicistronic gene cassette to contain (1 the d2r80A mutant, which is able to bind its ligand racloprid but unable to activate downstream signal transduction pathways, and (2 the tk39 mutant with enhanced enzymatic activity toward guanosine analogues fused to the green fluorescent protein gene (tk39gfp serving as a marker gene in cell culture. After infection of human Gli36dEGFR glioma cells with the HSV-d2r80AIREStk39gfp (HSV-DITG amplicon vector in cell culture, D2 receptor expression and its targeting to the cell surface were determined by Western blotting and immunolabeling. Vector application in vivo served for quantitative colocalization of d2r80A- and tk39gfp-derived PET signals employing the specific D2 receptor binding compound [11C]racloprid and the specific TK39 substrate 9-(4-[18F]fluoro-3-hydroxymethylbutylguanine. Our results demonstrate that for the range of gene expression studied in vivo, both enzymatic and receptor binding assays give comparable quantitative information on the level of vector-mediated gene expression in vivo. The d2r80A in combination with a specific binding compound passing the intact blood-brain barrier might be an alternative marker gene for the noninvasive assessment of vector

  4. Identification of novel candidate target genes in amplicons of Glioblastoma multiforme tumors detected by expression and CGH microarray profiling

    Directory of Open Access Journals (Sweden)

    Hernández-Moneo Jose-Luis

    2006-09-01

    Full Text Available Abstract Background Conventional cytogenetic and comparative genomic hybridization (CGH studies in brain malignancies have shown that glioblastoma multiforme (GBM is characterized by complex structural and numerical alterations. However, the limited resolution of these techniques has precluded the precise identification of detailed specific gene copy number alterations. Results We performed a genome-wide survey of gene copy number changes in 20 primary GBMs by CGH on cDNA microarrays. A novel amplicon at 4p15, and previously uncharacterized amplicons at 13q32-34 and 1q32 were detected and are analyzed here. These amplicons contained amplified genes not previously reported. Other amplified regions containg well-known oncogenes in GBMs were also detected at 7p12 (EGFR, 7q21 (CDK6, 4q12 (PDGFRA, and 12q13-15 (MDM2 and CDK4. In order to identify the putative target genes of the amplifications, and to determine the changes in gene expression levels associated with copy number change events, we carried out parallel gene expression profiling analyses using the same cDNA microarrays. We detected overexpression of the novel amplified genes SLA/LP and STIM2 (4p15, and TNFSF13B and COL4A2 (13q32-34. Some of the candidate target genes of amplification (EGFR, CDK6, MDM2, CDK4, and TNFSF13B were tested in an independent set of 111 primary GBMs by using FISH and immunohistological assays. The novel candidate 13q-amplification target TNFSF13B was amplified in 8% of the tumors, and showed protein expression in 20% of the GBMs. Conclusion This high-resolution analysis allowed us to propose novel candidate target genes such as STIM2 at 4p15, and TNFSF13B or COL4A2 at 13q32-34 that could potentially contribute to the pathogenesis of these tumors and which would require futher investigations. We showed that overexpression of the amplified genes could be attributable to gene dosage and speculate that deregulation of those genes could be important in the development

  5. Community analysis of picocyanobacteria in an oligotrophic lake by cloning 16S rRNA gene and 16S rRNA gene amplicon sequencing.

    Science.gov (United States)

    Fujimoto, Naoshi; Mizuno, Keigo; Yokoyama, Tomoki; Ohnishi, Akihiro; Suzuki, Masaharu; Watanabe, Satoru; Komatsu, Kenji; Sakata, Yoichi; Kishida, Naohiro; Akiba, Michihiro; Matsukura, Satoko

    2015-01-01

    In this study, the picocyanobacterial species composition of Lake Miyagase was examined by analyzing the 16S rRNA gene in a clone library and by amplicon sequencing using a benchtop next-generation sequencer. Five separate samples were analyzed from different days over a ten-month period. In the picocyanobacterial lineage, 9 and 12 OTUs were identified from a clone library and by amplicon sequencing, respectively. Both analyses suggested that a picocyanobacterium related to Synechococcus sp. MW6B4 was dominant in Lake Miyagase. Our findings suggest that 16S rRNA gene amplicon sequencing enables detailed evaluation of picocyanobacteria composition. One OTU identified was found to be a novel cluster that does not group with any of the known freshwater picocyanobacteria.

  6. HPV Genotyping of Modified General Primer-Amplicons Is More Analytically Sensitive and Specific by Sequencing than by Hybridization.

    Science.gov (United States)

    Meisal, Roger; Rounge, Trine Ballestad; Christiansen, Irene Kraus; Eieland, Alexander Kirkeby; Worren, Merete Molton; Molden, Tor Faksvaag; Kommedal, Øyvind; Hovig, Eivind; Leegaard, Truls Michael; Ambur, Ole Herman

    2017-01-01

    Sensitive and specific genotyping of human papillomaviruses (HPVs) is important for population-based surveillance of carcinogenic HPV types and for monitoring vaccine effectiveness. Here we compare HPV genotyping by Next Generation Sequencing (NGS) to an established DNA hybridization method. In DNA isolated from urine, the overall analytical sensitivity of NGS was found to be 22% higher than that of hybridization. NGS was also found to be the most specific method and expanded the detection repertoire beyond the 37 types of the DNA hybridization assay. Furthermore, NGS provided an increased resolution by identifying genetic variants of individual HPV types. The same Modified General Primers (MGP)-amplicon was used in both methods. The NGS method is described in detail to facilitate implementation in the clinical microbiology laboratory and includes suggestions for new standards for detection and calling of types and variants with improved resolution.

  7. HPV Genotyping of Modified General Primer-Amplicons Is More Analytically Sensitive and Specific by Sequencing than by Hybridization.

    Directory of Open Access Journals (Sweden)

    Roger Meisal

    Full Text Available Sensitive and specific genotyping of human papillomaviruses (HPVs is important for population-based surveillance of carcinogenic HPV types and for monitoring vaccine effectiveness. Here we compare HPV genotyping by Next Generation Sequencing (NGS to an established DNA hybridization method. In DNA isolated from urine, the overall analytical sensitivity of NGS was found to be 22% higher than that of hybridization. NGS was also found to be the most specific method and expanded the detection repertoire beyond the 37 types of the DNA hybridization assay. Furthermore, NGS provided an increased resolution by identifying genetic variants of individual HPV types. The same Modified General Primers (MGP-amplicon was used in both methods. The NGS method is described in detail to facilitate implementation in the clinical microbiology laboratory and includes suggestions for new standards for detection and calling of types and variants with improved resolution.

  8. Fluorescent quantification of melanin

    OpenAIRE

    Fernandes, Bruno Pacheco; Matamá, Maria Teresa; Guimarães, Diana Isabel Pereira; Gomes, Andreia; Cavaco-Paulo, Artur

    2016-01-01

    Melanin quantification is reportedly performed by absorption spectroscopy, commonly at 405 nm. Here, we propose the implementation of fluorescence spectroscopy for melanin assessment. In a typical in vitro assay to assess melanin production in response to an external stimulus, absorption spectroscopy clearly overvalues melanin content. This method is also incapable of distinguishing non-melanotic/amelanotic control cells from those that are actually capable of performing melanogenesis. Theref...

  9. Shotgun metagenomes and multiple primer pair-barcode combinations of amplicons reveal biases in metabarcoding analyses of fungi

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    Leho Tedersoo

    2015-05-01

    Full Text Available Rapid development of high-throughput (HTS molecular identification methods has revolutionized our knowledge about taxonomic diversity and ecology of fungi. However, PCR-based methods exhibit multiple technical shortcomings that may bias our understanding of the fungal kingdom. This study was initiated to quantify potential biases in fungal community ecology by comparing the relative performance of amplicon-free shotgun metagenomics and amplicons of nine primer pairs over seven nuclear ribosomal DNA (rDNA regions often used in metabarcoding analyses. The internal transcribed spacer (ITS barcodes ITS1 and ITS2 provided greater taxonomic and functional resolution and richness of operational taxonomic units (OTUs at the 97% similarity threshold compared to barcodes located within the ribosomal small subunit (SSU and large subunit (LSU genes. All barcode-primer pair combinations provided consistent results in ranking taxonomic richness and recovering the importance of floristic variables in driving fungal community composition in soils of Papua New Guinea. The choice of forward primer explained up to 2.0% of the variation in OTU-level analysis of the ITS1 and ITS2 barcode data sets. Across the whole data set, barcode-primer pair combination explained 37.6–38.1% of the variation, which surpassed any environmental signal. Overall, the metagenomics data set recovered a similar taxonomic overview, but resulted in much lower fungal rDNA sequencing depth, inability to infer OTUs, and high uncertainty in identification. We recommend the use of ITS2 or the whole ITS region for metabarcoding and we advocate careful choice of primer pairs in consideration of the relative proportion of fungal DNA and expected dominant groups.

  10. Bacterial diversity in Solenopsis invicta and Solenopsis geminata ant colonies characterized by 16S amplicon 454 pyrosequencing.

    Science.gov (United States)

    Ishak, Heather D; Plowes, Rob; Sen, Ruchira; Kellner, Katrin; Meyer, Eli; Estrada, Dora A; Dowd, Scot E; Mueller, Ulrich G

    2011-05-01

    Social insects harbor diverse assemblages of bacterial microbes, which may play a crucial role in the success or failure of biological invasions. The invasive fire ant Solenopsis invicta (Formicidae, Hymenoptera) is a model system for understanding the dynamics of invasive social insects and their biological control. However, little is known about microbes as biotic factors influencing the success or failure of ant invasions. This pilot study is the first attempt to characterize and compare microbial communities associated with the introduced S. invicta and the native Solenopsis geminata in the USA. Using 16S amplicon 454 pyrosequencing, bacterial communities of workers, brood, and soil from nest walls were compared between neighboring S. invicta and S. geminata colonies at Brackenridge Field Laboratory, Austin, Texas, with the aim of identifying potential pathogenic, commensal, or mutualistic microbial associates. Two samples of S. geminata workers showed high counts of Spiroplasma bacteria, a known pathogen or mutualist of other insects. A subsequent analysis using PCR and sequencing confirmed the presence of Spiroplasma in additional colonies of both Solenopsis species. Wolbachia was found in one alate sample of S. geminata, while one brood sample of S. invicta had a high count of Lactococcus. As expected, ant samples from both species showed much lower microbial diversity than the surrounding soil. Both ant species had similar overall bacterial diversities, although little overlap in specific microbes. To properly characterize a single bacterial community associated with a Solenopsis ant sample, rarefaction analyses indicate that it is necessary to obtain 5,000-10,000 sequences. Overall, 16S amplicon 454 pyrosequencing appears to be a cost-effective approach to screen whole microbial diversity associated with invasive ant species.

  11. Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform.

    Directory of Open Access Journals (Sweden)

    Hideyuki Tamaki

    Full Text Available BACKGROUND: 16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform METHODOLOGY/PRINCIPAL FINDINGS: The following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5-1.6 times more useable reads than the standard method (Method-1, after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management. CONCLUSIONS: Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies.

  12. Investigation of Microbial Diversity in Geothermal Hot Springs in Unkeshwar, India, Based on 16S rRNA Amplicon Metagenome Sequencing

    OpenAIRE

    Mehetre, Gajanan T.; Paranjpe, Aditi; Dastager, Syed G.; Dharne, Mahesh S.

    2016-01-01

    Microbial diversity in geothermal waters of the Unkeshwar hot springs in Maharashtra, India, was studied using 16S rRNA amplicon metagenomic sequencing. Taxonomic analysis revealed the presence of Bacteroidetes, Proteobacteria, Cyanobacteria, Actinobacteria, Archeae, and OD1 phyla. Metabolic function prediction analysis indicated a battery of biological information systems indicating rich and novel microbial diversity, with potential biotechnological applications in this niche.

  13. Obtaining representative community profiles of anaerobic digesters through optimisation of 16S rRNA amplicon sequencing protocols

    DEFF Research Database (Denmark)

    Kirkegaard, Rasmus Hansen; McIlroy, Simon Jon; Karst, Søren Michael

    A reliable and reproducible method for identification and quantification of the microorganisms involved in biogas production is important for the study and understanding of the microbial communities responsible for the function of anaerobic digester systems. DNA based identification using 16S rRN...

  14. Optimisation of 16S rDNA amplicon sequencing protocols for microbial community profiling of anaerobic digesters

    DEFF Research Database (Denmark)

    Kirkegaard, Rasmus Hansen; McIlroy, Simon Jon; Larsen, Poul

    A reliable and reproducible method for identification and quantification of the microorganisms involved in biogas production is important for the study and understanding of the microbial communities responsible for the function of anaerobic digester systems. DNA based identification using 16S rRN...

  15. KAT6A, a Chromatin Modifier from the 8p11-p12 Amplicon is a Candidate Oncogene in Luminal Breast Cancer

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    Brittany Turner-Ivey

    2014-08-01

    Full Text Available The chromosome 8p11-p12 amplicon is present in 12% to 15% of breast cancers, resulting in an increase in copy number and expression of several chromatin modifiers in these tumors, including KAT6A. Previous analyses in SUM-52 breast cancer cells showed amplification and overexpression of KAT6A, and subsequent RNAi screening identified KAT6A as a potential driving oncogene. KAT6A is a histone acetyltransferase previously identified as a fusion partner with CREB binding protein in acute myeloid leukemia. Knockdown of KAT6A in SUM-52 cells, a luminal breast cancer cell line harboring the amplicon, resulted in reduced growth rate compared to non-silencing controls and profound loss of clonogenic capacity both in mono-layer and in soft agar. The normal cell line MCF10A, however, did not exhibit slower growth with knockdown of KAT6A. SUM-52 cells with KAT6A knockdown formed fewer mammospheres in culture compared to controls, suggesting a possible role for KAT6A in self-renewal. Previous data from our laboratory identified FGFR2 as a driving oncogene in SUM-52 cells. The colony forming efficiency of SUM-52 KAT6A knockdown cells in the presence of FGFR inhibition was significantly reduced compared to cells with KAT6A knockdown only. These data suggest that KAT6A may be a novel oncogene in breast cancers bearing the 8p11-p12 amplicon. While there are other putative oncogenes in the amplicon, the identification of KAT6A as a driving oncogene suggests that chromatin-modifying enzymes are a key class of oncogenes in these cancers, and play an important role in the selection of this amplicon in luminal B breast cancers.

  16. Accident sequence quantification with KIRAP

    International Nuclear Information System (INIS)

    Kim, Tae Un; Han, Sang Hoon; Kim, Kil You; Yang, Jun Eon; Jeong, Won Dae; Chang, Seung Cheol; Sung, Tae Yong; Kang, Dae Il; Park, Jin Hee; Lee, Yoon Hwan; Hwang, Mi Jeong.

    1997-01-01

    The tasks of probabilistic safety assessment(PSA) consists of the identification of initiating events, the construction of event tree for each initiating event, construction of fault trees for event tree logics, the analysis of reliability data and finally the accident sequence quantification. In the PSA, the accident sequence quantification is to calculate the core damage frequency, importance analysis and uncertainty analysis. Accident sequence quantification requires to understand the whole model of the PSA because it has to combine all event tree and fault tree models, and requires the excellent computer code because it takes long computation time. Advanced Research Group of Korea Atomic Energy Research Institute(KAERI) has developed PSA workstation KIRAP(Korea Integrated Reliability Analysis Code Package) for the PSA work. This report describes the procedures to perform accident sequence quantification, the method to use KIRAP's cut set generator, and method to perform the accident sequence quantification with KIRAP. (author). 6 refs

  17. A one-step real-time multiplex PCR for screening Y-chromosomal microdeletions without downstream amplicon size analysis.

    Directory of Open Access Journals (Sweden)

    Viviana Kozina

    Full Text Available BACKGROUND: Y-chromosomal microdeletions (YCMD are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers. PRINCIPAL FINDINGS: The widely established "EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004" were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size. SIGNIFICANCE: With these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection.

  18. Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine.

    Science.gov (United States)

    Chaitanya, Lakshmi; Ralf, Arwin; van Oven, Mannis; Kupiec, Tomasz; Chang, Joseph; Lagacé, Robert; Kayser, Manfred

    2015-12-01

    Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylogenetic resolution. Most established protocols on the different massively parallel sequencing (MPS) platforms, however, invariably involve the PCR amplification of large fragments, typically several kilobases in size, which may fail due to mtDNA fragmentation in the available degraded materials. We introduce a MPS tiling approach for simultaneous whole human mt genome sequencing using 161 short overlapping amplicons (average 200 bp) with the Ion Torrent Personal Genome Machine. We illustrate the performance of this new method by sequencing 20 DNA samples belonging to different worldwide mtDNA haplogroups. Additional quality control, particularly regarding the potential detection of nuclear insertions of mtDNA (NUMTs), was performed by comparative MPS analysis using the conventional long-range amplification method. Preliminary sensitivity testing revealed that detailed haplogroup inference was feasible with 100 pg genomic input DNA. Complete mt genome coverage was achieved from DNA samples experimentally degraded down to genomic fragment sizes of about 220 bp, and up to 90% coverage from naturally degraded samples. Overall, we introduce a new approach for whole mt genome MPS analysis from degraded and nondegraded materials relevant to resolve and infer maternal genetic ancestry at complete resolution in anthropological, evolutionary, medical, and forensic applications. © 2015 The Authors. **Human Mutation published by Wiley Periodicals, Inc.

  19. Factors that affect large subunit ribosomal DNA amplicon sequencing studies of fungal communities: classification method, primer choice, and error.

    Directory of Open Access Journals (Sweden)

    Teresita M Porter

    Full Text Available Nuclear large subunit ribosomal DNA is widely used in fungal phylogenetics and to an increasing extent also amplicon-based environmental sequencing. The relatively short reads produced by next-generation sequencing, however, makes primer choice and sequence error important variables for obtaining accurate taxonomic classifications. In this simulation study we tested the performance of three classification methods: 1 a similarity-based method (BLAST + Metagenomic Analyzer, MEGAN; 2 a composition-based method (Ribosomal Database Project naïve bayesian classifier, NBC; and, 3 a phylogeny-based method (Statistical Assignment Package, SAP. We also tested the effects of sequence length, primer choice, and sequence error on classification accuracy and perceived community composition. Using a leave-one-out cross validation approach, results for classifications to the genus rank were as follows: BLAST + MEGAN had the lowest error rate and was particularly robust to sequence error; SAP accuracy was highest when long LSU query sequences were classified; and, NBC runs significantly faster than the other tested methods. All methods performed poorly with the shortest 50-100 bp sequences. Increasing simulated sequence error reduced classification accuracy. Community shifts were detected due to sequence error and primer selection even though there was no change in the underlying community composition. Short read datasets from individual primers, as well as pooled datasets, appear to only approximate the true community composition. We hope this work informs investigators of some of the factors that affect the quality and interpretation of their environmental gene surveys.

  20. Multilocus amplicon sequencing of Pseudomonas aeruginosa cystic fibrosis airways isolates collected prior to and after early antipseudomonal chemotherapy.

    Science.gov (United States)

    Fischer, Sebastian; Greipel, Leonie; Klockgether, Jens; Dorda, Marie; Wiehlmann, Lutz; Cramer, Nina; Tümmler, Burkhard

    2017-05-01

    Early antimicrobial chemotherapy can prevent or at least delay chronic cystic fibrosis (CF) airways infections with Pseudomonas aeruginosa. During a 10-year study period P. aeruginosa was detected for the first time in 54 CF patients regularly seen at the CF centre Hannover. Amplicon sequencing of 34 loci of the P. aeruginosa core genome was performed in baseline and post-treatment isolates of the 15 CF patients who had remained P. aeruginosa - positive after the first round of antipseudomonal chemotherapy. Deep sequencing uncovered coexisting alternative nucleotides at in total 33 of 55,284 examined genome positions including six non-synonymous polymorphisms in the lasR gene, a key regulator of quorum sensing. After early treatment 42 of 50 novel nucleotide substitutions had emerged in exopolysaccharide biosynthesis, efflux pump and porin genes. Early treatment selects pathoadaptive mutations in P. aeruginosa that are typical for chronic infections of CF lungs. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  1. Metagenomic Analysis of Slovak Bryndza Cheese Using Next-Generation 16S rDNA Amplicon Sequencing

    Directory of Open Access Journals (Sweden)

    Planý Matej

    2016-06-01

    Full Text Available Knowledge about diversity and taxonomic structure of the microbial population present in traditional fermented foods plays a key role in starter culture selection, safety improvement and quality enhancement of the end product. Aim of this study was to investigate microbial consortia composition in Slovak bryndza cheese. For this purpose, we used culture-independent approach based on 16S rDNA amplicon sequencing using next generation sequencing platform. Results obtained by the analysis of three commercial (produced on industrial scale in winter season and one traditional (artisanal, most valued, produced in May Slovak bryndza cheese sample were compared. A diverse prokaryotic microflora composed mostly of the genera Lactococcus, Streptococcus, Lactobacillus, and Enterococcus was identified. Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were the dominant taxons in all tested samples. Second most abundant species, detected in all bryndza cheeses, were Lactococcus fujiensis and Lactococcus taiwanensis, independently by two different approaches, using different reference 16S rRNA genes databases (Greengenes and NCBI respectively. They have been detected in bryndza cheese samples in substantial amount for the first time. The narrowest microbial diversity was observed in a sample made with a starter culture from pasteurised milk. Metagenomic analysis by high-throughput sequencing using 16S rRNA genes seems to be a powerful tool for studying the structure of the microbial population in cheeses.

  2. Fungi Sailing the Arctic Ocean: Speciose Communities in North Atlantic Driftwood as Revealed by High-Throughput Amplicon Sequencing.

    Science.gov (United States)

    Rämä, Teppo; Davey, Marie L; Nordén, Jenni; Halvorsen, Rune; Blaalid, Rakel; Mathiassen, Geir H; Alsos, Inger G; Kauserud, Håvard

    2016-08-01

    High amounts of driftwood sail across the oceans and provide habitat for organisms tolerating the rough and saline environment. Fungi have adapted to the extremely cold and saline conditions which driftwood faces in the high north. For the first time, we applied high-throughput sequencing to fungi residing in driftwood to reveal their taxonomic richness, community composition, and ecology in the North Atlantic. Using pyrosequencing of ITS2 amplicons obtained from 49 marine logs, we found 807 fungal operational taxonomic units (OTUs) based on clustering at 97 % sequence similarity cut-off level. The phylum Ascomycota comprised 74 % of the OTUs and 20 % belonged to Basidiomycota. The richness of basidiomycetes decreased with prolonged submersion in the sea, supporting the general view of ascomycetes being more extremotolerant. However, more than one fourth of the fungal OTUs remained unassigned to any fungal class, emphasising the need for better DNA reference data from the marine habitat. Different fungal communities were detected in coniferous and deciduous logs. Our results highlight that driftwood hosts a considerably higher fungal diversity than currently known. The driftwood fungal community is not a terrestrial relic but a speciose assemblage of fungi adapted to the stressful marine environment and different kinds of wooden substrates found in it.

  3. Direct quantification of fungal DNA from soil substrate using real-time PCR.

    Science.gov (United States)

    Filion, Martin; St-Arnaud, Marc; Jabaji-Hare, Suha H

    2003-04-01

    Detection and quantification of genomic DNA from two ecologically different fungi, the plant pathogen Fusarium solani f. sp. phaseoli and the arbuscular mycorrhizal fungus Glomus intraradices, was achieved from soil substrate. Specific primers targeting a 362-bp fragment from the SSU rRNA gene region of G. intraradices and a 562-bp fragment from the F. solani f. sp. phaseoli translation elongation factor 1 alpha gene were used in real-time polymerase chain reaction (PCR) assays conjugated with the fluorescent SYBR(R) Green I dye. Standard curves showed a linear relation (r(2)=0.999) between log values of fungal genomic DNA of each species and real-time PCR threshold cycles and were quantitative over 4-5 orders of magnitude. Real-time PCR assays were applied to in vitro-produced fungal structures and sterile and non-sterile soil substrate seeded with known propagule numbers of either fungi. Detection and genomic DNA quantification was obtained from the different treatments, while no amplicon was detected from non-seeded non-sterile soil samples, confirming the absence of cross-reactivity with the soil microflora DNA. A significant correlation (Pgenomic DNA of F. solani f. sp. phaseoli or G. intraradices detected and the number of fungal propagules present in seeded soil substrate. The DNA extraction protocol and real-time PCR quantification assay can be performed in less than 2 h and is adaptable to detect and quantify genomic DNA from other soilborne fungi.

  4. The practical analysis of food: the development of Sakalar quantification table of DNA (SQT-DNA).

    Science.gov (United States)

    Sakalar, Ergün

    2013-11-15

    Practical and highly sensitive Sakalar quantification table of DNA (SQT-DNA) has been developed for the detection% of species-specific DNA amount in food products. Cycle threshold (Ct) data were obtained from multiple curves of real-time qPCR. The statistical analysis was done to estimate the concentration of standard dilutions. Amplicon concentrations versus each Ct value were assessed by the predictions of targets at known concentrations. SQT-DNA was prepared by using the percentage versus each Ct values. The applicability of SQT-DNA to commercial foods was proved by using sausages containing varying ratios of beef, chicken, and soybean. The results showed that SQT-DNA can be used to directly quantify food DNA by a single PCR without the need to construct a standart curve in parallel with the samples every time the experiment is performed, and also quantification by SQT-DNA is as reliable as standard curve quantification for a wide range of DNA concentrations. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Combined amplicon pyrosequencing assays reveal presence of the apicomplexan "type-N" (cf. Gemmocystis cylindrus and Chromera velia on the Great Barrier Reef, Australia.

    Directory of Open Access Journals (Sweden)

    Jan Slapeta

    Full Text Available BACKGROUND: The coral is predominantly composed of the metabolically dependent coral host and the photosynthetic dinoflagellate Symbiodinium sp. The system as a whole interacts with symbiotic eukaryotes, bacteria and viruses. Gemmocystiscylindrus (cf. "type-N" symbiont belonging to the obligatory parasitic phylum Apicomplexa (Alveolata is ubiquitous in the Caribbean coral, but its presence in the Great Barrier Reef coral has yet to be documented. Approaches allowing identification of the healthy community from the pathogenic or saprobic organisms are needed for sustainable coral reef monitoring. METHODS & PRINCIPAL FINDINGS: We investigated the diversity of eukaryotes associated with a common reef-building corals from the southern Great Barrier Reef. We used three tag encoded 454 amplicon pyrosequencing assays targeting eukaryote small-subunit rRNA gene to demonstrate the presence of the apicomplexan type-N and a photosynthetic sister species to Apicomplexa-Chromeravelia. Amplicon pyrosequencing revealed presence of the small-subunit rRNA genes of known eukaryotic pathogens (Cryptosporidium and Cryptococcus. We therefore conducted bacterial tag encoded amplicon pyrosequencing assay for small-subunit rRNA gene to support effluent exposure of the coral. Bacteria of faecal origin (Enterobacteriales formed 41% of total sequences in contrast to 0-2% of the coral-associated bacterial communities with and without C. velia, respectively. SIGNIFICANCE: This is the first time apicomplexan type-N has been detected in the Great Barrier Reef. Eukaryote tag encoded amplicon pyrosequencing assays demonstrate presence of apicomplexan type-N and C. Velia in total coral DNA. The data highlight the need for combined approaches for eukaryotic diversity studies coupled with bacterial community assessment to achieve a more realistic goals of defining the holobiont community and assessing coral disease. With increasing evidence of Apicomplexa in coral reef

  6. Bacterial community in naturally fermented milk products of Arunachal Pradesh and Sikkim of India analysed by high-throughput amplicon sequencing

    OpenAIRE

    Shangpliang, H. Nakibapher Jones; Rai, Ranjita; Keisam, Santosh; Jeyaram, Kumaraswamy; Tamang, Jyoti Prakash

    2018-01-01

    Naturally fermented milk (NFM) products are popular ethnic fermented foods in Arunachal Pradesh and Sikkim states of India. The present study is the first to have documented the bacterial community in 54 samples of NFM products viz. chhurpi, churkam, dahi and gheu/mar by high-throughput Illumina amplicon sequencing. Metagenomic investigation showed that Firmicutes (Streptococcaceae, Lactobacillaceae) and Proteobacteria (Acetobacteraceae) were the two predominant members of the bacterial commu...

  7. FasL and FADD delivery by a glioma-specific and cell cycle-dependent HSV-1 amplicon virus enhanced apoptosis in primary human brain tumors

    Directory of Open Access Journals (Sweden)

    Lam Paula Y

    2010-10-01

    Full Text Available Abstract Background Glioblastoma multiforme is the most malignant cancer of the brain and is notoriously difficult to treat due to the highly proliferative and infiltrative nature of the cells. Herein, we explored the combination treatment of pre-established human glioma xenograft using multiple therapeutic genes whereby the gene expression is regulated by both cell-type and cell cycle-dependent transcriptional regulatory mechanism conferred by recombinant HSV-1 amplicon vectors. Results We demonstrated for the first time that Ki67-positive proliferating primary human glioma cells cultured from biopsy samples were effectively induced into cell death by the dual-specific function of the pG8-FasL amplicon vectors. These vectors were relatively stable and exhibited minimal cytotoxicity in vivo. Intracranial implantation of pre-transduced glioma cells resulted in better survival outcome when compared with viral vectors inoculated one week post-implantation of tumor cells, indicating that therapeutic efficacy is dependent on the viral spread and mode of viral vectors administration. We further showed that pG8-FasL amplicon vectors are functional in the presence of commonly used treatment regimens for human brain cancer. In fact, the combined therapies of pG8-FasL and pG8-FADD in the presence of temozolomide significantly improved the survival of mice bearing intracranial high-grade gliomas. Conclusion Taken together, our results showed that the glioma-specific and cell cycle-dependent HSV-1 amplicon vector is potentially useful as an adjuvant therapy to complement the current gene therapy strategy for gliomas.

  8. Using surface-enhanced Raman spectroscopy and electrochemically driven melting to discriminate Yersinia pestis from Y. pseudotuberculosis based on single nucleotide polymorphisms within unpurified polymerase chain reaction amplicons.

    Science.gov (United States)

    Papadopoulou, Evanthia; Goodchild, Sarah A; Cleary, David W; Weller, Simon A; Gale, Nittaya; Stubberfield, Michael R; Brown, Tom; Bartlett, Philip N

    2015-02-03

    The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. Herein, we use electrochemically driven denaturation assays monitored by surface-enhanced Raman spectroscopy (SERS) to target single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis. Two assays targeting SNPs within the groEL and metH genes of these two species have been successfully designed. Polymerase chain reaction (PCR) was used to produce Texas Red labeled single-stranded DNA (ssDNA) amplicons of 262 and 251 bases for the groEL and metH targets, respectively. These amplicons were used in an unpurified form to hybridize to immobilized probes then subjected to electrochemically driven melting. In all cases electrochemically driven melting was able to discriminate between fully homologous DNA and that containing SNPs. The metH assay was particularly challenging due to the presence of only a single base mismatch in the middle of the 251 base long PCR amplicon. However, manipulation of assay conditions (conducting the electrochemical experiments at 10 °C) resulted in greater discrimination between the complementary and mismatched DNA. Replicate data were collected and analyzed for each duplex on different days, using different batches of PCR product and different sphere segment void (SSV) substrates. Despite the variability introduced by these differences, the assays are shown to be reliable and robust providing a new platform for strain discrimination using unpurified PCR samples.

  9. Investigation of Microbial Diversity in Geothermal Hot Springs in Unkeshwar, India, Based on 16S rRNA Amplicon Metagenome Sequencing.

    Science.gov (United States)

    Mehetre, Gajanan T; Paranjpe, Aditi; Dastager, Syed G; Dharne, Mahesh S

    2016-02-25

    Microbial diversity in geothermal waters of the Unkeshwar hot springs in Maharashtra, India, was studied using 16S rRNA amplicon metagenomic sequencing. Taxonomic analysis revealed the presence of Bacteroidetes, Proteobacteria, Cyanobacteria, Actinobacteria, Archeae, and OD1 phyla. Metabolic function prediction analysis indicated a battery of biological information systems indicating rich and novel microbial diversity, with potential biotechnological applications in this niche. Copyright © 2016 Mehetre et al.

  10. Combined amplicon pyrosequencing assays reveal presence of the apicomplexan "type-N" (cf. Gemmocystis cylindrus) and Chromera velia on the Great Barrier Reef, Australia.

    Science.gov (United States)

    Slapeta, Jan; Linares, Marjorie C

    2013-01-01

    The coral is predominantly composed of the metabolically dependent coral host and the photosynthetic dinoflagellate Symbiodinium sp. The system as a whole interacts with symbiotic eukaryotes, bacteria and viruses. Gemmocystiscylindrus (cf. "type-N" symbiont) belonging to the obligatory parasitic phylum Apicomplexa (Alveolata) is ubiquitous in the Caribbean coral, but its presence in the Great Barrier Reef coral has yet to be documented. Approaches allowing identification of the healthy community from the pathogenic or saprobic organisms are needed for sustainable coral reef monitoring. We investigated the diversity of eukaryotes associated with a common reef-building corals from the southern Great Barrier Reef. We used three tag encoded 454 amplicon pyrosequencing assays targeting eukaryote small-subunit rRNA gene to demonstrate the presence of the apicomplexan type-N and a photosynthetic sister species to Apicomplexa-Chromeravelia. Amplicon pyrosequencing revealed presence of the small-subunit rRNA genes of known eukaryotic pathogens (Cryptosporidium and Cryptococcus). We therefore conducted bacterial tag encoded amplicon pyrosequencing assay for small-subunit rRNA gene to support effluent exposure of the coral. Bacteria of faecal origin (Enterobacteriales) formed 41% of total sequences in contrast to 0-2% of the coral-associated bacterial communities with and without C. velia, respectively. This is the first time apicomplexan type-N has been detected in the Great Barrier Reef. Eukaryote tag encoded amplicon pyrosequencing assays demonstrate presence of apicomplexan type-N and C. Velia in total coral DNA. The data highlight the need for combined approaches for eukaryotic diversity studies coupled with bacterial community assessment to achieve a more realistic goals of defining the holobiont community and assessing coral disease. With increasing evidence of Apicomplexa in coral reef environments, it is important not only to understand the evolution of these

  11. A One-Step Real-Time Multiplex PCR for Screening Y-Chromosomal Microdeletions without Downstream Amplicon Size Analysis

    Science.gov (United States)

    Kozina, Viviana; Cappallo-Obermann, Heike; Gromoll, Jörg; Spiess, Andrej-Nikolai

    2011-01-01

    Backgound Y-chromosomal microdeletions (YCMD) are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR) is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers. Principal Findings The widely established “EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004)” were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size. Significance With these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection. PMID:21887237

  12. Redefining the Human Oral Mycobiome with Improved Practices in Amplicon-based Taxonomy: Discovery of Malassezia as a Prominent Commensal

    Science.gov (United States)

    Dupuy, Amanda K.; David, Marika S.; Li, Lu; Heider, Thomas N.; Peterson, Jason D.; Montano, Elizabeth A.; Dongari-Bagtzoglou, Anna; Diaz, Patricia I.; Strausbaugh, Linda D.

    2014-01-01

    Fungi are a large, complex group, increasingly recognized as emerging threats. Their roles as modifiers of health mandate accurate portrayals of fungal communities in humans. As an entry point into the airways and gastrointestinal tract, fungi in the mouth are relevant to several biocompartments. We have revised current practices in sequence-based taxonomy assignments and employed the improvements to address the question of the fungal genera present in the healthy human mouth. The human oral mycobiome was surveyed using massively parallel, high throughput sequencing of internal transcribed spacer 1 (ITS1) amplicons from saliva following robust extraction methods. Taxonomy was assigned by comparison to a curated reference dataset, followed by filtering with an empirically determined BLAST E-value match statistic (10−42). Nomenclature corrections further refined results by conjoining redundant names for a single fungal genus. Following these curation steps, about two-thirds of the initially identified genera were eliminated. In comparison with the one similar metagenomic study and several earlier culture-based ones, our findings change the current conception of the oral mycobiome, especially with the discovery of the high prevalence and abundance of the genus Malassezia. Previously identified as an important pathogen of the skin, and recently reported as the predominant fungal genus at the nostril and backs of the head and ear, this is the first account of Malassezia in the human mouth. Findings from this study were in good agreement with others on the existence of many consensus members of the core mycobiome, and on unique patterns for individual subjects. This research offered a cautionary note about unconditional acceptance of lengthy lists of community members produced by automated assignments, provided a roadmap for enhancing the likely biological relevance of sequence-based fungal surveys, and built the foundation for understanding the role of fungi in health

  13. Nuclear species-diagnostic SNP markers mined from 454 amplicon sequencing reveal admixture genomic structure of modern citrus varieties.

    Directory of Open Access Journals (Sweden)

    Franck Curk

    Full Text Available Most cultivated Citrus species originated from interspecific hybridisation between four ancestral taxa (C. reticulata, C. maxima, C. medica, and C. micrantha with limited further interspecific recombination due to vegetative propagation. This evolution resulted in admixture genomes with frequent interspecific heterozygosity. Moreover, a major part of the phenotypic diversity of edible citrus results from the initial differentiation between these taxa. Deciphering the phylogenomic structure of citrus germplasm is therefore essential for an efficient utilization of citrus biodiversity in breeding schemes. The objective of this work was to develop a set of species-diagnostic single nucleotide polymorphism (SNP markers for the four Citrus ancestral taxa covering the nine chromosomes, and to use these markers to infer the phylogenomic structure of secondary species and modern cultivars. Species-diagnostic SNPs were mined from 454 amplicon sequencing of 57 gene fragments from 26 genotypes of the four basic taxa. Of the 1,053 SNPs mined from 28,507 kb sequence, 273 were found to be highly diagnostic for a single basic taxon. Species-diagnostic SNP markers (105 were used to analyse the admixture structure of varieties and rootstocks. This revealed C. maxima introgressions in most of the old and in all recent selections of mandarins, and suggested that C. reticulata × C. maxima reticulation and introgression processes were important in edible mandarin domestication. The large range of phylogenomic constitutions between C. reticulata and C. maxima revealed in mandarins, tangelos, tangors, sweet oranges, sour oranges, grapefruits, and orangelos is favourable for genetic association studies based on phylogenomic structures of the germplasm. Inferred admixture structures were in agreement with previous hypotheses regarding the origin of several secondary species and also revealed the probable origin of several acid citrus varieties. The developed species

  14. HeurAA: accurate and fast detection of genetic variations with a novel heuristic amplicon aligner program for next generation sequencing.

    Directory of Open Access Journals (Sweden)

    Lőrinc S Pongor

    Full Text Available Next generation sequencing (NGS of PCR amplicons is a standard approach to detect genetic variations in personalized medicine such as cancer diagnostics. Computer programs used in the NGS community often miss insertions and deletions (indels that constitute a large part of known human mutations. We have developed HeurAA, an open source, heuristic amplicon aligner program. We tested the program on simulated datasets as well as experimental data from multiplex sequencing of 40 amplicons in 12 oncogenes collected on a 454 Genome Sequencer from lung cancer cell lines. We found that HeurAA can accurately detect all indels, and is more than an order of magnitude faster than previous programs. HeurAA can compare reads and reference sequences up to several thousand base pairs in length, and it can evaluate data from complex mixtures containing reads of different gene-segments from different samples. HeurAA is written in C and Perl for Linux operating systems, the code and the documentation are available for research applications at http://sourceforge.net/projects/heuraa/

  15. Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap.

    Science.gov (United States)

    Conway, J E; Zolotukhin, S; Muzyczka, N; Hayward, G S; Byrne, B J

    1997-11-01

    Recombinant adeno-associated virus (AAV) type 2 (rAAV) vectors have recently been shown to have great utility as gene transfer agents both in vitro and in vivo. One of the problems associated with the use of rAAV vectors has been the difficulty of large-scale vector production. Low-efficiency plasmid transfection of the rAAV vector and complementing AAV type 2 (AAV-2) functions (rep and cap) followed by superinfection with adenovirus has been the standard approach to rAAV production. The objectives of this study were to demonstrate the ability of a recombinant herpes simplex virus type 1 (HSV-1) amplicon expressing AAV-2 Rep and Cap to support replication and packaging of rAAV vectors. HSV-1 amplicon vectors were constructed which contain the AAV-2 rep and cap genes under control of their native promoters (p5, p19, and p40). An HSV-1 amplicon vector, HSV-RC/KOS or HSV-RC/d27, was generated by supplying helper functions with either wild-type HSV-1 (KOS strain) or the ICP27-deleted mutant of HSV-1, d27-1, respectively. Replication of the amplicon stocks is not inhibited by the presence of AAV-2 Rep proteins, which highlights important differences between HSV-1 and adenovirus replication and the mechanism of providing helper function for productive AAV infection. Coinfection of rAAV and HSV-RC/KOS resulted in the replication and amplification of rAAV genomes. Similarly, rescue and replication of rAAV genomes occurred when rAAV vector plasmids were transfected into cells followed by HSV-RC/KOS infection and when two rAAV proviral cell lines were infected with HSV-RC/KOS or HSV-RC/d27. Production of infectious rAAV by rescue from two rAAV proviral cell lines has also been achieved with HSV-RC/KOS and HSV-RC/d27. The particle titer of rAAV produced with HSV-RC/d27 is equal to that achieved by supplying rep and cap by transfection followed by adenovirus superinfection. Importantly, no detectable wild-type AAV-2 is generated with this approach. These results demonstrate

  16. Polymicrobial nature of chronic diabetic foot ulcer biofilm infections determined using bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP.

    Directory of Open Access Journals (Sweden)

    Scot E Dowd

    Full Text Available BACKGROUND: Diabetic extremity ulcers are associated with chronic infections. Such ulcer infections are too often followed by amputation because there is little or no understanding of the ecology of such infections or how to control or eliminate this type of chronic infection. A primary impediment to the healing of chronic wounds is biofilm phenotype infections. Diabetic foot ulcers are the most common, disabling, and costly complications of diabetes. Here we seek to derive a better understanding of the polymicrobial nature of chronic diabetic extremity ulcer infections. METHODS AND FINDINGS: Using a new bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP approach we have evaluated the bacterial diversity of 40 chronic diabetic foot ulcers from different patients. The most prevalent bacterial genus associated with diabetic chronic wounds was Corynebacterium spp. Findings also show that obligate anaerobes including Bacteroides, Peptoniphilus, Fingoldia, Anaerococcus, and Peptostreptococcus spp. are ubiquitous in diabetic ulcers, comprising a significant portion of the wound biofilm communities. Other major components of the bacterial communities included commonly cultured genera such as Streptococcus, Serratia, Staphylococcus and Enterococcus spp. CONCLUSIONS: In this article, we highlight the patterns of population diversity observed in the samples and introduce preliminary evidence to support the concept of functional equivalent pathogroups (FEP. Here we introduce FEP as consortia of genotypically distinct bacteria that symbiotically produce a pathogenic community. According to this hypothesis, individual members of these communities when they occur alone may not cause disease but when they coaggregate or consort together into a FEP the synergistic effect provides the functional equivalence of well-known pathogens, such as Staphylococcus aureus, giving the biofilm community the factors necessary to maintain chronic biofilm infections

  17. RNA-Based Amplicon Sequencing Reveals Microbiota Development during Ripening of Artisanal versus Industrial Lard d'Arnad.

    Science.gov (United States)

    Ferrocino, Ilario; Bellio, Alberto; Romano, Angelo; Macori, Guerrino; Rantsiou, Kalliopi; Decastelli, Lucia; Cocolin, Luca

    2017-08-15

    Valle d'Aosta Lard d'Arnad is a protected designation of origin (PDO) product produced from fat of the shoulder and back of heavy pigs. Its manufacturing process can be very diverse, especially regarding the maturation temperature and the NaCl concentration used for the brine; thereby, the main goal of this study was to investigate the impact of those parameters on the microbiota developed during curing and ripening. Three farms producing Lard d'Arnad were selected. Two plants, reflecting the industrial process characterized either by low maturation temperature (plant A [10% NaCl, 2°C]) or by using a low NaCl concentration (plant B [2.5% NaCl, 4°C]), were selected, while the third was characterized by an artisanal process (plant C [30% NaCl, 8°C]). Lard samples were obtained at time 0 and after 7, 15, 30, 60, and 90 days of maturation. From each plant, 3 independent lots were analyzed. The diversity of live microbiota was evaluated by using classical plate counts and amplicon target sequencing of small subunit (SSU) rRNA. The main taxa identified by sequencing were Acinetobacter johnsonii , Psychrobacter , Staphylococcus equorum , Staphylococcus sciuri , Pseudomonas fragi , Brochothrix , Halomonas , and Vibrio , and differences in their relative abundances distinguished samples from the individual plants. The composition of the microbiota was more similar among plants A and B, and it was characterized by the higher presence of taxa recognized as undesired bacteria in food-processing environments. Oligotype analysis of Halomonas and Acinetobacter revealed the presence of several characteristic oligotypes associated with A and B samples. IMPORTANCE Changes in the food production process can drastically affect the microbial community structure, with a possible impact on the final characteristics of the products. The industrial processes of Lard d'Arnad production are characterized by a reduction in the salt concentration in the brines to address a consumer demand

  18. Strategy for the maximization of clinically relevant information from hepatitis C virus, RT-PCR quantification.

    LENUS (Irish Health Repository)

    Levis, J

    2012-02-03

    BACKGROUND: The increasing clinical application of viral load assays for monitoring viral infections has been an incentive for the development of standardized tests for the hepatitis C virus. OBJECTIVE: To develop a simple model for the prediction of baseline viral load in individuals infected with the hepatitis C virus. METHODOLOGY: Viral load quantification of each patient\\'s first sample was assessed by RT-PCR-ELISA using the Roche MONITOR assay in triplicate. Genotype of the infecting virus was identified by reverse line probe hybridization, using amplicons resulting from the qualitative HCV Roche AMPLICOR assay. RESULTS: Retrospective evaluation of first quantitative values suggested that 82.4% (n=168\\/204) of individuals had a viral load between 4.3 and 6.7 log(10) viral copies per ml. A few patients (3.4%; n=7\\/204) have a serum viremia less than the lower limit of the linear range of the RT-PCR assay. Subsequent, prospective evaluation of hepatitis C viral load of all new patients using a model based on the dynamic range of viral load in the retrospective group correctly predicted the dynamic range in 75.9% (n=33\\/54). CONCLUSION: The dynamic range of hepatitis C viremia extends beyond the linear range of the Roche MONITOR assay. Accurate determination of serum viremia is substantially improved by dilution of specimens prior to quantification.

  19. Detection of double-stranded PCR amplicons at the attomole level electrosprayed from low nanomolar solutions using FT-ICR mass spectrometry.

    Science.gov (United States)

    Hannis, J C; Muddiman, D C

    2001-02-01

    An 82-base-pair polymerase chain reaction (PCR) product was amplified from the tetranucleotide short tandem repeat locus within the human tyrosine hydroxylase gene. PCR amplification was carried out using 100 ng of human nuclear DNA obtained from an individual who is homozygotic for the 9.3 allele resulting in a 50.5 kDa amplicon. To generate sufficient material for these investigations, several reactions were pooled and subsequently purified and quantified using UV-vis spectrophotometry. A serial dilution was carried out from a 2 microM stock solution providing solution concentrations down to 5 nM. Measurements were made using hexapole accumulation and gated trapping strategies in a 4.7 Telsa Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) which facilitated detection of the amplicon at the attomole level when electrosprayed from a 5 nM solution with a single acquisition! The signal-to-noise ratio was determined to be 8.3 for the spectrum derived from the 5 nM solution using the magnitude-mode mass spectral peak height for the most abundant charge-state. This remarkable sensitivity for large PCR amplicons will dramatically improve the ability of electrospray ionization mass spectrometry to address important genetic questions for low copy number genes or when the amount of initial template is limited; the latter issue is commonly encountered in DNA forensics. Furthermore, these data represents over 2 orders of magnitude decrease in detection limits over other existing ESI-MS reports concerning PCR products, including those conducted using FTICR-MS.

  20. Microbial diversity of a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment: integration of 16S rRNA gene amplicon and shotgun metagenomic sequencing.

    Science.gov (United States)

    Delforno, Tiago Palladino; Lacerda Júnior, Gileno Vieira; Noronha, Melline F; Sakamoto, Isabel K; Varesche, Maria Bernadete A; Oliveira, Valéria M

    2017-06-01

    The 16S rRNA gene amplicon and whole-genome shotgun metagenomic (WGSM) sequencing approaches were used to investigate wide-spectrum profiles of microbial composition and metabolic diversity from a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment. The data were generated by using MiSeq 2 × 250 bp and HiSeq 2 × 150 bp Illumina sequencing platforms for 16S amplicon and WGSM sequencing, respectively. Each approach revealed a distinct microbial community profile, with Pseudomonas and Psychrobacter as predominant genus for the WGSM dataset and Clostridium and Methanosaeta for the 16S rRNA gene amplicon dataset. The virome characterization revealed the presence of two viral families with Bacteria and Archaea as host, Myoviridae, and Siphoviridae. A wide functional diversity was found with predominance of genes involved in the metabolism of acetone, butanol, and ethanol synthesis; and one-carbon metabolism (e.g., methanogenesis). Genes related to the acetotrophic methanogenesis pathways were more abundant than methylotrophic and hydrogenotrophic, corroborating the taxonomic results that showed the prevalence of the acetotrophic genus Methanosaeta. Moreover, the dataset indicated a variety of metabolic genes involved in sulfur, nitrogen, iron, and phosphorus cycles, with many genera able to act in all cycles. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) revealed that microbial community contained 43 different types of antibiotic resistance genes, some of them were associated with growth chicken promotion (e.g., bacitracin, tetracycline, and polymyxin). © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  1. Assessment of Capture and Amplicon-Based Approaches for the Development of a Targeted Next-Generation Sequencing Pipeline to Personalize Lymphoma Management.

    Science.gov (United States)

    Hung, Stacy S; Meissner, Barbara; Chavez, Elizabeth A; Ben-Neriah, Susana; Ennishi, Daisuke; Jones, Martin R; Shulha, Hennady P; Chan, Fong Chun; Boyle, Merrill; Kridel, Robert; Gascoyne, Randy D; Mungall, Andrew J; Marra, Marco A; Scott, David W; Connors, Joseph M; Steidl, Christian

    2018-03-01

    Targeted next-generation sequencing panels are increasingly used to assess the value of gene mutations for clinical diagnostic purposes. For assay development, amplicon-based methods have been preferentially used on the basis of short preparation time and small DNA input amounts. However, capture sequencing has emerged as an alternative approach because of high testing accuracy. We compared capture hybridization and amplicon sequencing approaches using fresh-frozen and formalin-fixed, paraffin-embedded tumor samples from eight lymphoma patients. Next, we developed a targeted sequencing pipeline using a 32-gene panel for accurate detection of actionable mutations in formalin-fixed, paraffin-embedded tumor samples of the most common lymphocytic malignancies: chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. We show that hybrid capture is superior to amplicon sequencing by providing deep more uniform coverage and yielding higher sensitivity for variant calling. Sanger sequencing of 588 variants identified specificity limits of thresholds for mutation calling, and orthogonal validation on 66 cases indicated 93% concordance with whole-genome sequencing. The developed pipeline and assay identified at least one actionable mutation in 91% of tumors from 219 lymphoma patients and revealed subtype-specific mutation patterns and frequencies consistent with the literature. This pipeline is an accurate and sensitive method for identifying actionable gene mutations in routinely acquired biopsy materials, suggesting further assessment of capture-based assays in the context of personalized lymphoma management. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  2. Illumina MiSeq 16S amplicon sequence analysis of bovine respiratory disease associated bacteria in lung and mediastinal lymph node tissue.

    Science.gov (United States)

    Johnston, Dayle; Earley, Bernadette; Cormican, Paul; Murray, Gerard; Kenny, David Anthony; Waters, Sinead Mary; McGee, Mark; Kelly, Alan Kieran; McCabe, Matthew Sean

    2017-05-02

    Bovine respiratory disease (BRD) is caused by growth of single or multiple species of pathogenic bacteria in lung tissue following stress and/or viral infection. Next generation sequencing of 16S ribosomal RNA gene PCR amplicons (NGS 16S amplicon analysis) is a powerful culture-independent open reference method that has recently been used to increase understanding of BRD-associated bacteria in the upper respiratory tract of BRD cattle. However, it has not yet been used to examine the microbiome of the bovine lower respiratory tract. The objective of this study was to use NGS 16S amplicon analysis to identify bacteria in post-mortem lung and lymph node tissue samples harvested from fatal BRD cases and clinically healthy animals. Cranial lobe and corresponding mediastinal lymph node post-mortem tissue samples were collected from calves diagnosed as BRD cases by veterinary laboratory pathologists and from clinically healthy calves. NGS 16S amplicon libraries, targeting the V3-V4 region of the bacterial 16S rRNA gene were prepared and sequenced on an Illumina MiSeq. Quantitative insights into microbial ecology (QIIME) was used to determine operational taxonomic units (OTUs) which corresponded to the 16S rRNA gene sequences. Leptotrichiaceae, Mycoplasma, Pasteurellaceae, and Fusobacterium were the most abundant OTUs identified in the lungs and lymph nodes of the calves which died from BRD. Leptotrichiaceae, Fusobacterium, Mycoplasma, Trueperella and Bacteroides had greater relative abundances in post-mortem lung samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Leptotrichiaceae, Mycoplasma and Pasteurellaceae showed higher relative abundances in post-mortem lymph node samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Two Leptotrichiaceae sequence contigs were subsequently assembled from bacterial DNA-enriched shotgun sequences

  3. Development of a quantitative competitive reverse transcriptase polymerase chain reaction for the quantification of growth hormone gene expression in pigs

    Directory of Open Access Journals (Sweden)

    Maurício Machaim Franco

    2003-01-01

    Full Text Available After the advent of the genome projects, followed by the discovery of DNA polymorphisms, basic understanding of gene expression is the next focus to explain the association between polymorphisms and the level of gene expression, as well as to demonstrate the interaction among genes. Among the various techniques for the investigation of transcriptional profiling involving patterns of gene expression, quantitative PCR is the simplest analytical laboratory technique. The objective of this work was to analyze two strategies of a competitive PCR technique for the quantification of the pig growth hormone (GH gene expression. A pair of primers was designed targeting exons 3 and 5, and two competitive PCR strategies were performed, one utilizing a specific amplicon as a competitor, and the other utilizing a low-stringency PCR amplicon as a competitor. The latter strategy proved to be easier and more efficient, offering an accessible tool that can be used in any kind of competitive reaction, facilitating the study of gene expression patterns for both genetics and diagnostics of infectious diseases.

  4. Digital quantification of gene methylation in stool DNA by emulsion-PCR coupled with hydrogel immobilized bead-array.

    Science.gov (United States)

    Liu, Yunlong; Wu, Haiping; Zhou, Qiang; Song, Qinxin; Rui, Jianzhong; Zou, Bingjie; Zhou, Guohua

    2017-06-15

    Aberrations of gene methylation in stool DNA (sDNA) is an effective biomarker for non-invasive colorectal cancer diagnosis. However, it is challenging to accurately quantitate the gene methylation levels in sDNA due to the low abundance and degradation of sDNA. In this study, a digital quantification strategy was proposed by combining emulsion PCR (emPCR) with hydrogel immobilized bead-array. The assay includes following steps: bisulfite conversion of sDNA, pre-amplification by PCR with specific primers containing 5' universal sequences, emPCR of pre-amplicons with beaded primers to achieve single-molecular amplification and identification of hydrogel embedding beads coated with amplicons. The sensitivity and the specificity of the method are high enough to pick up 0.05% methylated targets from unmethylated DNA background. The successful detection of hypermethylated vimentin gene in clinical stool samples suggests that the proposed method should be a potential tool for non-invasive colorectal cancer screening. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.

    Directory of Open Access Journals (Sweden)

    Yong Wang

    Full Text Available Bacterial 16S ribosomal DNA (rDNA amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90% were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519-539, E969-983, E1063-1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies.

  6. Cowpea mosaic virus RNA-1 acts as an amplicon whose effects can be counteracted by a RNA-2-encoded suppressor of silencing

    International Nuclear Information System (INIS)

    Liu Li; Grainger, Jef; Canizares, M. Carmen; Angell, Susan M.; Lomonossoff, George P.

    2004-01-01

    Lines of Nicotiana benthamiana transgenic for full-length copies of both Cowpea mosaic virus (CPMV) genomic RNAs, either singly or together, have been produced. Plants transgenic for both RNAs developed symptoms characteristic of a CPMV infection. When plants transgenic for RNA-1 were agro-inoculated with RNA-2, no infection developed and the plants were also resistant to challenge with CPMV. By contrast, plants transgenic for RNA-2 became infected when agro-inoculated with RNA-1 and were fully susceptible to CPMV infection. The resistance of RNA-1 transgenic plants was shown to be related to the ability of RNA-1 to self-replicate and act as an amplicon. The ability of transgenically expressed RNA-2 to counteract the amplicon effect suggested that it encodes a suppressor of posttranscriptional gene silencing (PTGS). By examining the ability of portions of RNA-2 to reverse PTGS in N. benthamiana, we have identified the small (S) coat protein as the CPMV RNA-2-encoded suppressor of PTGS

  7. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies

    KAUST Repository

    Wang, Yong

    2009-10-09

    Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519-539, E969- 983, E1063-1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies. © 2009 Wang, Qian.

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

    Science.gov (United States)

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

    2011-08-05

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

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

    Directory of Open Access Journals (Sweden)

    Lett Jean-Michel

    2011-08-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-07-06

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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  12. A need for standardization in drinking water analysis – an investigation of DNA extraction procedure, primer choice and detection limit of 16S rRNA amplicon sequencing

    DEFF Research Database (Denmark)

    Brandt, Jakob; Nielsen, Per Halkjær; Albertsen, Mads

    have been made to illuminate the effects specifically related to bacterial communities in drinking water. In this study, we investigated the impact of the DNA extraction and primer choice on the observed community structure, and we also estimated the detection limit of the 16S rRNA amplicon sequencing....... The PowerWater DNA Isolation Kit resulted in significantly higher amounts of isolated DNA compared to the FastDNA SPIN Kit for Soil. Furthermore, extraction with the PowerWater kit lead to detection of a significantly higher number of OTUs. Likewise, the primer experiment revealed great discrepancies in OTU....... coli could be detected in all samples. However, samples with 101 cells/mL had several contaminating OTUs, constituting approximately 8% of the read abundances. For 16S rRNA gene analysis in drinking water samples, we recommend using the PowerWater DNA Isolation Kit for DNA extraction in combination...

  13. Bacterial tag encoded FLX titanium amplicon pyrosequencing (bTEFAP based assessment of prokaryotic diversity in metagenome of Lonar soda lake, India

    Directory of Open Access Journals (Sweden)

    Pravin Dudhagara

    2015-06-01

    Full Text Available Bacterial diversity and archaeal diversity in metagenome of the Lonar soda lake sediment were assessed by bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP. Metagenome comprised 5093 sequences with 2,531,282 bp and 53 ± 2% G + C content. Metagenome sequence data are available at NCBI under the Bioproject database with accession no. PRJNA218849. Metagenome sequence represented the presence of 83.1% bacterial and 10.5% archaeal origin. A total of 14 different bacteria demonstrating 57 species were recorded with dominating species like Coxiella burnetii (17%, Fibrobacter intestinalis (12% and Candidatus Cloacamonas acidaminovorans (11%. Occurrence of two archaeal phyla representing 24 species, among them Methanosaeta harundinacea (35%, Methanoculleus chikugoensis (12% and Methanolinea tarda (11% were dominating species. Significant presence of 11% sequences as an unclassified indicated the possibilities for unknown novel prokaryotes from the metagenome.

  14. Large-scale benchmarking reveals false discoveries and count transformation sensitivity in 16S rRNA gene amplicon data analysis methods used in microbiome studies

    DEFF Research Database (Denmark)

    Thorsen, Jonathan; Brejnrod, Asker Daniel; Mortensen, Martin Steen

    2016-01-01

    detection power. For beta-diversity-based sample separation, we show that library size normalization has very little effect and that the distance metric is the most important factor in terms of separation power. CONCLUSIONS: Our results, generalizable to datasets from different sequencing platforms......, demonstrate how the choice of method considerably affects analysis outcome. Here, we give recommendations for tools that exhibit low false positive rates, have good retrieval power across effect sizes and case/control proportions, and have low sparsity bias. Result output from some commonly used methods......BACKGROUND: There is an immense scientific interest in the human microbiome and its effects on human physiology, health, and disease. A common approach for examining bacterial communities is high-throughput sequencing of 16S rRNA gene hypervariable regions, aggregating sequence-similar amplicons...

  15. Quantification of virtue in late Medieval Europe.

    Science.gov (United States)

    Kemp, Simon

    2018-02-01

    Fourteenth century Europe saw a growing interest in quantification. This interest has been well studied by historians of physical sciences, but medieval scholars were also interested in the quantification of psychological qualities. In general, the quantification issues addressed by medieval scholars were theoretical, even (by our standards) mathematical, rather than those of practical measurement. There was recognition that the seriousness of a sin and the penance laid down for it should be proportionate. A number of late medieval scholars were interested in the quantification of caritas, a Latin word that is translatable as charity or loving benevolence. The scholastic interest linked to the practical issue of how caritas might become habitual through the repeated performance of virtuous acts. Gregory of Rimini's treatment of caritas in his commentary on Peter Lombard's Sentences illustrates how one medieval scholar related the quantification of virtue to the quantification of physical qualities such as temperature and luminescence. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

  16. Simplified strategy for rapid first-line screening of fragile X syndrome: closed-tube triplet-primed PCR and amplicon melt peak analysis.

    Science.gov (United States)

    Rajan-Babu, Indhu-Shree; Law, Hai-Yang; Yoon, Chui-Sheun; Lee, Caroline G; Chong, Samuel S

    2015-05-04

    Premutation and full-mutation hyperexpansion of CGG-triplets in the X-linked Fragile X Mental Retardation 1 (FMR1) gene have been implicated in fragile X-associated tremor/ataxia syndrome, fragile X-associated primary ovarian insufficiency, and fragile X syndrome (FXS), respectively. The currently available molecular diagnostic tests are either costly or labour-intensive, which prohibits their application as a first-line FMR1 test in large-scale population-based screening programs. In this study, we demonstrate the utility of a simplified closed-tube strategy for rapid first-line screening of FXS based on melt peak temperature (Tm) analysis of direct triplet-primed polymerase chain reaction amplicons (dTP-PCR MCA). In addition, we also evaluated the correlation between Tm and CGG-repeat size based on capillary electrophoresis (CE) of dTP-PCR amplicons. The assays were initially tested on 29 FMR1 reference DNA samples, followed by a blinded validation on 107 previously characterised patient DNA samples. The dTP-PCR MCA produced distinct melt profiles of higher Tm for samples carrying an expanded allele. Among the samples tested, we also observed a good correlation between Tm and CGG-repeat size. In the blinded validation study, dTP-PCR MCA accurately classified all normal and expansion carriers, and the FMR1 genotypic classification of all samples was completely concordant with the previously determined genotypes as well as the dTP-PCR CE results. This simple and cost-effective MCA-based assay may be useful as a first-line FXS screening tool that could rapidly screen out the large majority of unaffected individuals, thus minimising the number of samples that need to be analysed by Southern blot analysis.

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

    Science.gov (United States)

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

    2012-01-01

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

  18. Comparison of five DNA quantification methods

    DEFF Research Database (Denmark)

    Nielsen, Karsten; Mogensen, Helle Smidt; Hedman, Johannes

    2008-01-01

    Six commercial preparations of human genomic DNA were quantified using five quantification methods: UV spectrometry, SYBR-Green dye staining, slot blot hybridization with the probe D17Z1, Quantifiler Human DNA Quantification kit and RB1 rt-PCR. All methods measured higher DNA concentrations than...... Quantification kit in two experiments. The measured DNA concentrations with Quantifiler were 125 and 160% higher than expected based on the manufacturers' information. When the Quantifiler human DNA standard (Raji cell line) was replaced by the commercial human DNA preparation G147A (Promega) to generate the DNA...... standard curve in the Quantifiler Human DNA Quantification kit, the DNA quantification results of the human DNA preparations were 31% higher than expected based on the manufacturers' information. The results indicate a calibration problem with the Quantifiler human DNA standard for its use...

  19. Advancing agricultural greenhouse gas quantification*

    Science.gov (United States)

    Olander, Lydia; Wollenberg, Eva; Tubiello, Francesco; Herold, Martin

    2013-03-01

    1. Introduction Better information on greenhouse gas (GHG) emissions and mitigation potential in the agricultural sector is necessary to manage these emissions and identify responses that are consistent with the food security and economic development priorities of countries. Critical activity data (what crops or livestock are managed in what way) are poor or lacking for many agricultural systems, especially in developing countries. In addition, the currently available methods for quantifying emissions and mitigation are often too expensive or complex or not sufficiently user friendly for widespread use. The purpose of this focus issue is to capture the state of the art in quantifying greenhouse gases from agricultural systems, with the goal of better understanding our current capabilities and near-term potential for improvement, with particular attention to quantification issues relevant to smallholders in developing countries. This work is timely in light of international discussions and negotiations around how agriculture should be included in efforts to reduce and adapt to climate change impacts, and considering that significant climate financing to developing countries in post-2012 agreements may be linked to their increased ability to identify and report GHG emissions (Murphy et al 2010, CCAFS 2011, FAO 2011). 2. Agriculture and climate change mitigation The main agricultural GHGs—methane and nitrous oxide—account for 10%-12% of anthropogenic emissions globally (Smith et al 2008), or around 50% and 60% of total anthropogenic methane and nitrous oxide emissions, respectively, in 2005. Net carbon dioxide fluxes between agricultural land and the atmosphere linked to food production are relatively small, although significant carbon emissions are associated with degradation of organic soils for plantations in tropical regions (Smith et al 2007, FAO 2012). Population growth and shifts in dietary patterns toward more meat and dairy consumption will lead to

  20. A performance evaluation of Nextera XT and KAPA HyperPlus for rapid Illumina library preparation of long-range mitogenome amplicons.

    Science.gov (United States)

    Ring, Joseph D; Sturk-Andreaggi, Kimberly; Peck, Michelle A; Marshall, Charla

    2017-07-01

    Next-generation sequencing (NGS) facilitates the rapid and high-throughput generation of human mitochondrial genome (mitogenome) data to build population and reference databases for forensic comparisons. To this end, long-range amplification provides an effective method of target enrichment that is amenable to library preparation assays employing DNA fragmentation. This study compared the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) and the KAPA HyperPlus Library Preparation Kit (Kapa Biosystems, Wilmington, MA) for enzymatic fragmentation and indexing of ∼8500bp mitogenome amplicons for Illumina sequencing. The Nextera XT libraries produced low-coverage regions that were consistent across all samples, while the HyperPlus libraries resulted in uniformly high coverage across the mitogenome, even with reduced-volume reaction conditions. The balanced coverage observed from KAPA HyperPlus libraries enables not only low-level variant calling across the mitogenome but also increased sample multiplexing for greater processing efficiency. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Tandem multiplication of the IS26-flanked amplicon with the bla(SHV-5) gene within plasmid p1658/97.

    Science.gov (United States)

    Zienkiewicz, Maksymilian; Kern-Zdanowicz, Izabela; Carattoli, Alessandra; Gniadkowski, Marek; Cegłowski, Piotr

    2013-04-01

    The IncF plasmid p1658/97 (c. 125 kb) from Escherichia coli isolates recovered during a clonal outbreak in a hospital in Warsaw, Poland, in 1997 contains the extended-spectrum β-lactamase (ESBL) gene bla(SHV-5), originated from the Klebsiella pneumoniae chromosome. A region containing the bla(SHV-5) gene is flanked by two IS26 copies and its copy number multiplies spontaneously within p1658/97 and RecA-deficient E. coli strains. Here, we demonstrate that the amplified IS26-bla(SHV-5) units were arranged in tandems, containing up to more than 10 units, which could raise ceftazidime MICs for host strains from 4 μg mL(-1) to more than 128 μg mL(-1). Successive deletions within p1658/97, located outside the amplifiable module and encompassing even as little as c. 15% of the plasmid, blocked the amplification. Moreover, the complementing re-introduction of the deleted fragments in trans did not restore the process. Similarly, insertions of a 1-kb DNA fragment into the amplicon inhibited its self-multiplication ability. The module was able to transmit into another IS26-containing plasmid by recombination. The results prompted us to speculate that local DNA structure, especially favorable in p1658/97, might have been responsible for the IS26-bla(SHV-5) multiplication ability. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  2. Application of long amplicon propidium monoazide-PCR to assess the effects of temperature and background microbiota on pathogens in river water.

    Science.gov (United States)

    Banihashemi, Avid; Van Dyke, Michele I; Huck, Peter M

    2017-06-01

    The decay rates of enteric waterborne pathogens were evaluated following the introduction of Yersinia enterocolitica, Salmonella enterica, Campylobacter jejuni and Arcobacter butzleri into river water at different temperatures (5, 15 and 25°C) for a period of 28 days. To improve the accuracy of the results a molecular viability assay, long amplicon propidium monoazide-polymerase chain reaction (PMA-PCR), was used to quantify the viable cell concentration and results from PCR with and without PMA were compared. As well, the effect of background microbiota was assessed for Y. enterocolitica and S. enterica by inoculating cells into sterile and non-sterile river water. Cell persistence was improved by up to 4 log for Y. enterocolitica and 4.5 log for S. enterica in sterile river water compared to natural river water, showing that the autochthonous biological activity in river water can accelerate the die-off of introduced bacteria. Results also showed that low temperature significantly improved the persistence of all four target bacteria in non-sterile river water. There was a more rapid decline in cell concentration in samples with PMA pretreatment; therefore using PMA-PCR analysis can provide more reliable data on viable/active enteric bacteria in aquatic microcosms and allows for improved assessment of pathogens in the environment.

  3. Identification of active denitrifiers by DNA-stable isotope probing and amplicon sequencing reveals Betaproteobacteria as responsible for attenuation of nitrate contamination in a low impacted aquifer.

    Science.gov (United States)

    Bellini, M Inés; Kumaresan, Deepak; Tarlera, Silvana; Murrell, J Colin; Fernández-Scavino, Ana

    2018-02-01

    Groundwater reservoirs constitute important freshwater resources. However, these ecosystems are highly vulnerable to contamination and have to rely on the resident microbiota to attenuate the impact of this contamination. Nitrate is one of the main contaminants found in groundwater, and denitrification is the main process that removes the compound. In this study, the response to nutrient load on indigenous microbial communities in groundwater from a low impacted aquifer in Uruguay was evaluated. Denitrification rates were measured in groundwater samples from three different sites with nitrate, acetate and pyrite amendments. Results showed that denitrification is feasible under in situ nitrate and electron donor concentrations, although the lack of readily available organic energy source would limit the attenuation of higher nitrate concentrations. DNA-stable isotope probing, combined with amplicon sequencing of 16S rRNA, nirS and nirK genes, was used to identify the active denitrifiers. Members of the phylum Betaproteobacteria were the dominant denitrifiers in two of three sites, with different families being observed; members of the genus Vogesella (Neisseriaceae) were key denitrifiers at one site, while the genera Dechloromonas (Rhodocyclaceae) and Comamonas (Comamonadaceae) were the main denitrifiers detected at the other sites. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. The use of genus-specific amplicon pyrosequencing to assess phytophthora species diversity using eDNA from soil and water in Northern Spain.

    Science.gov (United States)

    Català, Santiago; Pérez-Sierra, Ana; Abad-Campos, Paloma

    2015-01-01

    Phytophthora is one of the most important and aggressive plant pathogenic genera in agriculture and forestry. Early detection and identification of its pathways of infection and spread are of high importance to minimize the threat they pose to natural ecosystems. eDNA was extracted from soil and water from forests and plantations in the north of Spain. Phytophthora-specific primers were adapted for use in high-throughput Sequencing (HTS). Primers were tested in a control reaction containing eight Phytophthora species and applied to water and soil eDNA samples from northern Spain. Different score coverage threshold values were tested for optimal Phytophthora species separation in a custom-curated database and in the control reaction. Clustering at 99% was the optimal criteria to separate most of the Phytophthora species. Multiple Molecular Operational Taxonomic Units (MOTUs) corresponding to 36 distinct Phytophthora species were amplified in the environmental samples. Pyrosequencing of amplicons from soil samples revealed low Phytophthora diversity (13 species) in comparison with the 35 species detected in water samples. Thirteen of the MOTUs detected in rivers and streams showed no close match to sequences in international sequence databases, revealing that eDNA pyrosequencing is a useful strategy to assess Phytophthora species diversity in natural ecosystems.

  5. Gastrointestinal Bacterial and Methanogenic Archaea Diversity Dynamics Associated with Condensed Tannin-Containing Pine Bark Diet in Goats Using 16S rDNA Amplicon Pyrosequencing.

    Science.gov (United States)

    Min, Byeng R; Solaiman, Sandra; Shange, Raymon; Eun, Jong-Su

    2014-01-01

    Eighteen Kiko-cross meat goats (n = 6) were used to collect gastrointestinal (GI) bacteria and methanogenic archaea for diversity measures when fed condensed tannin-containing pine bark (PB). Three dietary treatments were tested: control diet (0% PB and 30% wheat straw (WS); 0.17% condensed tannins (CT) dry matter (DM)); 15% PB and 15% WS (1.6% CT DM), and 30% PB and 0% WS (3.2% CT DM). A 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing technique was used to characterize and elucidate changes in GI bacteria and methanogenic archaea diversity among the diets. Proteobacteria was the most dominant phylum in goats with mean relative abundance values ranging from 39.7 (30% PB) to 46.5% (control) and 47.1% (15% PB). Other phyla individually accounted for fewer than 25% of the relative abundance observed. Predominant methanogens were Methanobrevibacter (75, 72, and 49%), Methanosphaera (3.3, 2.3, and 3.4%), and Methanobacteriaceae (1.2, 0.6, and 0.7%) population in control, 15, and 30% PB, respectively. Among methanogens, Methanobrevibacter was linearly decreased (P = 0.05) with increasing PB supplementation. These results indicate that feeding PB selectively altered bacteria and methanogenic archaeal populations in the GI tract of goats.

  6. Gastrointestinal Bacterial and Methanogenic Archaea Diversity Dynamics Associated with Condensed Tannin-Containing Pine Bark Diet in Goats Using 16S rDNA Amplicon Pyrosequencing

    Directory of Open Access Journals (Sweden)

    Byeng R. Min

    2014-01-01

    Full Text Available Eighteen Kiko-cross meat goats (n=6 were used to collect gastrointestinal (GI bacteria and methanogenic archaea for diversity measures when fed condensed tannin-containing pine bark (PB. Three dietary treatments were tested: control diet (0% PB and 30% wheat straw (WS; 0.17% condensed tannins (CT dry matter (DM; 15% PB and 15% WS (1.6% CT DM, and 30% PB and 0% WS (3.2% CT DM. A 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing technique was used to characterize and elucidate changes in GI bacteria and methanogenic archaea diversity among the diets. Proteobacteria was the most dominant phylum in goats with mean relative abundance values ranging from 39.7 (30% PB to 46.5% (control and 47.1% (15% PB. Other phyla individually accounted for fewer than 25% of the relative abundance observed. Predominant methanogens were Methanobrevibacter (75, 72, and 49%, Methanosphaera (3.3, 2.3, and 3.4%, and Methanobacteriaceae (1.2, 0.6, and 0.7% population in control, 15, and 30% PB, respectively. Among methanogens, Methanobrevibacter was linearly decreased (P=0.05 with increasing PB supplementation. These results indicate that feeding PB selectively altered bacteria and methanogenic archaeal populations in the GI tract of goats.

  7. Size Matters: Assessing Optimum Soil Sample Size for Fungal and Bacterial Community Structure Analyses Using High Throughput Sequencing of rRNA Gene Amplicons

    Directory of Open Access Journals (Sweden)

    Christopher Ryan Penton

    2016-06-01

    Full Text Available We examined the effect of different soil sample sizes obtained from an agricultural field, under a single cropping system uniform in soil properties and aboveground crop responses, on bacterial and fungal community structure and microbial diversity indices. DNA extracted from soil sample sizes of 0.25, 1, 5 and 10 g using MoBIO kits and from 10 and 100 g sizes using a bead-beating method (SARDI were used as templates for high-throughput sequencing of 16S and 28S rRNA gene amplicons for bacteria and fungi, respectively, on the Illumina MiSeq and Roche 454 platforms. Sample size significantly affected overall bacterial and fungal community structure, replicate dispersion and the number of operational taxonomic units (OTUs retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. For the fungal data, smaller MoBIO extractions identified more unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while still capturing rarer taxa in concert with decreasing replicate variation.

  8. Uncertainty Quantification in Aerodynamics Simulations, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — The objective of the proposed work (Phases I and II) is to develop uncertainty quantification methodologies and software suitable for use in CFD simulations of...

  9. Uncertainty quantification theory, implementation, and applications

    CERN Document Server

    Smith, Ralph C

    2014-01-01

    The field of uncertainty quantification is evolving rapidly because of increasing emphasis on models that require quantified uncertainties for large-scale applications, novel algorithm development, and new computational architectures that facilitate implementation of these algorithms. Uncertainty Quantification: Theory, Implementation, and Applications provides readers with the basic concepts, theory, and algorithms necessary to quantify input and response uncertainties for simulation models arising in a broad range of disciplines. The book begins with a detailed discussion of applications where uncertainty quantification is critical for both scientific understanding and policy. It then covers concepts from probability and statistics, parameter selection techniques, frequentist and Bayesian model calibration, propagation of uncertainties, quantification of model discrepancy, surrogate model construction, and local and global sensitivity analysis. The author maintains a complementary web page where readers ca...

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

    Science.gov (United States)

    Liu, Jason Yingjie

    2014-11-01

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

  11. Inverse problems and uncertainty quantification

    KAUST Repository

    Litvinenko, Alexander

    2013-12-18

    In a Bayesian setting, inverse problems and uncertainty quantification (UQ)— the propagation of uncertainty through a computational (forward) model—are strongly connected. In the form of conditional expectation the Bayesian update becomes computationally attractive. This is especially the case as together with a functional or spectral approach for the forward UQ there is no need for time- consuming and slowly convergent Monte Carlo sampling. The developed sampling- free non-linear Bayesian update is derived from the variational problem associated with conditional expectation. This formulation in general calls for further discretisa- tion to make the computation possible, and we choose a polynomial approximation. After giving details on the actual computation in the framework of functional or spectral approximations, we demonstrate the workings of the algorithm on a number of examples of increasing complexity. At last, we compare the linear and quadratic Bayesian update on the small but taxing example of the chaotic Lorenz 84 model, where we experiment with the influence of different observation or measurement operators on the update.

  12. Stochastic approach for radionuclides quantification

    Science.gov (United States)

    Clement, A.; Saurel, N.; Perrin, G.

    2018-01-01

    Gamma spectrometry is a passive non-destructive assay used to quantify radionuclides present in more or less complex objects. Basic methods using empirical calibration with a standard in order to quantify the activity of nuclear materials by determining the calibration coefficient are useless on non-reproducible, complex and single nuclear objects such as waste packages. Package specifications as composition or geometry change from one package to another and involve a high variability of objects. Current quantification process uses numerical modelling of the measured scene with few available data such as geometry or composition. These data are density, material, screen, geometric shape, matrix composition, matrix and source distribution. Some of them are strongly dependent on package data knowledge and operator backgrounds. The French Commissariat à l'Energie Atomique (CEA) is developing a new methodology to quantify nuclear materials in waste packages and waste drums without operator adjustment and internal package configuration knowledge. This method suggests combining a global stochastic approach which uses, among others, surrogate models available to simulate the gamma attenuation behaviour, a Bayesian approach which considers conditional probability densities of problem inputs, and Markov Chains Monte Carlo algorithms (MCMC) which solve inverse problems, with gamma ray emission radionuclide spectrum, and outside dimensions of interest objects. The methodology is testing to quantify actinide activity in different kind of matrix, composition, and configuration of sources standard in terms of actinide masses, locations and distributions. Activity uncertainties are taken into account by this adjustment methodology.

  13. Inverse Problems and Uncertainty Quantification

    KAUST Repository

    Litvinenko, Alexander

    2014-01-06

    In a Bayesian setting, inverse problems and uncertainty quantification (UQ) - the propagation of uncertainty through a computational (forward) modelare strongly connected. In the form of conditional expectation the Bayesian update becomes computationally attractive. This is especially the case as together with a functional or spectral approach for the forward UQ there is no need for time- consuming and slowly convergent Monte Carlo sampling. The developed sampling- free non-linear Bayesian update is derived from the variational problem associated with conditional expectation. This formulation in general calls for further discretisa- tion to make the computation possible, and we choose a polynomial approximation. After giving details on the actual computation in the framework of functional or spectral approximations, we demonstrate the workings of the algorithm on a number of examples of increasing complexity. At last, we compare the linear and quadratic Bayesian update on the small but taxing example of the chaotic Lorenz 84 model, where we experiment with the influence of different observation or measurement operators on the update.

  14. Uncertainty Quantification in Numerical Aerodynamics

    KAUST Repository

    Litvinenko, Alexander

    2017-05-16

    We consider uncertainty quantification problem in aerodynamic simulations. We identify input uncertainties, classify them, suggest an appropriate statistical model and, finally, estimate propagation of these uncertainties into the solution (pressure, velocity and density fields as well as the lift and drag coefficients). The deterministic problem under consideration is a compressible transonic Reynolds-averaged Navier-Strokes flow around an airfoil with random/uncertain data. Input uncertainties include: uncertain angle of attack, the Mach number, random perturbations in the airfoil geometry, mesh, shock location, turbulence model and parameters of this turbulence model. This problem requires efficient numerical/statistical methods since it is computationally expensive, especially for the uncertainties caused by random geometry variations which involve a large number of variables. In numerical section we compares five methods, including quasi-Monte Carlo quadrature, polynomial chaos with coefficients determined by sparse quadrature and gradient-enhanced version of Kriging, radial basis functions and point collocation polynomial chaos, in their efficiency in estimating statistics of aerodynamic performance upon random perturbation to the airfoil geometry [D.Liu et al \\'17]. For modeling we used the TAU code, developed in DLR, Germany.

  15. Lung involvement quantification in chest radiographs

    International Nuclear Information System (INIS)

    Giacomini, Guilherme; Alvarez, Matheus; Oliveira, Marcela de; Miranda, Jose Ricardo A.; Pina, Diana R.; Pereira, Paulo C.M.; Ribeiro, Sergio M.

    2014-01-01

    Tuberculosis (TB) caused by Mycobacterium tuberculosis, is an infectious disease which remains a global health problem. The chest radiography is the commonly method employed to assess the TB's evolution. The methods for quantification of abnormalities of chest are usually performed on CT scans (CT). This quantification is important to assess the TB evolution and treatment and comparing different treatments. However, precise quantification is not feasible for the amount of CT scans required. The purpose of this work is to develop a methodology for quantification of lung damage caused by TB through chest radiographs. It was developed an algorithm for computational processing of exams in Matlab, which creates a lungs' 3D representation, with compromised dilated regions inside. The quantification of lung lesions was also made for the same patients through CT scans. The measurements from the two methods were compared and resulting in strong correlation. Applying statistical Bland and Altman, all samples were within the limits of agreement, with a confidence interval of 95%. The results showed an average variation of around 13% between the two quantification methods. The results suggest the effectiveness and applicability of the method developed, providing better risk-benefit to the patient and cost-benefit ratio for the institution. (author)

  16. Lactococcus lactis Diversity in Undefined Mixed Dairy Starter Cultures as Revealed by Comparative Genome Analyses and Targeted Amplicon Sequencing of epsD.

    Science.gov (United States)

    Frantzen, Cyril A; Kleppen, Hans Petter; Holo, Helge

    2018-02-01

    Undefined mesophilic mixed (DL) starter cultures are used in the production of continental cheeses and contain unknown strain mixtures of Lactococcus lactis and leuconostocs. The choice of starter culture affects the taste, aroma, and quality of the final product. To gain insight into the diversity of Lactococcus lactis strains in starter cultures, we whole-genome sequenced 95 isolates from three different starter cultures. Pan-genomic analyses, which included 30 publically available complete genomes, grouped the strains into 21 L. lactis subsp . lactis and 28 L. lactis subsp. cremoris lineages. Only one of the 95 isolates grouped with previously sequenced strains, and the three starter cultures showed no overlap in lineage distributions. The culture diversity was assessed by targeted amplicon sequencing using purR , a core gene, and epsD , present in 93 of the 95 starter culture isolates but absent in most of the reference strains. This enabled an unprecedented discrimination of starter culture Lactococcus lactis and revealed substantial differences between the three starter cultures and compositional shifts during the cultivation of cultures in milk. IMPORTANCE In contemporary cheese production, standardized frozen seed stock starter cultures are used to ensure production stability, reproducibility, and quality control of the product. The dairy industry experiences significant disruptions of cheese production due to phage attacks, and one commonly used countermeasure to phage attack is to employ a starter rotation strategy, in which two or more starters with minimal overlap in phage sensitivity are used alternately. A culture-independent analysis of the lactococcal diversity in complex undefined starter cultures revealed large differences between the three starter cultures and temporal shifts in lactococcal composition during the production of bulk starters. A better understanding of the lactococcal diversity in starter cultures will enable the development of

  17. Improving the genotyping resolution of Cryptosporidium hominis subtype IbA10G2 using one step PCR-based amplicon sequencing.

    Science.gov (United States)

    Beser, Jessica; Hallström, Björn M; Advani, Abdolreza; Andersson, Sofia; Östlund, Gabriel; Winiecka-Krusnell, Jadwiga; Lebbad, Marianne; Alm, Erik; Troell, Karin; Arrighi, Romanico B G

    2017-11-01

    Cryptosporidium hominis gp60 subtype IbA10G2 is a common cause of cryptosporidiosis. This subtype is responsible for many waterborne outbreaks as well as sporadic cases and is considered virulent and highly important in the epidemiology of cryptosporidiosis. Due to low heterogeneity within the genome of C. hominis it has been difficult to identify epidemiological markers with higher resolution than gp60. However, new markers are required in order to improve outbreak investigations and studies of the transmission dynamics of this clinically important subtype. Based on the whole genome sequences of 17 C. hominis isolates, we have identified several differential loci and developed a new sequence based typing panel with higher resolution than gp60. An amplicon sequencing method was also developed which is based on a one-step PCR which can be sequenced using a Next Generation Sequencing (NGS) platform. Such a system provides a rapid and high-throughput workflow. A panel of nine loci with 10 single nucleotide variants (SNV) was selected and evaluated using clinical IbA10G2 isolates from sporadic, cluster and outbreak associated cases. The specimens were separated into 10 different genetic profiles named sequence types (STs). All isolates within an outbreak or cluster belonged to the same ST, including several samples from the two large waterborne outbreaks which occurred in Sweden between 2010 and 2011 indicating that these outbreaks might be linked. The results demonstrate the methods suitability for improved genotyping of C. hominis IbA10G2. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Uncertainty quantification for environmental models

    Science.gov (United States)

    Hill, Mary C.; Lu, Dan; Kavetski, Dmitri; Clark, Martyn P.; Ye, Ming

    2012-01-01

    Environmental models are used to evaluate the fate of fertilizers in agricultural settings (including soil denitrification), the degradation of hydrocarbons at spill sites, and water supply for people and ecosystems in small to large basins and cities—to mention but a few applications of these models. They also play a role in understanding and diagnosing potential environmental impacts of global climate change. The models are typically mildly to extremely nonlinear. The persistent demand for enhanced dynamics and resolution to improve model realism [17] means that lengthy individual model execution times will remain common, notwithstanding continued enhancements in computer power. In addition, high-dimensional parameter spaces are often defined, which increases the number of model runs required to quantify uncertainty [2]. Some environmental modeling projects have access to extensive funding and computational resources; many do not. The many recent studies of uncertainty quantification in environmental model predictions have focused on uncertainties related to data error and sparsity of data, expert judgment expressed mathematically through prior information, poorly known parameter values, and model structure (see, for example, [1,7,9,10,13,18]). Approaches for quantifying uncertainty include frequentist (potentially with prior information [7,9]), Bayesian [13,18,19], and likelihood-based. A few of the numerous methods, including some sensitivity and inverse methods with consequences for understanding and quantifying uncertainty, are as follows: Bayesian hierarchical modeling and Bayesian model averaging; single-objective optimization with error-based weighting [7] and multi-objective optimization [3]; methods based on local derivatives [2,7,10]; screening methods like OAT (one at a time) and the method of Morris [14]; FAST (Fourier amplitude sensitivity testing) [14]; the Sobol' method [14]; randomized maximum likelihood [10]; Markov chain Monte Carlo (MCMC) [10

  19. Illumina MiSeq Phylogenetic Amplicon Sequencing Shows a Large Reduction of an Uncharacterised Succinivibrionaceae and an Increase of the Methanobrevibacter gottschalkii Clade in Feed Restricted Cattle.

    Directory of Open Access Journals (Sweden)

    Matthew Sean McCabe

    Full Text Available Periodic feed restriction is used in cattle production to reduce feed costs. When normal feed levels are resumed, cattle catch up to a normal weight by an acceleration of normal growth rate, known as compensatory growth, which is not yet fully understood. Illumina Miseq Phylogenetic marker amplicon sequencing of DNA extracted from rumen contents of 55 bulls showed that restriction of feed (70% concentrate, 30% grass silage for 125 days, to levels that caused a 60% reduction of growth rate, resulted in a large increase of relative abundance of Methanobrevibacter gottschalkii clade (designated as OTU-M7, and a large reduction of an uncharacterised Succinivibrionaceae species (designated as OTU-S3004. There was a strong negative Spearman correlation (ρ = -0.72, P = <1x10(-20 between relative abundances of OTU-3004 and OTU-M7 in the liquid rumen fraction. There was also a significant increase in acetate:propionate ratio (A:P in feed restricted animals that showed a negative Spearman correlation (ρ = -0.69, P = <1x10(-20 with the relative abundance of OTU-S3004 in the rumen liquid fraction but not the solid fraction, and a strong positive Spearman correlation with OTU-M7 in the rumen liquid (ρ = 0.74, P = <1x10(-20 and solid (ρ = 0.69, P = <1x10(-20 fractions. Reduced A:P ratios in the rumen are associated with increased feed efficiency and reduced production of methane which has a global warming potential (GWP 100 years of 28. Succinivibrionaceae growth in the rumen was previously suggested to reduce methane emissions as some members of this family utilise hydrogen, which is also utilised by methanogens for methanogenesis, to generate succinate which is converted to propionate. Relative abundance of OTU-S3004 showed a positive Spearman correlation with propionate (ρ = 0.41, P = <0.01 but not acetate in the liquid rumen fraction.

  20. Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR.

    Science.gov (United States)

    Tatti, Enrico; McKew, Boyd A; Whitby, Corrine; Smith, Cindy J

    2016-06-11

    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

  1. Molecular quantification of genes encoding for green-fluorescent proteins

    DEFF Research Database (Denmark)

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

    2003-01-01

    A quantitative PCR approach is presented to analyze the amount of recombinant green fluorescent protein (gfp) genes in environmental DNA samples. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification...

  2. Distortion of genetically modified organism quantification in processed foods: influence of particle size compositions and heat-induced DNA degradation.

    Science.gov (United States)

    Moreano, Francisco; Busch, Ulrich; Engel, Karl-Heinz

    2005-12-28

    Milling fractions from conventional and transgenic corn were prepared at laboratory scale and used to study the influence of sample composition and heat-induced DNA degradation on the relative quantification of genetically modified organisms (GMO) in food products. Particle size distributions of the obtained fractions (coarse grits, regular grits, meal, and flour) were characterized using a laser diffraction system. The application of two DNA isolation protocols revealed a strong correlation between the degree of comminution of the milling fractions and the DNA yield in the extracts. Mixtures of milling fractions from conventional and transgenic material (1%) were prepared and analyzed via real-time polymerase chain reaction. Accurate quantification of the adjusted GMO content was only possible in mixtures containing conventional and transgenic material in the form of analogous milling fractions, whereas mixtures of fractions exhibiting different particle size distributions delivered significantly over- and underestimated GMO contents depending on their compositions. The process of heat-induced nucleic acid degradation was followed by applying two established quantitative assays showing differences between the lengths of the recombinant and reference target sequences (A, deltal(A) = -25 bp; B, deltal(B) = +16 bp; values related to the amplicon length of the reference gene). Data obtained by the application of method A resulted in underestimated recoveries of GMO contents in the samples of heat-treated products, reflecting the favored degradation of the longer target sequence used for the detection of the transgene. In contrast, data yielded by the application of method B resulted in increasingly overestimated recoveries of GMO contents. The results show how commonly used food technological processes may lead to distortions in the results of quantitative GMO analyses.

  3. Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms

    Directory of Open Access Journals (Sweden)

    Žel Jana

    2008-03-01

    Full Text Available Abstract Background The real-time polymerase chain reaction is currently the method of choice for quantifying nucleic acids in different DNA based quantification applications. It is widely used also for detecting and quantifying genetically modified components in food and feed, predominantly employing TaqMan® and SYBR® Green real-time PCR chemistries. In our study four alternative chemistries: Lux™, Plexor™, Cycling Probe Technology and LNA® were extensively evaluated and compared using TaqMan® chemistry as a reference system. Results Amplicons were designed on the maize invertase gene and the 5'-junction of inserted transgene and plant genomic DNA in MON 810 event. Real-time assays were subsequently compared for their efficiency in PCR amplification, limits of detection and quantification, repeatability and accuracy to test the performance of the assays. Additionally, the specificity of established assays was checked on various transgenic and non-transgenic plant species. The overall applicability of the designed assays was evaluated, adding practicability and costs issues to the performance characteristics. Conclusion Although none of the chemistries significantly outperformed the others, there are certain characteristics that suggest that LNA® technology is an alternative to TaqMan® when designing assays for quantitative analysis. Because LNA® probes are much shorter they might be especially appropriate when high specificity is required and where the design of a common TaqMan® probe is difficult or even impossible due to sequence characteristics. Plexor™ on the other hand might be a method of choice for qualitative analysis when sensitivity, low cost and simplicity of use prevail.

  4. TaqMan probe real-time polymerase chain reaction assay for the quantification of canine DNA in chicken nugget.

    Science.gov (United States)

    Rahman, Md Mahfujur; Hamid, Sharifah Bee Abd; Basirun, Wan Jefrey; Bhassu, Subha; Rashid, Nur Raifana Abdul; Mustafa, Shuhaimi; Mohd Desa, Mohd Nasir; Ali, Md Eaqub

    2016-01-01

    This paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R(2) = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.

  5. HPC Analytics Support. Requirements for Uncertainty Quantification Benchmarks

    Energy Technology Data Exchange (ETDEWEB)

    Paulson, Patrick R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Purohit, Sumit [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Rodriguez, Luke R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2015-05-01

    This report outlines techniques for extending benchmark generation products so they support uncertainty quantification by benchmarked systems. We describe how uncertainty quantification requirements can be presented to candidate analytical tools supporting SPARQL. We describe benchmark data sets for evaluating uncertainty quantification, as well as an approach for using our benchmark generator to produce data sets for generating benchmark data sets.

  6. Quantification analysis of CT for aphasic patients

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  7. Quantification of Cannabinoid Content in Cannabis

    Science.gov (United States)

    Tian, Y.; Zhang, F.; Jia, K.; Wen, M.; Yuan, Ch.

    2015-09-01

    Cannabis is an economically important plant that is used in many fields, in addition to being the most commonly consumed illicit drug worldwide. Monitoring the spatial distribution of cannabis cultivation and judging whether it is drug- or fiber-type cannabis is critical for governments and international communities to understand the scale of the illegal drug trade. The aim of this study was to investigate whether the cannabinoids content in cannabis could be spectrally quantified using a spectrometer and to identify the optimal wavebands for quantifying the cannabinoid content. Spectral reflectance data of dried cannabis leaf samples and the cannabis canopy were measured in the laboratory and in the field, respectively. Correlation analysis and the stepwise multivariate regression method were used to select the optimal wavebands for cannabinoid content quantification based on the laboratory-measured spectral data. The results indicated that the delta-9-tetrahydrocannabinol (THC) content in cannabis leaves could be quantified using laboratory-measured spectral reflectance data and that the 695 nm band is the optimal band for THC content quantification. This study provides prerequisite information for designing spectral equipment to enable immediate quantification of THC content in cannabis and to discriminate drug- from fiber-type cannabis based on THC content quantification in the field.

  8. Source Specific Quantification, Characterisation and Management of ...

    African Journals Online (AJOL)

    The most important aspect of solid waste management is the quantity and characteristics of waste to be managed. Lapai town lacks data on quantity of waste generated and their characteristics for efficient and sustainable waste management. This study is the quantification, characterisation and management of solid waste ...

  9. Recurrence quantification analysis in Liu's attractor

    International Nuclear Information System (INIS)

    Balibrea, Francisco; Caballero, M. Victoria; Molera, Lourdes

    2008-01-01

    Recurrence Quantification Analysis is used to detect transitions chaos to periodical states or chaos to chaos in a new dynamical system proposed by Liu et al. This system contains a control parameter in the second equation and was originally introduced to investigate the forming mechanism of the compound structure of the chaotic attractor which exists when the control parameter is zero

  10. Colour thresholding and objective quantification in bioimaging

    Science.gov (United States)

    Fermin, C. D.; Gerber, M. A.; Torre-Bueno, J. R.

    1992-01-01

    Computer imaging is rapidly becoming an indispensable tool for the quantification of variables in research and medicine. Whilst its use in medicine has largely been limited to qualitative observations, imaging in applied basic sciences, medical research and biotechnology demands objective quantification of the variables in question. In black and white densitometry (0-256 levels of intensity) the separation of subtle differences between closely related hues from stains is sometimes very difficult. True-colour and real-time video microscopy analysis offer choices not previously available with monochrome systems. In this paper we demonstrate the usefulness of colour thresholding, which has so far proven indispensable for proper objective quantification of the products of histochemical reactions and/or subtle differences in tissue and cells. In addition, we provide interested, but untrained readers with basic information that may assist decisions regarding the most suitable set-up for a project under consideration. Data from projects in progress at Tulane are shown to illustrate the advantage of colour thresholding over monochrome densitometry and for objective quantification of subtle colour differences between experimental and control samples.

  11. Model Uncertainty Quantification Methods In Data Assimilation

    Science.gov (United States)

    Pathiraja, S. D.; Marshall, L. A.; Sharma, A.; Moradkhani, H.

    2017-12-01

    Data Assimilation involves utilising observations to improve model predictions in a seamless and statistically optimal fashion. Its applications are wide-ranging; from improving weather forecasts to tracking targets such as in the Apollo 11 mission. The use of Data Assimilation methods in high dimensional complex geophysical systems is an active area of research, where there exists many opportunities to enhance existing methodologies. One of the central challenges is in model uncertainty quantification; the outcome of any Data Assimilation study is strongly dependent on the uncertainties assigned to both observations and models. I focus on developing improved model uncertainty quantification methods that are applicable to challenging real world scenarios. These include developing methods for cases where the system states are only partially observed, where there is little prior knowledge of the model errors, and where the model error statistics are likely to be highly non-Gaussian.

  12. Acidity: Modes of characterization and quantification.

    Science.gov (United States)

    Ruthenberg, Klaus; Chang, Hasok

    This paper provides an account of early historical developments in the characterization and quantification of acidity, which may be considered preliminary steps leading to the measurement of acidity. In this "pre-history" of acidity measurement, emphasis is laid on the relative independence of the rich empirical knowledge about acids from theories of acidity. Many attempts were made to compare and assess the strengths of various acids, based on concrete laboratory operations. However, at least until the arrival of the pH measure, the quantification attempts failed to produce anything qualifying as a measurement scale of a recognizable type. It is doubtful whether even pH qualifies as a true measure of acidity, when the full meaning of acidity is taken into account. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Intracranial Volume Quantification from 3D Photography

    OpenAIRE

    Tu, Liyun; Porras, Antonio R.; Ensel, Scott; Tsering, Deki; Paniagua, Beatriz; Enquobahrie, Andinet; Oh, Albert; Keating, Robert; Rogers, Gary F.; Linguraru, Marius George

    2017-01-01

    3D photography offers non-invasive, radiation-free, and anesthetic-free evaluation of craniofacial morphology. However, intracranial volume (ICV) quantification is not possible with current non-invasive imaging systems in order to evaluate brain development in children with cranial pathology. The aim of this study is to develop an automated, radiation-free framework to estimate ICV. Pairs of computed tomography (CT) images and 3D photographs were aligned using registration. We used the real I...

  14. Tentacle: distributed quantification of genes in metagenomes

    OpenAIRE

    Boulund, Fredrik; Sjögren, Anders; Kristiansson, Erik

    2015-01-01

    Background In metagenomics, microbial communities are sequenced at increasingly high resolution, generating datasets with billions of DNA fragments. Novel methods that can efficiently process the growing volumes of sequence data are necessary for the accurate analysis and interpretation of existing and upcoming metagenomes. Findings Here we present Tentacle, which is a novel framework that uses distributed computational resources for gene quantification in metagenomes. Tentacle is implemented...

  15. Uncertainty quantification for hyperbolic and kinetic equations

    CERN Document Server

    Pareschi, Lorenzo

    2017-01-01

    This book explores recent advances in uncertainty quantification for hyperbolic, kinetic, and related problems. The contributions address a range of different aspects, including: polynomial chaos expansions, perturbation methods, multi-level Monte Carlo methods, importance sampling, and moment methods. The interest in these topics is rapidly growing, as their applications have now expanded to many areas in engineering, physics, biology and the social sciences. Accordingly, the book provides the scientific community with a topical overview of the latest research efforts.

  16. Artifacts Quantification of Metal Implants in MRI

    Science.gov (United States)

    Vrachnis, I. N.; Vlachopoulos, G. F.; Maris, T. G.; Costaridou, L. I.

    2017-11-01

    The presence of materials with different magnetic properties, such as metal implants, causes distortion of the magnetic field locally, resulting in signal voids and pile ups, i.e. susceptibility artifacts in MRI. Quantitative and unbiased measurement of the artifact is prerequisite for optimization of acquisition parameters. In this study an image gradient based segmentation method is proposed for susceptibility artifact quantification. The method captures abrupt signal alterations by calculation of the image gradient. Then the artifact is quantified in terms of its extent by an automated cross entropy thresholding method as image area percentage. The proposed method for artifact quantification was tested in phantoms containing two orthopedic implants with significantly different magnetic permeabilities. The method was compared against a method proposed in the literature, considered as a reference, demonstrating moderate to good correlation (Spearman’s rho = 0.62 and 0.802 in case of titanium and stainless steel implants). The automated character of the proposed quantification method seems promising towards MRI acquisition parameter optimization.

  17. Uncertainty Quantification with Applications to Engineering Problems

    DEFF Research Database (Denmark)

    Bigoni, Daniele

    The systematic quantification of the uncertainties affecting dynamical systems and the characterization of the uncertainty of their outcomes is critical for engineering design and analysis, where risks must be reduced as much as possible. Uncertainties stem naturally from our limitations in measu......The systematic quantification of the uncertainties affecting dynamical systems and the characterization of the uncertainty of their outcomes is critical for engineering design and analysis, where risks must be reduced as much as possible. Uncertainties stem naturally from our limitations...... in measurements, predictions and manufacturing, and we can say that any dynamical system used in engineering is subject to some of these uncertainties. The first part of this work presents an overview of the mathematical framework used in Uncertainty Quantification (UQ) analysis and introduces the spectral tensor...... functions and on an elliptic problem with random inputs. This work will also present three active research directions aimed at improving the efficiency of the STT-decomposition. In this context, we propose three new strategies for solving the ordering problem suffered by the tensor-train decomposition...

  18. Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water.

    Science.gov (United States)

    Ahmed, W; Staley, C; Sadowsky, M J; Gyawali, P; Sidhu, J P S; Palmer, A; Beale, D J; Toze, S

    2015-10-01

    In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. Digital PCR for direct quantification of viruses without DNA extraction.

    Science.gov (United States)

    Pavšič, Jernej; Žel, Jana; Milavec, Mojca

    2016-01-01

    DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration material and it has higher tolerance to inhibitors. DNA quantification without an extraction step (i.e. direct quantification) was performed here using dPCR and two different human cytomegalovirus whole-virus materials. Two dPCR platforms were used for this direct quantification of the viral DNA, and these were compared with quantification of the extracted viral DNA in terms of yield and variability. Direct quantification of both whole-virus materials present in simple matrices like cell lysate or Tris-HCl buffer provided repeatable measurements of virus concentrations that were probably in closer agreement with the actual viral load than when estimated through quantification of the extracted DNA. Direct dPCR quantification of other viruses, reference materials and clinically relevant matrices is now needed to show the full versatility of this very promising and cost-efficient development in virus quantification.

  20. Quantification procedures in micro X-ray fluorescence analysis

    International Nuclear Information System (INIS)

    Kanngiesser, Birgit

    2003-01-01

    For the quantification in micro X-ray fluorescence analysis standardfree quantification procedures have become especially important. An introduction to the basic concepts of these quantification procedures is given, followed by a short survey of the procedures which are available now and what kind of experimental situations and analytical problems are addressed. The last point is extended by the description of an own development for the fundamental parameter method, which renders the inclusion of nonparallel beam geometries possible. Finally, open problems for the quantification procedures are discussed

  1. Stereo-particle image velocimetry uncertainty quantification

    Science.gov (United States)

    Bhattacharya, Sayantan; Charonko, John J.; Vlachos, Pavlos P.

    2017-01-01

    Particle image velocimetry (PIV) measurements are subject to multiple elemental error sources and thus estimating overall measurement uncertainty is challenging. Recent advances have led to a posteriori uncertainty estimation methods for planar two-component PIV. However, no complete methodology exists for uncertainty quantification in stereo PIV. In the current work, a comprehensive framework is presented to quantify the uncertainty stemming from stereo registration error and combine it with the underlying planar velocity uncertainties. The disparity in particle locations of the dewarped images is used to estimate the positional uncertainty of the world coordinate system, which is then propagated to the uncertainty in the calibration mapping function coefficients. Next, the calibration uncertainty is combined with the planar uncertainty fields of the individual cameras through an uncertainty propagation equation and uncertainty estimates are obtained for all three velocity components. The methodology was tested with synthetic stereo PIV data for different light sheet thicknesses, with and without registration error, and also validated with an experimental vortex ring case from 2014 PIV challenge. Thorough sensitivity analysis was performed to assess the relative impact of the various parameters to the overall uncertainty. The results suggest that in absence of any disparity, the stereo PIV uncertainty prediction method is more sensitive to the planar uncertainty estimates than to the angle uncertainty, although the latter is not negligible for non-zero disparity. Overall the presented uncertainty quantification framework showed excellent agreement between the error and uncertainty RMS values for both the synthetic and the experimental data and demonstrated reliable uncertainty prediction coverage. This stereo PIV uncertainty quantification framework provides the first comprehensive treatment on the subject and potentially lays foundations applicable to volumetric

  2. Advances in forensic DNA quantification: a review.

    Science.gov (United States)

    Lee, Steven B; McCord, Bruce; Buel, Eric

    2014-11-01

    This review focuses upon a critical step in forensic biology: detection and quantification of human DNA from biological samples. Determination of the quantity and quality of human DNA extracted from biological evidence is important for several reasons. Firstly, depending on the source and extraction method, the quality (purity and length), and quantity of the resultant DNA extract can vary greatly. This affects the downstream method as the quantity of input DNA and its relative length can determine which genotyping procedure to use-standard short-tandem repeat (STR) typing, mini-STR typing or mitochondrial DNA sequencing. Secondly, because it is important in forensic analysis to preserve as much of the evidence as possible for retesting, it is important to determine the total DNA amount available prior to utilizing any destructive analytical method. Lastly, results from initial quantitative and qualitative evaluations permit a more informed interpretation of downstream analytical results. Newer quantitative techniques involving real-time PCR can reveal the presence of degraded DNA and PCR inhibitors, that provide potential reasons for poor genotyping results and may indicate methods to use for downstream typing success. In general, the more information available, the easier it is to interpret and process the sample resulting in a higher likelihood of successful DNA typing. The history of the development of quantitative methods has involved two main goals-improving precision of the analysis and increasing the information content of the result. This review covers advances in forensic DNA quantification methods and recent developments in RNA quantification. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Stereo-particle image velocimetry uncertainty quantification

    International Nuclear Information System (INIS)

    Bhattacharya, Sayantan; Vlachos, Pavlos P; Charonko, John J

    2017-01-01

    Particle image velocimetry (PIV) measurements are subject to multiple elemental error sources and thus estimating overall measurement uncertainty is challenging. Recent advances have led to a posteriori uncertainty estimation methods for planar two-component PIV. However, no complete methodology exists for uncertainty quantification in stereo PIV. In the current work, a comprehensive framework is presented to quantify the uncertainty stemming from stereo registration error and combine it with the underlying planar velocity uncertainties. The disparity in particle locations of the dewarped images is used to estimate the positional uncertainty of the world coordinate system, which is then propagated to the uncertainty in the calibration mapping function coefficients. Next, the calibration uncertainty is combined with the planar uncertainty fields of the individual cameras through an uncertainty propagation equation and uncertainty estimates are obtained for all three velocity components. The methodology was tested with synthetic stereo PIV data for different light sheet thicknesses, with and without registration error, and also validated with an experimental vortex ring case from 2014 PIV challenge. Thorough sensitivity analysis was performed to assess the relative impact of the various parameters to the overall uncertainty. The results suggest that in absence of any disparity, the stereo PIV uncertainty prediction method is more sensitive to the planar uncertainty estimates than to the angle uncertainty, although the latter is not negligible for non-zero disparity. Overall the presented uncertainty quantification framework showed excellent agreement between the error and uncertainty RMS values for both the synthetic and the experimental data and demonstrated reliable uncertainty prediction coverage. This stereo PIV uncertainty quantification framework provides the first comprehensive treatment on the subject and potentially lays foundations applicable to volumetric

  4. Uncertainty quantification and stochastic modeling with Matlab

    CERN Document Server

    Souza de Cursi, Eduardo

    2015-01-01

    Uncertainty Quantification (UQ) is a relatively new research area which describes the methods and approaches used to supply quantitative descriptions of the effects of uncertainty, variability and errors in simulation problems and models. It is rapidly becoming a field of increasing importance, with many real-world applications within statistics, mathematics, probability and engineering, but also within the natural sciences. Literature on the topic has up until now been largely based on polynomial chaos, which raises difficulties when considering different types of approximation and does no

  5. Linking probe thermodynamics to microarray quantification

    International Nuclear Information System (INIS)

    Li, Shuzhao; Pozhitkov, Alexander; Brouwer, Marius

    2010-01-01

    Understanding the difference in probe properties holds the key to absolute quantification of DNA microarrays. So far, Langmuir-like models have failed to link sequence-specific properties to hybridization signals in the presence of a complex hybridization background. Data from washing experiments indicate that the post-hybridization washing has no major effect on the specifically bound targets, which give the final signals. Thus, the amount of specific targets bound to probes is likely determined before washing, by the competition against nonspecific binding. Our competitive hybridization model is a viable alternative to Langmuir-like models. (comment)

  6. Image cytometry: nuclear and chromosomal DNA quantification.

    Science.gov (United States)

    Carvalho, Carlos Roberto; Clarindo, Wellington Ronildo; Abreu, Isabella Santiago

    2011-01-01

    Image cytometry (ICM) associates microscopy, digital image and software technologies, and has been particularly useful in spatial and densitometric cytological analyses, such as DNA ploidy and DNA content measurements. Basically, ICM integrates methodologies of optical microscopy calibration, standard density filters, digital CCD camera, and image analysis softwares for quantitative applications. Apart from all system calibration and setup, cytological protocols must provide good slide preparations for efficient and reliable ICM analysis. In this chapter, procedures for ICM applications employed in our laboratory are described. Protocols shown here for human DNA ploidy determination and quantification of nuclear and chromosomal DNA content in plants could be used as described, or adapted for other studies.

  7. Automated quantification and analysis of mandibular asymmetry

    DEFF Research Database (Denmark)

    Darvann, T. A.; Hermann, N. V.; Larsen, P.

    2010-01-01

    We present an automated method of spatially detailed 3D asymmetry quantification in mandibles extracted from CT and apply it to a population of infants with unilateral coronal synostosis (UCS). An atlas-based method employing non-rigid registration of surfaces is used for determining deformation ...... after mirroring the mandible across the MSP. A principal components analysis of asymmetry characterizes the major types of asymmetry in the population, and successfully separates the asymmetric UCS mandibles from a number of less asymmetric mandibles from a control population....

  8. Adjoint-Based Uncertainty Quantification with MCNP

    Energy Technology Data Exchange (ETDEWEB)

    Seifried, Jeffrey E. [Univ. of California, Berkeley, CA (United States)

    2011-09-01

    This work serves to quantify the instantaneous uncertainties in neutron transport simulations born from nuclear data and statistical counting uncertainties. Perturbation and adjoint theories are used to derive implicit sensitivity expressions. These expressions are transformed into forms that are convenient for construction with MCNP6, creating the ability to perform adjoint-based uncertainty quantification with MCNP6. These new tools are exercised on the depleted-uranium hybrid LIFE blanket, quantifying its sensitivities and uncertainties to important figures of merit. Overall, these uncertainty estimates are small (< 2%). Having quantified the sensitivities and uncertainties, physical understanding of the system is gained and some confidence in the simulation is acquired.

  9. Targeted Amplicon Sequencing for Single-Nucleotide-Polymorphism Genotyping of Attaching and Effacing Escherichia coli O26:H11 Cattle Strains via a High-Throughput Library Preparation Technique.

    Science.gov (United States)

    Ison, Sarah A; Delannoy, Sabine; Bugarel, Marie; Nagaraja, Tiruvoor G; Renter, David G; den Bakker, Henk C; Nightingale, Kendra K; Fach, Patrick; Loneragan, Guy H

    2016-01-15

    Enterohemorrhagic Escherichia coli (EHEC) O26:H11, a serotype within Shiga toxin-producing E. coli (STEC) that causes severe human disease, has been considered to have evolved from attaching and effacing E. coli (AEEC) O26:H11 through the acquisition of a Shiga toxin-encoding gene. Targeted amplicon sequencing using next-generation sequencing technology of 48 phylogenetically informative single-nucleotide polymorphisms (SNPs) and three SNPs differentiating Shiga toxin-positive (stx-positive) strains from Shiga toxin-negative (stx-negative) strains were used to infer the phylogenetic relationships of 178 E. coli O26:H11 strains (6 stx-positive strains and 172 stx-negative AEEC strains) from cattle feces to 7 publically available genomes of human clinical strains. The AEEC cattle strains displayed synonymous SNP genotypes with stx2-positive sequence type 29 (ST29) human O26:H11 strains, while stx1 ST21 human and cattle strains clustered separately, demonstrating the close phylogenetic relatedness of these Shiga toxin-negative AEEC cattle strains and human clinical strains. With the exception of seven stx-negative strains, five of which contained espK, three stx-related SNPs differentiated the STEC strains from non-STEC strains, supporting the hypothesis that these AEEC cattle strains could serve as a potential reservoir for new or existing pathogenic human strains. Our results support the idea that targeted amplicon sequencing for SNP genotyping expedites strain identification and genetic characterization of E. coli O26:H11, which is important for food safety and public health. Copyright © 2016 Ison et al.

  10. Quantification of complex modular architecture in plants.

    Science.gov (United States)

    Reeb, Catherine; Kaandorp, Jaap; Jansson, Fredrik; Puillandre, Nicolas; Dubuisson, Jean-Yves; Cornette, Raphaël; Jabbour, Florian; Coudert, Yoan; Patiño, Jairo; Flot, Jean-François; Vanderpoorten, Alain

    2018-04-01

    Morphometrics, the assignment of quantities to biological shapes, is a powerful tool to address taxonomic, evolutionary, functional and developmental questions. We propose a novel method for shape quantification of complex modular architecture in thalloid plants, whose extremely reduced morphologies, combined with the lack of a formal framework for thallus description, have long rendered taxonomic and evolutionary studies extremely challenging. Using graph theory, thalli are described as hierarchical series of nodes and edges, allowing for accurate, homologous and repeatable measurements of widths, lengths and angles. The computer program MorphoSnake was developed to extract the skeleton and contours of a thallus and automatically acquire, at each level of organization, width, length, angle and sinuosity measurements. Through the quantification of leaf architecture in Hymenophyllum ferns (Polypodiopsida) and a fully worked example of integrative taxonomy in the taxonomically challenging thalloid liverwort genus Riccardia, we show that MorphoSnake is applicable to all ramified plants. This new possibility of acquiring large numbers of quantitative traits in plants with complex modular architectures opens new perspectives of applications, from the development of rapid species identification tools to evolutionary analyses of adaptive plasticity. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

  11. Virus detection and quantification using electrical parameters

    Science.gov (United States)

    Ahmad, Mahmoud Al; Mustafa, Farah; Ali, Lizna M.; Rizvi, Tahir A.

    2014-10-01

    Here we identify and quantitate two similar viruses, human and feline immunodeficiency viruses (HIV and FIV), suspended in a liquid medium without labeling, using a semiconductor technique. The virus count was estimated by calculating the impurities inside a defined volume by observing the change in electrical parameters. Empirically, the virus count was similar to the absolute value of the ratio of the change of the virus suspension dopant concentration relative to the mock dopant over the change in virus suspension Debye volume relative to mock Debye volume. The virus type was identified by constructing a concentration-mobility relationship which is unique for each kind of virus, allowing for a fast (within minutes) and label-free virus quantification and identification. For validation, the HIV and FIV virus preparations were further quantified by a biochemical technique and the results obtained by both approaches corroborated well. We further demonstrate that the electrical technique could be applied to accurately measure and characterize silica nanoparticles that resemble the virus particles in size. Based on these results, we anticipate our present approach to be a starting point towards establishing the foundation for label-free electrical-based identification and quantification of an unlimited number of viruses and other nano-sized particles.

  12. Seed shape quantification in the order Cucurbitales

    Directory of Open Access Journals (Sweden)

    Emilio Cervantes

    2018-02-01

    Full Text Available Seed shape quantification in diverse species of the families belonging to the order Cucurbitales is done based on the comparison of seed images with geometric figures. Quantification of seed shape is a useful tool in plant description for phenotypic characterization and taxonomic analysis. J index gives the percent of similarity of the image of a seed with a geometric figure and it is useful in taxonomy for the study of relationships between plant groups. Geometric figures used as models in the Cucurbitales are the ovoid, two ellipses with different x/y ratios and the outline of the Fibonacci spiral. The images of seeds have been compared with these figures and values of J index obtained. The results obtained for 29 species in the family Cucurbitaceae support a relationship between seed shape and species ecology. Simple seed shape, with images resembling simple geometric figures like the ovoid, ellipse or the Fibonacci spiral, may be a feature in the basal clades of taxonomic groups.

  13. Quantification of ontogenetic allometry in ammonoids.

    Science.gov (United States)

    Korn, Dieter

    2012-01-01

    Ammonoids are well-known objects used for studies on ontogeny and phylogeny, but a quantification of ontogenetic change has not yet been carried out. Their planispirally coiled conchs allow for a study of "longitudinal" ontogenetic data, that is data of ontogenetic trajectories that can be obtained from a single specimen. Therefore, they provide a good model for ontogenetic studies of geometry in other shelled organisms. Using modifications of three cardinal conch dimensions, computer simulations can model artificial conchs. The trajectories of ontogenetic allometry of these simulations can be analyzed in great detail in a theoretical morphospace. A method for the classification of conch ontogeny and quantification of the degree of allometry is proposed. Using high-precision cross-sections, the allometric conch growth of real ammonoids can be documented and compared. The members of the Ammonoidea show a wide variety of allometric growth, ranging from near isometry to monophasic, biphasic, or polyphasic allometry. Selected examples of Palaeozoic and Mesozoic ammonoids are shown with respect to their degree of change during ontogeny of the conch. © 2012 Wiley Periodicals, Inc.

  14. Poisson Plus Quantification for Digital PCR Systems.

    Science.gov (United States)

    Majumdar, Nivedita; Banerjee, Swapnonil; Pallas, Michael; Wessel, Thomas; Hegerich, Patricia

    2017-08-29

    Digital PCR, a state-of-the-art nucleic acid quantification technique, works by spreading the target material across a large number of partitions. The average number of molecules per partition is estimated using Poisson statistics, and then converted into concentration by dividing by partition volume. In this standard approach, identical partition sizing is assumed. Violations of this assumption result in underestimation of target quantity, when using Poisson modeling, especially at higher concentrations. The Poisson-Plus Model accommodates for this underestimation, if statistics of the volume variation are well characterized. The volume variation was measured on the chip array based QuantStudio 3D Digital PCR System using the ROX fluorescence level as a proxy for effective load volume per through-hole. Monte Carlo simulations demonstrate the efficacy of the proposed correction. Empirical measurement of model parameters characterizing the effective load volume on QuantStudio 3D Digital PCR chips is presented. The model was used to analyze digital PCR experiments and showed improved accuracy in quantification. At the higher concentrations, the modeling must take effective fill volume variation into account to produce accurate estimates. The extent of the difference from the standard to the new modeling is positively correlated to the extent of fill volume variation in the effective load of your reactions.

  15. Dystrophin quantification: Biological and translational research implications.

    Science.gov (United States)

    Anthony, Karen; Arechavala-Gomeza, Virginia; Taylor, Laura E; Vulin, Adeline; Kaminoh, Yuuki; Torelli, Silvia; Feng, Lucy; Janghra, Narinder; Bonne, Gisèle; Beuvin, Maud; Barresi, Rita; Henderson, Matt; Laval, Steven; Lourbakos, Afrodite; Campion, Giles; Straub, Volker; Voit, Thomas; Sewry, Caroline A; Morgan, Jennifer E; Flanigan, Kevin M; Muntoni, Francesco

    2014-11-25

    We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials. Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels. Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable. Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval. © 2014 American Academy of Neurology.

  16. CT quantification of central airway in tracheobronchomalacia

    Energy Technology Data Exchange (ETDEWEB)

    Im, Won Hyeong; Jin, Gong Yong; Han, Young Min; Kim, Eun Young [Dept. of Radiology, Chonbuk National University Hospital, Jeonju (Korea, Republic of)

    2016-05-15

    To know which factors help to diagnose tracheobronchomalacia (TBM) using CT quantification of central airway. From April 2013 to July 2014, 19 patients (68.0 ± 15.0 years; 6 male, 13 female) were diagnosed as TBM on CT. As case-matching, 38 normal subjects (65.5 ± 21.5 years; 6 male, 13 female) were selected. All 57 subjects underwent CT with end-inspiration and end-expiration. Airway parameters of trachea and both main bronchus were assessed using software (VIDA diagnostic). Airway parameters of TBM patients and normal subjects were compared using the Student t-test. In expiration, both wall perimeter and wall thickness in TBM patients were significantly smaller than normal subjects (wall perimeter: trachea, 43.97 mm vs. 49.04 mm, p = 0.020; right main bronchus, 33.52 mm vs. 42.69 mm, p < 0.001; left main bronchus, 26.76 mm vs. 31.88 mm, p = 0.012; wall thickness: trachea, 1.89 mm vs. 2.22 mm, p = 0.017; right main bronchus, 1.64 mm vs. 1.83 mm, p = 0.021; left main bronchus, 1.61 mm vs. 1.75 mm, p = 0.016). Wall thinning and decreased perimeter of central airway of expiration by CT quantification would be a new diagnostic indicators in TBM.

  17. Development of a VHH-Based Erythropoietin Quantification Assay

    DEFF Research Database (Denmark)

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

    2015-01-01

    human EPO was evaluated as a capturing antibody in a label-free biolayer interferometry-based quantification assay. Human recombinant EPO can be specifically detected in Chinese hamster ovary cell supernatants in a sensitive and pH-dependent manner. This method enables rapid and robust quantification...

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

    African Journals Online (AJOL)

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

  19. Quantification of Material State using Reflectance FTIR Spectroscopy (Preprint)

    Science.gov (United States)

    2017-12-08

    AFRL-RX-WP-JA-2017-0517 QUANTIFICATION OF MATERIAL STATE USING REFLECTANCE FTIR SPECTROSCOPY (PREPRINT) Amanda K. Criner and...Statement A. Approved for public release: distribution unlimited. (STINFO COPY) AIR FORCE RESEARCH LABORATORY MATERIALS AND MANUFACTURING...March 2014 – 8 November 2017 4. TITLE AND SUBTITLE QUANTIFICATION OF MATERIAL STATE USING REFLECTANCE FTIR SPECTROSCOPY (PREPRINT) 5a. CONTRACT

  20. Chlorophyll a interference in phycocyanin and allophycocyanin spectrophotometric quantification

    Directory of Open Access Journals (Sweden)

    Rosaria Lauceri

    2017-09-01

    Full Text Available The accurate quantification of cyanobacteria phycobiliproteins is an important aspect in various research topics, such as cyanobacteria ecology and physiology studies, and especially to calibrate algorithms used in remote sensing of cyanobacterial blooms. Here we present a spectroscopic approach, exploiting spectrophotometric equations, aimed at improving the phycocyanin and allophycocyanin quantification when chlorophyll a is present in the phycobiliprotein aqueous extract.

  1. In vivo MRS metabolite quantification using genetic optimization

    Science.gov (United States)

    Papakostas, G. A.; Karras, D. A.; Mertzios, B. G.; van Ormondt, D.; Graveron-Demilly, D.

    2011-11-01

    The in vivo quantification of metabolites' concentrations, revealed in magnetic resonance spectroscopy (MRS) spectra, constitutes the main subject under investigation in this work. Significant contributions based on artificial intelligence tools, such as neural networks (NNs), with good results have been presented lately but have shown several drawbacks, regarding their quantification accuracy under difficult conditions. A general framework that encounters the quantification procedure as an optimization problem, which is solved using a genetic algorithm (GA), is proposed in this paper. Two different lineshape models are examined, while two GA configurations are applied on artificial data. Moreover, the introduced quantification technique deals with metabolite peaks' overlapping, a considerably difficult situation occurring under real conditions. Appropriate experiments have proved the efficiency of the introduced methodology, in artificial MRS data, by establishing it as a generic metabolite quantification procedure.

  2. In vivo MRS metabolite quantification using genetic optimization

    International Nuclear Information System (INIS)

    Papakostas, G A; Mertzios, B G; Karras, D A; Van Ormondt, D; Graveron-Demilly, D

    2011-01-01

    The in vivo quantification of metabolites' concentrations, revealed in magnetic resonance spectroscopy (MRS) spectra, constitutes the main subject under investigation in this work. Significant contributions based on artificial intelligence tools, such as neural networks (NNs), with good results have been presented lately but have shown several drawbacks, regarding their quantification accuracy under difficult conditions. A general framework that encounters the quantification procedure as an optimization problem, which is solved using a genetic algorithm (GA), is proposed in this paper. Two different lineshape models are examined, while two GA configurations are applied on artificial data. Moreover, the introduced quantification technique deals with metabolite peaks' overlapping, a considerably difficult situation occurring under real conditions. Appropriate experiments have proved the efficiency of the introduced methodology, in artificial MRS data, by establishing it as a generic metabolite quantification procedure

  3. Quantification of viable Giardia cysts and Cryptosporidium oocysts in wastewater using propidium monoazide quantitative real-time PCR.

    Science.gov (United States)

    Alonso, José L; Amorós, Inmaculada; Guy, Rebecca A

    2014-07-01

    Real-time PCR (qPCR) is a rapid tool to quantify pathogens in the aquatic environment; however, it quantifies all pathogens, including both viable and nonviable. Propidium monoazide (PMA) is a membrane-impairment dye that penetrates only membrane-damaged cells. Once inside the cell, PMA is covalently cross-linked to DNA through light photoactivation, and PCR amplification is strongly inhibited. The goal of this study was to evaluate PMA-qPCR assays for rapid quantification of viable and heat-treated Giardia cysts and Cryptosporidium oocysts in wastewater. We observed a reduction in detection of heat-treated Giardia duodenalis cysts of 83.2, 89.9, 98.2, or 97% with PMA-qPCR assays amplifying a 75 base-pair (bp) β-giardin target, 77-bp triosephosphate isomerase (tpi), 133-bp glutamate dehydrogenase (GDH), and 143-bp β-giardin gene target, respectively. Thus, the exclusion of dead cysts was more effective when qPCR assays that produced larger amplicons were used. The PMA treatment of Cryptosporidium oocysts plus/minus heat treatment abolished the fluorescent signal for dead oocysts with a PMA-qPCR assay amplifying a Cryptosporidium parvum (150-bp) oocyst wall protein (COWP) gene. The PMA-qPCR 143-bp β-giardin assay for Giardia and the PMA-qPCR 150-bp COWP assay for Cryptosporidium accurately quantified live oo(cysts), and failed to detect dead oo(cysts), when phosphate-buffered saline and tertiary effluent wastewater were spiked with concentrations of 10(3) or 10(2) dead oo(cysts), respectively. Therefore, these assays are suitable for the detection of viable parasites that are typically present in tertiary wastewater effluents at concentrations of <10(3) oo(cysts)/l and can provide rapid risk assessments of environmental water.

  4. Survey and Evaluate Uncertainty Quantification Methodologies

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Guang; Engel, David W.; Eslinger, Paul W.

    2012-02-01

    The Carbon Capture Simulation Initiative (CCSI) is a partnership among national laboratories, industry and academic institutions that will develop and deploy state-of-the-art computational modeling and simulation tools to accelerate the commercialization of carbon capture technologies from discovery to development, demonstration, and ultimately the widespread deployment to hundreds of power plants. The CCSI Toolset will provide end users in industry with a comprehensive, integrated suite of scientifically validated models with uncertainty quantification, optimization, risk analysis and decision making capabilities. The CCSI Toolset will incorporate commercial and open-source software currently in use by industry and will also develop new software tools as necessary to fill technology gaps identified during execution of the project. The CCSI Toolset will (1) enable promising concepts to be more quickly identified through rapid computational screening of devices and processes; (2) reduce the time to design and troubleshoot new devices and processes; (3) quantify the technical risk in taking technology from laboratory-scale to commercial-scale; and (4) stabilize deployment costs more quickly by replacing some of the physical operational tests with virtual power plant simulations. The goal of CCSI is to deliver a toolset that can simulate the scale-up of a broad set of new carbon capture technologies from laboratory scale to full commercial scale. To provide a framework around which the toolset can be developed and demonstrated, we will focus on three Industrial Challenge Problems (ICPs) related to carbon capture technologies relevant to U.S. pulverized coal (PC) power plants. Post combustion capture by solid sorbents is the technology focus of the initial ICP (referred to as ICP A). The goal of the uncertainty quantification (UQ) task (Task 6) is to provide a set of capabilities to the user community for the quantification of uncertainties associated with the carbon

  5. A newly developed real-time PCR assay for detection and quantification of Fusarium oxysporum and its use in compatible and incompatible interactions with grafted melon genotypes.

    Science.gov (United States)

    Haegi, Anita; Catalano, Valentina; Luongo, Laura; Vitale, Salvatore; Scotton, Michele; Ficcadenti, Nadia; Belisario, Alessandra

    2013-08-01

    A reliable and species-specific real-time quantitative polymerase chain reaction (qPCR) assay was developed for detection of the complex soilborne anamorphic fungus Fusarium oxysporum. The new primer pair, designed on the translation elongation factor 1-α gene with an amplicon of 142 bp, was highly specific to F. oxysporum without cross reactions with other Fusarium spp. The protocol was applied to grafted melon plants for the detection and quantification of F. oxysporum f. sp. melonis, a devastating pathogen of this cucurbit. Grafting technologies are widely used in melon to confer resistance against new virulent races of F. oxysporum f. sp. melonis, while maintaining the properties of valuable commercial varieties. However, the effects on the vascular pathogen colonization have not been fully investigated. Analyses were performed on 'Charentais-T' (susceptible) and 'Nad-1' (resistant) melon cultivars, both used either as rootstock and scion, and inoculated with F. oxysporum f. sp. melonis race 1 and race 1,2. Pathogen development was compared using qPCR and isolations from stem tissues. Early asymptomatic melon infections were detected with a quantification limit of 1 pg of fungal DNA. The qPCR protocol clearly showed that fungal development was highly affected by host-pathogen interaction (compatible or incompatible) and time (days postinoculation). The principal significant effect (P ≤ 0.01) on fungal development was due to the melon genotype used as rootstock, and this effect had a significant interaction with time and F. oxysporum f. sp. melonis race. In particular, the amount of race 1,2 DNA was significantly higher compared with that estimated for race 1 in the incompatible interaction at 18 days postinoculation. The two fungal races were always present in both the rootstock and scion of grafted plants in either the compatible or incompatible interaction.

  6. Multicentric evaluation of a new real-time PCR assay for quantification of Cryptosporidium spp. and identification of Cryptosporidium parvum and Cryptosporidium hominis.

    Science.gov (United States)

    Mary, C; Chapey, E; Dutoit, E; Guyot, K; Hasseine, L; Jeddi, F; Menotti, J; Paraud, C; Pomares, C; Rabodonirina, M; Rieux, A; Derouin, F

    2013-08-01

    Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidium-positive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.

  7. Quantitative PCR for the specific quantification of Lactococcus lactis and Lactobacillus paracasei and its interest for Lactococcus lactis in cheese samples.

    Science.gov (United States)

    Achilleos, Christine; Berthier, Françoise

    2013-12-01

    The first objective of this work was to develop real-time quantitative PCR (qPCR) assays to quantify two species of mesophilic lactic acid bacteria technologically active in food fermentation, including cheese making: Lactococcus lactis and Lactobacillus paracasei. The second objective was to compare qPCR and plate counts of these two species in cheese samples. Newly designed primers efficiently amplified a region of the tuf gene from the target species. Sixty-three DNA samples from twenty different bacterial species, phylogenetically related or commonly found in raw milk and dairy products, were selected as positive and negative controls. Target DNA was successfully amplified showing a single peak on the amplicon melting curve; non-target DNA was not amplified. Quantification was linear over 5 log units (R(2) > 0.990), down to 22 gene copies/μL per well for Lc. lactis and 73 gene copies/μL per well for Lb. paracasei. qPCR efficiency ranged from 82.9% to 93.7% for Lc. lactis and from 81.1% to 99.5% for Lb. paracasei. At two stages of growth, Lc. lactis was quantified in 12 soft cheeses and Lb. paracasei in 24 hard cooked cheeses. qPCR proved to be useful for quantifying Lc. lactis, but not Lb. paracasei. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Quantification practices in the nuclear industry

    International Nuclear Information System (INIS)

    1986-01-01

    In this chapter the quantification of risk practices adopted by the nuclear industries in Germany, Britain and France are examined as representative of the practices adopted throughout Europe. From this examination a number of conclusions are drawn about the common features of the practices adopted. In making this survey, the views expressed in the report of the Task Force on Safety Goals/Objectives appointed by the Commission of the European Communities, are taken into account. For each country considered, the legal requirements for presentation of quantified risk assessment as part of the licensing procedure are examined, and the way in which the requirements have been developed for practical application are then examined. (author)

  9. Towards enhanced PET quantification in clinical oncology

    DEFF Research Database (Denmark)

    Zaidi, Habib; Karakatsanis, Nicolas

    2018-01-01

    Positron emission tomography (PET) has, since its inception, established itself as the imaging modality of choice for the in vivo quantitative assessment of molecular targets in a wide range of biochemical processes underlying tumour physiology. PET image quantification enables to ascertain...... a direct link between the time-varying activity concentration in organs/tissues and the fundamental parameters portraying the biological processes at the cellular level being assessed. However, the quantitative potential of PET may be affected by a number of factors related to physical effects, hardware...... and software system specifications, tracer kinetics, motion, scan protocol design and limitations in current image-derived PET metrics. Given the relatively large number of PET metrics reported in the literature, the selection of the best metric for fulfilling a specific task in a particular application...

  10. QUANTIFICATION OF OCHRATOXIN A IN MOLDAVIAN WINES

    Directory of Open Access Journals (Sweden)

    RODICA STURZA

    2017-10-01

    Full Text Available The level of the carcinogenic mycotoxin ochratoxin A (OTA in wines produced in Moldova, including bottled and raw material wines, was analyzed by enzyme-linked immunosorbent assay (ELISA and high performance liquid chromatography (HPLC analysis with fluorescence detection after immunoaffinity column clean-up. The study was conducted on wine samples from vintage 2006 - 2016. Our results confirm previously published reports that the levels of OTA are considerably higher in red wines than those in white ones. It was found that OTA levels of analyzed samples differ significantly depending on the harvest year of the wine. Thus, 48 % of studied wine samples from vintage 2006 were contaminated with OTA, including 10 % samples with concentration of OTA higher than 2 µg‧L-1. In contrast, levels of OTA detected in samples from 2016, haven’t exceed 0.05 µg‧L-1 (quantification limit of HPLC method.

  11. Recurrence quantification analysis theory and best practices

    CERN Document Server

    Jr, Jr; Marwan, Norbert

    2015-01-01

    The analysis of recurrences in dynamical systems by using recurrence plots and their quantification is still an emerging field.  Over the past decades recurrence plots have proven to be valuable data visualization and analysis tools in the theoretical study of complex, time-varying dynamical systems as well as in various applications in biology, neuroscience, kinesiology, psychology, physiology, engineering, physics, geosciences, linguistics, finance, economics, and other disciplines.   This multi-authored book intends to comprehensively introduce and showcase recent advances as well as established best practices concerning both theoretical and practical aspects of recurrence plot based analysis.  Edited and authored by leading researcher in the field, the various chapters address an interdisciplinary readership, ranging from theoretical physicists to application-oriented scientists in all data-providing disciplines.

  12. Recurrence quantification analysis of global stock markets

    Science.gov (United States)

    Bastos, João A.; Caiado, Jorge

    2011-04-01

    This study investigates the presence of deterministic dependencies in international stock markets using recurrence plots and recurrence quantification analysis (RQA). The results are based on a large set of free float-adjusted market capitalization stock indices, covering a period of 15 years. The statistical tests suggest that the dynamics of stock prices in emerging markets is characterized by higher values of RQA measures when compared to their developed counterparts. The behavior of stock markets during critical financial events, such as the burst of the technology bubble, the Asian currency crisis, and the recent subprime mortgage crisis, is analyzed by performing RQA in sliding windows. It is shown that during these events stock markets exhibit a distinctive behavior that is characterized by temporary decreases in the fraction of recurrence points contained in diagonal and vertical structures.

  13. Kinetic quantification of plyometric exercise intensity.

    Science.gov (United States)

    Ebben, William P; Fauth, McKenzie L; Garceau, Luke R; Petushek, Erich J

    2011-12-01

    Ebben, WP, Fauth, ML, Garceau, LR, and Petushek, EJ. Kinetic quantification of plyometric exercise intensity. J Strength Cond Res 25(12): 3288-3298, 2011-Quantification of plyometric exercise intensity is necessary to understand the characteristics of these exercises and the proper progression of this mode of exercise. The purpose of this study was to assess the kinetic characteristics of a variety of plyometric exercises. This study also sought to assess gender differences in these variables. Twenty-six men and 23 women with previous experience in performing plyometric training served as subjects. The subjects performed a variety of plyometric exercises including line hops, 15.24-cm cone hops, squat jumps, tuck jumps, countermovement jumps (CMJs), loaded CMJs equal to 30% of 1 repetition maximum squat, depth jumps normalized to the subject's jump height (JH), and single leg jumps. All plyometric exercises were assessed with a force platform. Outcome variables associated with the takeoff, airborne, and landing phase of each plyometric exercise were evaluated. These variables included the peak vertical ground reaction force (GRF) during takeoff, the time to takeoff, flight time, JH, peak power, landing rate of force development, and peak vertical GRF during landing. A 2-way mixed analysis of variance with repeated measures for plyometric exercise type demonstrated main effects for exercise type and all outcome variables (p ≤ 0.05) and for the interaction between gender and peak vertical GRF during takeoff (p ≤ 0.05). Bonferroni-adjusted pairwise comparisons identified a number of differences between the plyometric exercises for the outcome variables assessed (p ≤ 0.05). These findings can be used to guide the progression of plyometric training by incorporating exercises of increasing intensity over the course of a program.

  14. Convex geometry of quantum resource quantification

    Science.gov (United States)

    Regula, Bartosz

    2018-01-01

    We introduce a framework unifying the mathematical characterisation of different measures of general quantum resources and allowing for a systematic way to define a variety of faithful quantifiers for any given convex quantum resource theory. The approach allows us to describe many commonly used measures such as matrix norm-based quantifiers, robustness measures, convex roof-based measures, and witness-based quantifiers together in a common formalism based on the convex geometry of the underlying sets of resource-free states. We establish easily verifiable criteria for a measure to possess desirable properties such as faithfulness and strong monotonicity under relevant free operations, and show that many quantifiers obtained in this framework indeed satisfy them for any considered quantum resource. We derive various bounds and relations between the measures, generalising and providing significantly simplified proofs of results found in the resource theories of quantum entanglement and coherence. We also prove that the quantification of resources in this framework simplifies for pure states, allowing us to obtain more easily computable forms of the considered measures, and show that many of them are in fact equal on pure states. Further, we investigate the dual formulation of resource quantifiers, which provide a characterisation of the sets of resource witnesses. We present an explicit application of the results to the resource theories of multi-level coherence, entanglement of Schmidt number k, multipartite entanglement, as well as magic states, providing insight into the quantification of the four resources by establishing novel quantitative relations and introducing new quantifiers, such as a measure of entanglement of Schmidt number k which generalises the convex roof–extended negativity, a measure of k-coherence which generalises the \

  15. Definitive differentiation between single and mixed mycobacterial infections in red deer (Cervus elaphus) by a combination of duplex amplification of p34 and f57 sequences and Hpy188I enzymatic restriction of duplex amplicons.

    Science.gov (United States)

    Godfroid, Jacques; Delcorps, Cathy; Irenge, Leonid M; Walravens, Karl; Marché, Sylvie; Gala, Jean-Luc

    2005-09-01

    Severe emaciation and mortalities suggestive of mycobacterial infections were recently reported for both adult and young wild red deer (Cervus elaphus) in the southeastern part of Belgium. In deer, tuberculous lesions are not pathognomonic of Mycobacterium bovis infection due to gross and microscopic similarities with lesions caused by Mycobacterium avium subsp. paratuberculosis or M. avium subsp. avium. The aim of this study was to improve molecular methods for the species-specific identification of M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis in mycobacterial infections of deer. DNA banding patterns were assessed prior to and after Hpy188I restriction of f57-upstream (us)-p34 duplex amplicons. The duplex f57-us-p34 PCR differentiated M. bovis from M. avium subsp. paratuberculosis and M. avium subsp. avium infections, whereas the restriction step differentiated single M. avium subsp. paratuberculosis or M. avium subsp. avium infections from mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections. The endonuclease Hpy188I cleaves DNA between nucleotides N and G in the unique TCNGA sequence. This restriction site was found at position 168 upstream of the us-p34 initiation codon in all M. avium subsp. avium strains tested, regardless of their origin and the results of IS901 PCR. In contrast, the restriction site was abrogated in all M. avium subsp. paratuberculosis strains tested, independent of their origin, Mycobactin J dependency, and IS900 PCR results. Consequently, a two-step strategy, i.e., duplex us-p34-f57 PCR and Hpy188I restriction, allowed us to exclude M. bovis infection and to identify single (M. avium subsp. paratuberculosis or M. avium subsp. avium) or mixed (M. avium subsp. paratuberculosis/M. avium subsp. avium) infections in wild red deer in Belgium. Accordingly, we propose to integrate, in a functional molecular definition of M. avium subsp. paratuberculosis, the absence of the Hpy188I restriction site from

  16. Application of Fuzzy Comprehensive Evaluation Method in Trust Quantification

    Directory of Open Access Journals (Sweden)

    Shunan Ma

    2011-10-01

    Full Text Available Trust can play an important role for the sharing of resources and information in open network environments. Trust quantification is thus an important issue in dynamic trust management. By considering the fuzziness and uncertainty of trust, in this paper, we propose a fuzzy comprehensive evaluation method to quantify trust along with a trust quantification algorithm. Simulation results show that the trust quantification algorithm that we propose can effectively quantify trust and the quantified value of an entity's trust is consistent with the behavior of the entity.

  17. Exploring Heterogeneous Multicore Architectures for Advanced Embedded Uncertainty Quantification.

    Energy Technology Data Exchange (ETDEWEB)

    Phipps, Eric T.; Edwards, Harold C.; Hu, Jonathan J.

    2014-09-01

    We explore rearrangements of classical uncertainty quantification methods with the aim of achieving higher aggregate performance for uncertainty quantification calculations on emerging multicore and manycore architectures. We show a rearrangement of the stochastic Galerkin method leads to improved performance and scalability on several computational architectures whereby un- certainty information is propagated at the lowest levels of the simulation code improving memory access patterns, exposing new dimensions of fine grained parallelism, and reducing communica- tion. We also develop a general framework for implementing such rearrangements for a diverse set of uncertainty quantification algorithms as well as computational simulation codes to which they are applied.

  18. Extraction, quantification and degree of polymerization of yacon ...

    African Journals Online (AJOL)

    Extraction, quantification and degree of polymerization of yacon (Smallanthus sonchifolia) fructans. EWN da Fonseca Contado, E de Rezende Queiroz, DA Rocha, RM Fraguas, AA Simao, LNS Botelho, A de Fatima Abreu, MABCMP de Abreu ...

  19. Uncertainty Quantification for Production Navier-Stokes Solvers, Phase II

    Data.gov (United States)

    National Aeronautics and Space Administration — The uncertainty quantification methods developed under this program are designed for use with current state-of-the-art flow solvers developed by and in use at NASA....

  20. A Micropillar Compression Methodology for Ductile Damage Quantification

    NARCIS (Netherlands)

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

    2012-01-01

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

  1. Quantification of propionic acid from Scutellaria baicalensis roots

    Directory of Open Access Journals (Sweden)

    Eunjung Son

    2017-03-01

    Conclusion: This study is the first to report that propionic acid exists in S. baicalensis roots and also provides a useful ultra performance liquid chromatography analysis method for its quantification.

  2. Quantification of Uncertainties in Integrated Spacecraft System Models, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — The proposed effort is to investigate a novel uncertainty quantification (UQ) approach based on non-intrusive polynomial chaos (NIPC) for computationally efficient...

  3. Synthesis and Review: Advancing agricultural greenhouse gas quantification

    Science.gov (United States)

    Olander, Lydia P.; Wollenberg, Eva; Tubiello, Francesco N.; Herold, Martin

    2014-07-01

    Reducing emissions of agricultural greenhouse gases (GHGs), such as methane and nitrous oxide, and sequestering carbon in the soil or in living biomass can help reduce the impact of agriculture on climate change while improving productivity and reducing resource use. There is an increasing demand for improved, low cost quantification of GHGs in agriculture, whether for national reporting to the United Nations Framework Convention on Climate Change (UNFCCC), underpinning and stimulating improved practices, establishing crediting mechanisms, or supporting green products. This ERL focus issue highlights GHG quantification to call attention to our existing knowledge and opportunities for further progress. In this article we synthesize the findings of 21 papers on the current state of global capability for agricultural GHG quantification and visions for its improvement. We conclude that strategic investment in quantification can lead to significant global improvement in agricultural GHG estimation in the near term.

  4. Quantification of Uncertainties in Integrated Spacecraft System Models, Phase II

    Data.gov (United States)

    National Aeronautics and Space Administration — The objective for the Phase II effort will be to develop a comprehensive, efficient, and flexible uncertainty quantification (UQ) framework implemented within a...

  5. Quantification of propionic acid from Scutellaria baicalensis roots

    OpenAIRE

    Eunjung Son; Ho Kyoung Kim; Hyun Sik Kim; Mee Ree Kim; Dong-Seon Kim

    2017-01-01

    Background: Propionic acid is a widely used preservative and has been mainly formed by artificial synthesis or fermentation. In the case of natural products, the presence of propionic acid is viewed as a sign that an additive has been introduced for antimicrobial effects. Methods: In this work, the propionic acid that occurs in Scutellaria baicalensis roots was studied. A quantification method was developed, validated, and showed good linearity, low limit of detection, and limit of quantif...

  6. Installation Restoration Program. Confirmation/Quantification Stage 1. Phase 2

    Science.gov (United States)

    1985-03-07

    follows: Phase I - Problem Identification/Records Search Phase II - Problem Confirmation/QuantificationI Phase III - Technology Base Development Phase IV...Identification/Records Search 3 Phase II-- Problem Confirmation/Quantification Phase III -- Technology Base Development 3 Phase IV -- Operations...United States Air Force Occupational and Environmental Helth Laboratory 3 VADOSE ZONE: That area below the ground surface and above the water table WATER

  7. FRANX. Application for analysis and quantification of the APS fire

    International Nuclear Information System (INIS)

    Snchez, A.; Osorio, F.; Ontoso, N.

    2014-01-01

    The FRANX application has been developed by EPRI within the Risk and Reliability User Group in order to facilitate the process of quantification and updating APS Fire (also covers floods and earthquakes). By applying fire scenarios are quantified in the central integrating the tasks performed during the APS fire. This paper describes the main features of the program to allow quantification of an APS Fire. (Author)

  8. Standardless quantification methods in electron probe microanalysis

    Energy Technology Data Exchange (ETDEWEB)

    Trincavelli, Jorge, E-mail: trincavelli@famaf.unc.edu.ar [Facultad de Matemática, Astronomía y Física, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba (Argentina); Instituto de Física Enrique Gaviola, Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina, Medina Allende s/n, Ciudad Universitaria, 5000 Córdoba (Argentina); Limandri, Silvina, E-mail: s.limandri@conicet.gov.ar [Facultad de Matemática, Astronomía y Física, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba (Argentina); Instituto de Física Enrique Gaviola, Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina, Medina Allende s/n, Ciudad Universitaria, 5000 Córdoba (Argentina); Bonetto, Rita, E-mail: bonetto@quimica.unlp.edu.ar [Centro de Investigación y Desarrollo en Ciencias Aplicadas Dr. Jorge Ronco, Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina, Facultad de Ciencias Exactas, de la Universidad Nacional de La Plata, Calle 47 N° 257, 1900 La Plata (Argentina)

    2014-11-01

    The elemental composition of a solid sample can be determined by electron probe microanalysis with or without the use of standards. The standardless algorithms are quite faster than the methods that require standards; they are useful when a suitable set of standards is not available or for rough samples, and also they help to solve the problem of current variation, for example, in equipments with cold field emission gun. Due to significant advances in the accuracy achieved during the last years, product of the successive efforts made to improve the description of generation, absorption and detection of X-rays, the standardless methods have increasingly become an interesting option for the user. Nevertheless, up to now, algorithms that use standards are still more precise than standardless methods. It is important to remark, that care must be taken with results provided by standardless methods that normalize the calculated concentration values to 100%, unless an estimate of the errors is reported. In this work, a comprehensive discussion of the key features of the main standardless quantification methods, as well as the level of accuracy achieved by them is presented. - Highlights: • Standardless methods are a good alternative when no suitable standards are available. • Their accuracy reaches 10% for 95% of the analyses when traces are excluded. • Some of them are suitable for the analysis of rough samples.

  9. Uncertainty quantification in volumetric Particle Image Velocimetry

    Science.gov (United States)

    Bhattacharya, Sayantan; Charonko, John; Vlachos, Pavlos

    2016-11-01

    Particle Image Velocimetry (PIV) uncertainty quantification is challenging due to coupled sources of elemental uncertainty and complex data reduction procedures in the measurement chain. Recent developments in this field have led to uncertainty estimation methods for planar PIV. However, no framework exists for three-dimensional volumetric PIV. In volumetric PIV the measurement uncertainty is a function of reconstructed three-dimensional particle location that in turn is very sensitive to the accuracy of the calibration mapping function. Furthermore, the iterative correction to the camera mapping function using triangulated particle locations in space (volumetric self-calibration) has its own associated uncertainty due to image noise and ghost particle reconstructions. Here we first quantify the uncertainty in the triangulated particle position which is a function of particle detection and mapping function uncertainty. The location uncertainty is then combined with the three-dimensional cross-correlation uncertainty that is estimated as an extension of the 2D PIV uncertainty framework. Finally the overall measurement uncertainty is quantified using an uncertainty propagation equation. The framework is tested with both simulated and experimental cases. For the simulated cases the variation of estimated uncertainty with the elemental volumetric PIV error sources are also evaluated. The results show reasonable prediction of standard uncertainty with good coverage.

  10. Low cost high performance uncertainty quantification

    KAUST Repository

    Bekas, C.

    2009-01-01

    Uncertainty quantification in risk analysis has become a key application. In this context, computing the diagonal of inverse covariance matrices is of paramount importance. Standard techniques, that employ matrix factorizations, incur a cubic cost which quickly becomes intractable with the current explosion of data sizes. In this work we reduce this complexity to quadratic with the synergy of two algorithms that gracefully complement each other and lead to a radically different approach. First, we turned to stochastic estimation of the diagonal. This allowed us to cast the problem as a linear system with a relatively small number of multiple right hand sides. Second, for this linear system we developed a novel, mixed precision, iterative refinement scheme, which uses iterative solvers instead of matrix factorizations. We demonstrate that the new framework not only achieves the much needed quadratic cost but in addition offers excellent opportunities for scaling at massively parallel environments. We based our implementation on BLAS 3 kernels that ensure very high processor performance. We achieved a peak performance of 730 TFlops on 72 BG/P racks, with a sustained performance 73% of theoretical peak. We stress that the techniques presented in this work are quite general and applicable to several other important applications. Copyright © 2009 ACM.

  11. Uncertainty quantification in flood risk assessment

    Science.gov (United States)

    Blöschl, Günter; Hall, Julia; Kiss, Andrea; Parajka, Juraj; Perdigão, Rui A. P.; Rogger, Magdalena; Salinas, José Luis; Viglione, Alberto

    2017-04-01

    Uncertainty is inherent to flood risk assessments because of the complexity of the human-water system, which is characterised by nonlinearities and interdependencies, because of limited knowledge about system properties and because of cognitive biases in human perception and decision-making. On top of the uncertainty associated with the assessment of the existing risk to extreme events, additional uncertainty arises because of temporal changes in the system due to climate change, modifications of the environment, population growth and the associated increase in assets. Novel risk assessment concepts are needed that take into account all these sources of uncertainty. They should be based on the understanding of how flood extremes are generated and how they change over time. They should also account for the dynamics of risk perception of decision makers and population in the floodplains. In this talk we discuss these novel risk assessment concepts through examples from Flood Frequency Hydrology, Socio-Hydrology and Predictions Under Change. We believe that uncertainty quantification in flood risk assessment should lead to a robust approach of integrated flood risk management aiming at enhancing resilience rather than searching for optimal defense strategies.

  12. Verification Validation and Uncertainty Quantification for CGS

    Energy Technology Data Exchange (ETDEWEB)

    Rider, William J. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Kamm, James R. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Weirs, V. Gregory [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2015-01-01

    The overall conduct of verification, validation and uncertainty quantification (VVUQ) is discussed through the construction of a workflow relevant to computational modeling including the turbulence problem in the coarse grained simulation (CGS) approach. The workflow contained herein is defined at a high level and constitutes an overview of the activity. Nonetheless, the workflow represents an essential activity in predictive simulation and modeling. VVUQ is complex and necessarily hierarchical in nature. The particular characteristics of VVUQ elements depend upon where the VVUQ activity takes place in the overall hierarchy of physics and models. In this chapter, we focus on the differences between and interplay among validation, calibration and UQ, as well as the difference between UQ and sensitivity analysis. The discussion in this chapter is at a relatively high level and attempts to explain the key issues associated with the overall conduct of VVUQ. The intention is that computational physicists can refer to this chapter for guidance regarding how VVUQ analyses fit into their efforts toward conducting predictive calculations.

  13. Tentacle: distributed quantification of genes in metagenomes.

    Science.gov (United States)

    Boulund, Fredrik; Sjögren, Anders; Kristiansson, Erik

    2015-01-01

    In metagenomics, microbial communities are sequenced at increasingly high resolution, generating datasets with billions of DNA fragments. Novel methods that can efficiently process the growing volumes of sequence data are necessary for the accurate analysis and interpretation of existing and upcoming metagenomes. Here we present Tentacle, which is a novel framework that uses distributed computational resources for gene quantification in metagenomes. Tentacle is implemented using a dynamic master-worker approach in which DNA fragments are streamed via a network and processed in parallel on worker nodes. Tentacle is modular, extensible, and comes with support for six commonly used sequence aligners. It is easy to adapt Tentacle to different applications in metagenomics and easy to integrate into existing workflows. Evaluations show that Tentacle scales very well with increasing computing resources. We illustrate the versatility of Tentacle on three different use cases. Tentacle is written for Linux in Python 2.7 and is published as open source under the GNU General Public License (v3). Documentation, tutorials, installation instructions, and the source code are freely available online at: http://bioinformatics.math.chalmers.se/tentacle.

  14. [Quantification of acetabular coverage in normal adult].

    Science.gov (United States)

    Lin, R M; Yang, C Y; Yu, C Y; Yang, C R; Chang, G L; Chou, Y L

    1991-03-01

    Quantification of acetabular coverage is important and can be expressed by superimposition of cartilage tracings on the maximum cross-sectional area of the femoral head. A practical Autolisp program on PC AutoCAD has been developed by us to quantify the acetabular coverage through numerical expression of the images of computed tomography. Thirty adults (60 hips) with normal center-edge angle and acetabular index in plain X ray were randomly selected for serial drops. These slices were prepared with a fixed coordination and in continuous sections of 5 mm in thickness. The contours of the cartilage of each section were digitized into a PC computer and processed by AutoCAD programs to quantify and characterize the acetabular coverage of normal and dysplastic adult hips. We found that a total coverage ratio of greater than 80%, an anterior coverage ratio of greater than 75% and a posterior coverage ratio of greater than 80% can be categorized in a normal group. Polar edge distance is a good indicator for the evaluation of preoperative and postoperative coverage conditions. For standardization and evaluation of acetabular coverage, the most suitable parameters are the total coverage ratio, anterior coverage ratio, posterior coverage ratio and polar edge distance. However, medial coverage and lateral coverage ratios are indispensable in cases of dysplastic hip because variations between them are so great that acetabuloplasty may be impossible. This program can also be used to classify precisely the type of dysplastic hip.

  15. Characterization and quantification of biochar alkalinity.

    Science.gov (United States)

    Fidel, Rivka B; Laird, David A; Thompson, Michael L; Lawrinenko, Michael

    2017-01-01

    Lack of knowledge regarding the nature of biochar alkalis has hindered understanding of pH-sensitive biochar-soil interactions. Here we investigate the nature of biochar alkalinity and present a cohesive suite of methods for its quantification. Biochars produced from cellulose, corn stover and wood feedstocks had significant low-pK a organic structural (0.03-0.34 meq g -1 ), other organic (0-0.92 meq g -1 ), carbonate (0.02-1.5 meq g -1 ), and other inorganic (0-0.26 meq g -1 ) alkalinities. All four categories of biochar alkalinity contributed to total biochar alkalinity and are therefore relevant to pH-sensitive soil processes. Total biochar alkalinity was strongly correlated with base cation concentration, but biochar alkalinity was not a simple function of elemental composition, soluble ash, fixed carbon, or volatile matter content. More research is needed to characterize soluble biochar alkalis other than carbonates and to establish predictive relationships among biochar production parameters and the composition of biochar alkalis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. On uncertainty quantification in hydrogeology and hydrogeophysics

    Science.gov (United States)

    Linde, Niklas; Ginsbourger, David; Irving, James; Nobile, Fabio; Doucet, Arnaud

    2017-12-01

    Recent advances in sensor technologies, field methodologies, numerical modeling, and inversion approaches have contributed to unprecedented imaging of hydrogeological properties and detailed predictions at multiple temporal and spatial scales. Nevertheless, imaging results and predictions will always remain imprecise, which calls for appropriate uncertainty quantification (UQ). In this paper, we outline selected methodological developments together with pioneering UQ applications in hydrogeology and hydrogeophysics. The applied mathematics and statistics literature is not easy to penetrate and this review aims at helping hydrogeologists and hydrogeophysicists to identify suitable approaches for UQ that can be applied and further developed to their specific needs. To bypass the tremendous computational costs associated with forward UQ based on full-physics simulations, we discuss proxy-modeling strategies and multi-resolution (Multi-level Monte Carlo) methods. We consider Bayesian inversion for non-linear and non-Gaussian state-space problems and discuss how Sequential Monte Carlo may become a practical alternative. We also describe strategies to account for forward modeling errors in Bayesian inversion. Finally, we consider hydrogeophysical inversion, where petrophysical uncertainty is often ignored leading to overconfident parameter estimation. The high parameter and data dimensions encountered in hydrogeological and geophysical problems make UQ a complicated and important challenge that has only been partially addressed to date.

  17. Cross recurrence quantification for cover song identification

    International Nuclear Information System (INIS)

    Serra, Joan; Serra, Xavier; Andrzejak, Ralph G

    2009-01-01

    There is growing evidence that nonlinear time series analysis techniques can be used to successfully characterize, classify, or process signals derived from real-world dynamics even though these are not necessarily deterministic and stationary. In the present study, we proceed in this direction by addressing an important problem our modern society is facing, the automatic classification of digital information. In particular, we address the automatic identification of cover songs, i.e. alternative renditions of a previously recorded musical piece. For this purpose, we here propose a recurrence quantification analysis measure that allows the tracking of potentially curved and disrupted traces in cross recurrence plots (CRPs). We apply this measure to CRPs constructed from the state space representation of musical descriptor time series extracted from the raw audio signal. We show that our method identifies cover songs with a higher accuracy as compared to previously published techniques. Beyond the particular application proposed here, we discuss how our approach can be useful for the characterization of a variety of signals from different scientific disciplines. We study coupled Roessler dynamics with stochastically modulated mean frequencies as one concrete example to illustrate this point.

  18. Cross recurrence quantification for cover song identification

    Energy Technology Data Exchange (ETDEWEB)

    Serra, Joan; Serra, Xavier; Andrzejak, Ralph G [Department of Information and Communication Technologies, Universitat Pompeu Fabra, Roc Boronat 138, 08018 Barcelona (Spain)], E-mail: joan.serraj@upf.edu

    2009-09-15

    There is growing evidence that nonlinear time series analysis techniques can be used to successfully characterize, classify, or process signals derived from real-world dynamics even though these are not necessarily deterministic and stationary. In the present study, we proceed in this direction by addressing an important problem our modern society is facing, the automatic classification of digital information. In particular, we address the automatic identification of cover songs, i.e. alternative renditions of a previously recorded musical piece. For this purpose, we here propose a recurrence quantification analysis measure that allows the tracking of potentially curved and disrupted traces in cross recurrence plots (CRPs). We apply this measure to CRPs constructed from the state space representation of musical descriptor time series extracted from the raw audio signal. We show that our method identifies cover songs with a higher accuracy as compared to previously published techniques. Beyond the particular application proposed here, we discuss how our approach can be useful for the characterization of a variety of signals from different scientific disciplines. We study coupled Roessler dynamics with stochastically modulated mean frequencies as one concrete example to illustrate this point.

  19. Tissue quantification for development of pediatric phantom

    International Nuclear Information System (INIS)

    Alves, A.F.F.; Miranda, J.R.A.; Pina, D.R.

    2013-01-01

    The optimization of the risk- benefit ratio is a major concern in the pediatric radiology, due to the greater vulnerability of children to the late somatic effects and genetic effects of exposure to radiation compared to adults. In Brazil, it is estimated that the causes of death from head trauma are 18 % for the age group between 1-5 years and the radiograph is the primary diagnostic test for the detection of skull fracture . Knowing that the image quality is essential to ensure the identification of structures anatomical and minimizing errors diagnostic interpretation, this paper proposed the development and construction of homogeneous phantoms skull, for the age group 1-5 years. The construction of the phantoms homogeneous was performed using the classification and quantification of tissue present in the skull of pediatric patients. In this procedure computational algorithms were used, using Matlab, to quantify distinct biological tissues present in the anatomical regions studied , using pictures retrospective CT scans. Preliminary data obtained from measurements show that between the ages of 1-5 years, assuming an average anteroposterior diameter of the pediatric skull region of the 145.73 ± 2.97 mm, can be represented by 92.34 mm ± 5.22 of lucite and 1.75 ± 0:21 mm of aluminum plates of a provision of PEP (Pacient equivalent phantom). After its construction, the phantoms will be used for image and dose optimization in pediatric protocols process to examinations of computerized radiography

  20. Quantification of food intake in Drosophila.

    Directory of Open Access Journals (Sweden)

    Richard Wong

    2009-06-01

    Full Text Available Measurement of food intake in the fruit fly Drosophila melanogaster is often necessary for studies of behaviour, nutrition and drug administration. There is no reliable and agreed method for measuring food intake of flies in undisturbed, steady state, and normal culture conditions. We report such a method, based on measurement of feeding frequency by proboscis-extension, validated by short-term measurements of food dye intake. We used the method to demonstrate that (a female flies feed more frequently than males, (b flies feed more often when housed in larger groups and (c fly feeding varies at different times of the day. We also show that alterations in food intake are not induced by dietary restriction or by a null mutation of the fly insulin receptor substrate chico. In contrast, mutation of takeout increases food intake by increasing feeding frequency while mutation of ovo(D increases food intake by increasing the volume of food consumed per proboscis-extension. This approach provides a practical and reliable method for quantification of food intake in Drosophila under normal, undisturbed culture conditions.

  1. Delignification of high kappa number soda-ODiMAQ pulps with a polyoxometalate

    Science.gov (United States)

    Edward L. Springer; Aziz Ahmed

    2001-01-01

    Increased use of pressure sensitive adhesives for labels and stamps has introduced another contaminant into the office paper stream: silicone- coated release liners. This study examines methods and conditions for removal of contaminants, including these liners, from a typical batch of discarded office papers. Removal of contaminants contained in the furnish were...

  2. The Maqāṣid approach and rethinking political rights in modern society

    Directory of Open Access Journals (Sweden)

    Louay Safi

    2010-12-01

    Full Text Available This paper examines political rights in Islam by focusing on freedom of religion and the extent to which the state is empowered to enforce faith and religious law on society. It starts by comparing the notion of law in both Western and Islamic traditions, and then analyzes the difference between the ethical and legal within Sharī‘ah. The paper illustrates how Islamic law grew historically by working to limit the power of the state, and points out the need to maintain the distinction between the state and civil society for the proper implementation of Sharī‘ah. The paper also contends that those who call on the state to enforce all rules of Sharī‘ah on society rely on a faulty theory of right and concludes that Islamic law fully recognizes the right of individuals to adopt and practice their faith freely. Freedom of religion, it stresses, is an intrinsic aspect of Islamic law and all efforts to limit this freedom is bound to violate its purpose and dictates.

  3. Topic-Focus Structure and Quantification of Dou 'all'

    Directory of Open Access Journals (Sweden)

    Joonho Shin

    2007-06-01

    Full Text Available This paper examines a type of dou quantification found in wh-questions such as ta dou mai le shenme? ‘What are all the things that he bought?’ This type is different from the well-known dou quantification in that the leftness condition cannot be applied to the former. I propose that the former type of quantification is subject to the topic-focus structure rather than to the syntactic structure, which means that the domain of the quantification is determined in relation to 'old' and 'new' information of a sentence. Sentences including dou can be divided into topic and focus, and each part is mapped onto the restrictor and the nuclear scope in a tripartite structure of dou quantification. This analysis accounts for the reason why a list answer is appropriate to questions with dou, why wh-words in the questions cannot be quantity expressions, and why wh-words should either have a plural interpretation or take the plural form. This analysis also explains the distribution of dou, i.e., dou should c-command a focused phrase. Finally, I point out that the analysis can extend to declaratives which are rare but still observable, and that the two types of dou quantification can arise simultaneously.

  4. Volumetric motion quantification by 3D tissue phase mapped CMR

    Directory of Open Access Journals (Sweden)

    Lutz Anja

    2012-10-01

    Full Text Available Abstract Background The objective of this study was the quantification of myocardial motion from 3D tissue phase mapped (TPM CMR. Recent work on myocardial motion quantification by TPM has been focussed on multi-slice 2D acquisitions thus excluding motion information from large regions of the left ventricle. Volumetric motion assessment appears an important next step towards the understanding of the volumetric myocardial motion and hence may further improve diagnosis and treatments in patients with myocardial motion abnormalities. Methods Volumetric motion quantification of the complete left ventricle was performed in 12 healthy volunteers and two patients applying a black-blood 3D TPM sequence. The resulting motion field was analysed regarding motion pattern differences between apical and basal locations as well as for asynchronous motion pattern between different myocardial segments in one or more slices. Motion quantification included velocity, torsion, rotation angle and strain derived parameters. Results All investigated motion quantification parameters could be calculated from the 3D-TPM data. Parameters quantifying hypokinetic or asynchronous motion demonstrated differences between motion impaired and healthy myocardium. Conclusions 3D-TPM enables the gapless volumetric quantification of motion abnormalities of the left ventricle, which can be applied in future application as additional information to provide a more detailed analysis of the left ventricular function.

  5. GPU-accelerated voxelwise hepatic perfusion quantification.

    Science.gov (United States)

    Wang, H; Cao, Y

    2012-09-07

    Voxelwise quantification of hepatic perfusion parameters from dynamic contrast enhanced (DCE) imaging greatly contributes to assessment of liver function in response to radiation therapy. However, the efficiency of the estimation of hepatic perfusion parameters voxel-by-voxel in the whole liver using a dual-input single-compartment model requires substantial improvement for routine clinical applications. In this paper, we utilize the parallel computation power of a graphics processing unit (GPU) to accelerate the computation, while maintaining the same accuracy as the conventional method. Using compute unified device architecture-GPU, the hepatic perfusion computations over multiple voxels are run across the GPU blocks concurrently but independently. At each voxel, nonlinear least-squares fitting the time series of the liver DCE data to the compartmental model is distributed to multiple threads in a block, and the computations of different time points are performed simultaneously and synchronically. An efficient fast Fourier transform in a block is also developed for the convolution computation in the model. The GPU computations of the voxel-by-voxel hepatic perfusion images are compared with ones by the CPU using the simulated DCE data and the experimental DCE MR images from patients. The computation speed is improved by 30 times using a NVIDIA Tesla C2050 GPU compared to a 2.67 GHz Intel Xeon CPU processor. To obtain liver perfusion maps with 626 400 voxels in a patient's liver, it takes 0.9 min with the GPU-accelerated voxelwise computation, compared to 110 min with the CPU, while both methods result in perfusion parameters differences less than 10(-6). The method will be useful for generating liver perfusion images in clinical settings.

  6. Quantification of water in hydrous ringwoodite

    Directory of Open Access Journals (Sweden)

    Sylvia-Monique eThomas

    2015-01-01

    Full Text Available Ringwoodite, γ-(Mg,Fe2SiO4, in the lower 150 km of Earth’s mantle transition zone (410-660 km depth can incorporate up to 1.5-2 wt% H2O as hydroxyl defects. We present a mineral-specific IR calibration for the absolute water content in hydrous ringwoodite by combining results from Raman spectroscopy, secondary ion mass spectrometery (SIMS and proton-proton (pp-scattering on a suite of synthetic Mg- and Fe-bearing hydrous ringwoodites. H2O concentrations in the crystals studied here range from 0.46 to 1.7 wt% H2O (absolute methods, with the maximum H2O in the same sample giving 2.5 wt% by SIMS calibration. Anchoring our spectroscopic results to absolute H-atom concentrations from pp-scattering measurements, we report frequency-dependent integrated IR-absorption coefficients for water in ringwoodite ranging from 78180 to 158880 L mol-1cm-2, depending upon frequency of the OH absorption. We further report a linear wavenumber IR calibration for H2O quantification in hydrous ringwoodite across the Mg2SiO4-Fe2SiO4 solid solution, which will lead to more accurate estimations of the water content in both laboratory-grown and naturally occurring ringwoodites. Re-evaluation of the IR spectrum for a natural hydrous ringwoodite inclusion in diamond from the study of Pearson et al. (2014 indicates the crystal contains 1.43 ± 0.27 wt% H2O, thus confirming near-maximum amounts of H2O for this sample from the transition zone.

  7. GPU-Accelerated Voxelwise Hepatic Perfusion Quantification

    Science.gov (United States)

    Wang, H; Cao, Y

    2012-01-01

    Voxelwise quantification of hepatic perfusion parameters from dynamic contrast enhanced (DCE) imaging greatly contributes to assessment of liver function in response to radiation therapy. However, the efficiency of the estimation of hepatic perfusion parameters voxel-by-voxel in the whole liver using a dual-input single-compartment model requires substantial improvement for routine clinical applications. In this paper, we utilize the parallel computation power of a graphics processing unit (GPU) to accelerate the computation, while maintaining the same accuracy as the conventional method. Using CUDA-GPU, the hepatic perfusion computations over multiple voxels are run across the GPU blocks concurrently but independently. At each voxel, non-linear least squares fitting the time series of the liver DCE data to the compartmental model is distributed to multiple threads in a block, and the computations of different time points are performed simultaneously and synchronically. An efficient fast Fourier transform in a block is also developed for the convolution computation in the model. The GPU computations of the voxel-by-voxel hepatic perfusion images are compared with ones by the CPU using the simulated DCE data and the experimental DCE MR images from patients. The computation speed is improved by 30 times using a NVIDIA Tesla C2050 GPU compared to a 2.67 GHz Intel Xeon CPU processor. To obtain liver perfusion maps with 626400 voxels in a patient’s liver, it takes 0.9 min with the GPU-accelerated voxelwise computation, compared to 110 min with the CPU, while both methods result in perfusion parameters differences less than 10−6. The method will be useful for generating liver perfusion images in clinical settings. PMID:22892645

  8. Uncertainty Quantification in Geomagnetic Field Modeling

    Science.gov (United States)

    Chulliat, A.; Nair, M. C.; Alken, P.; Meyer, B.; Saltus, R.; Woods, A.

    2017-12-01

    Geomagnetic field models are mathematical descriptions of the various sources of the Earth's magnetic field, and are generally obtained by solving an inverse problem. They are widely used in research to separate and characterize field sources, but also in many practical applications such as aircraft and ship navigation, smartphone orientation, satellite attitude control, and directional drilling. In recent years, more sophisticated models have been developed, thanks to the continuous availability of high quality satellite data and to progress in modeling techniques. Uncertainty quantification has become an integral part of model development, both to assess the progress made and to address specific users' needs. Here we report on recent advances made by our group in quantifying the uncertainty of geomagnetic field models. We first focus on NOAA's World Magnetic Model (WMM) and the International Geomagnetic Reference Field (IGRF), two reference models of the main (core) magnetic field produced every five years. We describe the methods used in quantifying the model commission error as well as the omission error attributed to various un-modeled sources such as magnetized rocks in the crust and electric current systems in the atmosphere and near-Earth environment. A simple error model was derived from this analysis, to facilitate usage in practical applications. We next report on improvements brought by combining a main field model with a high resolution crustal field model and a time-varying, real-time external field model, like in NOAA's High Definition Geomagnetic Model (HDGM). The obtained uncertainties are used by the directional drilling industry to mitigate health, safety and environment risks.

  9. Quantification of nanowire uptake by live cells

    KAUST Repository

    Margineanu, Michael B.

    2015-05-01

    Nanostructures fabricated by different methods have become increasingly important for various applications at the cellular level. In order to understand how these nanostructures “behave” and for studying their internalization kinetics, several attempts have been made at tagging and investigating their interaction with living cells. In this study, magnetic iron nanowires with an iron oxide layer are coated with (3-Aminopropyl)triethoxysilane (APTES), and subsequently labeled with a fluorogenic pH-dependent dye pHrodo™ Red, covalently bound to the aminosilane surface. Time-lapse live imaging of human colon carcinoma HCT 116 cells interacting with the labeled iron nanowires is performed for 24 hours. As the pHrodo™ Red conjugated nanowires are non-fluorescent outside the cells but fluoresce brightly inside, internalized nanowires are distinguished from non-internalized ones and their behavior inside the cells can be tracked for the respective time length. A machine learning-based computational framework dedicated to automatic analysis of live cell imaging data, Cell Cognition, is adapted and used to classify cells with internalized and non-internalized nanowires and subsequently determine the uptake percentage by cells at different time points. An uptake of 85 % by HCT 116 cells is observed after 24 hours incubation at NW-to-cell ratios of 200. While the approach of using pHrodo™ Red for internalization studies is not novel in the literature, this study reports for the first time the utilization of a machine-learning based time-resolved automatic analysis pipeline for quantification of nanowire uptake by cells. This pipeline has also been used for comparison studies with nickel nanowires coated with APTES and labeled with pHrodo™ Red, and another cell line derived from the cervix carcinoma, HeLa. It has thus the potential to be used for studying the interaction of different types of nanostructures with potentially any live cell types.

  10. Visceral adipose tissue quantification using Lunar Prodigy.

    Science.gov (United States)

    Ergun, David L; Rothney, Megan P; Oates, Mary K; Xia, Yi; Wacker, Wynn K; Binkley, Neil C

    2013-01-01

    A dual-energy X-ray absorptiometry (DXA) application to measure visceral adipose tissue (VAT) in the android region of a total body DXA scan has recently been developed. This new application, CoreScan, has been validated on the Lunar iDXA (GE Healthcare, Madison, WI) densitometer against volumetric computed tomography. The geometric assumptions underlying the CoreScan model are the same on the Prodigy (GE Healthcare, Madison, WI) densitometer. However, differences between the peak X-ray voltage and detector array configurations may lead to differences in VAT quantification. The purpose of this study was to evaluate the agreement of Prodigy and iDXA CoreScan values and to characterize differences in VAT precision between the instruments. Data from volunteers with paired Prodigy and iDXA measurements were used to define empirical adjustments to the VAT algorithm parameters (n=59) and validate performance on Prodigy (n=62). Prodigy VAT measurements were highly correlated to iDXA (r=0.984). The mean of the Prodigy-iDXA VAT volume differences was -13.8cm³ with a 95% confidence interval of -45 to +17cm³. The Bland-Altman 95% limits of agreement for the 2 methods were -252 to +224cm³. Measurement of short-term precision showed that measurement error variance on iDXA was smaller (pProdigy (coefficient of variance: 7.3% vs 9.8%). Precision results are in agreement with previous reports on the differences between Prodigy and iDXA for body composition measures. Prodigy and iDXA measures of VAT are similar, but the lower precision of the Prodigy may require investigators to target larger changes in VAT. Copyright © 2013 The International Society for Clinical Densitometry. Published by Elsevier Inc. All rights reserved.

  11. Quantification of microvessels in canine lymph nodes.

    Science.gov (United States)

    Tonar, Zbynĕk; Egger, Gunter F; Witter, Kirsti; Wolfesberger, Birgitt

    2008-10-01

    Quantification of microvessels in tumors is mostly based on counts of vessel profiles in tumor hot spots. Drawbacks of this method include low reproducibility and large interobserver variance, mainly as a result of individual differences in sampling of image fields for analysis. Our aim was to test an unbiased method for quantifying microvessels in healthy and tumorous lymph nodes of dogs. The endothelium of blood vessels was detected in paraffin sections by a combination of immunohistochemistry (von Willebrand factor) and lectin histochemistry (wheat germ agglutinin) in comparison with detection of basal laminae by laminin immunohistochemistry or silver impregnation. Systematic uniform random sampling of 50 image fields was performed during photo-documentation. An unbiased counting frame (area 113,600 microm(2)) was applied to each micrograph. The total area sampled from each node was 5.68 mm(2). Vessel profiles were counted according to stereological counting rules. Inter- and intraobserver variabilities were tested. The application of systematic uniform random sampling was compared with the counting of vessel profiles in hot spots. The unbiased estimate of the number of vessel profiles per unit area ranged from 100.5 +/- 44.0/mm(2) to 442.6 +/- 102.5/mm(2) in contrast to 264 +/- 72.2/mm(2) to 771.0 +/- 108.2/mm(2) in hot spots. The advantage of using systematic uniform random sampling is its reproducibility, with reasonable interobserver and low intraobserver variance. This method also allows for the possibility of using archival material, because staining quality is not limiting as it is for image analysis, and artifacts can easily be excluded. However, this method is comparatively time-consuming. (c) 2008 Wiley-Liss, Inc.

  12. Towards enhanced PET quantification in clinical oncology.

    Science.gov (United States)

    Zaidi, Habib; Karakatsanis, Nicolas

    2018-01-01

    Positron emission tomography (PET) has, since its inception, established itself as the imaging modality of choice for the in vivo quantitative assessment of molecular targets in a wide range of biochemical processes underlying tumour physiology. PET image quantification enables to ascertain a direct link between the time-varying activity concentration in organs/tissues and the fundamental parameters portraying the biological processes at the cellular level being assessed. However, the quantitative potential of PET may be affected by a number of factors related to physical effects, hardware and software system specifications, tracer kinetics, motion, scan protocol design and limitations in current image-derived PET metrics. Given the relatively large number of PET metrics reported in the literature, the selection of the best metric for fulfilling a specific task in a particular application is still a matter of debate. Quantitative PET has advanced elegantly during the last two decades and is now reaching the maturity required for clinical exploitation, particularly in oncology where it has the capability to open many avenues for clinical diagnosis, assessment of response to treatment and therapy planning. Therefore, the preservation and further enhancement of the quantitative features of PET imaging is crucial to ensure that the full clinical value of PET imaging modality is utilized in clinical oncology. Recent advancements in PET technology and methodology have paved the way for faster PET acquisitions of enhanced sensitivity to support the clinical translation of highly quantitative four-dimensional (4D) parametric imaging methods in clinical oncology. In this report, we provide an overview of recent advances and future trends in quantitative PET imaging in the context of clinical oncology. The pros/cons of the various image-derived PET metrics will be discussed and the promise of novel methodologies will be highlighted.

  13. Technology Credit Scoring Based on a Quantification Method

    Directory of Open Access Journals (Sweden)

    Yonghan Ju

    2017-06-01

    Full Text Available Credit scoring models are usually formulated by fitting the probability of loan default as a function of individual evaluation attributes. Typically, these attributes are measured using a Likert-type scale, but are treated as interval scale explanatory variables to predict loan defaults. Existing models also do not distinguish between types of default, although they vary: default by an insolvent company and default by an insolvent debtor. This practice can bias the results. In this paper, we applied Quantification Method II, a categorical version of canonical correlation analysis, to determine the relationship between two sets of categorical variables: a set of default types and a set of evaluation attributes. We distinguished between two types of loan default patterns based on quantification scores. In the first set of quantification scores, we found knowledge management, new technology development, and venture registration as important predictors of default from non-default status. Based on the second quantification score, we found that the technology and profitability factors influence loan defaults due to an insolvent company. Finally, we proposed a credit-risk rating model based on the quantification score.

  14. Quantitative Proteomics via High Resolution MS Quantification: Capabilities and Limitations.

    Science.gov (United States)

    Higgs, Richard E; Butler, Jon P; Han, Bomie; Knierman, Michael D

    2013-01-01

    Recent improvements in the mass accuracy and resolution of mass spectrometers have led to renewed interest in label-free quantification using data from the primary mass spectrum (MS1) acquired from data-dependent proteomics experiments. The capacity for higher specificity quantification of peptides from samples enriched for proteins of biological interest offers distinct advantages for hypothesis generating experiments relative to immunoassay detection methods or prespecified peptide ions measured by multiple reaction monitoring (MRM) approaches. Here we describe an evaluation of different methods to post-process peptide level quantification information to support protein level inference. We characterize the methods by examining their ability to recover a known dilution of a standard protein in background matrices of varying complexity. Additionally, the MS1 quantification results are compared to a standard, targeted, MRM approach on the same samples under equivalent instrument conditions. We show the existence of multiple peptides with MS1 quantification sensitivity similar to the best MRM peptides for each of the background matrices studied. Based on these results we provide recommendations on preferred approaches to leveraging quantitative measurements of multiple peptides to improve protein level inference.

  15. GMO quantification: valuable experience and insights for the future.

    Science.gov (United States)

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

    2014-10-01

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

  16. Uncertainty Quantification in Climate Modeling and Projection

    Energy Technology Data Exchange (ETDEWEB)

    Qian, Yun; Jackson, Charles; Giorgi, Filippo; Booth, Ben; Duan, Qingyun; Forest, Chris; Higdon, Dave; Hou, Z. Jason; Huerta, Gabriel

    2016-05-01

    The projection of future climate is one of the most complex problems undertaken by the scientific community. Although scientists have been striving to better understand the physical basis of the climate system and to improve climate models, the overall uncertainty in projections of future climate has not been significantly reduced (e.g., from the IPCC AR4 to AR5). With the rapid increase of complexity in Earth system models, reducing uncertainties in climate projections becomes extremely challenging. Since uncertainties always exist in climate models, interpreting the strengths and limitations of future climate projections is key to evaluating risks, and climate change information for use in Vulnerability, Impact, and Adaptation (VIA) studies should be provided with both well-characterized and well-quantified uncertainty. The workshop aimed at providing participants, many of them from developing countries, information on strategies to quantify the uncertainty in climate model projections and assess the reliability of climate change information for decision-making. The program included a mixture of lectures on fundamental concepts in Bayesian inference and sampling, applications, and hands-on computer laboratory exercises employing software packages for Bayesian inference, Markov Chain Monte Carlo methods, and global sensitivity analyses. The lectures covered a range of scientific issues underlying the evaluation of uncertainties in climate projections, such as the effects of uncertain initial and boundary conditions, uncertain physics, and limitations of observational records. Progress in quantitatively estimating uncertainties in hydrologic, land surface, and atmospheric models at both regional and global scales was also reviewed. The application of Uncertainty Quantification (UQ) concepts to coupled climate system models is still in its infancy. The Coupled Model Intercomparison Project (CMIP) multi-model ensemble currently represents the primary data for

  17. Quantification model for energy consumption in edification

    Directory of Open Access Journals (Sweden)

    Mercader, Mª P.

    2012-12-01

    Full Text Available The research conducted in this paper focuses on the generation of a model for the quantification of energy consumption in building. This is to be done through one of the most relevant environmental impact indicators associated with weight per m2 of construction, as well as the energy consumption resulting from the manufacturing process of materials used in building construction. The practical application of the proposed model on different buildings typologies in Seville, will provide information regarding the building materials, the subsystems and the most relevant construction elements. Hence, we will be able to observe the impact the built surface has on the environment. The results obtained aim to reference the scientific community, providing quantitative data comparable to other types of buildings and geographical areas. Furthermore, it may also allow the analysis and the characterization of feasible solutions to reduce the environmental impact generated by the different materials, subsystems and construction elements commonly used in the different building types defined in this study.

    La investigación realizada en el presente trabajo plantea la generación de un modelo de cuantificación del consumo energético en edificación, a través de uno de los indicadores de impacto ambiental más relevantes asociados al peso por m2 de construcción, el consumo energético derivado del proceso de fabricación de los materiales de construcción empleados en edificación. La aplicación práctica del modelo propuesto sobre diferentes tipologías edificatorias en Sevilla aportará información respecto a los materiales de construcción, subsistemas y elementos constructivos más impactantes, permitiendo visualizar la influencia que presenta la superficie construida en cuanto al impacto ambiental generado. Los resultados obtenidos pretenden servir de referencia a la comunidad científica, aportando datos num

  18. Symmetry quantification and mapping using convergent beam electron diffraction.

    Science.gov (United States)

    Kim, Kyou-Hyun; Zuo, Jian-Min

    2013-01-01

    We propose a new algorithm to quantify symmetry recorded in convergent beam electron diffraction (CBED) patterns and use it for symmetry mapping in materials applications. We evaluate the effectiveness of the profile R-factor (R(p)) and the normalized cross-correlation coefficient (γ) for quantifying the amount of symmetry in a CBED pattern. The symmetry quantification procedures are automated and the algorithm is implemented as a DM (Digital Micrograph(©)) script. Experimental and simulated CBED patterns recorded from a Si single crystal are used to calibrate the proposed algorithm for the symmetry quantification. The proposed algorithm is then applied to a Si sample with defects to test the sensitivity of symmetry quantification to defects. Using the mirror symmetry as an example, we demonstrate that the normalized cross-correlation coefficient provides an effective and robust measurement of the symmetry recorded in experimental CBED patterns. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Stereological quantification of mast cells in human synovium

    DEFF Research Database (Denmark)

    Damsgaard, T E; Sørensen, Flemming Brandt; Herlin, T

    1999-01-01

    Mast cells participate in both the acute allergic reaction as well as in chronic inflammatory diseases. Earlier studies have revealed divergent results regarding the quantification of mast cells in the human synovium. The aim of the present study was therefore to quantify these cells in the human...... synovium, using stereological techniques. Different methods of staining and quantification have previously been used for mast cell quantification in human synovium. Stereological techniques provide precise and unbiased information on the number of cell profiles in two-dimensional tissue sections of......, in this case, human synovium. In 10 patients suffering from osteoarthritis a median of 3.6 mast cells/mm2 synovial membrane was found. The total number of cells (synoviocytes, fibroblasts, lymphocytes, leukocytes) present was 395.9 cells/mm2 (median). The mast cells constituted 0.8% of all the cell profiles...

  20. Benchmarking common quantification strategies for large-scale phosphoproteomics

    DEFF Research Database (Denmark)

    Hogrebe, Alexander; von Stechow, Louise; Bekker-Jensen, Dorte B

    2018-01-01

    Comprehensive mass spectrometry (MS)-based proteomics is now feasible, but reproducible quantification remains challenging, especially for post-translational modifications such as phosphorylation. Here, we compare the most popular quantification techniques for global phosphoproteomics: label......-free quantification (LFQ), stable isotope labeling by amino acids in cell culture (SILAC) and MS2- and MS3-measured tandem mass tags (TMT). In a mixed species comparison with fixed phosphopeptide ratios, we find LFQ and SILAC to be the most accurate techniques. MS2-based TMT yields the highest precision but lowest...... accuracy due to ratio compression, which MS3-based TMT can partly rescue. However, MS2-based TMT outperforms MS3-based TMT when analyzing phosphoproteome changes in the DNA damage response, since its higher precision and larger identification numbers allow detection of a greater number of significantly...

  1. Superlattice band structure: New and simple energy quantification condition

    Energy Technology Data Exchange (ETDEWEB)

    Maiz, F., E-mail: fethimaiz@gmail.com [University of Cartage, Nabeul Engineering Preparatory Institute, Merazka, 8000 Nabeul (Tunisia); King Khalid University, Faculty of Science, Physics Department, P.O. Box 9004, Abha 61413 (Saudi Arabia)

    2014-10-01

    Assuming an approximated effective mass and using Bastard's boundary conditions, a simple method is used to calculate the subband structure for periodic semiconducting heterostructures. Our method consists to derive and solve the energy quantification condition (EQC), this is a simple real equation, composed of trigonometric and hyperbolic functions, and does not need any programming effort or sophistic machine to solve it. For less than ten wells heterostructures, we have derived and simplified the energy quantification conditions. The subband is build point by point; each point presents an energy level. Our simple energy quantification condition is used to calculate the subband structure of the GaAs/Ga{sub 0.5}Al{sub 0.5}As heterostructures, and build its subband point by point for 4 and 20 wells. Our finding shows a good agreement with previously published results.

  2. Molecular quantification of environmental DNA using microfluidics and digital PCR.

    Science.gov (United States)

    Hoshino, Tatsuhiko; Inagaki, Fumio

    2012-09-01

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

  3. Large-Scale Inverse Problems and Quantification of Uncertainty

    CERN Document Server

    Biegler, Lorenz; Ghattas, Omar

    2010-01-01

    Large-scale inverse problems and associated uncertainty quantification has become an important area of research, central to a wide range of science and engineering applications. Written by leading experts in the field, Large-scale Inverse Problems and Quantification of Uncertainty focuses on the computational methods used to analyze and simulate inverse problems. The text provides PhD students, researchers, advanced undergraduate students, and engineering practitioners with the perspectives of researchers in areas of inverse problems and data assimilation, ranging from statistics and large-sca

  4. Detection and quantification of chimerism by droplet digital PCR.

    Science.gov (United States)

    George, David; Czech, Juliann; John, Bobby; Yu, Min; Jennings, Lawrence J

    2013-01-01

    Accurate quantification of chimerism and microchimerism is proving to be increasingly valuable for hematopoietic cell transplantation as well as non-transplant conditions. However, methods that are available to quantify low-level chimerism lack accuracy. Therefore, we developed and validated a method for quantifying chimerism based on digital PCR technology. We demonstrate accurate quantification that far exceeds what is possible with analog qPCR down to 0.01% with the potential to go even lower. Also, this method is inherently more informative than qPCR. We expect the advantages of digital PCR will make it the preferred method for chimerism analysis.

  5. Uncertainty Quantification for Safety Verification Applications in Nuclear Power Plants

    Science.gov (United States)

    Boafo, Emmanuel

    There is an increasing interest in computational reactor safety analysis to systematically replace the conservative calculations by best estimate calculations augmented by quantitative uncertainty analysis methods. This has been necessitated by recent regulatory requirements that have permitted the use of such methods in reactor safety analysis. Stochastic uncertainty quantification methods have shown great promise, as they are better suited to capture the complexities in real engineering problems. This study proposes a framework for performing uncertainty quantification based on the stochastic approach, which can be applied to enhance safety analysis. (Abstract shortened by ProQuest.).

  6. Quantification is Neither Necessary Nor Sufficient for Measurement

    International Nuclear Information System (INIS)

    Mari, Luca; Maul, Andrew; Torres Irribarra, David; Wilson, Mark

    2013-01-01

    Being an infrastructural, widespread activity, measurement is laden with stereotypes. Some of these concern the role of measurement in the relation between quality and quantity. In particular, it is sometimes argued or assumed that quantification is necessary for measurement; it is also sometimes argued or assumed that quantification is sufficient for or synonymous with measurement. To assess the validity of these positions the concepts of measurement and quantitative evaluation should be independently defined and their relationship analyzed. We contend that the defining characteristic of measurement should be the structure of the process, not a feature of its results. Under this perspective, quantitative evaluation is neither sufficient nor necessary for measurement

  7. Molecular quantification of genes encoding for green-fluorescent proteins

    DEFF Research Database (Denmark)

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

    2003-01-01

    A quantitative PCR approach is presented to analyze the amount of recombinant green fluorescent protein (gfp) genes in environmental DNA samples. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification...... is provided by a competitively coamplified DNA standard constructed by point mutation PCR. A single base difference was introduced to achieve a suitable migration difference in TGGE between the original target DNA and the modified standard without altering the PCR amplification efficiency. This competitive...... PCR strategy is a highly specific and sensitive way to monitor recombinant DNA in environments like the efflux of a biotechnological plant....

  8. Objective quantification of mode competition in THz BWO optimization

    Science.gov (United States)

    Bao, Rong; Wang, Hongguang; Zhang, Ruxia; Li, Yongdong; Liu, Chunliang

    2017-11-01

    The corrugated rectangular waveguide slow-wave structure (CRWSWS) terahertz (THz) backward wave oscillator (BWO) is suitable for many applications because of its compactness and portability. An analytical model is proposed for calculating the cold and hot parameters of a CRWSWS THz BWO. Two forms of mode competition are quantified using the analytical model. The quantifications are used as objectives when optimizing the CRWSWS THz BWO. The optimization is performed based on the analytical model and a local search algorithm. The optimization results are verified using CST Microwave Studio and Particle Studio. The influence of one of the quantifications on the convergence of the optimization algorithm is also investigated.

  9. A quick colorimetric method for total lipid quantification in microalgae.

    Science.gov (United States)

    Byreddy, Avinesh R; Gupta, Adarsha; Barrow, Colin J; Puri, Munish

    2016-06-01

    Discovering microalgae with high lipid productivity are among the key milestones for achieving sustainable biodiesel production. Current methods of lipid quantification are time intensive and costly. A rapid colorimetric method based on sulfo-phospho-vanillin (SPV) reaction was developed for the quantification of microbial lipids to facilitate screening for lipid producing microalgae. This method was successfully tested on marine thraustochytrid strains and vegetable oils. The colorimetric method results correlated well with gravimetric method estimates. The new method was less time consuming than gravimetric analysis and is quantitative for lipid determination, even in the presence of carbohydrates, proteins and glycerol. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Reliability quantification and visualization for electric microgrids

    Science.gov (United States)

    Panwar, Mayank

    and parallel with the area Electric Power Systems (EPS), (3) includes the local EPS and may include portions of the area EPS, and (4) is intentionally planned. A more reliable electric power grid requires microgrids to operate in tandem with the EPS. The reliability can be quantified through various metrics for performance measure. This is done through North American Electric Reliability Corporation (NERC) metrics in North America. The microgrid differs significantly from the traditional EPS, especially at asset level due to heterogeneity in assets. Thus, the performance cannot be quantified by the same metrics as used for EPS. Some of the NERC metrics are calculated and interpreted in this work to quantify performance for a single asset and group of assets in a microgrid. Two more metrics are introduced for system level performance quantification. The next step is a better representation of the large amount of data generated by the microgrid. Visualization is one such form of representation which is explored in detail and a graphical user interface (GUI) is developed as a deliverable tool to the operator for informative decision making and planning. Electronic appendices-I and II contain data and MATLAB© program codes for analysis and visualization for this work.

  11. Quantification and Negation in Event Semantics

    Directory of Open Access Journals (Sweden)

    Lucas Champollion

    2010-12-01

    Full Text Available Recently, it has been claimed that event semantics does not go well together with quantification, especially if one rejects syntactic, LF-based approaches to quantifier scope. This paper shows that such fears are unfounded, by presenting a simple, variable-free framework which combines a Neo-Davidsonian event semantics with a type-shifting based account of quantifier scope. The main innovation is that the event variable is bound inside the verbal denotation, rather than at sentence level by existential closure. Quantifiers can then be interpreted in situ. The resulting framework combines the strengths of event semantics and type-shifting accounts of quantifiers and thus does not force the semanticist to posit either a default underlying word order or a syntactic LF-style level. It is therefore well suited for applications to languages where word order is free and quantifier scope is determined by surface order. As an additional benefit, the system leads to a straightforward account of negation, which has also been claimed to be problematic for event-based frameworks.ReferencesBarker, Chris. 2002. ‘Continuations and the nature of quantification’. Natural Language Semantics 10: 211–242.http://dx.doi.org/10.1023/A:1022183511876Barker, Chris & Shan, Chung-chieh. 2008. ‘Donkey anaphora is in-scope binding’. Semantics and Pragmatics 1: 1–46.Beaver, David & Condoravdi, Cleo. 2007. ‘On the logic of verbal modification’. In Maria Aloni, Paul Dekker & Floris Roelofsen (eds. ‘Proceedings of the Sixteenth Amsterdam Colloquium’, 3–9. Amsterdam, Netherlands: University of Amsterdam.Beghelli, Filippo & Stowell, Tim. 1997. ‘Distributivity and negation: The syntax of each and every’. In Anna Szabolcsi (ed. ‘Ways of scope taking’, 71–107. Dordrecht, Netherlands: Kluwer.Brasoveanu, Adrian. 2010. ‘Modified Numerals as Post-Suppositions’. In Maria Aloni, Harald Bastiaanse, Tikitu de Jager & Katrin Schulz (eds. ‘Logic, Language

  12. Improved Strategies and Optimization of Calibration Models for Real-time PCR Absolute Quantification

    Science.gov (United States)

    Real-time PCR absolute quantification applications rely on the use of standard curves to make estimates of DNA target concentrations in unknown samples. Traditional absolute quantification approaches dictate that a standard curve must accompany each experimental run. However, t...

  13. Comparison of Quantifiler(®) Trio and InnoQuant™ human DNA quantification kits for detection of DNA degradation in developed and aged fingerprints.

    Science.gov (United States)

    Goecker, Zachary C; Swiontek, Stephen E; Lakhtakia, Akhlesh; Roy, Reena

    2016-06-01

    The development techniques employed to visualize fingerprints collected from crime scenes as well as post-development ageing may result in the degradation of the DNA present in low quantities in such evidence samples. Amplification of the DNA samples with short tandem repeat (STR) amplification kits may result in partial DNA profiles. A comparative study of two commercially available quantification kits, Quantifiler(®) Trio and InnoQuant™, was performed on latent fingerprint samples that were either (i) developed using one of three different techniques and then aged in ambient conditions or (ii) undeveloped and then aged in ambient conditions. The three fingerprint development techniques used were: cyanoacrylate fuming, dusting with black powder, and the columnar-thin-film (CTF) technique. In order to determine the differences between the expected quantities and actual quantities of DNA, manually degraded samples generated by controlled exposure of DNA standards to ultraviolet radiation were also analyzed. A total of 144 fingerprint and 42 manually degraded DNA samples were processed in this study. The results indicate that the InnoQuant™ kit is capable of producing higher degradation ratios compared to the Quantifiler(®) Trio kit. This was an expected result since the degradation ratio is a relative value specific for a kit based on the length and extent of amplification of the two amplicons that vary from one kit to the other. Additionally, samples with lower concentrations of DNA yielded non-linear relationships of degradation ratio with the duration of aging, whereas samples with higher concentrations of DNA yielded quasi-linear relationships. None of the three development techniques produced a noticeably different degradation pattern when compared to undeveloped fingerprints, and therefore do not impede downstream DNA analysis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. Damage Localization and Quantification of Earthquake Excited RC-Frames

    DEFF Research Database (Denmark)

    Skjærbæk, P.S.; Nielsen, Søren R.K.; Kirkegaard, Poul Henning

    In the paper a recently proposed method for damage localization and quantification of RC-structures from response measurements is tested on experimental data. The method investigated requires at least one response measurement along the structure and the ground surface acceleration. Further, the t...

  15. Quantification in dynamic and small-animal positron emission tomography

    NARCIS (Netherlands)

    Disselhorst, Johannes Antonius

    2011-01-01

    This thesis covers two aspects of positron emission tomography (PET) quantification. The first section addresses the characterization and optimization of a small-animal PET/CT scanner. The sensitivity and resolution as well as various parameters affecting image quality (reconstruction settings, type

  16. Investigation on feasibility of recurrence quantification analysis for ...

    African Journals Online (AJOL)

    The RQA parameters such as percent recurrence (REC), trapping time (TT), percent laminarity (LAM) and entropy (ENT), and also the recurrence plots color patterns for different flank wear, can be used in detecting insert wear in face milling. Keywords: milling, flank wear, recurrence plot, recurrence quantification analysis.

  17. Dissolution and Quantification of Tantalum-Containing Compounds ...

    African Journals Online (AJOL)

    NICO

    The 100 % recovery for both the halide salts clearly indicates the complete dissolution and accurate quantification of the tantalum compounds using nitric acid or a methanol/nitric acid mixture. The small standard deviation also points to good precision in these analyses. The recovery results for Ta2O5 and Ta metal powder ...

  18. Current Stereological Methods and Tools for Simple Quantification ...

    African Journals Online (AJOL)

    Current Stereological Methods and Tools for Simple Quantification of Biological Structure: A short Review. ... are estimated using sampling probe has random position and whenever appropriate, random orientation, recent design-based stereology enjoys the advantage of being unbiased that is without systematic error.

  19. Real-Time PCR for Universal Phytoplasma Detection and Quantification

    DEFF Research Database (Denmark)

    Christensen, Nynne Meyn; Nyskjold, Henriette; Nicolaisen, Mogens

    2013-01-01

    Currently, the most efficient detection and precise quantification of phytoplasmas is by real-time PCR. Compared to nested PCR, this method is less sensitive to contamination and is less work intensive. Therefore, a universal real-time PCR method will be valuable in screening programs and in other...

  20. Striga hermonthica seed bank dynamics: process quantification and modelling

    NARCIS (Netherlands)

    Mourik, van T.A.

    2007-01-01

    Key words: Weed control, integrated management, parasitic weed, population, sorghum, millet. This thesis presents a study on the quantification of seed bank dynamics of the parasitic weed Striga hermonthica. The main objectives were to quantify transition rates between different stages of the

  1. Machine Learning for Quantification of Small Vessel Disease Imaging Biomarkers

    NARCIS (Netherlands)

    Ghafoorian, M.

    2018-01-01

    This thesis is devoted to developing fully automated methods for quantification of small vessel disease imaging bio-markers, namely WMHs and lacunes, using vari- ous machine learning/deep learning and computer vision techniques. The rest of the thesis is organized as follows: Chapter 2 describes

  2. Quantification of the sequestration of indium 111 labelled platelets

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-01-01

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

  3. Methane emission quantification from landfills using a double tracer approach

    DEFF Research Database (Denmark)

    Scheutz, Charlotte; Samuelsson, J.; Fredenslund, Anders Michael

    2007-01-01

    A tracer method was successfully used for quantification of the whole methane (CH4) emission from Fakse landfill. By using two different tracers the emission from different sections of the landfill could be quantified. Furthermore, is was possible to determine the emissions from local on site...

  4. The Wiener-Khinchin theorem and recurrence quantification

    Energy Technology Data Exchange (ETDEWEB)

    Zbilut, Joseph P. [Department of Molecular Biophysics and Physiology, Rush University Medical Center, 1653 W. Congress, Chicago, IL 60612 (United States)], E-mail: jzbilut@rush.edu; Marwan, Norbert [Potsdam Institute for Climate Impact Research (PIK), 14412 Potsdam (Germany)

    2008-10-27

    The Wiener-Khinchin theorem states that the power spectrum is the Fourier transform of the autocovariance function. One form of the autocovariance function can be obtained through recurrence quantification. We show that the advantage of defining the autocorrelation function with recurrences can demonstrate higher dimensional dynamics.

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

    DEFF Research Database (Denmark)

    Parikh, Harsh; Spataru, Sergiu; Sera, Dezso

    2018-01-01

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

  6. Data-driven Demand Response Characterization and Quantification

    DEFF Research Database (Denmark)

    Le Ray, Guillaume; Pinson, Pierre; Larsen, Emil Mahler

    2017-01-01

    Analysis of load behavior in demand response (DR) schemes is important to evaluate the performance of participants. Very few real-world experiments have been carried out and quantification and characterization of the response is a difficult task. Nevertheless it will be a necessary tool...

  7. Identification and Quantification Soil Redoximorphic Features by Digital Image Processing

    Science.gov (United States)

    Soil redoximorphic features (SRFs) have provided scientists and land managers with insight into relative soil moisture for approximately 60 years. The overall objective of this study was to develop a new method of SRF identification and quantification from soil cores using a digital camera and imag...

  8. Quantification and Correlation of Oral Candida with Caries Index ...

    African Journals Online (AJOL)

    Quantification and Correlation of Oral Candida with. Caries Index Among Different Age Groups of School. Children: A Case–Control Study. Naidu BV, Reginald BA1. Department of Oral Pathology, Anil Neerukonda Institute of Dental Sciences, Sanghivalasa, Visakhapatnam, 1Department of Oral and Maxillofacial Pathology, ...

  9. Quantification of Rhodium in a Series of Inorganic and ...

    African Journals Online (AJOL)

    An analytical method for the quantification of rhodium using inductively coupled plasma optical emission spectrometry (ICP-OES) and cobalt as internal standard was developed. Rhodium recovery was determined in different samples, which included a certified reference material (CRM), pure rhodium metal, inorganic ...

  10. Uncertainty quantification in nanomechanical measurements using the atomic force microscope

    Science.gov (United States)

    Ryan Wagner; Robert Moon; Jon Pratt; Gordon Shaw; Arvind Raman

    2011-01-01

    Quantifying uncertainty in measured properties of nanomaterials is a prerequisite for the manufacture of reliable nanoengineered materials and products. Yet, rigorous uncertainty quantification (UQ) is rarely applied for material property measurements with the atomic force microscope (AFM), a widely used instrument that can measure properties at nanometer scale...

  11. Quantification of motility of carabid beetles in farmland

    NARCIS (Netherlands)

    Allema, A.B.; Werf, van der W.; Groot, J.C.J.; Hemerik, L.; Gort, G.; Rossing, W.A.H.; Lenteren, van J.C.

    2015-01-01

    Quantification of the movement of insects at field and landscape levels helps us to understand their ecology and ecological functions. We conducted a meta-analysis on movement of carabid beetles (Coleoptera: Carabidae), to identify key factors affecting movement and population redistribution. We

  12. Quantification of water usage at a South African platinum processing ...

    African Journals Online (AJOL)

    Quantification of water usage at a South African platinum processing plant. EL Haggard1, CM Sheridan1 and KG Harding1*. 1Industrial and Mining Water Research Unit (IMWaRU), School of Chemical and Metallurgical Engineering, University of the Witwatersrand,. Johannesburg, Private Bag 3, Wits 2050, South Africa.

  13. Comparison of DNA Quantification Methods for Next Generation Sequencing.

    Science.gov (United States)

    Robin, Jérôme D; Ludlow, Andrew T; LaRanger, Ryan; Wright, Woodring E; Shay, Jerry W

    2016-04-06

    Next Generation Sequencing (NGS) is a powerful tool that depends on loading a precise amount of DNA onto a flowcell. NGS strategies have expanded our ability to investigate genomic phenomena by referencing mutations in cancer and diseases through large-scale genotyping, developing methods to map rare chromatin interactions (4C; 5C and Hi-C) and identifying chromatin features associated with regulatory elements (ChIP-seq, Bis-Seq, ChiA-PET). While many methods are available for DNA library quantification, there is no unambiguous gold standard. Most techniques use PCR to amplify DNA libraries to obtain sufficient quantities for optical density measurement. However, increased PCR cycles can distort the library's heterogeneity and prevent the detection of rare variants. In this analysis, we compared new digital PCR technologies (droplet digital PCR; ddPCR, ddPCR-Tail) with standard methods for the titration of NGS libraries. DdPCR-Tail is comparable to qPCR and fluorometry (QuBit) and allows sensitive quantification by analysis of barcode repartition after sequencing of multiplexed samples. This study provides a direct comparison between quantification methods throughout a complete sequencing experiment and provides the impetus to use ddPCR-based quantification for improvement of NGS quality.

  14. Reproducibility of Two 3-D Ultrasound Carotid Plaque Quantification Methods

    DEFF Research Database (Denmark)

    Graebe, Martin; Entrekin, Robert; Collet-Billon, Antoine

    2014-01-01

    Compared with single 2-D images, emerging 3-D ultrasound technologies hold the promise of reducing variability and increasing sensitivity in the quantification of carotid plaques for individual cardiovascular risk stratification. Inter- and intra-observer agreement between a manual, cross-section...

  15. Recognition and quantification of pain in horses: A tutorial review

    DEFF Research Database (Denmark)

    Gleerup, Karina Charlotte Bech; Lindegaard, Casper

    2016-01-01

    Pain management is dependent on the quality of the pain evaluation. Ideally, pain evaluation is objective, pain-specific and easily incorporated into a busy equine clinic. This paper reviews the existing knowledge base regarding the identification and quantification of pain in horses. Behavioural...

  16. Uncertainty Quantification of Heavy Gas Release Over a Barrier

    NARCIS (Netherlands)

    P. Shoeibi Omrani; T. O'Mahoney; A. Mack; J.A.S. Witteveen (Jeroen)

    2015-01-01

    htmlabstractIn this study a procedure for input uncertainty quantification (UQ) in computational fluid dynamics (CFD) simulations is proposed. The suggested procedure has been applied to a test case. The test case concerns the modeling of a heavy gas release into an atmospheric boundary layer over

  17. Quantification of Spectrum Utilization | Adeloye | Nigeria Journal of ...

    African Journals Online (AJOL)

    ... to substantially reduce the effect of assumptions and approximations of the expression, BAT, (which generally defines spectrum utilization, U) on the quantification of spectrum utilization, U. The derived expression is applied to VHF television transmitters in Nigeria. (Nigeria Journal of Pure and Applied Physics: 2003 2 (1): ...

  18. Detection and quantification of probiotic bacteria using optimized ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-03-08

    Mar 8, 2010 ... The aim of this study is to optimize molecular detection and quantification methods of probiotic bacteria in complex microbial communities that have long been difficult for traditional culture-based methods. Traditional and real-time PCR were optimized to detect and quantify Lactobacillus spp. and.

  19. MM98.57 Quantification of Combined Strain Paths

    DEFF Research Database (Denmark)

    Nielsen, Morten Sturgård; Wanheim, Tarras

    1998-01-01

    Hitherto the quantification of material properties of a material submitted to a deformation has been taken as a function of one or more internal variables. When making measurements of yield properties as a function of a deformation a convenient way of describing the deformation in an experiment i...

  20. Tractable Quantification of Metastability for Robust Bipedal Locomotion

    Science.gov (United States)

    2015-06-01

    Mathematics GPA 4.0/4.0 2007-2011 Sabanci University, Istanbul B.S. in Mechatronics Engineering Minor in Mathematics GPA 3.88/4.0 Major GPA: 3.96/4.00...on Mechatronics (ICM), pages 997-1002, April 2011. viii Abstract Tractable Quantification of Metastability for Robust Bipedal Locomotion by Cenk Oguz

  1. 15 CFR 990.52 - Injury assessment-quantification.

    Science.gov (United States)

    2010-01-01

    ... (Continued) NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE OIL POLLUTION ACT..., trustees must quantify the degree, and spatial and temporal extent of such injuries relative to baseline. (b) Quantification approaches. Trustees may quantify injuries in terms of: (1) The degree, and...

  2. A posteriori uncertainty quantification of PIV-based pressure data

    NARCIS (Netherlands)

    Azijli, I.; Sciacchitano, A.; Ragni, D.; Palha Da Silva Clérigo, A.; Dwight, R.P.

    2016-01-01

    A methodology for a posteriori uncertainty quantification of pressure data retrieved from particle image velocimetry (PIV) is proposed. It relies upon the Bayesian framework, where the posterior distribution (probability distribution of the true velocity, given the PIV measurements) is obtained from

  3. Temperature dependence of postmortem MR quantification for soft tissue discrimination

    International Nuclear Information System (INIS)

    Zech, Wolf-Dieter; Schwendener, Nicole; Jackowski, Christian; Persson, Anders; Warntjes, Marcel J.

    2015-01-01

    To investigate and correct the temperature dependence of postmortem MR quantification used for soft tissue characterization and differentiation in thoraco-abdominal organs. Thirty-five postmortem short axis cardiac 3-T MR examinations were quantified using a quantification sequence. Liver, spleen, left ventricular myocardium, pectoralis muscle and subcutaneous fat were analysed in cardiac short axis images to obtain mean T1, T2 and PD tissue values. The core body temperature was measured using a rectally inserted thermometer. The tissue-specific quantitative values were related to the body core temperature. Equations to correct for temperature differences were generated. In a 3D plot comprising the combined data of T1, T2 and PD, different organs/tissues could be well differentiated from each other. The quantitative values were influenced by the temperature. T1 in particular exhibited strong temperature dependence. The correction of quantitative values to a temperature of 37 C resulted in better tissue discrimination. Postmortem MR quantification is feasible for soft tissue discrimination and characterization of thoraco-abdominal organs. This provides a base for computer-aided diagnosis and detection of tissue lesions. The temperature dependence of the T1 values challenges postmortem MR quantification. Equations to correct for the temperature dependence are provided. (orig.)

  4. A novel quantification method for low-density gel dosimeter.

    Science.gov (United States)

    Nedaie, Hasan Ali; Pak, Farideh; Vaezzadeh, Vahid; Eqlimi, Ehsan; Takavar, Abas; Saligheh Rad, Hamid Reza; Mosleh Shirazi, Mohammad Amin; Mirheydari, Mona

    2018-01-01

    Low signal-to-noise ratio (SNR) images of lung-like (low-density [LD]) gel dosimeters, compared to unit-density (UD) gels, necessitate the use of different quantification methods. In this study, a new method is introduced based on noise correction and exponential (NCEXP) fitting. The feasibility of NCEXP method for quantifying dose absorption in LD gels is evaluated. Sensitivity, dose resolution, detectable dynamic range, and correlation of the calibration curve for both UD and LD gel dosimeters are the parameters, which we analyze to investigate the consequences of new method. Results of NCEXP method are compared to maximum likelihood estimation of rician distribution (MLE-R) and variable echo number (VAREC) quantification methods. Dose response of LD gel dosimeter shows wider detectable dynamic range as compared to UD gel. Using NCEXP method for both LD and UD dosimeter gels, a more sensitive calibration curve with a superior dose resolution is obtained. The advantage of new quantification method is more significant for LD dosimeter gel analysis, where SNR decreases as a result of higher absorbed doses (≥10 Gy). Despite the inverse effect of the VAREC method on detectable dose range of UD gel, no specific changes are observed in dynamic dose range of LD gel dosimeter with different quantification methods. The correlations obtained with different methods were approximately of the same order for UD and LD gels. NCEXP method seems to be more effective than the MLE-R and VAREC methods for quantification of LD dosimeter gel, especially where high-dose absorption and steep-dose gradients exist such as those in intensity-modulated radiation therapy and stereotactic radiosurgery.

  5. Performance of the Real-Q EBV Quantification Kit for Epstein-Barr Virus DNA Quantification in Whole Blood.

    Science.gov (United States)

    Huh, Hee Jae; Park, Jong Eun; Kim, Ji Youn; Yun, Sun Ae; Lee, Myoung Keun; Lee, Nam Yong; Kim, Jong Won; Ki, Chang Seok

    2017-03-01

    There has been increasing interest in standardized and quantitative Epstein-Barr virus (EBV) DNA testing for the management of EBV disease. We evaluated the performance of the Real-Q EBV Quantification Kit (BioSewoom, Korea) in whole blood (WB). Nucleic acid extraction and real-time PCR were performed by using the MagNA Pure 96 (Roche Diagnostics, Germany) and 7500 Fast real-time PCR system (Applied Biosystems, USA), respectively. Assay sensitivity, linearity, and conversion factor were determined by using the World Health Organization international standard diluted in EBV-negative WB. We used 81 WB clinical specimens to compare performance of the Real-Q EBV Quantification Kit and artus EBV RG PCR Kit (Qiagen, Germany). The limit of detection (LOD) and limit of quantification (LOQ) for the Real-Q kit were 453 and 750 IU/mL, respectively. The conversion factor from EBV genomic copies to IU was 0.62. The linear range of the assay was from 750 to 10⁶ IU/mL. Viral load values measured with the Real-Q assay were on average 0.54 log₁₀ copies/mL higher than those measured with the artus assay. The Real-Q assay offered good analytical performance for EBV DNA quantification in WB.

  6. Uncertainty Quantification for Large-Scale Ice Sheet Modeling

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-02-05

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

  7. Analytical Methods for the Quantification of Histamine and Histamine Metabolites.

    Science.gov (United States)

    Bähre, Heike; Kaever, Volkhard

    2017-01-01

    The endogenous metabolite histamine (HA) is synthesized in various mammalian cells but can also be ingested from exogenous sources. It is involved in a plethora of physiological and pathophysiological processes. So far, four different HA receptors (H 1 R-H 4 R) have been described and numerous HAR antagonists have been developed. Contemporary investigations regarding the various roles of HA and its main metabolites have been hampered by the lack of highly specific and sensitive analytic methods for all of these analytes. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is the method of choice for identification and sensitive quantification of many low-molecular weight endogenous metabolites. In this chapter, different methodological aspects of HA quantification as well as recommendations for LC-MS/MS methods suitable for analysis of HA and its main metabolites are summarized.

  8. Metering error quantification under voltage and current waveform distortion

    Science.gov (United States)

    Wang, Tao; Wang, Jia; Xie, Zhi; Zhang, Ran

    2017-09-01

    With integration of more and more renewable energies and distortion loads into power grid, the voltage and current waveform distortion results in metering error in the smart meters. Because of the negative effects on the metering accuracy and fairness, it is an important subject to study energy metering combined error. In this paper, after the comparing between metering theoretical value and real recorded value under different meter modes for linear and nonlinear loads, a quantification method of metering mode error is proposed under waveform distortion. Based on the metering and time-division multiplier principles, a quantification method of metering accuracy error is proposed also. Analyzing the mode error and accuracy error, a comprehensive error analysis method is presented which is suitable for new energy and nonlinear loads. The proposed method has been proved by simulation.

  9. Improved perfusion quantification in FAIR imaging by offset correction

    DEFF Research Database (Denmark)

    Sidaros, Karam; Andersen, Irene Klærke; Gesmar, Henrik

    2001-01-01

    Perfusion quantification using pulsed arterial spin labeling has been shown to be sensitive to the RF pulse slice profiles. Therefore, in Flow-sensitive Alternating-Inversion Recovery (FAIR) imaging the slice selective (ss) inversion slab is usually three to four times thicker than the imaging...... slice. However, this reduces perfusion sensitivity due to the increased transit delay of the incoming blood with unperturbed spins. In the present article, the dependence of the magnetization on the RF pulse slice profiles is inspected both theoretically and experimentally. A perfusion quantification...... model is presented that allows the use of thinner ss inversion slabs by taking into account the offset of RF slice profiles between ss and nonselective inversion slabs. This model was tested in both phantom and human studies. Magn Reson Med 46:193-197, 2001...

  10. Compositional Solution Space Quantification for Probabilistic Software Analysis

    Science.gov (United States)

    Borges, Mateus; Pasareanu, Corina S.; Filieri, Antonio; d'Amorim, Marcelo; Visser, Willem

    2014-01-01

    Probabilistic software analysis aims at quantifying how likely a target event is to occur during program execution. Current approaches rely on symbolic execution to identify the conditions to reach the target event and try to quantify the fraction of the input domain satisfying these conditions. Precise quantification is usually limited to linear constraints, while only approximate solutions can be provided in general through statistical approaches. However, statistical approaches may fail to converge to an acceptable accuracy within a reasonable time. We present a compositional statistical approach for the efficient quantification of solution spaces for arbitrarily complex constraints over bounded floating-point domains. The approach leverages interval constraint propagation to improve the accuracy of the estimation by focusing the sampling on the regions of the input domain containing the sought solutions. Preliminary experiments show significant improvement on previous approaches both in results accuracy and analysis time.

  11. Techniques of biomolecular quantification through AMS detection of radiocarbon

    International Nuclear Information System (INIS)

    Vogel, S.J.; Turteltaub, K.W.; Frantz, C.; Felton, J.S.; Gledhill, B.L.

    1992-01-01

    Accelerator mass spectrometry offers a large gain over scintillation counting in sensitivity for detecting radiocarbon in biomolecular tracing. Application of this sensitivity requires new considerations of procedures to extract or isolate the carbon fraction to be quantified, to inventory all carbon in the sample, to prepare graphite from the sample for use in the spectrometer, and to derive a meaningful quantification from the measured isotope ratio. These procedures need to be accomplished without contaminating the sample with radiocarbon, which may be ubiquitous in laboratories and on equipment previously used for higher dose, scintillation experiments. Disposable equipment, materials and surfaces are used to control these contaminations. Quantification of attomole amounts of labeled substances are possible through these techniques

  12. The necessity of operational risk management and quantification

    Directory of Open Access Journals (Sweden)

    Barbu Teodora Cristina

    2008-04-01

    Full Text Available Beginning with the fact that performant strategies of the financial institutions have programmes and management procedures for the banking risks, which have as main objective to minimize the probability of risk generation and the bank’s potential exposure, this paper wants to present the operational risk management and quantification methods. Also it presents the modality of minimum capital requirement for the operational risk. Therefore, the first part presents the conceptual approach of the operational risks through the point of view of the financial institutions exposed to this type of risk. The second part describes the management and evaluation methods for the operational risk. The final part of this article presents the approach assumed by a financial institution with a precise purpose: the quantification of the minimum capital requirements of the operational risk.

  13. A Point-Wise Quantification of Asymmetry Using Deformation Fields

    DEFF Research Database (Denmark)

    Ólafsdóttir, Hildur; Lanche, Stephanie; Darvann, Tron Andre

    2007-01-01

    of the resulting displacement vectors on the left and right side of the symmetry plane, gives a point-wise measure of asymmetry. The asymmetry measure was applied to the study of Crouzon syndrome using Micro CT scans of genetically modified mice. Crouzon syndrome is characterised by the premature fusion of cranial...... sutures, which gives rise to a highly asymmetric growth. Quantification and localisation of this asymmetry is of high value with respect to surgery planning and treatment evaluation. Using the proposed method, asymmetry was calculated in each point of the surface of Crouzon mice and wild-type mice...... (controls). Asymmetry appeared in similar regions for the two groups but the Crouzon mice were found significantly more asymmetric. The localisation ability of the method was in good agreement with ratings from a clinical expert. Validating the quantification ability is a less trivial task due to the lack...

  14. Quantification of Si in silicone oils by ICP-OES

    DEFF Research Database (Denmark)

    Wang, Qian; Yang, Zhenyu

    2014-01-01

    A new simple and low cost method of quantification of trace amounts of silicon (Si) in silicone oils has been developed by combining the silicone emulsion and inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis. Silicone oils that contained phenyl groups in the viscosity...... range from 20 to 1000 mPa.s formed stable oil in water emulsions in the presence of Tween 80 surfactant and methylisobutylketone (MIBK). The Si in the emulsions was further quantified by ICP-OES. The calibration was performed using spiked inorganic silicon standard in the emulsions and the method...... (LOD) in the emulsion as 0.5 ppm Si. Compared to the Si determination by the direct organic solvent ICP-OES, this method is much more convenient, where a regular ICP-OES instrument can be directly used for the quantification of Si in the silicone oils obtained via extraction by organic solvents from...

  15. Quantification of the effects of dependence on human error probabilities

    International Nuclear Information System (INIS)

    Bell, B.J.; Swain, A.D.

    1980-01-01

    In estimating the probabilities of human error in the performance of a series of tasks in a nuclear power plant, the situation-specific characteristics of the series must be considered. A critical factor not to be overlooked in this estimation is the dependence or independence that pertains to any of the several pairs of task performances. In discussing the quantification of the effects of dependence, the event tree symbology described will be used. In any series of tasks, the only dependence considered for quantification in this document will be that existing between the task of interest and the immediately preceeding task. Tasks performed earlier in the series may have some effect on the end task, but this effect is considered negligible

  16. Quantification of uranyl in presence of citric acid

    International Nuclear Information System (INIS)

    Garcia G, N.; Barrera D, C.E.; Ordonez R, E.

    2007-01-01

    To determine the influence that has the organic matter of the soil on the uranyl sorption on some solids is necessary to have a detection technique and quantification of uranyl that it is reliable and sufficiently quick in the obtaining of results. For that in this work, it intends to carry out the uranyl quantification in presence of citric acid modifying the Fluorescence induced by UV-Vis radiation technique. Since the uranyl ion is very sensitive to the medium that contains it, (speciation, pH, ionic forces, etc.) it was necessary to develop an analysis technique that stands out the fluorescence of uranyl ion avoiding the out one that produce the organic acids. (Author)

  17. Collaborative framework for PIV uncertainty quantification: comparative assessment of methods

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  18. Surface landmark quantification of embryonic mouse craniofacial morphogenesis

    OpenAIRE

    Percival, Christopher J; Green, Rebecca; Marcucio, Ralph; Hallgrímsson, Benedikt

    2014-01-01

    Background Morphometric quantification of subtle craniofacial variation in studies of experimentally modified embryonic mice has proved valuable in determining the effects of developmental perturbations on craniofacial morphogenesis. The direct comparison of landmark coordinate data from embryos of many different mouse strains and mouse models can advance our understanding of the bases for craniofacial variation. We propose a standard set of craniofacial surface landmarks, for use with embryo...

  19. Synthesis and Review: Advancing agricultural greenhouse gas quantification

    OpenAIRE

    Olander, L.P.; Wollenberg, E.; Tubiello, F.N.; Herold, M.

    2014-01-01

    Reducing emissions of agricultural greenhouse gases (GHGs), such as methane and nitrous oxide, and sequestering carbon in the soil or in living biomass can help reduce the impact of agriculture on climate change while improving productivity and reducing resource use. There is an increasing demand for improved, low cost quantification of GHGs in agriculture, whether for national reporting to the United Nations Framework Convention on Climate Change (UNFCCC), underpinning and stimulating improv...

  20. Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA

    OpenAIRE

    Szwagierczak, Aleksandra; Bultmann, Sebastian; Schmidt, Christine S.; Spada, Fabio; Leonhardt, Heinrich

    2010-01-01

    The recent discovery of genomic 5-hydroxymethylcytosine (hmC) and mutations affecting the respective Tet hydroxylases in leukemia raises fundamental questions about this epigenetic modification. We present a sensitive method for fast quantification of genomic hmC based on specific transfer of radiolabeled glucose to hmC by a purified glucosyltransferase. We determined hmC levels in various adult tissues and differentiating embryonic stem cells and show a correlation with differential expressi...

  1. Digital PCR for direct quantification of viruses without DNA extraction

    OpenAIRE

    Pav?i?, Jernej; ?el, Jana; Milavec, Mojca

    2015-01-01

    DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration mat...

  2. Detection and quantification of Leveillula taurica growth in pepper leaves.

    Science.gov (United States)

    Zheng, Zheng; Nonomura, Teruo; Bóka, Károly; Matsuda, Yoshinori; Visser, Richard G F; Toyoda, Hideyoshi; Kiss, Levente; Bai, Yuling

    2013-06-01

    Leveillula taurica is an obligate fungal pathogen that causes powdery mildew disease on a broad range of plants, including important crops such as pepper, tomato, eggplant, onion, cotton, and so on. The early stage of this disease is difficult to diagnose and the disease can easily spread unobserved; for example, in pepper and tomato production fields and greenhouses. The objective of this study was to develop a detection and quantification method of L. taurica biomass in pepper leaves with special regard to the early stages of infection. We monitored the development of the disease to time the infection process on the leaf surface as well as inside the pepper leaves. The initial and final steps of the infection taking place on the leaf surface were consecutively observed using a dissecting microscope and a scanning electron microscope. The development of the intercellular mycelium in the mesophyll was followed by light and transmission electron microscopy. A pair of L. taurica-specific primers was designed based on the internal transcribed spacer sequence of L. taurica and used in real-time polymerase chain reaction (PCR) assay to quantify the fungal DNA during infection. The specificity of this assay was confirmed by testing the primer pair with DNA from host plants and also from another powdery mildew species, Oidium neolycopersici, infecting tomato. A standard curve was obtained for absolute quantification of L. taurica biomass. In addition, we tested a relative quantification method by using a plant gene as reference and the obtained results were compared with the visual disease index scoring. The real-time PCR assay for L. taurica provides a valuable tool for detection and quantification of this pathogen in breeding activities as well in plant-microbe interaction studies.

  3. Leishmania parasite detection and quantification using PCR-ELISA

    Czech Academy of Sciences Publication Activity Database

    Kobets, Tetyana; Badalová, Jana; Grekov, Igor; Havelková, Helena; Lipoldová, Marie

    2010-01-01

    Roč. 5, č. 6 (2010), s. 1074-1080 ISSN 1754-2189 R&D Projects: GA ČR GA310/08/1697; GA MŠk(CZ) LC06009 Institutional research plan: CEZ:AV0Z50520514 Keywords : polymerase chain reaction * Leishmania major infection * parasite quantification Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.362, year: 2010

  4. A fast and robust hepatocyte quantification algorithm including vein processing

    Directory of Open Access Journals (Sweden)

    Homeyer André

    2010-03-01

    Full Text Available Abstract Background Quantification of different types of cells is often needed for analysis of histological images. In our project, we compute the relative number of proliferating hepatocytes for the evaluation of the regeneration process after partial hepatectomy in normal rat livers. Results Our presented automatic approach for hepatocyte (HC quantification is suitable for the analysis of an entire digitized histological section given in form of a series of images. It is the main part of an automatic hepatocyte quantification tool that allows for the computation of the ratio between the number of proliferating HC-nuclei and the total number of all HC-nuclei for a series of images in one processing run. The processing pipeline allows us to obtain desired and valuable results for a wide range of images with different properties without additional parameter adjustment. Comparing the obtained segmentation results with a manually retrieved segmentation mask which is considered to be the ground truth, we achieve results with sensitivity above 90% and false positive fraction below 15%. Conclusions The proposed automatic procedure gives results with high sensitivity and low false positive fraction and can be applied to process entire stained sections.

  5. (1) H-MRS processing parameters affect metabolite quantification

    DEFF Research Database (Denmark)

    Bhogal, Alex A; Schür, Remmelt R; Houtepen, Lotte C

    2017-01-01

    Proton magnetic resonance spectroscopy ((1) H-MRS) can be used to quantify in vivo metabolite levels, such as lactate, γ-aminobutyric acid (GABA) and glutamate (Glu). However, there are considerable analysis choices which can alter the accuracy or precision of (1) H-MRS metabolite quantification....... reinforce the notion that standard practices should be established to regularize outcomes of (1) H-MRS studies, and that basis sets used for processing should be made available to the scientific community....... individualized and optimal parameter settings using LCModel software. Data were processed a second time in one group using an independent software package (NMRWizard) for an additional comparison with a different post-processing platform. Correlations across research groups of the ratio between the highest and...... + NAAG/Cr + PCr and Glu/Cr + PCr, respectively. Metabolite quantification using identical (1) H-MRS data was influenced by processing parameters, basis sets and software choice. Locally preferred processing choices affected metabolite quantification, even when using identical software. Our results...

  6. The Use of Auxin Quantification for Understanding Clonal Tree Propagation

    Directory of Open Access Journals (Sweden)

    Carlos A. Stuepp

    2017-01-01

    Full Text Available Qualitative and quantitative hormone analyses have been essential for understanding the metabolic, physiological, and morphological processes that are influenced by plant hormones. Auxins are key hormones in the control of many aspects of plant growth and development and their endogenous levels are considered critical in the process of adventitious root induction. Exogenous auxins are used extensively in the clonal propagation of tree species by cuttings or tissue culture. Understanding of auxin effects has advanced with the development of increasingly accurate methods for auxin quantification. However, auxin analysis has been challenging because auxins typically occur at low concentrations, while compounds that interfere with their detection often occur at high concentrations, in plant tissues. Interference from other compounds has been addressed by extensive purification of plant extracts prior to auxin analysis, although this means that quantification methods have been limited by their expense. This review explores the extraction, purification, and quantification of auxins and the application of these techniques in developing improved methods for the clonal propagation of forestry trees.

  7. Quantification of propionic acid fromScutellaria baicalensisroots.

    Science.gov (United States)

    Son, Eunjung; Kim, Ho Kyoung; Kim, Hyun Sik; Kim, Mee Ree; Kim, Dong-Seon

    2017-03-01

    Propionic acid is a widely used preservative and has been mainly formed by artificial synthesis or fermentation. In the case of natural products, the presence of propionic acid is viewed as a sign that an additive has been introduced for antimicrobial effects. In this work, the propionic acid that occurs in Scutellaria baicalensis roots was studied. A quantification method was developed, validated, and showed good linearity, low limit of detection, and limit of quantification values, as well as fine precision and recovery rate. The developed method was applied to the analysis of S. baicalensis roots collected in South Korea and China. The detection rate for all samples was 94.0%. The average concentration was 0.41 ± 0.24 mg/g from the China sample and 0.76 ± 0.28 mg/g from the Korea sample. This study is the first to report that propionic acid exists in S. baicalensis roots and also provides a useful ultra performance liquid chromatography analysis method for its quantification.

  8. Initial water quantification results using neutron computed tomography

    Energy Technology Data Exchange (ETDEWEB)

    Heller, A.K. [Department of Mechanical and Nuclear Engineering, Pennsylvania State University (United States)], E-mail: axh174@psu.edu; Shi, L.; Brenizer, J.S.; Mench, M.M. [Department of Mechanical and Nuclear Engineering, Pennsylvania State University (United States)

    2009-06-21

    Neutron computed tomography is an important imaging tool in the field of non-destructive testing and in fundamental research for many engineering applications. Contrary to X-rays, neutrons can be attenuated by some light materials, such as hydrogen, but can penetrate many heavy materials. Thus, neutron computed tomography is useful in obtaining important three-dimensional information about a sample's interior structure and material properties that other traditional methods cannot provide. The neutron computed tomography system at Pennsylvania State University's Radiation Science and Engineering Center is being utilized to develop a water quantification technique for investigation of water distribution in fuel cells under normal conditions. A hollow aluminum cylinder test sample filled with a known volume of water was constructed for purposes of testing the quantification technique. Transmission images of the test sample at different angles were easily acquired through the synthesis of a dedicated image acquisition computer driving a rotary table controller and an in-house developed synchronization software package. After data acquisition, Octopus (version 8.2) and VGStudio Max (version 1.2) were used to perform cross-sectional and three-dimensional reconstructions of the sample, respectively. The initial reconstructions and water quantification results are presented.

  9. [DNA quantification of blood samples pre-treated with pyramidon].

    Science.gov (United States)

    Zhu, Chuan-Hong; Zheng, Dao-Li; Ni, Rao-Zhi; Wang, Hai-Sheng; Ning, Ping; Fang, Hui; Liu, Yan

    2014-06-01

    To study DNA quantification and STR typing of samples pre-treated with pyramidon. The blood samples of ten unrelated individuals were anticoagulated in EDTA. The blood stains were made on the filter paper. The experimental groups were divided into six groups in accordance with the storage time, 30 min, 1 h, 3 h, 6 h, 12 h and 24h after pre-treated with pyramidon. DNA was extracted by three methods: magnetic bead-based extraction, QIAcube DNA purification method and Chelex-100 method. The quantification of DNA was made by fluorescent quantitative PCR. STR typing was detected by PCR-STR fluorescent technology. In the same DNA extraction method, the sample DNA decreased gradually with times after pre-treatment with pyramidon. In the same storage time, the DNA quantification in different extraction methods had significant differences. Sixteen loci DNA typing were detected in 90.56% of samples. Pyramidon pre-treatment could cause DNA degradation, but effective STR typing can be achieved within 24 h. The magnetic bead-based extraction is the best method for STR profiling and DNA extraction.

  10. Nuclear and mitochondrial DNA quantification of various forensic materials.

    Science.gov (United States)

    Andréasson, H; Nilsson, M; Budowle, B; Lundberg, H; Allen, M

    2006-12-01

    Due to the different types and quality of forensic evidence materials, their DNA content can vary substantially, and particularly low quantities can impact the results in an identification analysis. In this study, the quantity of mitochondrial and nuclear DNA was determined in a variety of materials using a previously described real-time PCR method. DNA quantification in the roots and distal sections of plucked and shed head hairs revealed large variations in DNA content particularly between the root and the shaft of plucked hairs. Also large intra- and inter-individual variations were found among hairs. In addition, DNA content was estimated in samples collected from fingerprints and accessories. The quantification of DNA on various items also displayed large variations, with some materials containing large amounts of nuclear DNA while no detectable nuclear DNA and only limited amounts of mitochondrial DNA were seen in others. Using this sensitive real-time PCR quantification assay, a better understanding was obtained regarding DNA content and variation in commonly analysed forensic evidence materials and this may guide the forensic scientist as to the best molecular biology approach for analysing various forensic evidence materials.

  11. Automatic quantification in lung scintigraphy: functional atlas; Quantification automatique en scintigraphie pulmonaire: elaboration d'atlas fonctionnels

    Energy Technology Data Exchange (ETDEWEB)

    Ledee, R.; Therain, F. [Orleans Univ., Lab. d' Electronique - Signaux - Images (LESI), 45 (France); Debrun, D. [Centre Hospitalier Regional de la Source (CHRO), Medecine Nucleaire, 45 - Orleans (France)

    2003-06-01

    In spite of the development of new techniques, the ventilation perfusion lung scintigraphy keeps a good place for the diagnosis of pulmonary embolism (PE). In the context of an improvement of reliability and reproducibility of the diagnosis, this study proposes to realize an automatic quantification of the distribution of the radioactive tracers by pulmonary segments. Measurements are made following a procedure of non-rigid matching of morphological 2-D charts of the lungs on the scintigraphic images. The adaptation of these charts to the patients' morphology is carried out by exploiting iso-contour information of the images and using Fourier descriptors to determine the parameters of the transformation. The study was performed on a population of 30 patients with a probability of nil of the pulmonary embolism. After a study of the robustness of the quantification, 2-D segmental functional reference charts (according to the conditions of acquisition) were proposed. In the perfusion case and four views, the following lobar distribution, in relative value, is measured: Right Inferior Lobar = 23,39%, Medial Lobar = 10,41 %, Right Superior Lobar = 20,37%, Left Inferior Lobar 20,6% and Left Superior Lobar = 25.6% with culmen = 18,8% and lingula = 6,8%; values comparable with those of the publication. The process of quantification is adaptable to the ventilation lung scans. The segmental quantifications of a patient carried out under the same conditions of acquisition as the functional reference charts, could be compared with the reference data and provide indicators for the diagnosis but also for patient follow-up and preoperative evaluation of lung cancers. (authors)

  12. Quantification of pharmaceutical peptides in human plasma by LC-ICP-MS sulfur detection

    DEFF Research Database (Denmark)

    Møller, Laura Hyrup; Macherius, André; Hansen, Thomas Hesselhøj

    2016-01-01

    -DRC-MS instrument and subsequent quantification by post-column isotope dilution (IDA). Plasma proteins were precipitated prior to analysis. Analytical figures of merit including linearity, precision, LOD, LOQ and accuracy were considered satisfactory for analysis of plasma samples. The selectivity of the developed...... demonstrated that LC-ICP-MS post-column IDA may constitute a valuable additional tool in quantification of non-labelled peptides in the early drug development offering absolute quantification without need of species specific standards....

  13. Efficient Quantification of Uncertainties in Complex Computer Code Results, Phase II

    Data.gov (United States)

    National Aeronautics and Space Administration — Propagation of parameter uncertainties through large computer models can be very resource intensive. Frameworks and tools for uncertainty quantification are...

  14. Efficient Quantification of Uncertainties in Complex Computer Code Results, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — This proposal addresses methods for efficient quantification of margins and uncertainties (QMU) for models that couple multiple, large-scale commercial or...

  15. Aerodynamic Modeling with Heterogeneous Data Assimilation and Uncertainty Quantification, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — Clear Science Corp. proposes to develop an aerodynamic modeling tool that assimilates data from different sources and facilitates uncertainty quantification. The...

  16. Effects of humic acid on DNA quantification with Quantifiler® Human DNA Quantification kit and short tandem repeat amplification efficiency.

    Science.gov (United States)

    Seo, Seung Bum; Lee, Hye Young; Zhang, Ai Hua; Kim, Hye Yeon; Shin, Dong Hoon; Lee, Soong Deok

    2012-11-01

    Correct DNA quantification is an essential part to obtain reliable STR typing results. Forensic DNA analysts often use commercial kits for DNA quantification; among them, real-time-based DNA quantification kits are most frequently used. Incorrect DNA quantification due to the presence of PCR inhibitors may affect experiment results. In this study, we examined the alteration degree of DNA quantification results estimated in DNA samples containing a PCR inhibitor by using a Quantifiler® Human DNA Quantification kit. For experiments, we prepared approximately 0.25 ng/μl DNA samples containing various concentrations of humic acid (HA). The quantification results were 0.194-0.303 ng/μl at 0-1.6 ng/μl HA (final concentration in the Quantifiler reaction) and 0.003-0.168 ng/μl at 2.4-4.0 ng/μl HA. Most DNA quantity was undetermined when HA concentration was higher than 4.8 ng/μl HA. The C (T) values of an internal PCR control (IPC) were 28.0-31.0, 36.5-37.1, and undetermined at 0-1.6, 2.4, and 3.2 ng/μl HA. These results indicate that underestimated DNA quantification results may be obtained in the DNA sample with high C (T) values of IPC. Thus, researchers should carefully interpret the DNA quantification results. We additionally examined the effects of HA on the STR amplification by using an Identifiler® kit and a MiniFiler™ kit. Based on the results of this study, it is thought that a better understanding of various effects of HA would help researchers recognize and manipulate samples containing HA.

  17. Two-stream Convolutional Neural Network for Methane Emissions Quantification

    Science.gov (United States)

    Wang, J.; Ravikumar, A. P.; McGuire, M.; Bell, C.; Tchapmi, L. P.; Brandt, A. R.

    2017-12-01

    Methane, a key component of natural gas, has a 25x higher global warming potential than carbon dioxide on a 100-year basis. Accurately monitoring and mitigating methane emissions require cost-effective detection and quantification technologies. Optical gas imaging, one of the most commonly used leak detection technology, adopted by Environmental Protection Agency, cannot estimate leak-sizes. In this work, we harness advances in computer science to allow for rapid and automatic leak quantification. Particularly, we utilize two-stream deep Convolutional Networks (ConvNets) to estimate leak-size by capturing complementary spatial information from still plume frames, and temporal information from plume motion between frames. We build large leak datasets for training and evaluating purposes by collecting about 20 videos (i.e. 397,400 frames) of leaks. The videos were recorded at six distances from the source, covering 10 -60 ft. Leak sources included natural gas well-heads, separators, and tanks. All frames were labeled with a true leak size, which has eight levels ranging from 0 to 140 MCFH. Preliminary analysis shows that two-stream ConvNets provides significant accuracy advantage over single steam ConvNets. Spatial stream ConvNet can achieve an accuracy of 65.2%, by extracting important features, including texture, plume area, and pattern. Temporal stream, fed by the results of optical flow analysis, results in an accuracy of 58.3%. The integration of the two-stream ConvNets gives a combined accuracy of 77.6%. For future work, we will split the training and testing datasets in distinct ways in order to test the generalization of the algorithm for different leak sources. Several analytic metrics, including confusion matrix and visualization of key features, will be used to understand accuracy rates and occurrences of false positives. The quantification algorithm can help to find and fix super-emitters, and improve the cost-effectiveness of leak detection and repair

  18. Quantification of breast arterial calcification using full field digital mammography

    International Nuclear Information System (INIS)

    Molloi, Sabee; Xu Tong; Ducote, Justin; Iribarren, Carlos

    2008-01-01

    Breast arterial calcification is commonly detected on some mammograms. Previous studies indicate that breast arterial calcification is evidence of general atherosclerotic vascular disease and it may be a useful marker of coronary artery disease. It can potentially be a useful tool for assessment of coronary artery disease in women since mammography is widely used as a screening tool for early detection of breast cancer. However, there are currently no available techniques for quantification of calcium mass using mammography. The purpose of this study was to determine whether it is possible to quantify breast arterial calcium mass using standard digital mammography. An anthropomorphic breast phantom along with a vessel calcification phantom was imaged using a full field digital mammography system. Densitometry was used to quantify calcium mass. A calcium calibration measurement was performed at each phantom thickness and beam energy. The known (K) and measured (M) calcium mass on 5 and 9 cm thickness phantoms were related by M=0.964K-0.288 mg (r=0.997 and SEE=0.878 mg) and M=1.004K+0.324 mg (r=0.994 and SEE=1.32 mg), respectively. The results indicate that accurate calcium mass measurements can be made without correction for scatter glare as long as careful calcium calibration is made for each breast thickness. The results also indicate that composition variations and differences of approximately 1 cm between calibration phantom and breast thickness introduce only minimal error in calcium measurement. The uncertainty in magnification is expected to cause up to 5% and 15% error in calcium mass for 5 and 9 cm breast thicknesses, respectively. In conclusion, a densitometry technique for quantification of breast arterial calcium mass was validated using standard full field digital mammography. The results demonstrated the feasibility and potential utility of the densitometry technique for accurate quantification of breast arterial calcium mass using standard digital

  19. Final Report: Quantification of Uncertainty in Extreme Scale Computations (QUEST)

    Energy Technology Data Exchange (ETDEWEB)

    Marzouk, Youssef [Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Conrad, Patrick [Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Bigoni, Daniele [Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Parno, Matthew [Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)

    2017-06-09

    QUEST (\\url{www.quest-scidac.org}) is a SciDAC Institute that is focused on uncertainty quantification (UQ) in large-scale scientific computations. Our goals are to (1) advance the state of the art in UQ mathematics, algorithms, and software; and (2) provide modeling, algorithmic, and general UQ expertise, together with software tools, to other SciDAC projects, thereby enabling and guiding a broad range of UQ activities in their respective contexts. QUEST is a collaboration among six institutions (Sandia National Laboratories, Los Alamos National Laboratory, the University of Southern California, Massachusetts Institute of Technology, the University of Texas at Austin, and Duke University) with a history of joint UQ research. Our vision encompasses all aspects of UQ in leadership-class computing. This includes the well-founded setup of UQ problems; characterization of the input space given available data/information; local and global sensitivity analysis; adaptive dimensionality and order reduction; forward and inverse propagation of uncertainty; handling of application code failures, missing data, and hardware/software fault tolerance; and model inadequacy, comparison, validation, selection, and averaging. The nature of the UQ problem requires the seamless combination of data, models, and information across this landscape in a manner that provides a self-consistent quantification of requisite uncertainties in predictions from computational models. Accordingly, our UQ methods and tools span an interdisciplinary space across applied math, information theory, and statistics. The MIT QUEST effort centers on statistical inference and methods for surrogate or reduced-order modeling. MIT personnel have been responsible for the development of adaptive sampling methods, methods for approximating computationally intensive models, and software for both forward uncertainty propagation and statistical inverse problems. A key software product of the MIT QUEST effort is the MIT

  20. QUANTIFICATION OF GENETICALLY MODIFIED MAIZE MON 810 IN PROCESSED FOODS

    Directory of Open Access Journals (Sweden)

    Peter Siekel

    2012-12-01

    Full Text Available 800x600 Normal 0 21 false false false SK X-NONE X-NONE MicrosoftInternetExplorer4 Maize MON 810 (Zea mays L. represents the majority of genetically modified food crops. It is the only transgenic cultivar grown in the EU (European Union countries and food products with its content higher than 0.9 % must be labelled. This study was aimed at impact of food processing (temperature, pH and pressure on DNA degradation and quantification of the genetically modified maize MON 810. The transgenic DNA was quantified by the real-time polymerase chain reaction method. Processing as is high temperature (121 °C, elevated pressure (0.1 MPa and low pH 2.25 fragmented DNA. A consequence of two order difference in the species specific gene content compared to the transgenic DNA content in plant materials used has led to false negative results in the quantification of transgenic DNA. The maize containing 4.2 % of the transgene after processing appeared to be as low as 3.0 % (100 °C and 1.9 % (121 °C, 0.1 MPa. The 2.1 % amount of transgene dropped at 100 °C to 1.0 % and at 121 °C, 0.1 MPa to 0.6 %. Under such make up the DNA degradation of transgenic content showed up 2 or 3 time higher decrease a consequence of unequal gene presence. Such genes disparity is expressed as considerable decrease of transgenic content while the decrease of species specific gene content remains unnoticed. Based on our findings we conclude that high degree of processing might have led to false negative results of the transgenic constituent quantification. Determination of GMO content in processed foods may leads to incorrect statement and labelling in these cases could misleads consumers.doi:10.5219/212

  1. Targeted Proteomic Quantification on Quadrupole-Orbitrap Mass Spectrometer*

    Science.gov (United States)

    Gallien, Sebastien; Duriez, Elodie; Crone, Catharina; Kellmann, Markus; Moehring, Thomas; Domon, Bruno

    2012-01-01

    There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein

  2. Improved Method for PD-Quantification in Power Cables

    DEFF Research Database (Denmark)

    Holbøll, Joachim T.; Villefrance, Rasmus; Henriksen, Mogens

    1999-01-01

    n this paper, a method is described for improved quantification of partial discharges(PD) in power cables. The method is suitable for PD-detection and location systems in the MHz-range, where pulse attenuation and distortion along the cable cannot be neglected. The system transfer function...... was calculated and measured in order to form basis for magnitude calculation after each measurements. --- Limitations and capabilities of the method will be discussed and related to relevant field applications of high frequent PD-measurements. --- Methods for increased signal/noise ratio are easily implemented...

  3. Quantification in histopathology-Can magnetic particles help?

    International Nuclear Information System (INIS)

    Mitchels, John; Hawkins, Peter; Luxton, Richard; Rhodes, Anthony

    2007-01-01

    Every year, more than 270,000 people are diagnosed with cancer in the UK alone; this means that one in three people worldwide contract cancer within their lifetime. Histopathology is the principle method for confirming cancer and directing treatment. In this paper, a novel application of magnetic particles is proposed to help address the problem of subjectivity in histopathology. Preliminary results indicate that magnetic nanoparticles cannot only be used to assist diagnosis through improving quantification but also potentially increase throughput, hence offering a way of dramatically reducing costs within the routine histopathology laboratory

  4. Rapid quantification of cardiolipin and DOPC lipid and vesicle concentration.

    Science.gov (United States)

    Elmer-Dixon, Margaret M; Bowler, Bruce E

    2017-03-01

    A novel approach to quantification of cardiolipin and DOPC lipid and vesicle concentration that is rapid and inexpensive is described. Traditional approaches to quantifying vesicle concentration destroy sample and are often time consuming. Using common laboratory equipment and software, lipid vesicles were reliably quantified allowing for immediate use without significant sample loss. Once calibrated, only absorbance measurements with a UV-Vis spectrophotometer are necessary as input into a Matlab program, which calculates the corresponding vesicle and lipid concentration. Fast and accurate concentration determination for preparations of vesicles is essential for analytical titration experiments necessary for protein/vesicle binding curves. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Qu’enseigner des sciences sociales en quantification ?

    OpenAIRE

    Tralongo, Stephanie

    2015-01-01

    International audience; Journée d'études « Enseigner le quanti. Les sciences sociales face aux nombres », Paris, 5 juin 2015 Titre : « Qu'enseigner des sciences sociales en quantification ? » Résumé : La communication porte sur une licence professionnelle intitulée Chargé d'études statistiques. Elle vise à présenter la demande faite à la sociologie, de confronter les étudiants « à une problématique sociologique pour les aider à construire une culture sociologique, à acquérir un regard objecti...

  6. Multi data reservior history matching and uncertainty quantification framework

    KAUST Repository

    Katterbauer, Klemens

    2015-11-26

    A multi-data reservoir history matching and uncertainty quantification framework is provided. The framework can utilize multiple data sets such as production, seismic, electromagnetic, gravimetric and surface deformation data for improving the history matching process. The framework can consist of a geological model that is interfaced with a reservoir simulator. The reservoir simulator can interface with seismic, electromagnetic, gravimetric and surface deformation modules to predict the corresponding observations. The observations can then be incorporated into a recursive filter that subsequently updates the model state and parameters distributions, providing a general framework to quantify and eventually reduce with the data, uncertainty in the estimated reservoir state and parameters.

  7. NEW MODEL FOR QUANTIFICATION OF ICT DEPENDABLE ORGANIZATIONS RESILIENCE

    Directory of Open Access Journals (Sweden)

    Zora Arsovski

    2011-03-01

    Full Text Available Business environment today demands high reliable organizations in every segment to be competitive on the global market. Beside that, ICT sector is becoming irreplaceable in many fields of business, from the communication to the complex systems for process control and production. To fulfill those requirements and to develop further, many organizations worldwide are implementing business paradigm called - organizations resilience. Although resilience is well known term in many science fields, it is not well studied due to its complex nature. This paper is dealing with developing the new model for assessment and quantification of ICT dependable organizations resilience.

  8. Review of some aspects of human reliability quantification

    International Nuclear Information System (INIS)

    Lydell, B.O.Y.; Spurgin, A.J.; Hannaman, G.W.; Lukic, Y.D.

    1986-01-01

    An area in systems reliability considered to be weak, is the characterization and quantification of the role of the operations and maintenance staff in combatting accidents. Several R and D programs are underway to improve the modeling of human interactions and some progress has been made. This paper describes a specific aspect of human reliability analysis which is referred to as modeling of cognitive processes. In particular, the basis for the so- called Human Cognitive Reliability (HCR) model is described and the focus is on its validation and on its benefits and limitations

  9. Preliminary Results on Uncertainty Quantification for Pattern Analytics

    Energy Technology Data Exchange (ETDEWEB)

    Stracuzzi, David John [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States); Brost, Randolph [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States); Chen, Maximillian Gene [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States); Malinas, Rebecca [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States); Peterson, Matthew Gregor [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States); Phillips, Cynthia A. [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States); Robinson, David G. [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States); Woodbridge, Diane [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States)

    2015-09-01

    This report summarizes preliminary research into uncertainty quantification for pattern ana- lytics within the context of the Pattern Analytics to Support High-Performance Exploitation and Reasoning (PANTHER) project. The primary focus of PANTHER was to make large quantities of remote sensing data searchable by analysts. The work described in this re- port adds nuance to both the initial data preparation steps and the search process. Search queries are transformed from does the specified pattern exist in the data? to how certain is the system that the returned results match the query? We show example results for both data processing and search, and discuss a number of possible improvements for each.

  10. Hyperspectral chemical plume quantification via background radiance estimation

    Science.gov (United States)

    Niu, Sidi; Golowich, Steven E.; Ingle, Vinay K.; Manolakis, Dimitris G.

    2013-05-01

    Existing chemical plume quantification algorithms assume that the off-plume radiance of a pixel containing the plume signal is unobservable. When the problem is limited to a single gas, the off-plume radiance may be estimated from the bands in which the gas absorption is nearly zero. It is then possible to compute the difference between the on- and off-plume radiances and solve for the plume strength from Beer's Law. The major advantage of this proposed method is that the gas strength can be resolved from the radiance difference so that the estimation error remains small for thick plumes.

  11. Quantification of Lung Metastases from In Vivo Mouse Models

    DEFF Research Database (Denmark)

    Chang, Joan; Erler, Janine T

    2016-01-01

    Cancer research has made significant progress in terms of understanding and targeting primary tumors; however, the challenge remains for the successful treatment of metastatic cancers. This highlights the importance to use in vivo models to study the metastatic process, as well as for preclinical...... testing of compounds that could inhibit metastasis. As a result, proper quantification of metastases from in vivo models is of the utmost significance. Here, we provide a detailed protocol for collecting and handling lung tissues from mice, and guidance for subsequent analysis of metastases, as well...

  12. DNA imaging and quantification using chemi-luminescent probes

    International Nuclear Information System (INIS)

    Dorner, G.; Redjdal, N.; Laniece, P.; Siebert, R.; Tricoire, H.; Valentin, L.

    1999-01-01

    During this interdisciplinary study we have developed an ultra sensitive and reliable imaging system of DNA labelled by chemiluminescence. Based on a liquid nitrogen cooled CCD, the system achieves sensitivities down to 10 fg/mm 2 labelled DNA over a surface area of 25 x 25 cm 2 with a sub-millimeter resolution. Commercially available chemi-luminescent - and enhancer molecules are compared and their reaction conditions optimized for best signal-to-noise ratios. Double labelling was performed to verify quantification with radioactive probes. (authors)

  13. HPLC Quantification of Cytotoxic Compounds from Aspergillus niger

    Directory of Open Access Journals (Sweden)

    Paula Karina S. Uchoa

    2017-01-01

    Full Text Available A high-performance liquid chromatography method was developed and validated for the quantification of the cytotoxic compounds produced by a marine strain of Aspergillus niger. The fungus was grown in malt peptone dextrose (MPD, potato dextrose yeast (PDY, and mannitol peptone yeast (MnPY media during 7, 14, 21, and 28 days, and the natural products were identified by standard compounds. The validation parameters obtained were selectivity, linearity (coefficient of correlation > 0.99, precision (relative standard deviation below 5%, and accuracy (recovery > 96.

  14. Simple quantification of ultrasonic intensity using aqueous solution of phenolphthalein.

    Science.gov (United States)

    Rong, L; Kojima, Y; Koda, S; Nomura, H

    2001-01-01

    Aqueous phenolphthalein solution under sonication was investigated for use as a chemical dosimeter. The fading time of aqueous phenolphthalein solution under sonication depended on the concentration of phenolphthalein and the pH values of solutions. The fading time was correlated to the ultrasonic intensity in a reaction vessel that is estimated on the basis of decomposition of porphyrin. The relation between the fading time and the ultrasonic intensity for different frequencies is expressed by a single curve. From these results, it is indicated that aqueous solutions of phenolphthalein is useful for simple quantification of ultrasonic intensity for practical use, and one can regard it as one of the ultrasonic intensity indicators.

  15. Prospective comparison of liver stiffness measurements between two point wave elastography methods: Virtual ouch quantification and elastography point quantification

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Hyun Suk; Lee, Jeong Min; Yoon, Jeong Hee; Lee, Dong Ho; Chang, Won; Han, Joon Koo [Seoul National University Hospital, Seoul (Korea, Republic of)

    2016-09-15

    To prospectively compare technical success rate and reliable measurements of virtual touch quantification (VTQ) elastography and elastography point quantification (ElastPQ), and to correlate liver stiffness (LS) measurements obtained by the two elastography techniques. Our study included 85 patients, 80 of whom were previously diagnosed with chronic liver disease. The technical success rate and reliable measurements of the two kinds of point shear wave elastography (pSWE) techniques were compared by χ{sup 2} analysis. LS values measured using the two techniques were compared and correlated via Wilcoxon signed-rank test, Spearman correlation coefficient, and 95% Bland-Altman limit of agreement. The intraobserver reproducibility of ElastPQ was determined by 95% Bland-Altman limit of agreement and intraclass correlation coefficient (ICC). The two pSWE techniques showed similar technical success rate (98.8% for VTQ vs. 95.3% for ElastPQ, p = 0.823) and reliable LS measurements (95.3% for VTQ vs. 90.6% for ElastPQ, p = 0.509). The mean LS measurements obtained by VTQ (1.71 ± 0.47 m/s) and ElastPQ (1.66 ± 0.41 m/s) were not significantly different (p = 0.209). The LS measurements obtained by the two techniques showed strong correlation (r = 0.820); in addition, the 95% limit of agreement of the two methods was 27.5% of the mean. Finally, the ICC of repeat ElastPQ measurements was 0.991. Virtual touch quantification and ElastPQ showed similar technical success rate and reliable measurements, with strongly correlated LS measurements. However, the two methods are not interchangeable due to the large limit of agreement.

  16. A Spanish model for quantification and management of construction waste.

    Science.gov (United States)

    Solís-Guzmán, Jaime; Marrero, Madelyn; Montes-Delgado, Maria Victoria; Ramírez-de-Arellano, Antonio

    2009-09-01

    Currently, construction and demolition waste (C&D waste) is a worldwide issue that concerns not only governments but also the building actors involved in construction activity. In Spain, a new national decree has been regulating the production and management of C&D waste since February 2008. The present work describes the waste management model that has inspired this decree: the Alcores model implemented with good results in Los Alcores Community (Seville, Spain). A detailed model is also provided to estimate the volume of waste that is expected to be generated on the building site. The quantification of C&D waste volume, from the project stage, is essential for the building actors to properly plan and control its disposal. This quantification model has been developed by studying 100 dwelling projects, especially their bill of quantities, and defining three coefficients to estimate the demolished volume (CT), the wreckage volume (CR) and the packaging volume (CE). Finally, two case studies are included to illustrate the usefulness of the model to estimate C&D waste volume in both new construction and demolition projects.

  17. Cell-based quantification of molecular biomarkers in histopathology specimens.

    Science.gov (United States)

    Al-Kofahi, Yousef; Lassoued, Wiem; Grama, Kedar; Nath, Sumit K; Zhu, Jianliang; Oueslati, Ridha; Feldman, Michael; Lee, William M F; Roysam, Badrinath

    2011-07-01

    To investigate the use of a computer-assisted technology for objective, cell-based quantification of molecular biomarkers in specified cell types in histopathology specimens, with the aim of advancing current visual estimation and pixel-level (rather than cell-based) quantification methods. Tissue specimens were multiplex-immunostained to reveal cell structures, cell type markers, and analytes, and imaged with multispectral microscopy. The image data were processed with novel software that automatically delineates and types each cell in the field, measures morphological features, and quantifies analytes in different subcellular compartments of specified cells.The methodology was validated with the use of cell blocks composed of differentially labelled cultured cells mixed in known proportions, and evaluated on human breast carcinoma specimens for quantifying human epidermal growth factor receptor 2, estrogen receptor, progesterone receptor, Ki67, phospho-extracellular signal-related kinase, and phospho-S6. Automated cell-level analyses closely matched human assessments, but, predictably, differed from pixel-level analyses of the same images. Our method reveals the type, distribution, morphology and biomarker state of each cell in the field, and allows multiple biomarkers to be quantified over specified cell types, regardless of their abundance. It is ideal for studying specimens from patients in clinical trials of targeted therapeutic agents, for investigating minority stromal cell subpopulations, and for phenotypic characterization to personalize therapy and prognosis. © 2011 Blackwell Publishing Limited.

  18. A critical view on microplastic quantification in aquatic organisms

    International Nuclear Information System (INIS)

    Vandermeersch, Griet; Van Cauwenberghe, Lisbeth; Janssen, Colin R.; Marques, Antonio; Granby, Kit; Fait, Gabriella; Kotterman, Michiel J.J.; Diogène, Jorge; Bekaert, Karen; Robbens, Johan; Devriese, Lisa

    2015-01-01

    Microplastics, plastic particles and fragments smaller than 5 mm, are ubiquitous in the marine environment. Ingestion and accumulation of microplastics have previously been demonstrated for diverse marine species ranging from zooplankton to bivalves and fish, implying the potential for microplastics to accumulate in the marine food web. In this way, microplastics can potentially impact food safety and human health. Although a few methods to quantify microplastics in biota have been described, no comparison and/or intercalibration of these techniques have been performed. Here we conducted a literature review on all available extraction and quantification methods. Two of these methods, involving wet acid destruction, were used to evaluate the presence of microplastics in field-collected mussels (Mytilus galloprovincialis) from three different “hotspot” locations in Europe (Po estuary, Italy; Tagus estuary, Portugal; Ebro estuary, Spain). An average of 0.18±0.14 total microplastics g −1 w.w. for the Acid mix Method and 0.12±0.04 total microplastics g −1 w.w. for the Nitric acid Method was established. Additionally, in a pilot study an average load of 0.13±0.14 total microplastics g −1 w.w. was recorded in commercial mussels (Mytilus edulis and M. galloprovincialis) from five European countries (France, Italy, Denmark, Spain and The Netherlands). A detailed analysis and comparison of methods indicated the need for further research to develop a standardised operating protocol for microplastic quantification and monitoring.

  19. Mixture quantification using PLS in plastic scintillation measurements

    Energy Technology Data Exchange (ETDEWEB)

    Bagan, H.; Tarancon, A.; Rauret, G. [Departament de Quimica Analitica, Universitat de Barcelona, Diagonal 647, E-08028 Barcelona (Spain); Garcia, J.F., E-mail: jfgarcia@ub.ed [Departament de Quimica Analitica, Universitat de Barcelona, Diagonal 647, E-08028 Barcelona (Spain)

    2011-06-15

    This article reports the capability of plastic scintillation (PS) combined with multivariate calibration (Partial least squares; PLS) to detect and quantify alpha and beta emitters in mixtures. While several attempts have been made with this purpose in mind using liquid scintillation (LS), no attempt was done using PS that has the great advantage of not producing mixed waste after the measurements are performed. Following this objective, ternary mixtures of alpha and beta emitters ({sup 241}Am, {sup 137}Cs and {sup 90}Sr/{sup 90}Y) have been quantified. Procedure optimisation has evaluated the use of the net spectra or the sample spectra, the inclusion of different spectra obtained at different values of the Pulse Shape Analysis parameter and the application of the PLS1 or PLS2 algorithms. The conclusions show that the use of PS+PLS2 applied to the sample spectra, without the use of any pulse shape discrimination, allows quantification of the activities with relative errors less than 10% in most of the cases. This procedure not only allows quantification of mixtures but also reduces measurement time (no blanks are required) and the application of this procedure does not require detectors that include the pulse shape analysis parameter.

  20. Biomass to energy : GHG reduction quantification protocols and case study

    International Nuclear Information System (INIS)

    Reusing, G.; Taylor, C.; Nolan, W.; Kerr, G.

    2009-01-01

    With the growing concerns over greenhouses gases and their contribution to climate change, it is necessary to find ways of reducing environmental impacts by diversifying energy sources to include non-fossil fuel energy sources. Among the fastest growing green energy sources is energy from waste facilities that use biomass that would otherwise be landfilled or stockpiled. The quantification of greenhouse gas reductions through the use of biomass to energy systems can be calculated using various protocols and methodologies. This paper described each of these methodologies and presented a case study comparing some of these quantification methodologies. A summary and comparison of biomass to energy greenhouse gas reduction protocols in use or under development by the United Nations, the European Union, the Province of Alberta and Environment Canada was presented. It was concluded that regulatory, environmental pressures, and public policy will continue to impact the practices associated with biomass processing or landfill operations, such as composting, or in the case of landfills, gas collection systems, thus reducing the amount of potential credit available for biomass to energy facility offset projects. 10 refs., 2 tabs., 6 figs

  1. Evaluation of semi-automatic arterial stenosis quantification

    Energy Technology Data Exchange (ETDEWEB)

    Hernandez Hoyos, M. [CREATIS Research Unit, CNRS, INSERM, INSA, Lyon (France); Universite Claude Bernard Lyon 1, 69 - Villeurbanne (France). INSA; Univ. de los Andes, Bogota (Colombia). Grupo de Ingenieria Biomedica; Serfaty, J.M.; Douek, P.C. [CREATIS Research Unit, CNRS, INSERM, INSA, Lyon (France); Universite Claude Bernard Lyon 1, 69 - Villeurbanne (France). INSA; Hopital Cardiovasculaire et Pneumologique L. Pradel, Bron (France). Dept. de Radiologie; Maghiar, A. [Hopital Cardiovasculaire et Pneumologique L. Pradel, Bron (France). Dept. de Radiologie; Mansard, C.; Orkisz, M.; Magnin, I. [CREATIS Research Unit, CNRS, INSERM, INSA, Lyon (France); Universite Claude Bernard Lyon 1, 69 - Villeurbanne (France). INSA

    2006-11-15

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

  2. Simple and inexpensive quantification of ammonia in whole blood.

    Science.gov (United States)

    Ayyub, Omar B; Behrens, Adam M; Heligman, Brian T; Natoli, Mary E; Ayoub, Joseph J; Cunningham, Gary; Summar, Marshall; Kofinas, Peter

    2015-01-01

    Quantification of ammonia in whole blood has applications in the diagnosis and management of many hepatic diseases, including cirrhosis and rare urea cycle disorders, amounting to more than 5 million patients in the United States. Current techniques for ammonia measurement suffer from limited range, poor resolution, false positives or large, complex sensor set-ups. Here we demonstrate a technique utilizing inexpensive reagents and simple methods for quantifying ammonia in 100 μL of whole blood. The sensor comprises a modified form of the indophenol reaction, which resists sources of destructive interference in blood, in conjunction with a cation-exchange membrane. The presented sensing scheme is selective against other amine containing molecules such as amino acids and has a shelf life of at least 50 days. Additionally, the resulting system has high sensitivity and allows for the accurate reliable quantification of ammonia in whole human blood samples at a minimum range of 25 to 500 μM, which is clinically for rare hyperammonemic disorders and liver disease. Furthermore, concentrations of 50 and 100 μM ammonia could be reliably discerned with p = 0.0001. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Sludge quantification at water treatment plant and its management scenario.

    Science.gov (United States)

    Ahmad, Tarique; Ahmad, Kafeel; Alam, Mehtab

    2017-08-15

    Large volume of sludge is generated at the water treatment plants during the purification of surface water for potable supplies. Handling and disposal of sludge require careful attention from civic bodies, plant operators, and environmentalists. Quantification of the sludge produced at the treatment plants is important to develop suitable management strategies for its economical and environment friendly disposal. Present study deals with the quantification of sludge using empirical relation between turbidity, suspended solids, and coagulant dosing. Seasonal variation has significant effect on the raw water quality received at the water treatment plants so forth sludge generation also varies. Yearly production of the sludge in a water treatment plant at Ghaziabad, India, is estimated to be 29,700 ton. Sustainable disposal of such a quantity of sludge is a challenging task under stringent environmental legislation. Several beneficial reuses of sludge in civil engineering and constructional work have been identified globally such as raw material in manufacturing cement, bricks, and artificial aggregates, as cementitious material, and sand substitute in preparing concrete and mortar. About 54 to 60% sand, 24 to 28% silt, and 16% clay constitute the sludge generated at the water treatment plant under investigation. Characteristics of the sludge are found suitable for its potential utilization as locally available construction material for safe disposal. An overview of the sustainable management scenario involving beneficial reuses of the sludge has also been presented.

  4. Quantification of HEV RNA by Droplet Digital PCR

    Directory of Open Access Journals (Sweden)

    Florence Nicot

    2016-08-01

    Full Text Available The sensitivity of real-time PCR for hepatitis E virus (HEV RNA quantification differs greatly among techniques. Standardized tools that measure the real quantity of virus are needed. We assessed the performance of a reverse transcription droplet digital PCR (RT-ddPCR assay that gives absolute quantities of HEV RNA. Analytical and clinical validation was done on HEV genotypes 1, 3 and 4, and was based on open reading frame (ORF3 amplification. The within-run and between-run reproducibilities were very good, the analytical sensitivity was 80 HEV RNA international units (IU/mL and linearities of HEV genotype 1, 3 and 4 were very similar. Clinical validation based on 45 samples of genotype 1, 3 or 4 gave results that correlated well with a validated reverse transcription quantitative PCR (RT-qPCR assay (Spearman rs = 0.89, p < 0.0001. The RT-ddPCR assay is a sensitive method and could be a promising tool for standardizing HEV RNA quantification in various sample types.

  5. First approach to radionuclide mixtures quantification by using plastic scintillators

    Energy Technology Data Exchange (ETDEWEB)

    Tarancon, A. [Departament de Quimica Analitica, Universitat de Barcelona, Diagonal 647, E-08028 Barcelona (Spain); Garcia, J.F. [Departament de Pintura, Universitat de Barcelona, Pau Gargallo 4, E-08028 Barcelona (Spain)]. E-mail: jfgarcia@ub.edu; Rauret, G. [Departament de Quimica Analitica, Universitat de Barcelona, Diagonal 647, E-08028 Barcelona (Spain)

    2007-05-08

    Recent studies have evaluated the capability of plastic scintillation (PS) as an alternative to liquid scintillation (LS) in radionuclide activity determination without mixed waste production. In order to complete the comparison, we now assess the extent to which PS can be used to quantify mixtures of radionuclides and the influence of the diameter of the plastic scintillation beads in detection efficiency. The results show that the detection efficiency decreases and the spectrum shrink to lower energies when the size of the plastic scintillation beads increases, and that the lower the energy of the beta particle, the greater the variation takes place. Similar behaviour has been observed for beta-gamma and alpha emitters. Two scenarios for the quantification of mixtures are considered, one including two radionuclides ({sup 14}C and {sup 60}Co) whose spectra do not overlap significantly, and the other including two radionuclides ({sup 137}Cs and {sup 90}Sr/{sup 90}Y), where the spectra of one the isotopes is totally overlapped by the other The calculation has been performed by using the conventional window selection procedure and a new approach in which the selected windows correspond to those with lower quantification errors. Relative errors obtained using the proposed approach (less than 10%) are lower than those of the conventional procedure, even when a radionuclide is completely overlapped, except for those samples with extreme activity ratios that were not included in the window optimization process.

  6. Evaluation of semi-automatic arterial stenosis quantification

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  7. Dimensionality reduction for uncertainty quantification of nuclear engineering models.

    Energy Technology Data Exchange (ETDEWEB)

    Roderick, O.; Wang, Z.; Anitescu, M. (Mathematics and Computer Science)

    2011-01-01

    The task of uncertainty quantification consists of relating the available information on uncertainties in the model setup to the resulting variation in the outputs of the model. Uncertainty quantification plays an important role in complex simulation models of nuclear engineering, where better understanding of uncertainty results in greater confidence in the model and in the improved safety and efficiency of engineering projects. In our previous work, we have shown that the effect of uncertainty can be approximated by polynomial regression with derivatives (PRD): a hybrid regression method that uses first-order derivatives of the model output as additional fitting conditions for a polynomial expansion. Numerical experiments have demonstrated the advantage of this approach over classical methods of uncertainty analysis: in precision, computational efficiency, or both. To obtain derivatives, we used automatic differentiation (AD) on the simulation code; hand-coded derivatives are acceptable for simpler models. We now present improvements on the method. We use a tuned version of the method of snapshots, a technique based on proper orthogonal decomposition (POD), to set up the reduced order representation of essential information on uncertainty in the model inputs. The automatically obtained sensitivity information is required to set up the method. Dimensionality reduction in combination with PRD allows analysis on a larger dimension of the uncertainty space (>100), at modest computational cost.

  8. An uncertainty inventory demonstration - a primary step in uncertainty quantification

    Energy Technology Data Exchange (ETDEWEB)

    Langenbrunner, James R. [Los Alamos National Laboratory; Booker, Jane M [Los Alamos National Laboratory; Hemez, Francois M [Los Alamos National Laboratory; Salazar, Issac F [Los Alamos National Laboratory; Ross, Timothy J [UNM

    2009-01-01

    Tools, methods, and theories for assessing and quantifying uncertainties vary by application. Uncertainty quantification tasks have unique desiderata and circumstances. To realistically assess uncertainty requires the engineer/scientist to specify mathematical models, the physical phenomena of interest, and the theory or framework for assessments. For example, Probabilistic Risk Assessment (PRA) specifically identifies uncertainties using probability theory, and therefore, PRA's lack formal procedures for quantifying uncertainties that are not probabilistic. The Phenomena Identification and Ranking Technique (PIRT) proceeds by ranking phenomena using scoring criteria that results in linguistic descriptors, such as importance ranked with words, 'High/Medium/Low.' The use of words allows PIRT to be flexible, but the analysis may then be difficult to combine with other uncertainty theories. We propose that a necessary step for the development of a procedure or protocol for uncertainty quantification (UQ) is the application of an Uncertainty Inventory. An Uncertainty Inventory should be considered and performed in the earliest stages of UQ.

  9. Recurrence Quantification for the Analysis of Coupled Processes in Aging.

    Science.gov (United States)

    Brick, Timothy R; Gray, Allison L; Staples, Angela D

    2017-12-15

    Aging is a complex phenomenon, with numerous simultaneous processes that interact with each other on a moment-to-moment basis. One way to quantify the interactions of these processes is by measuring how much a process is similar to its own past states or the past states of another system through the analysis of recurrence. This paper presents an introduction to recurrence quantification analysis (RQA) and cross-recurrence quantification analysis (CRQA), two dynamical systems analysis techniques that provide ways to characterize the self-similar nature of each process and the properties of their mutual temporal co-occurrence. We present RQA and CRQA and demonstrate their effectiveness with an example of conversational movements across age groups. RQA and CRQA provide methods of analyzing the repetitive processes that occur in day-to-day life, describing how different processes co-occur, synchronize, or predict each other and comparing the characteristics of those processes between groups. With intensive longitudinal data becoming increasingly available, it is possible to examine how the processes of aging unfold. RQA and CRQA provide information about how one process may show patterns of internal repetition or echo the patterning of another process and how those characteristics may change across the process of aging. © The Author 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA.

    Science.gov (United States)

    Shahal, Tamar; Gilat, Noa; Michaeli, Yael; Redy-Keisar, Orit; Shabat, Doron; Ebenstein, Yuval

    2014-08-19

    5-Hydroxymethylcytosine (5hmC), a modified form of the DNA base cytosine, is an important epigenetic mark linked to regulation of gene expression in development, and tumorigenesis. We have developed a spectroscopic method for a global quantification of 5hmC in genomic DNA. The assay is performed within a multiwell plate, which allows simultaneous recording of up to 350 samples. Our quantification procedure of 5hmC is direct, simple, and rapid. It relies on a two-step protocol that consists of enzymatic glucosylation of 5hmC with an azide-modified glucose, followed by a "click reaction" with an alkyne-fluorescent tag. The fluorescence intensity recorded from the DNA sample is proportional to its 5hmC content and can be quantified by a simple plate reader measurement. This labeling technique is specific and highly sensitive, allowing detection of 5hmC down to 0.002% of the total nucleotides. Our results reveal significant variations in the 5hmC content obtained from different mouse tissues, in agreement with previously reported data.

  11. Quantification of 5-methyl-2'-deoxycytidine in the DNA.

    Science.gov (United States)

    Giel-Pietraszuk, Małgorzata; Insińska-Rak, Małgorzata; Golczak, Anna; Sikorski, Marek; Barciszewska, Mirosława; Barciszewski, Jan

    2015-01-01

    Methylation at position 5 of cytosine (Cyt) at the CpG sequences leading to formation of 5-methyl-cytosine (m(5)Cyt) is an important element of epigenetic regulation of gene expression. Modification of the normal methylation pattern, unique to each organism, leads to the development of pathological processes and diseases, including cancer. Therefore, quantification of the DNA methylation and analysis of changes in the methylation pattern is very important from a practical point of view and can be used for diagnostic purposes, as well as monitoring of the treatment progress. In this paper we present a new method for quantification of 5-methyl-2'deoxycytidine (m(5)C) in the DNA. The technique is based on conversion of m(5)C into fluorescent 3,N(4)-etheno-5-methyl-2'deoxycytidine (εm(5)C) and its identification by reversed-phase high-performance liquid chromatography (RP-HPLC). The assay was used to evaluate m(5)C concentration in DNA of calf thymus and peripheral blood of cows bred under different conditions. This approach can be applied for measuring of 5-methylcytosine in cellular DNA from different cells and tissues.

  12. Accurate quantification of supercoiled DNA by digital PCR

    Science.gov (United States)

    Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

    2016-01-01

    Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry. PMID:27063649

  13. A Spanish model for quantification and management of construction waste

    International Nuclear Information System (INIS)

    Solis-Guzman, Jaime; Marrero, Madelyn; Montes-Delgado, Maria Victoria; Ramirez-de-Arellano, Antonio

    2009-01-01

    Currently, construction and demolition waste (C and D waste) is a worldwide issue that concerns not only governments but also the building actors involved in construction activity. In Spain, a new national decree has been regulating the production and management of C and D waste since February 2008. The present work describes the waste management model that has inspired this decree: the Alcores model implemented with good results in Los Alcores Community (Seville, Spain). A detailed model is also provided to estimate the volume of waste that is expected to be generated on the building site. The quantification of C and D waste volume, from the project stage, is essential for the building actors to properly plan and control its disposal. This quantification model has been developed by studying 100 dwelling projects, especially their bill of quantities, and defining three coefficients to estimate the demolished volume (CT), the wreckage volume (CR) and the packaging volume (CE). Finally, two case studies are included to illustrate the usefulness of the model to estimate C and D waste volume in both new construction and demolition projects.

  14. MORPHOLOGICAL QUANTIFICATION OF AORTIC CALCIFICATION FROM LOW MAGNIFICATION IMAGES

    Directory of Open Access Journals (Sweden)

    Jesús Angulo

    2011-05-01

    Full Text Available Atherosclerotic and medial vascular calcifications are frequent in chronic renal failure patiens and predict their increased cardiovascular mortality. Experimental models for mice have been recently developed in order to study these disorders. The aim of this paper is to present the morphological image processing algorithms developed for the semi-automated measurement of calcification from sections of aorta stained using von Kossa's silver nitrate procedure and acquired at low magnification power (x 2.5 on colour images. The approach is separated into two sequential phases. First, the segmentation is aimed to extract the calcification structures and on the other hand to demarcate the region of the atherosclerotic lesion within the tissue. The segmentation yields the image data which is the input to the second phase, the quantification. Calcified structures are measured inside and outside the lesion using a granulometric curve which allows the calculation of statistical parameters of size. The same operator computes the shape of the lesion. The relative proportion of the area of calcification is also calculated respectively for the atherosclerotic lesion area and the area outside such lesions. In conclusion, the here developed method allows quantification of vascular calcified deposits in mouse aorta. This method will be useful for the quantitative assessment of pathological vascular changes in animals and man.

  15. Guided Wave Delamination Detection and Quantification With Wavefield Data Analysis

    Science.gov (United States)

    Tian, Zhenhua; Campbell Leckey, Cara A.; Seebo, Jeffrey P.; Yu, Lingyu

    2014-01-01

    Unexpected damage can occur in aerospace composites due to impact events or material stress during off-nominal loading events. In particular, laminated composites are susceptible to delamination damage due to weak transverse tensile and inter-laminar shear strengths. Developments of reliable and quantitative techniques to detect delamination damage in laminated composites are imperative for safe and functional optimally-designed next-generation composite structures. In this paper, we investigate guided wave interactions with delamination damage and develop quantification algorithms by using wavefield data analysis. The trapped guided waves in the delamination region are observed from the wavefield data and further quantitatively interpreted by using different wavenumber analysis methods. The frequency-wavenumber representation of the wavefield shows that new wavenumbers are present and correlate to trapped waves in the damage region. These new wavenumbers are used to detect and quantify the delamination damage through the wavenumber analysis, which can show how the wavenumber changes as a function of wave propagation distance. The location and spatial duration of the new wavenumbers can be identified, providing a useful means not only for detecting the presence of delamination damage but also allowing for estimation of the delamination size. Our method has been applied to detect and quantify real delamination damage with complex geometry (grown using a quasi-static indentation technique). The detection and quantification results show the location, size, and shape of the delamination damage.

  16. Complex Empiricism and the Quantification of Uncertainty in Paleoclimate Reconstructions

    Science.gov (United States)

    Brumble, K. C.

    2014-12-01

    Because the global climate cannot be observed directly, and because of vast and noisy data sets, climate science is a rich field to study how computational statistics informs what it means to do empirical science. Traditionally held virtues of empirical science and empirical methods like reproducibility, independence, and straightforward observation are complicated by representational choices involved in statistical modeling and data handling. Examining how climate reconstructions instantiate complicated empirical relationships between model, data, and predictions reveals that the path from data to prediction does not match traditional conceptions of empirical inference either. Rather, the empirical inferences involved are "complex" in that they require articulation of a good deal of statistical processing wherein assumptions are adopted and representational decisions made, often in the face of substantial uncertainties. Proxy reconstructions are both statistical and paleoclimate science activities aimed at using a variety of proxies to reconstruct past climate behavior. Paleoclimate proxy reconstructions also involve complex data handling and statistical refinement, leading to the current emphasis in the field on the quantification of uncertainty in reconstructions. In this presentation I explore how the processing needed for the correlation of diverse, large, and messy data sets necessitate the explicit quantification of the uncertainties stemming from wrangling proxies into manageable suites. I also address how semi-empirical pseudo-proxy methods allow for the exploration of signal detection in data sets, and as intermediary steps for statistical experimentation.

  17. Within-day repeatability for absolute quantification of Lawsonia intracellularis bacteria in feces from growing pigs

    DEFF Research Database (Denmark)

    Pedersen, Ken Steen; Pedersen, Klaus H.; Hjulsager, Charlotte Kristiane

    2012-01-01

    Absolute quantification of Lawsonia intracellularis by real-time polymerase chain reaction (PCR) is now possible on a routine basis. Poor repeatability of quantification can result in disease status misclassification of individual pigs when a single fecal sample is obtained. The objective...

  18. PCR amplification of repetitive sequences as a possible approach in relative species quantification

    DEFF Research Database (Denmark)

    Ballin, Nicolai Zederkopff; Vogensen, Finn Kvist; Karlsson, Anders H

    2012-01-01

    Abstract Both relative and absolute quantifications are possible in species quantification when single copy genomic DNA is used. However, amplification of single copy genomic DNA does not allow a limit of detection as low as one obtained from amplification of repetitive sequences. Amplification...... of repetitive sequences is therefore frequently used in absolute quantification but problems occur in relative quantification as the number of repetitive sequences is unknown. A promising approach was developed where data from amplification of repetitive sequences were used in relative quantification of species...... to relatively quantify the amount of chicken DNA in a binary mixture of chicken DNA and pig DNA. However, the designed PCR primers lack the specificity required for regulatory species control....

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

    Science.gov (United States)

    Klosterman, Steven J

    2012-01-01

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

  20. Concentration of bioaerosols in composting plants using different quantification methods.

    Science.gov (United States)

    van Kampen, Vera; Sander, Ingrid; Liebers, Verena; Deckert, Anja; Neumann, Heinz-Dieter; Buxtrup, Martin; Willer, Eckart; Felten, Christian; Jäckel, Udo; Klug, Kerstin; Brüning, Thomas; Raulf, Monika; Bünger, Jürgen

    2014-07-01

    Bioaerosols (organic dusts) containing viable and non-viable microorganisms and their metabolic products can lead to adverse health effects in exposed workers. Standard quantification methods of airborne microorganisms are mainly based on cultivation, which often underestimates the microbial burden. The aim of the study was to determine the microbial load in German composting plants with different, mainly cultivation-independent, methods. Second purpose was to evaluate which working areas are associated with higher or lower bioaerosol concentrations. A total of 124 inhalable dust samples were collected at different workplaces in 31 composting plants. Besides the determination of inhalable dust, particles, and total cell numbers, antigen quantification for moulds (Aspergillus fumigatus, Aspergillus versicolor, Penicillium chrysogenum, and Cladosporium spp.) and mites was performed. Concentrations of β-glucans as well as endotoxin and pyrogenic activities were also measured. The number of colony forming units (cfu) was determined by cultivation of moulds and actinomycetes in 36 additional dust samples. With the exception of particle numbers, concentrations of all determined parameters showed significant correlations (P parameters were measured highest in dusty working areas like next to the shredder and during processing with the exception of Cladosporium antigens that were found in the highest concentrations in the delivery area. The lowest concentrations of dust, particles, antigens, and pyrogenic activity were determined in wheel loader cabins (WLCs), which were equipped with an air filtration system. It was possible to assess the microbial load of air in composting plants with different quantification methods. Since allergic and toxic reactions may be also caused by nonliving microorganisms, cultivation-independent methods may provide additional information about bioaerosol composition. In general, air filtration reduced the bioaerosol exposure shown in WLCs

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

    Science.gov (United States)

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

    2006-01-01

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

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

    Science.gov (United States)

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

    2006-08-14

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

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

    Directory of Open Access Journals (Sweden)

    Žel Jana

    2006-08-01

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

  4. Reliability and discriminatory power of methods for dental plaque quantification

    Directory of Open Access Journals (Sweden)

    Daniela Prócida Raggio

    2010-04-01

    Full Text Available OBJECTIVE: This in situ study evaluated the discriminatory power and reliability of methods of dental plaque quantification and the relationship between visual indices (VI and fluorescence camera (FC to detect plaque. MATERIAL AND METHODS: Six volunteers used palatal appliances with six bovine enamel blocks presenting different stages of plaque accumulation. The presence of plaque with and without disclosing was assessed using VI. Images were obtained with FC and digital camera in both conditions. The area covered by plaque was assessed. Examinations were done by two independent examiners. Data were analyzed by Kruskal-Wallis and Kappa tests to compare different conditions of samples and to assess the inter-examiner reproducibility. RESULTS: Some methods presented adequate reproducibility. The Turesky index and the assessment of area covered by disclosed plaque in the FC images presented the highest discriminatory powers. CONCLUSION: The Turesky index and images with FC with disclosing present good reliability and discriminatory power in quantifying dental plaque.

  5. UQTools: The Uncertainty Quantification Toolbox - Introduction and Tutorial

    Science.gov (United States)

    Kenny, Sean P.; Crespo, Luis G.; Giesy, Daniel P.

    2012-01-01

    UQTools is the short name for the Uncertainty Quantification Toolbox, a software package designed to efficiently quantify the impact of parametric uncertainty on engineering systems. UQTools is a MATLAB-based software package and was designed to be discipline independent, employing very generic representations of the system models and uncertainty. Specifically, UQTools accepts linear and nonlinear system models and permits arbitrary functional dependencies between the system s measures of interest and the probabilistic or non-probabilistic parametric uncertainty. One of the most significant features incorporated into UQTools is the theoretical development centered on homothetic deformations and their application to set bounding and approximating failure probabilities. Beyond the set bounding technique, UQTools provides a wide range of probabilistic and uncertainty-based tools to solve key problems in science and engineering.

  6. Quantification of cell density in rat Achilles tendon

    DEFF Research Database (Denmark)

    Couppé, Christian; Svensson, René B; Heinemeier, Katja M

    2017-01-01

    Increased tendon cell nuclei density (TCND) has been proposed to induce tendon mechanical adaptations. However, it is unknown whether TCND is increased in tendon tissue after mechanical loading and whether such an increase can be quantified in a reliable manner. The aim of this study was to develop...... a reliable method for quantification of TCND and to investigate potential changes in TCND in rat Achilles tendons in response to 12 weeks of running. Eight adult male Sprague-Dawley rats ran (RUN) on a treadmill with 10° incline, 1 h/day, 5 days/wk (17-20 m/min) for 12 weeks (which improved tendon mechanical...... properties) and were compared with 11 control rats (SED). Tissue-Tek-embedded cryosections (10 µm) from the mid region of the Achilles tendon were cut longitudinally on a cryostat. Sections were stained with alcian blue and picrosirius red. One blinded investigator counted the number of tendon cell nuclei 2...

  7. Structural Health Monitoring 2007: Quantification, Validation, and Implementation

    Science.gov (United States)

    2008-07-01

    11.00 p, 1734 P~rkr.1It , Han-’Slill Slnl its for Slruclunlllnlllll MOllilorin WI",1est Nen.’orb 11:00-11:20 Vinod R. Challa . M.G Prasad and Frank T ...6th International Workshop on Structural Health Monitoring is "Quantification, Validation, and Implementation." Similar to the l’\\ T ))E community, the...ofTok\\’ll. J : Serdai’ IJckun. NASA. USA T ~o l’ur P I’UIlnd futu"" Pn>s ’" i"SII)1 09 OS -09:40 Jill D. Achc:obach. Nonhwe51ern Unt\\’ffJll • USA On t

  8. Quantification of Uncertainty in Extreme Scale Computations (QUEST)

    Energy Technology Data Exchange (ETDEWEB)

    Ghanem, Roger [Univ. of Southern California, Los Angeles, CA (United States)

    2017-04-18

    QUEST was a SciDAC Institute comprising Sandia National Laboratories, Los Alamos National Laboratory, the University of Southern California, the Massachusetts Institute of Technology, the University of Texas at Austin, and Duke University. The mission of QUEST is to: (1) develop a broad class of uncertainty quantification (UQ) methods/tools, and (2) provide UQ expertise and software to other SciDAC projects, thereby enabling/guiding their UQ activities. The USC effort centered on the development of reduced models and efficient algorithms for implementing various components of the UQ pipeline. USC personnel were responsible for the development of adaptive bases, adaptive quadrature, and reduced models to be used in estimation and inference.

  9. Quantification of stable isotope label in metabolites via mass spectrometry.

    Science.gov (United States)

    Huege, Jan; Goetze, Jan; Dethloff, Frederik; Junker, Bjoern; Kopka, Joachim

    2014-01-01

    Isotope labelling experiments with stable or radioactive isotopes have long been an integral part of biological and medical research. Labelling experiments led to the discovery of new metabolic pathways and made it possible to calculate the fluxes responsible for a metabolic phenotype, i.e., the qualitative and quantitative composition of metabolites in a biological system. Prerequisite for efficient isotope labelling experiments is a reliable and precise method to analyze the redistribution of isotope label in a metabolic network. Here we describe the use of the CORRECTOR program, which utilizes matrix calculations to correct mass spectral data from stable isotope labelling experiments for the distorting effect of naturally occurring stable isotopes (NOIs). CORRECTOR facilitates and speeds up the routine quantification of experimentally introduced isotope label from multiple mass spectral readouts, which are generated by routine metabolite profiling when combined with stable isotope labelling experiments.

  10. Quantification of benzoxazinoids and their metabolites in Nordic breads

    DEFF Research Database (Denmark)

    Dihm, Katharina; Vendelbo Lind, Mads; Sundén, Henrik

    2017-01-01

    Benzoxazinoids (Bx) and their metabolites are molecules with suggested health effects in humans, found in cereal grains and consequently in cereal foods. However, to date little is known about the amount of Bx in our diet. In this study, deuterated standards 2-hydroxy-1,4-benzoxazin-3-one (HBOA-d4......) and 2-hydroxy-N-(2-hydroxyphenyl) acetamide (HHPAA-d4) were synthesized, to allow quantification of nine Bx and their metabolites in 30 breads and flours from Nordic countries by UHPLC-MS/MS. Samples containing rye had larger amounts of Bx (143–3560 µg/g DM) than the ones containing wheat (11–449 µg...

  11. Protocol for Quantification of Defects in Natural Fibres for Composites

    DEFF Research Database (Denmark)

    Mortensen, Ulrich Andreas; Madsen, Bo

    2014-01-01

    on the experimental method of optical microscopy and the image analysis algorithms of the seeded region growing method and Otsu’s method. The use of the protocol is demonstrated by examining two types of differently processed flax fibres to give mean defect contents of 6.9 and 3.9%, a difference which is tested......Natural bast-type plant fibres are attracting increasing interest for being used for structural composite applications where high quality fibres with good mechanical properties are required. A protocol for the quantification of defects in natural fibres is presented. The protocol is based...... to be statistically significant. The protocol is evaluated with respect to the selection of image analysis algorithms, and Otsu’s method is found to be a more appropriate method than the alternative coefficient of variation method. The traditional way of defining defect size by area is compared to the definition...

  12. Quantification of cellulase activity using cellulose-azure.

    Science.gov (United States)

    Lai, Takwai E; Pullammanappallil, Pratap C; Clarke, William P

    2006-03-15

    Despite wide application of cellulose-azure as a substrate for measuring cellulase activity, there is no quantification of hydrolysis rate or enzymatic activities using this substrate. The aim of this study was to quantify the hydrolysis rate in terms of product formation and dye released using cellulose-azure. The amount of dye released was correlated with the production of glucose and the enzyme concentrations. It is shown that the lack of correlation can be due to (1) repression of the release of the azure-dye when azure-dye accumulates, (2) presence of degradable substrates in the cellulase powder which inflate the glucose measurements and (3) the degradation of cellulose which is not linked to the dye in the cellulose-azure. Based on the lack of correlation, it is recommended that cellulose-azure should only be applied in assays when the aim is to compare relative activities of different enzymatic systems.

  13. Quantification of chromatin condensation level by image processing.

    Science.gov (United States)

    Irianto, Jerome; Lee, David A; Knight, Martin M

    2014-03-01

    The level of chromatin condensation is related to the silencing/activation of chromosomal territories and therefore impacts on gene expression. Chromatin condensation changes during cell cycle, progression and differentiation, and is influenced by various physicochemical and epigenetic factors. This study describes a validated experimental technique to quantify chromatin condensation. A novel image processing procedure is developed using Sobel edge detection to quantify the level of chromatin condensation from nuclei images taken by confocal microscopy. The algorithm was developed in MATLAB and used to quantify different levels of chromatin condensation in chondrocyte nuclei achieved through alteration in osmotic pressure. The resulting chromatin condensation parameter (CCP) is in good agreement with independent multi-observer qualitative visual assessment. This image processing technique thereby provides a validated unbiased parameter for rapid and highly reproducible quantification of the level of chromatin condensation. Copyright © 2013 IPEM. Published by Elsevier Ltd. All rights reserved.

  14. Uncertainty Quantification for Combined Polynomial Chaos Kriging Surrogate Models

    Science.gov (United States)

    Weinmeister, Justin; Gao, Xinfeng; Krishna Prasad, Aditi; Roy, Sourajeet

    2017-11-01

    Surrogate modeling techniques are currently used to perform uncertainty quantification on computational fluid dynamics (CFD) models for their ability to identify the most impactful parameters on CFD simulations and help reduce computational cost in engineering design process. The accuracy of these surrogate models depends on a number of factors, such as the training data created from the CFD simulations, the target functions, the surrogate model framework, and so on. Recently, we have combined polynomial chaos expansions (PCE) and Kriging to produce a more accurate surrogate model, polynomial chaos Kriging (PCK). In this talk, we analyze the error convergence rate for the Kriging, PCE, and PCK model on a convection-diffusion-reaction problem, and validate the statistical measures and performance of the PCK method for its application to practical CFD simulations.

  15. Quantification methodology for the French 900 MW PWR PRA

    International Nuclear Information System (INIS)

    Ducamp, F.; Lanore, J.M.; Duchemin, B.; De Villeneuve, M.J.

    1985-02-01

    This paper develops some improvements brought to provide to the classical way of risk assessment. The calculation of the contribution to the risk of one peculiar sequence of an event tree is composed of four stages: creation of a fault tree for each system which appears in the event trees, in terms of component faults; simplification of these fault trees into smaller ones, in terms of macrocomponents; creation of one ''super-tree'' by regrouping the fault trees of down systems (systems which fail in the sequence) under an AND gate and calculation of minimal cut sets of this super-tree, taking into account the up systems (systems that do not fail in the sequence) and peculiarities related to the initiating event if needed; quantification of the minimal cut sets so obtained, taking into account the duration of the scenario depicted by the sequence and the possibilities of repair. Each of these steps is developed in this article

  16. Systematic Quantification of Biogas Potential in Urban Organic Waste

    DEFF Research Database (Denmark)

    Fitamo, Temesgen Mathewos

    of biogas from organic waste rather than incineration and landfilling. The production of biogas from urban organic waste is expected to contribute to reaching the EU target of 20% of overall energy production and 10% of vehicle fuel derived from renewable sources by 2020. The Danish energy strategy...... is for Demark to become a 100% fossil fuel-free nation by 2050. However, existing technical challenges and barriers must be overcome to make the production of biogas more attractive. In this respect, a systematic quantification of the biogas production potential of various urban organic waste sources...... is necessary, in order to analyse and improve processes for biogas production. Conventionally, the potential biogas production of organic waste sources is quantified through biochemical methane potential (BMP) analysis and anaerobic digestion in biogas reactors. However, the determination of BMP in batch...

  17. Automated image analysis for quantification of filamentous bacteria

    DEFF Research Database (Denmark)

    Fredborg, M.; Rosenvinge, F. S.; Spillum, E.

    2015-01-01

    Background: Antibiotics of the beta-lactam group are able to alter the shape of the bacterial cell wall, e.g. filamentation or a spheroplast formation. Early determination of antimicrobial susceptibility may be complicated by filamentation of bacteria as this can be falsely interpreted as growth...... displaying different resistant profiles and differences in filamentation kinetics were used to study a novel image analysis algorithm to quantify length of bacteria and bacterial filamentation. A total of 12 beta-lactam antibiotics or beta-lactam-beta-lactamase inhibitor combinations were analyzed...... in systems relying on colorimetry or turbidometry (such as Vitek-2, Phoenix, MicroScan WalkAway). The objective was to examine an automated image analysis algorithm for quantification of filamentous bacteria using the 3D digital microscopy imaging system, oCelloScope. Results: Three E. coli strains...

  18. Study on quantification of HBs-antibody by immunoradiometric assay

    International Nuclear Information System (INIS)

    Kondo, Yuichi; Itoi, Yoshihiro; Kajiyama, Shizuo

    1989-01-01

    Quantification of HBs-antibody assay was carried out using a commercialized assay kit and standard solutions of HBs-antibody recognised as 1 st reference preparation of hepatitis B immunogloblin by WHO. Standard curve of HBs-antibody was drawn with the function of 3D-spline and the correlation factor was obtained as r = 0.999. Coefficient of intra-assay variance was 3.8 % and that of inter-assay variance was 7.8 %. Dilution tests showed satisfactory results in the range of 2-16 times. Correlation between value of cut-off indices and concentration of HBs-antibody was obtained as the formula of y = 2.599 x-3.894 (r = 0.992) and 2.1 of cut-off index corresponded to about 5 mIU/ml of HBs-antibody concentration. (author)

  19. Temporal and spatial quantification of farm and landscape functions

    DEFF Research Database (Denmark)

    Andersen, Peter Stubkjær

    This PhD thesis presents a study on the spatial distribution of agricultural functions at farm and landscape levels. The study focuses on conceptualization of multifunctionality. The concrete conceptual steps include: identification of indicators of four farm and landscape functions – production......, residence, habitat, and recreation; development of a method for quantifying farm functionality and assessing multifunctionality; and definition of a farm typology based on multifunctionality strategies. Empirical data from farm interviews were used in the study to test the developed methods. The results...... is generally decreases and a tendency of increased segregation of the rural landscape is observed. In perspective, further studies on quantification in tangible units, synergies and trade-offs between functions at different scales, and correlations between structures and functions are needed....

  20. Characterisation and quantification of regional diurnal SST cycles from SEVIRI

    DEFF Research Database (Denmark)

    Karagali, Ioanna; Høyer, J.L.

    2014-01-01

    Hourly SST (sea surface temperature) fields from the geostationary Spinning Enhanced Visible and Infrared Imager (SEVIRI) offer a unique opportunity for the characterisation and quantification of the diurnal cycle of SST in the Atlantic Ocean, the Mediterranean Sea and the northern European shelf....... The different methodologies tested for the foundation temperature fields reveal variability introduced by averaging night-time SSTs over many days compared to single-day, pre-dawn values. Diurnal warming is most pronounced in the Mediterranean and Baltic seas while weaker diurnal signals are found...... in the tropics. Longer diurnal warming duration is identified in the high latitudes compared to the tropics. The maximum monthly mean diurnal signal can be up to 0.5K in specific regions....

  1. Microplastics in Baltic bottom sediments: Quantification procedures and first results.

    Science.gov (United States)

    Zobkov, M; Esiukova, E

    2017-01-30

    Microplastics in the marine environment are known as a global ecological problem but there are still no standardized analysis procedures for their quantification. The first breakthrough in this direction was the NOAA Laboratory Methods for quantifying synthetic particles in water and sediments, but fibers numbers have been found to be underestimated with this approach. We propose modifications for these methods that will allow us to analyze microplastics in bottom sediments, including small fibers. Addition of an internal standard to sediment samples and occasional empty runs are advised for analysis quality control. The microplastics extraction efficiency using the proposed modifications is 92±7%. Distribution of microplastics in bottom sediments of the Russian part of the Baltic Sea is presented. Microplastic particles were found in all of the samples with an average concentration of 34±10 items/kg DW and have the same order of magnitude as neighbor studies reported. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Uncertainty quantification in computational fluid dynamics and aircraft engines

    CERN Document Server

    Montomoli, Francesco; D'Ammaro, Antonio; Massini, Michela; Salvadori, Simone

    2015-01-01

    This book introduces novel design techniques developed to increase the safety of aircraft engines. The authors demonstrate how the application of uncertainty methods can overcome problems in the accurate prediction of engine lift, caused by manufacturing error. This in turn ameliorates the difficulty of achieving required safety margins imposed by limits in current design and manufacturing methods. This text shows that even state-of-the-art computational fluid dynamics (CFD) are not able to predict the same performance measured in experiments; CFD methods assume idealised geometries but ideal geometries do not exist, cannot be manufactured and their performance differs from real-world ones. By applying geometrical variations of a few microns, the agreement with experiments improves dramatically, but unfortunately the manufacturing errors in engines or in experiments are unknown. In order to overcome this limitation, uncertainty quantification considers the probability density functions of manufacturing errors...

  3. High-Throughput Thermodynamic Modeling and Uncertainty Quantification for ICME

    Science.gov (United States)

    Otis, Richard A.; Liu, Zi-Kui

    2017-05-01

    One foundational component of the integrated computational materials engineering (ICME) and Materials Genome Initiative is the computational thermodynamics based on the calculation of phase diagrams (CALPHAD) method. The CALPHAD method pioneered by Kaufman has enabled the development of thermodynamic, atomic mobility, and molar volume databases of individual phases in the full space of temperature, composition, and sometimes pressure for technologically important multicomponent engineering materials, along with sophisticated computational tools for using the databases. In this article, our recent efforts will be presented in terms of developing new computational tools for high-throughput modeling and uncertainty quantification based on high-throughput, first-principles calculations and the CALPHAD method along with their potential propagations to downstream ICME modeling and simulations.

  4. Raman spectroscopy for DNA quantification in cell nucleus.

    Science.gov (United States)

    Okotrub, K A; Surovtsev, N V; Semeshin, V F; Omelyanchuk, L V

    2015-01-01

    Here we demonstrate the feasibility of a novel approach to quantify DNA in cell nuclei. This approach is based on spectroscopy analysis of Raman light scattering, and avoids the problem of nonstoichiometric binding of dyes to DNA, as it directly measures the signal from DNA. Quantitative analysis of nuclear DNA contribution to Raman spectrum could be reliably performed using intensity of a phosphate mode at 1096 cm(-1) . When compared to the known DNA standards from cells of different animals, our results matched those values at error of 10%. We therefore suggest that this approach will be useful to expand the list of DNA standards, to properly adjust the duration of hydrolysis in Feulgen staining, to assay the applicability of fuchsines for DNA quantification, as well as to measure DNA content in cells with complex hydrolysis patterns, when Feulgen densitometry is inappropriate. © 2014 International Society for Advancement of Cytometry.

  5. Enhanced peptide quantification using spectral count clustering and cluster abundance

    Directory of Open Access Journals (Sweden)

    Lee Seungmook

    2011-10-01

    Full Text Available Abstract Background Quantification of protein expression by means of mass spectrometry (MS has been introduced in various proteomics studies. In particular, two label-free quantification methods, such as spectral counting and spectra feature analysis have been extensively investigated in a wide variety of proteomic studies. The cornerstone of both methods is peptide identification based on a proteomic database search and subsequent estimation of peptide retention time. However, they often suffer from restrictive database search and inaccurate estimation of the liquid chromatography (LC retention time. Furthermore, conventional peptide identification methods based on the spectral library search algorithms such as SEQUEST or SpectraST have been found to provide neither the best match nor high-scored matches. Lastly, these methods are limited in the sense that target peptides cannot be identified unless they have been previously generated and stored into the database or spectral libraries. To overcome these limitations, we propose a novel method, namely Quantification method based on Finding the Identical Spectral set for a Homogenous peptide (Q-FISH to estimate the peptide's abundance from its tandem mass spectrometry (MS/MS spectra through the direct comparison of experimental spectra. Intuitively, our Q-FISH method compares all possible pairs of experimental spectra in order to identify both known and novel proteins, significantly enhancing identification accuracy by grouping replicated spectra from the same peptide targets. Results We applied Q-FISH to Nano-LC-MS/MS data obtained from human hepatocellular carcinoma (HCC and normal liver tissue samples to identify differentially expressed peptides between the normal and disease samples. For a total of 44,318 spectra obtained through MS/MS analysis, Q-FISH yielded 14,747 clusters. Among these, 5,777 clusters were identified only in the HCC sample, 6,648 clusters only in the normal tissue sample

  6. Imaging and Quantification of Extracellular Vesicles by Transmission Electron Microscopy.

    Science.gov (United States)

    Linares, Romain; Tan, Sisareuth; Gounou, Céline; Brisson, Alain R

    2017-01-01

    Extracellular vesicles (EVs) are cell-derived vesicles that are present in blood and other body fluids. EVs raise major interest for their diverse physiopathological roles and their potential biomedical applications. However, the characterization and quantification of EVs constitute major challenges, mainly due to their small size and the lack of methods adapted for their study. Electron microscopy has made significant contributions to the EV field since their initial discovery. Here, we describe the use of two transmission electron microscopy (TEM) techniques for imaging and quantifying EVs. Cryo-TEM combined with receptor-specific gold labeling is applied to reveal the morphology, size, and phenotype of EVs, while their enumeration is achieved after high-speed sedimentation on EM grids.

  7. Capacitive immunosensor for C-reactive protein quantification

    KAUST Repository

    Sapsanis, Christos

    2015-08-02

    We report an agglutination-based immunosensor for the quantification of C-reactive protein (CRP). The developed immunoassay sensor requires approximately 15 minutes of assay time per sample and provides a sensitivity of 0.5 mg/L. We have measured the capacitance of interdigitated electrodes (IDEs) and quantified the concentration of added analyte. The proposed method is a label free detection method and hence provides rapid measurement preferable in diagnostics. We have so far been able to quantify the concentration to as low as 0.5 mg/L and as high as 10 mg/L. By quantifying CRP in serum, we can assess whether patients are prone to cardiac diseases and monitor the risk associated with such diseases. The sensor is a simple low cost structure and it can be a promising device for rapid and sensitive detection of disease markers at the point-of-care stage.

  8. Plasticity models of material variability based on uncertainty quantification techniques

    Energy Technology Data Exchange (ETDEWEB)

    Jones, Reese E. [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Rizzi, Francesco [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Boyce, Brad [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Templeton, Jeremy Alan [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Ostien, Jakob [Sandia National Lab. (SNL-CA), Livermore, CA (United States)

    2017-11-01

    The advent of fabrication techniques like additive manufacturing has focused attention on the considerable variability of material response due to defects and other micro-structural aspects. This variability motivates the development of an enhanced design methodology that incorporates inherent material variability to provide robust predictions of performance. In this work, we develop plasticity models capable of representing the distribution of mechanical responses observed in experiments using traditional plasticity models of the mean response and recently developed uncertainty quantification (UQ) techniques. Lastly, we demonstrate that the new method provides predictive realizations that are superior to more traditional ones, and how these UQ techniques can be used in model selection and assessing the quality of calibrated physical parameters.

  9. Tannins quantification in barks of Mimosa tenuiflora and Acacia mearnsii

    Directory of Open Access Journals (Sweden)

    Leandro Calegari

    2016-03-01

    Full Text Available Due to its chemical complexity, there are several methodologies for vegetable tannins quantification. Thus, this work aims at quantifying both tannin and non-tannin substances present in the barks of Mimosa tenuiflora and Acacia mearnsii by two different methods. From bark particles of both species, analytical solutions were produced by using a steam-jacketed extractor. The solution was analyzed by Stiasny and hide-powder (no chromed methods. For both species, tannin levels were superior when analyzed by hide-powder method, reaching 47.8% and 24.1% for A. mearnsii and M. tenuiflora, respectively. By Stiasny method, the tannins levels considered were 39.0% for A. mearnsii, and 15.5% for M. tenuiflora. Despite the best results presented by A. mearnsii, the bark of M. tenuiflora also showed great potential due to its considerable amount of tannin and the availability of the species at Caatinga biome.

  10. Nanodiamond arrays on glass for quantification and fluorescence characterisation.

    Science.gov (United States)

    Heffernan, Ashleigh H; Greentree, Andrew D; Gibson, Brant C

    2017-08-23

    Quantifying the variation in emission properties of fluorescent nanodiamonds is important for developing their wide-ranging applicability. Directed self-assembly techniques show promise for positioning nanodiamonds precisely enabling such quantification. Here we show an approach for depositing nanodiamonds in pre-determined arrays which are used to gather statistical information about fluorescent lifetimes. The arrays were created via a layer of photoresist patterned with grids of apertures using electron beam lithography and then drop-cast with nanodiamonds. Electron microscopy revealed a 90% average deposition yield across 3,376 populated array sites, with an average of 20 nanodiamonds per site. Confocal microscopy, optimised for nitrogen vacancy fluorescence collection, revealed a broad distribution of fluorescent lifetimes in agreement with literature. This method for statistically quantifying fluorescent nanoparticles provides a step towards fabrication of hybrid photonic devices for applications from quantum cryptography to sensing.

  11. Multiscale Modeling and Uncertainty Quantification for Nuclear Fuel Performance

    Energy Technology Data Exchange (ETDEWEB)

    Estep, Donald [Colorado State Univ., Fort Collins, CO (United States); El-Azab, Anter [Florida State Univ., Tallahassee, FL (United States); Pernice, Michael [Idaho National Lab. (INL), Idaho Falls, ID (United States); Peterson, John W. [Idaho National Lab. (INL), Idaho Falls, ID (United States); Polyakov, Peter [Univ. of Wyoming, Laramie, WY (United States); Tavener, Simon [Colorado State Univ., Fort Collins, CO (United States); Xiu, Dongbin [Purdue Univ., West Lafayette, IN (United States); Univ. of Utah, Salt Lake City, UT (United States)

    2017-03-23

    In this project, we will address the challenges associated with constructing high fidelity multiscale models of nuclear fuel performance. We (*) propose a novel approach for coupling mesoscale and macroscale models, (*) devise efficient numerical methods for simulating the coupled system, and (*) devise and analyze effective numerical approaches for error and uncertainty quantification for the coupled multiscale system. As an integral part of the project, we will carry out analysis of the effects of upscaling and downscaling, investigate efficient methods for stochastic sensitivity analysis of the individual macroscale and mesoscale models, and carry out a posteriori error analysis for computed results. We will pursue development and implementation of solutions in software used at Idaho National Laboratories on models of interest to the Nuclear Energy Advanced Modeling and Simulation (NEAMS) program.

  12. Improved semiquantitative Western blot technique with increased quantification range.

    Science.gov (United States)

    Heidebrecht, F; Heidebrecht, A; Schulz, I; Behrens, S-E; Bader, A

    2009-06-30

    With the development of new interdisciplinary fields such as systems biology, the quantitative analysis of protein expression in biological samples gains more and more importance. Although the most common method for this is ELISA, Western blot also has advantages: The separation of proteins by size allows the evaluation of only specifically bound protein. This work examines the Western blot signal chain, determines some of the parameters relevant for quantitative analysis and proposes a mathematical model of the reaction kinetics. Using this model, a semiquantitative Western blot method for simultaneous quantification of different proteins using a hyperbolic calibration curve was developed. A program was written for the purpose of hyperbolic regression that allows quick determination of the calibration curve coefficients. This program can be used also for approximation of calibration curves in other applications such as ELISA, BCA or Bradford assays.

  13. Quantification of rutile in anatase by X-ray diffraction

    International Nuclear Information System (INIS)

    Chavez R, A.

    2001-01-01

    Nowadays the discovering of new and better materials required in all areas of the industry has been lead to the human being to introduce him to this small and great world. The crystalline materials, have properties markedly directional. When it is necessary to realize a quantitative analysis to these materials the task is not easy. The main objective of this work is the research of a real problem, its solution and perfecting of a technique involving the theoretical and experimental principles which allow the quantification of crystalline phases. The chapter 1 treats about the study of crystalline state during the last century, by means of the X-ray diffraction technique. The chapter 2 studies the nature and production of X-rays, the chapter 3 expounds the principles of the diffraction technique which to carry out when it is satisfied the Bragg law studying the powder diffraction method and its applications. In the chapter 4 it is explained how the intensities of the beams diffracted are determined by the atoms positions inside of the elemental cell of the crystal. The properties of the crystalline samples of anatase and rutile are described in the chapter 5. The results of this last analysis are the information which will be processed by means of the auxiliary software: Diffrac AT, Axum and Peakfit as well as the TAFOR and CUANTI software describing this part with more detail in the chapters 6 and 7 where it is mentioned step by step the function of each software until to reach the quantification of crystalline phases, objective of this work. Finally, in the chapter 8 there are a results analysis and conclusions. The contribution of this work is for those learned institutions of limited resources which can tackle in this way the characterization of materials. (Author)

  14. Quantification of the genetic risk of environmental mutagens

    International Nuclear Information System (INIS)

    Ehling, U.H.

    1988-01-01

    Screening methods are used for hazard identification. Assays for heritable mutations in mammals are used for the confirmation of short-term test results and for the quantification of the genetic risk. There are two main approaches in making genetic risk estimates. One of these, termed the direct method, expresses risk in terms of the expected frequency of genetic changes induced per unit. The other, referred to as the doubling dose method or the indirect method, expresses risk in relation to the observed incidence of genetic disorders now present in man. The indirect method uses experimental data only for the calculation of the doubling dose. The quality of the risk estimation depends on the assumption of persistence of the induced mutations and the ability to determine the current incidence of genetic diseases. The difficulties of improving the estimates of current incidences of genetic diseases or the persistence of the genes in the population led them to the development of an alternative method, the direct estimation of the genetic risk. The direct estimation uses experimental data for the induced frequency for dominant mutations in mice. For the verification of these quantifications one can use the data of Hiroshima and Nagasaki. According to the estimation with the direct method, one would expect less than 1 radiation-induced dominant cataract in 19,000 children with one or both parents exposed. The expected overall frequency of dominant mutations in the first generation would be 20-25, based on radiation-induced dominant cataract mutations. It is estimated that 10 times more recessive than dominant mutations are induced. The same approaches can be used to determine the impact of chemical mutagens

  15. Surface landmark quantification of embryonic mouse craniofacial morphogenesis.

    Science.gov (United States)

    Percival, Christopher J; Green, Rebecca; Marcucio, Ralph; Hallgrímsson, Benedikt

    2014-07-24

    Morphometric quantification of subtle craniofacial variation in studies of experimentally modified embryonic mice has proved valuable in determining the effects of developmental perturbations on craniofacial morphogenesis. The direct comparison of landmark coordinate data from embryos of many different mouse strains and mouse models can advance our understanding of the bases for craniofacial variation. We propose a standard set of craniofacial surface landmarks, for use with embryonic day (E) 10.5-12.5 mice, to serve as the foundation for this type of data compilation and analysis. We quantify the intra- and inter-observer landmark placement variation associated with each landmark and determine how the results of a simple ontogenetic analysis might be influenced by selection of landmark set. Intraobserver landmark placement error for experienced landmarkers generally remains below 0.1 mm, with some landmarks exhibiting higher values at E11.5 and E12.5. Interobserver error tends to increase with embryonic age and those landmarks defined on wide inflections of curves or facial processes exhibit the highest error. Landmarks with highest intra- or inter-observer are identified and we determine that their removal from the dataset does not significantly change the vectors of craniofacial shape change associated with an ontogenetic regression. Our quantification of landmark placement error demonstrates that it is preferable for a single observer to identify all landmark coordinates within a single study and that significant training and experience are necessary before a landmarker can produce data for use in larger meta-analyses. However, we are confident that this standard landmark set, once landmarks with higher error are removed, can serve as a foundation for a comparative dataset of facial morphogenesis across various mouse populations to help identify the developmental bases for phenotypic variation in the craniofacial complex.

  16. Bayesian Proteoform Modeling Improves Protein Quantification of Global Proteomic Measurements

    Energy Technology Data Exchange (ETDEWEB)

    Webb-Robertson, Bobbie-Jo M.; Matzke, Melissa M.; Datta, Susmita; Payne, Samuel H.; Kang, Jiyun; Bramer, Lisa M.; Nicora, Carrie D.; Shukla, Anil K.; Metz, Thomas O.; Rodland, Karin D.; Smith, Richard D.; Tardiff, Mark F.; McDermott, Jason E.; Pounds, Joel G.; Waters, Katrina M.

    2014-12-01

    As the capability of mass spectrometry-based proteomics has matured, tens of thousands of peptides can be measured simultaneously, which has the benefit of offering a systems view of protein expression. However, a major challenge is that with an increase in throughput, protein quantification estimation from the native measured peptides has become a computational task. A limitation to existing computationally-driven protein quantification methods is that most ignore protein variation, such as alternate splicing of the RNA transcript and post-translational modifications or other possible proteoforms, which will affect a significant fraction of the proteome. The consequence of this assumption is that statistical inference at the protein level, and consequently downstream analyses, such as network and pathway modeling, have only limited power for biomarker discovery. Here, we describe a Bayesian model (BP-Quant) that uses statistically derived peptides signatures to identify peptides that are outside the dominant pattern, or the existence of multiple over-expressed patterns to improve relative protein abundance estimates. It is a research-driven approach that utilizes the objectives of the experiment, defined in the context of a standard statistical hypothesis, to identify a set of peptides exhibiting similar statistical behavior relating to a protein. This approach infers that changes in relative protein abundance can be used as a surrogate for changes in function, without necessarily taking into account the effect of differential post-translational modifications, processing, or splicing in altering protein function. We verify the approach using a dilution study from mouse plasma samples and demonstrate that BP-Quant achieves similar accuracy as the current state-of-the-art methods at proteoform identification with significantly better specificity. BP-Quant is available as a MatLab ® and R packages at https://github.com/PNNL-Comp-Mass-Spec/BP-Quant.

  17. Longitudinal MRI quantification of muscle degeneration in Duchenne muscular dystrophy.

    Science.gov (United States)

    Godi, Claudia; Ambrosi, Alessandro; Nicastro, Francesca; Previtali, Stefano C; Santarosa, Corrado; Napolitano, Sara; Iadanza, Antonella; Scarlato, Marina; Natali Sora, Maria Grazia; Tettamanti, Andrea; Gerevini, Simonetta; Cicalese, Maria Pia; Sitzia, Clementina; Venturini, Massimo; Falini, Andrea; Gatti, Roberto; Ciceri, Fabio; Cossu, Giulio; Torrente, Yvan; Politi, Letterio S

    2016-08-01

    The aim of this study was to evaluate the usefulness of magnetic resonance imaging (MRI) in detecting the progression of Duchenne muscular dystrophy (DMD) by quantification of fat infiltration (FI) and muscle volume index (MVI, a residual-to-total muscle volume ratio). Twenty-six patients (baseline age: 5-12 years) with genetically proven DMD were longitudinally analyzed with lower limb 3T MRI, force measurements, and functional tests (Gowers, 10-m time, North Star Ambulatory Assessment, 6-min walking test). Five age-matched controls were also examined, with a total of 85 MRI studies. Semiquantitative (scores) and quantitative MRI (qMRI) analyses (signal intensity ratio - SIR, lower limb MVI, and individual muscle MVI) were carried out. Permutation and regression analyses according to both age and functional test-outcomes were calculated. Age-related quantitative reference curves of SIRs and MVIs were generated. FI was present on glutei and adductor magnus in all patients since the age of 5, with a proximal-to-distal progression and selective sparing of sartorius and gracilis. Patients' qMRI measures were significantly different from controls' and among age classes. qMRI were more sensitive than force measurements and functional tests in assessing disease progression, allowing quantification also after loss of ambulation. Age-related curves with percentile values were calculated for SIRs and MVIs, to provide a reference background for future experimental therapy trials. SIRs and MVIs significantly correlated with all clinical measures, and could reliably predict functional outcomes and loss of ambulation. qMRI-based indexes are sensitive measures that can track the progression of DMD and represent a valuable tool for follow-up and clinical studies.

  18. Uncertainty quantification applied to the radiological characterization of radioactive waste.

    Science.gov (United States)

    Zaffora, B; Magistris, M; Saporta, G; Chevalier, J-P

    2017-09-01

    This paper describes the process adopted at the European Organization for Nuclear Research (CERN) to quantify uncertainties affecting the characterization of very-low-level radioactive waste. Radioactive waste is a by-product of the operation of high-energy particle accelerators. Radioactive waste must be characterized to ensure its safe disposal in final repositories. Characterizing radioactive waste means establishing the list of radionuclides together with their activities. The estimated activity levels are compared to the limits given by the national authority of the waste disposal. The quantification of the uncertainty affecting the concentration of the radionuclides is therefore essential to estimate the acceptability of the waste in the final repository but also to control the sorting, volume reduction and packaging phases of the characterization process. The characterization method consists of estimating the activity of produced radionuclides either by experimental methods or statistical approaches. The uncertainties are estimated using classical statistical methods and uncertainty propagation. A mixed multivariate random vector is built to generate random input parameters for the activity calculations. The random vector is a robust tool to account for the unknown radiological history of legacy waste. This analytical technique is also particularly useful to generate random chemical compositions of materials when the trace element concentrations are not available or cannot be measured. The methodology was validated using a waste population of legacy copper activated at CERN. The methodology introduced here represents a first approach for the uncertainty quantification (UQ) of the characterization process of waste produced at particle accelerators. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Event-Specific Quantification of Radiation Belt Radial Diffusion

    Science.gov (United States)

    Tu, W.; Sarris, T. E.; Ozeke, L.

    2016-12-01

    Recently, there has been a great emphasis on developing event-specific inputs for radiation belt models, since they are proven critical for reproducing the observed radiation belt dynamics during strong events. For example, our DREAM3D simulation of the 8-9 October 2012 storm demonstrates that event-specific chorus wave model and seed population are critical to reproduce the strong enhancement of MeV electrons in this event. However, the observed fast electron dropout preceding the enhancement was not captured by the simulation, which could be due to the combined effects of fast outward radial diffusion of radiation belt electrons with magnetopause shadowing and enhanced electron precipitation. Without an event-specific quantification of radial diffusion, we cannot resolve the relative contribution of outward radial diffusion and precipitation to the observed electron dropout or realistically reproduce the dynamics during the event. In this work, we provide physical quantification of radial diffusion specific to the October 2012 event by including both real-time and global distributions of ULF waves from a constellation of wave measurements and event-specific estimation of ULF wave mode structure. The global maps of ULF waves during the event are constructed by combining the real-time measurements from the Van Allen Probes, THEMIS, and GOES satellites in space and a large array of ground magnetometers. The real-time ULF wave mode structure is then estimated using the new Cross-Wavelet Transform technique, applied to various azimuthally aligned pairs of ULF wave measurements that are located at the same L shells. The cross power and phase differences between the time series are calculated using the technique, based on which the wave power per mode number is estimated. Finally, the physically estimated radial diffusion coefficients specific to the event are applied to the DREAM3D model to quantify the relative contribution of radial diffusion to the electron dynamics

  20. A novel immunological assay for hepcidin quantification in human serum.

    Directory of Open Access Journals (Sweden)

    Vasiliki Koliaraki

    Full Text Available BACKGROUND: Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera. METHODS AND FINDINGS: An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10-1500 microg/L and a detection limit of 5.4 microg/L. The intra- and interassay coefficients of variance ranged from 8-15% and 5-16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 microg/L and 10 patients with iron deficiency anemia (15.7 microg/L and higher in 7 patients with Hodgkin lymphoma (116.7 microg/L compared to 32 age-matched healthy controls (42.7 microg/L. CONCLUSIONS: We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.

  1. Biventricular MR volumetric analysis and MR flow quantification in the ascending aorta and pulmonary trunk for quantification of valvular regurgitation

    International Nuclear Information System (INIS)

    Rominger, M.B.

    2004-01-01

    Purpose: To test the value of biventricular volumetric analysis and the combination of biventricular volumetric analysis with flow quantification in the ascending aorta (Ao) and pulmonary trunk (Pu) for quantification of regurgitation volume and cardiac function in valvular regurgitation (VR) according to location and presence of single or multivalvular disease. Materials and Methods: In 106 patients, the stroke volumes were assessed by measuring the biventricular volumes and the forward-stroke volumes in the great and small circulation by measuring the flow in the Ao and Pu. Valve regurgitation volumes and quotients were calculated for single and multivalvular disease and correlated with semiquantitative 2D-echocardiography (grade I-IV). For the assessment of the cardiac function in VR, the volumetric parameters of ejection fraction and end-diastolic (EDV) and end-systolic (ESV) volumes were determined. Results: The detection rate was 49% for left ventricular (LV) VR and 42% for right ventricular (RV) VR. Low LV VR and RV VR usually could not be detected quantitatively, with the detection rate improving with echocardiographically higher insufficiency grades. Quantitative MRI could detect a higher grade solitary aortic valve insufficiency (≥2) in 11 of 12 patients and higher grade mitral valve insufficiency in 4 of 10 patients. A significant increase in RV and LV ventricular EDV and ESV was seen more often with increased MR regurgitation volumes. Aortic stenosis did not interfere with flow measurements in the Ao. Conclusions: Biventricular volumetry combined with flow measurements in Ao and Pu is a robust, applicable and simple method to assess higher grade regurgitation volumes and the cardiac function in single and multivalvular regurgitation at different locations. It is an important application for the diagnosis of VR by MRI [de

  2. Quantification of cellular uptake of DNA nanostructures by qPCR

    DEFF Research Database (Denmark)

    Okholm, Anders Hauge; Nielsen, Jesper Sejrup; Vinther, Mathias

    2014-01-01

    interactions and structural and functional features of the DNA delivery device must be thoroughly investigated. Here, we present a rapid and robust method for the precise quantification of the component materials of DNA origami structures capable of entering cells in vitro. The quantification is performed...... is suitable for quantification of in vitro uptake studies but should easily be extended to quantify DNA nanostructures in blood or tissue samples......DNA nanostructures facilitating drug delivery are likely soon to be realized. In the past few decades programmed self-assembly of DNA building blocks have successfully been employed to construct sophisticated nanoscale objects. By conjugating functionalities to DNA, other molecules such as peptides...

  3. Quantification of rice bran oil in oil blends

    Directory of Open Access Journals (Sweden)

    Mishra, R.

    2012-03-01

    Full Text Available Blends consisting of physically refined rice bran oil (PRBO: sunflower oil (SnF and PRBO: safflower oil (SAF in different proportions were analyzed for various physicochemical parameters. The quantification of pure rice bran oil in the blended oils was carried out using different methods including gas chromatographic, HPLC, ultrasonic velocity and methods based on physico-chemical parameters. The physicochemical parameters such as ultrasonic velocity, relative association and acoustic impedance at 2 MHz, iodine value, palmitic acid content and oryzanol content reflected significant changes with increased proportions of PRBO in the blended oils. These parameters were selected as dependent parameters and % PRBO proportion was selected as independent parameters. The study revealed that regression equations based on the oryzanol content, palmitic acid composition, ultrasonic velocity, relative association, acoustic impedance, and iodine value can be used for the quantification of rice bran oil in blended oils. The rice bran oil can easily be quantified in the blended oils based on the oryzanol content by HPLC even at a 1% level. The palmitic acid content in blended oils can also be used as an indicator to quantify rice bran oil at or above the 20% level in blended oils whereas the method based on ultrasonic velocity, acoustic impedance and relative association showed initial promise in the quantification of rice bran oil.

    Se analizaron diversos parámetros físico-químicos para la evaluación de mezclas de aceites en diferentes proporciones que incluyen: aceite de salvado de arroz físícamente refinado (PRBO: aceite de girasol (SNF y las mezclas PRBO: aceite de cártamo (SAF en diferentes proporciones. La cuantificación de la presencia del aceite de salvado de arroz en las mezclas se llevó a cabo por diferentes métodos, como cromatografía de gases (GC, cromatografía líquida (HPLC, ultrasonidos y métodos basados en otros parámetros f

  4. Preclinical imaging characteristics and quantification of Platinum-195m SPECT

    Energy Technology Data Exchange (ETDEWEB)

    Aalbersberg, E.A.; Wit-van der Veen, B.J. de; Vegt, E.; Vogel, Wouter V. [The Netherlands Cancer Institute (NKI-AVL), Department of Nuclear Medicine, Amsterdam (Netherlands); Zwaagstra, O.; Codee-van der Schilden, K. [Nuclear Research and Consultancy Group (NRG), Petten (Netherlands)

    2017-08-15

    In vivo biodistribution imaging of platinum-based compounds may allow better patient selection for treatment with chemo(radio)therapy. Radiolabeling with Platinum-195m ({sup 195m}Pt) allows SPECT imaging, without altering the chemical structure or biological activity of the compound. We have assessed the feasibility of {sup 195m}Pt SPECT imaging in mice, with the aim to determine the image quality and accuracy of quantification for current preclinical imaging equipment. Enriched (>96%) {sup 194}Pt was irradiated in the High Flux Reactor (HFR) in Petten, The Netherlands (NRG). A 0.05 M HCl {sup 195m}Pt-solution with a specific activity of 33 MBq/mg was obtained. Image quality was assessed for the NanoSPECT/CT (Bioscan Inc., Washington DC, USA) and U-SPECT{sup +}/CT (MILabs BV, Utrecht, the Netherlands) scanners. A radioactivity-filled rod phantom (rod diameter 0.85-1.7 mm) filled with 1 MBq {sup 195m}Pt was scanned with different acquisition durations (10-120 min). Four healthy mice were injected intravenously with 3-4 MBq {sup 195m}Pt. Mouse images were acquired with the NanoSPECT for 120 min at 0, 2, 4, or 24 h after injection. Organs were delineated to quantify {sup 195m}Pt concentrations. Immediately after scanning, the mice were sacrificed, and the platinum concentration was determined in organs using a gamma counter and graphite furnace - atomic absorption spectroscopy (GF-AAS) as reference standards. A 30-min acquisition of the phantom provided visually adequate image quality for both scanners. The smallest visible rods were 0.95 mm in diameter on the NanoSPECT and 0.85 mm in diameter on the U-SPECT{sup +}. The image quality in mice was visually adequate. Uptake was seen in the kidneys with excretion to the bladder, and in the liver, blood, and intestine. No uptake was seen in the brain. The Spearman correlation between SPECT and gamma counter was 0.92, between SPECT and GF-AAS it was 0.84, and between GF-AAS and gamma counter it was0.97 (all p < 0

  5. 3C-digital PCR for quantification of chromatin interactions.

    Science.gov (United States)

    Du, Meijun; Wang, Liang

    2016-12-06

    Chromosome conformation capture (3C) is a powerful and widely used technique for detecting the physical interactions between chromatin regions in vivo. The principle of 3C is to convert physical chromatin interactions into specific DNA ligation products, which are then detected by quantitative polymerase chain reaction (qPCR). However, 3C-qPCR assays are often complicated by the necessity of normalization controls to correct for amplification biases. In addition, qPCR is often limited to a certain cycle number, making it difficult to detect fragment ligations with low frequency. Recently, digital PCR (dPCR) technology has become available, which allows for highly sensitive nucleic acid quantification. Main advantage of dPCR is its high precision of absolute nucleic acid quantification without requirement of normalization controls. To demonstrate the utility of dPCR in quantifying chromatin interactions, we examined two prostate cancer risk loci at 8q24 and 2p11.2 for their interaction target genes MYC and CAPG in LNCaP cell line. We designed anchor and testing primers at known regulatory element fragments and target gene regions, respectively. dPCR results showed that interaction frequency between the regulatory element and MYC gene promoter was 0.7 (95% CI 0.40-1.10) copies per 1000 genome copies while other regions showed relatively low ligation frequencies. The dPCR results also showed that the ligation frequencies between the regulatory element and two EcoRI fragments containing CAPG gene promoter were 1.9 copies (95% CI 1.41-2.47) and 1.3 copies per 1000 genome copies (95% CI 0.76-1.92), respectively, while the interaction signals were reduced on either side of the promoter region of CAPG gene. Additionally, we observed comparable results from 3C-dPCR and 3C-qPCR at 2p11.2 in another cell line (DU145). Compared to traditional 3C-qPCR, our results show that 3C-dPCR is much simpler and more sensitive to detect weak chromatin interactions. It may eliminate

  6. Coral Pigments: Quantification Using HPLC and Detection by Remote Sensing

    Science.gov (United States)

    Cottone, Mary C.

    1995-01-01

    Widespread coral bleaching (loss of pigments of symbiotic dinoflagellates), and the corresponding decline in coral reef health worldwide, mandates the monitoring of coral pigmentation. Samples of the corals Porites compressa and P. lobata were collected from a healthy reef at Puako, Hawaii, and chlorophyll (chl) a, peridinin, and Beta-carotene (Beta-car) were quantified using reverse-phase high performance liquid chromatography (HPLC). Detailed procedures are presented for the extraction of the coral pigments in 90% acetone, and the separation, identification, and quantification of the major zooxanthellar pigments using spectrophotometry and a modification of the HPLC system described by Mantoura and Llewellyn (1983). Beta-apo-8-carotenal was found to be inadequate as in internal standard, due to coelution with chl b and/or chl a allomer in the sample extracts. Improvements are suggested, which may result in better resolution of the major pigments and greater accuracy in quantification. Average concentrations of peridinin, chl a, and Beta-car in corals on the reef were 5.01, 8.59, and 0.29, micro-grams/cm(exp 2), respectively. Average concentrations of peridinin and Beta-car did not differ significantly between the two coral species sampled; however, the mean chl a concentration in P. compressa specimens (7.81 ,micro-grams/cm(exp 2) was significantly lower than that in P. lobata specimens (9.96 11g/cm2). Chl a concentrations determined spectrophotometrically were significantly higher than those generated through HPLC, suggesting that spectrophotometry overestimates chl a concentrations. The average ratio of chl a-to-peridinin concentrations was 1.90, with a large (53%) coefficient of variation and a significant difference between the two species sampled. Additional data are needed before conclusions can be drawn regarding average pigment concentrations in healthy corals and the consistency of the chl a/peridinin ratio. The HPLC pigment concentration values

  7. Computer-assisted radiological quantification of rheumatoid arthritis

    International Nuclear Information System (INIS)

    Peloschek, P.L.

    2000-03-01

    Specific objective was to develop the layout and structure of a platform for effective quantification of rheumatoid arthritis (RA). A fully operative Java stand-alone application software (RheumaCoach) was developed to support the efficacy of the scoring process in RA (Web address: http://www.univie.ac.at/radio/radio.htm). Addressed as potential users of such a program are physicians enrolled in clinical trials to evaluate the course of RA and its modulation with drug therapies and scientists developing new scoring modalities. The software 'RheumaCoach' consists of three major modules: The Tutorial starts with 'Rheumatoid Arthritis', to teach the basic pathology of the disease. Afterwards the section 'Imaging Standards' explains how to produce proper radiographs. 'Principles - How to use the 'Larsen Score', 'Radiographic Findings' and 'Quantification by Scoring' explain the requirements for unbiased scoring of RA. At the Data Input Sheet care was taken to follow the radiologist's approach in analysing films as published previously. At the compute sheet the calculated Larsen-Score may be compared with former scores and the further possibilities (calculate, export, print, send) are easily accessible. In a first pre-clinical study the system was tested in an unstructured. Two structured evaluations (30 fully documented and blinded cases of RA, four radiologists scored hands and feet with or without the RheumaCoach) followed. Between the evaluations we permanently improved the software. For all readers the usage of the RheumaCoach fastened the procedure, all together the scoring without computer-assistance needed about 20 % percent more time. Availability of the programme via the internet provides common access for potential quality control in multi-center studies. Documentation of results in a specifically designed printout improves communication between radiologists and rheumatologists. The possibilities of direct export to other programmes and electronic

  8. Modeling transport phenomena and uncertainty quantification in solidification processes

    Science.gov (United States)

    Fezi, Kyle S.

    Direct chill (DC) casting is the primary processing route for wrought aluminum alloys. This semicontinuous process consists of primary cooling as the metal is pulled through a water cooled mold followed by secondary cooling with a water jet spray and free falling water. To gain insight into this complex solidification process, a fully transient model of DC casting was developed to predict the transport phenomena of aluminum alloys for various conditions. This model is capable of solving mixture mass, momentum, energy, and species conservation equations during multicomponent solidification. Various DC casting process parameters were examined for their effect on transport phenomena predictions in an alloy of commercial interest (aluminum alloy 7050). The practice of placing a wiper to divert cooling water from the ingot surface was studied and the results showed that placement closer to the mold causes remelting at the surface and increases susceptibility to bleed outs. Numerical models of metal alloy solidification, like the one previously mentioned, are used to gain insight into physical phenomena that cannot be observed experimentally. However, uncertainty in model inputs cause uncertainty in results and those insights. The analysis of model assumptions and probable input variability on the level of uncertainty in model predictions has not been calculated in solidification modeling as yet. As a step towards understanding the effect of uncertain inputs on solidification modeling, uncertainty quantification (UQ) and sensitivity analysis were first performed on a transient solidification model of a simple binary alloy (Al-4.5wt.%Cu) in a rectangular cavity with both columnar and equiaxed solid growth models. This analysis was followed by quantifying the uncertainty in predictions from the recently developed transient DC casting model. The PRISM Uncertainty Quantification (PUQ) framework quantified the uncertainty and sensitivity in macrosegregation, solidification

  9. Uncertainty Quantification Bayesian Framework for Porous Media Flows

    Science.gov (United States)

    Demyanov, V.; Christie, M.; Erbas, D.

    2005-12-01

    Uncertainty quantification is an increasingly important aspect of many areas of applied science, where the challenge is to make reliable predictions about the performance of complex physical systems in the absence of complete or reliable data. Predicting flows of fluids through undersurface reservoirs is an example of a complex system where accuracy in prediction is needed (e.g. in oil industry it is essential for financial reasons). Simulation of fluid flow in oil reservoirs is usually carried out using large commercially written finite difference simulators solving conservation equations describing the multi-phase flow through the porous reservoir rocks, which is a highly computationally expensive task. This work examines a Bayesian Framework for uncertainty quantification in porous media flows that uses a stochastic sampling algorithm to generate models that match observed time series data. The framework is flexible for a wide range of general physical/statistical parametric models, which are used to describe the underlying hydro-geological process in its temporal dynamics. The approach is based on exploration of the parameter space and update of the prior beliefs about what the most likely model definitions are. Optimization problem for a highly parametric physical model usually have multiple solutions, which impact the uncertainty of the made predictions. Stochastic search algorithm (e.g. genetic algorithm) allows to identify multiple "good enough" models in the parameter space. Furthermore, inference of the generated model ensemble via MCMC based algorithm evaluates the posterior probability of the generated models and quantifies uncertainty of the predictions. Machine learning algorithm - Artificial Neural Networks - are used to speed up the identification of regions in parameter space where good matches to observed data can be found. Adaptive nature of ANN allows to develop different ways of integrating them into the Bayesian framework: as direct time

  10. Development of hydrate risk quantification in oil and gas production

    Science.gov (United States)

    Chaudhari, Piyush N.

    order to reduce the parametric study that may require a long duration of time using The Colorado School of Mines Hydrate Kinetic Model (CSMHyK). The evolution of the hydrate plugging risk along flowline-riser systems is modeled for steady state and transient operations considering the effect of several critical parameters such as oil-hydrate slip, duration of shut-in, and water droplet size on a subsea tieback system. This research presents a novel platform for quantification of the hydrate plugging risk, which in-turn will play an important role in improving and optimizing current hydrate management strategies. The predictive strength of the hydrate risk quantification and hydrate prediction models will have a significant impact on flow assurance engineering and design with respect to building safe and efficient hydrate management techniques for future deep-water developments.

  11. Aerosol-type retrieval and uncertainty quantification from OMI data

    Science.gov (United States)

    Kauppi, Anu; Kolmonen, Pekka; Laine, Marko; Tamminen, Johanna

    2017-11-01

    We discuss uncertainty quantification for aerosol-type selection in satellite-based atmospheric aerosol retrieval. The retrieval procedure uses precalculated aerosol microphysical models stored in look-up tables (LUTs) and top-of-atmosphere (TOA) spectral reflectance measurements to solve the aerosol characteristics. The forward model approximations cause systematic differences between the modelled and observed reflectance. Acknowledging this model discrepancy as a source of uncertainty allows us to produce more realistic uncertainty estimates and assists the selection of the most appropriate LUTs for each individual retrieval.This paper focuses on the aerosol microphysical model selection and characterisation of uncertainty in the retrieved aerosol type and aerosol optical depth (AOD). The concept of model evidence is used as a tool for model comparison. The method is based on Bayesian inference approach, in which all uncertainties are described as a posterior probability distribution. When there is no single best-matching aerosol microphysical model, we use a statistical technique based on Bayesian model averaging to combine AOD posterior probability densities of the best-fitting models to obtain an averaged AOD estimate. We also determine the shared evidence of the best-matching models of a certain main aerosol type in order to quantify how plausible it is that it represents the underlying atmospheric aerosol conditions.The developed method is applied to Ozone Monitoring Instrument (OMI) measurements using a multiwavelength approach for retrieving the aerosol type and AOD estimate with uncertainty quantification for cloud-free over-land pixels. Several larger pixel set areas were studied in order to investigate the robustness of the developed method. We evaluated the retrieved AOD by comparison with ground-based measurements at example sites. We found that the uncertainty of AOD expressed by posterior probability distribution reflects the difficulty in model

  12. Aerosol-type retrieval and uncertainty quantification from OMI data

    Directory of Open Access Journals (Sweden)

    A. Kauppi

    2017-11-01

    Full Text Available We discuss uncertainty quantification for aerosol-type selection in satellite-based atmospheric aerosol retrieval. The retrieval procedure uses precalculated aerosol microphysical models stored in look-up tables (LUTs and top-of-atmosphere (TOA spectral reflectance measurements to solve the aerosol characteristics. The forward model approximations cause systematic differences between the modelled and observed reflectance. Acknowledging this model discrepancy as a source of uncertainty allows us to produce more realistic uncertainty estimates and assists the selection of the most appropriate LUTs for each individual retrieval.This paper focuses on the aerosol microphysical model selection and characterisation of uncertainty in the retrieved aerosol type and aerosol optical depth (AOD. The concept of model evidence is used as a tool for model comparison. The method is based on Bayesian inference approach, in which all uncertainties are described as a posterior probability distribution. When there is no single best-matching aerosol microphysical model, we use a statistical technique based on Bayesian model averaging to combine AOD posterior probability densities of the best-fitting models to obtain an averaged AOD estimate. We also determine the shared evidence of the best-matching models of a certain main aerosol type in order to quantify how plausible it is that it represents the underlying atmospheric aerosol conditions.The developed method is applied to Ozone Monitoring Instrument (OMI measurements using a multiwavelength approach for retrieving the aerosol type and AOD estimate with uncertainty quantification for cloud-free over-land pixels. Several larger pixel set areas were studied in order to investigate the robustness of the developed method. We evaluated the retrieved AOD by comparison with ground-based measurements at example sites. We found that the uncertainty of AOD expressed by posterior probability distribution reflects the

  13. Collaborative framework for PIV uncertainty quantification: the experimental database

    International Nuclear Information System (INIS)

    Neal, Douglas R; Sciacchitano, Andrea; Scarano, Fulvio; Smith, Barton L

    2015-01-01

    The uncertainty quantification of particle image velocimetry (PIV) measurements has recently become a topic of great interest as shown by the recent appearance of several different methods within the past few years. These approaches have different working principles, merits and limitations, which have been speculated upon in subsequent studies. This paper reports a unique experiment that has been performed specifically to test the efficacy of PIV uncertainty methods. The case of a rectangular jet, as previously studied by Timmins et al (2012) and Wilson and Smith (2013b), is used. The novel aspect of the experiment is simultaneous velocity measurements using two different time-resolved PIV systems and a hot-wire anemometry (HWA) system. The first PIV system, called the PIV measurement system (‘PIV-MS’), is intended for nominal measurements of which the uncertainty is to be evaluated. It is based on a single camera and features a dynamic velocity range (DVR) representative of typical PIV experiments. The second PIV system, called the ‘PIV-HDR’ (high dynamic range) system, features a significantly higher DVR obtained with a higher digital imaging resolution. The hot-wire is placed in close proximity to the PIV measurement domain. The three measurement systems were carefully set to simultaneously measure the flow velocity at the same time and location. The comparison between the PIV-HDR system and the HWA provides an estimate of the measurement precision of the reference velocity for evaluation of the instantaneous error in the measurement system. The discrepancy between the PIV-MS and the reference data provides the measurement error, which is later used to assess the different uncertainty quantification methods proposed in the literature. A detailed comparison of the uncertainty estimation methods based on the present datasets is presented in a second paper from Sciacchitano et al (2015). Furthermore, this database offers the potential to be used for

  14. Optofluidic interferometry chip designs of differential NIR absorbance based sensors for identification and quantification of electrolytes

    NARCIS (Netherlands)

    Steen, Gerrit W.; Wexler, Adam D.; Offerhaus, Herman L.

    2014-01-01

    Design and optimization of integrated photonic NIR absorbance based sensors for identification and quantification of aqueous electrolytes was performed by simulation in MATLAB and Optodesigner. Ten designs are presented and compared for suitability.

  15. The Effect of AOP on Software Engineering, with Particular Attention to OIF and Event Quantification

    Science.gov (United States)

    Havelund, Klaus; Filman, Robert; Korsmeyer, David (Technical Monitor)

    2003-01-01

    We consider the impact of Aspect-Oriented Programming on Software Engineering, and, in particular, analyze two AOP systems, one of which does component wrapping and the other, quantification over events, for their software engineering effects.

  16. Optimisation of chloride quantification in cementitious mortars using energy-dispersive X-ray analysis

    NARCIS (Netherlands)

    Pacheco, J.; Çopuroǧlu, O.; Polder, R.B.

    2013-01-01

    Chlorides are responsible for initiating steel corrosion in reinforced concrete, the economically most important deterioration mechanism in concrete infrastructure. The quantification of chlorides is commonly performed by wet chemical analysis, e.g. acid dissolution and Volhard's titration.

  17. 3.8 Proposed approach to uncertainty quantification and sensitivity analysis in the next PA

    Energy Technology Data Exchange (ETDEWEB)

    Flach, Greg [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL); Wohlwend, Jen [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

    2017-10-02

    This memorandum builds upon Section 3.8 of SRNL (2016) and Flach (2017) by defining key error analysis, uncertainty quantification, and sensitivity analysis concepts and terms, in preparation for the next E-Area Performance Assessment (WSRC 2008) revision.

  18. A SIMPLE METHOD FOR THE EXTRACTION AND QUANTIFICATION OF PHOTOPIGMENTS FROM SYMBIODINIUM SPP.

    Science.gov (United States)

    John E. Rogers and Dragoslav Marcovich. Submitted. Simple Method for the Extraction and Quantification of Photopigments from Symbiodinium spp.. Limnol. Oceanogr. Methods. 19 p. (ERL,GB 1192). We have developed a simple, mild extraction procedure using methanol which, when...

  19. Quantification of collateral flow in humans: a comparison of angiographic, electrocardiographic and hemodynamic variables

    NARCIS (Netherlands)

    van Liebergen, R. A.; Piek, J. J.; Koch, K. T.; de Winter, R. J.; Schotborgh, C. E.; Lie, K. I.

    1999-01-01

    Evaluation of collateral vascular circulation according to hemodynamic variables and its relation to myocardial ischemia. There is limited information regarding the hemodynamic quantification of recruitable collateral vessels. Angiography of the donor coronary artery was performed before and during

  20. Clinical performance of the VERIS HCV assay for hepatitis C virus RNA quantification.

    Science.gov (United States)

    Izquierdo, Laure; Prégermain, Corinne; Hottelet, Corinne; Decombe, Gwenaëlle; Roque-Afonso, Anne-Marie

    2017-08-01

    Diagnosis of hepatitis C virus (HCV) infection and treatment monitoring rely on detection/quantification of HCV RNA and real-time polymerase chain reaction (PCR) techniques are expected to equivalently quantify the different HCV genotypes. The clinical performance of the VERIS HCV assay for HCV RNA quantification was compared to that of the Abbott RealTime HCV assay. Qualitative concordance and quantitative comparison were evaluated on a first panel of 286 clinical samples containing HCV genotypes 1-6. Forty additional genotype 4 samples were tested to explore genotype 4 HCV RNA underquantification. Qualitative discrepancies were observed for low viral loads (HCV RNA quantification by the VERIS HCV assay and the Abbott RealTime HCV assay was well correlated for all HCV genotypes, except genotype 4 where 5' UTR RNA folding may impact quantification. Nevertheless, this underestimation of HCV RNA levels had no impact on clinical use. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

    Fischer, Andreia Caroline Fischer da Silveira

    2014-01-01

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

  2. Combined model-based segmentation and elastic registration for accurate quantification of the aortic arch.

    Science.gov (United States)

    Biesdorf, Andreas; Rohr, Karl; von Tengg-Kobligk, Hendrik; Wörz, Stefan

    2010-01-01

    Accurate quantification of the morphology of vessels is important for diagnosis and treatment of cardiovascular diseases. We introduce a new approach for the quantification of the aortic arch morphology that combines 3D model-based segmentation with elastic image registration. The performance of the approach has been evaluated using 3D synthetic images and clinically relevant 3D CTA images including pathologies. We also performed a comparison with a previous approach.

  3. Detection and quantification of beef and pork materials in meat products by duplex droplet digital PCR

    OpenAIRE

    Cai, Yicun; He, Yuping; Lv, Rong; Chen, Hongchao; Wang, Qiang; Pan, Liangwen

    2017-01-01

    Meat products often consist of meat from multiple animal species, and inaccurate food product adulteration and mislabeling can negatively affect consumers. Therefore, a cost-effective and reliable method for identification and quantification of animal species in meat products is required. In this study, we developed a duplex droplet digital PCR (dddPCR) detection and quantification system to simultaneously identify and quantify the source of meat in samples containing a mixture of beef (Bos t...

  4. Extraction and Simultaneous Quantification of Endocannabinoids and Endocannabinoid-Like Lipids in Biological Tissues.

    Science.gov (United States)

    Bindila, Laura; Lutz, Beat

    2016-01-01

    Extraction and quantification of endocannabinoids (eCBs) from biological tissues are essential to unravel their changes in physiological and pathophysiological conditions. We describe here an analytical protocol for extraction of endocannabinoids, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), endocannabinoid-like lipids such as palmitoyl ethanolamide (PEA) and oleoyl ethanolamide (OEA), as well as arachidonic acid (AA) from biological tissues using liquid-liquid extraction method and simultaneous quantification by liquid chromatography multiple reaction monitoring (LC/MRM).

  5. Simultaneous Quantification of Multiple Food- and Waterborne Pathogens by Use of Microfluidic Quantitative PCR

    OpenAIRE

    Ishii, Satoshi; Segawa, Takahiro; Okabe, Satoshi

    2013-01-01

    The direct quantification of multiple pathogens has been desired for diagnostic and public health purposes for a long time. In this study, we applied microfluidic quantitative PCR (qPCR) technology to the simultaneous detection and quantification of multiple food- and waterborne pathogens. In this system, multiple singleplex qPCR assays were run under identical detection conditions in nanoliter-volume chambers that are present in high densities on a chip. First, we developed 18 TaqMan qPCR as...

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

    Science.gov (United States)

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

    2017-04-01

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

  7. Absolute quantification in multi-pinhole micro-SPECT for different isotopes

    OpenAIRE

    Vandeghinste, Bert; Vanhove, Christian; De Beenhouwer, Jan; Van Holen, Roel; Vandenberghe, Stefaan; Staelens, Steven

    2011-01-01

    In preclinical Single Photon Emission Tomography (SPECT), absolute quantification is interesting, expressed in percentage of injected radioactive dose per gram of tissue. This allows for accurate evaluation of disease progression and precise follow-up studies without the need for sacrificing animals. Accurate modeling of image degrading effects is currently under development for isotopes different from 99mTc. The aim of this work is to develop absolute micro-SPECT quantification for three dif...

  8. Probabilistic risk assessment course documentation. Volume 5. System reliability and analysis techniques Session D - quantification

    International Nuclear Information System (INIS)

    Lofgren, E.V.

    1985-08-01

    This course in System Reliability and Analysis Techniques focuses on the probabilistic quantification of accident sequences and the link between accident sequences and consequences. Other sessions in this series focus on the quantification of system reliability and the development of event trees and fault trees. This course takes the viewpoint that event tree sequences or combinations of system failures and success are available and that Boolean equations for system fault trees have been developed and are available. 93 figs., 11 tabs

  9. Specificity and affinity quantification of protein-protein interactions.

    Science.gov (United States)

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

    2013-05-01

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

  10. Temporal Delineation and Quantification of Short Term Clustered Mining Seismicity

    Science.gov (United States)

    Woodward, Kyle; Wesseloo, Johan; Potvin, Yves

    2017-07-01

    The assessment of the temporal characteristics of seismicity is fundamental to understanding and quantifying the seismic hazard associated with mining, the effectiveness of strategies and tactics used to manage seismic hazard, and the relationship between seismicity and changes to the mining environment. This article aims to improve the accuracy and precision in which the temporal dimension of seismic responses can be quantified and delineated. We present a review and discussion on the occurrence of time-dependent mining seismicity with a specific focus on temporal modelling and the modified Omori law (MOL). This forms the basis for the development of a simple weighted metric that allows for the consistent temporal delineation and quantification of a seismic response. The optimisation of this metric allows for the selection of the most appropriate modelling interval given the temporal attributes of time-dependent mining seismicity. We evaluate the performance weighted metric for the modelling of a synthetic seismic dataset. This assessment shows that seismic responses can be quantified and delineated by the MOL, with reasonable accuracy and precision, when the modelling is optimised by evaluating the weighted MLE metric. Furthermore, this assessment highlights that decreased weighted MLE metric performance can be expected if there is a lack of contrast between the temporal characteristics of events associated with different processes.

  11. Serendipity: Global Detection and Quantification of Plant Stress

    Science.gov (United States)

    Schimel, D.; Verma, M.; Drewry, D.

    2016-12-01

    Detecting and quantifying plant stress is a grand challenge for remote sensing, and is important for understanding climate impacts on ecosystems broadly and also for early warning systems supporting food security. The long record from moderate resolution sensors providing frequent data has allowed using phenology to detect stress in forest and agroecosystems, but can fail or give ambiguous results when stress occurs during later phases of growth and in high leaf area systems. The recent recognition that greenhouse gas satellites such as GOSAT and OCO-2 observe Solar-Induced Fluorescence has added a new and complementary tool for the quantification of stress but algorithms to detect and quantify stress using SIF are in their infancy. Here we report new results showing a more complex response of SIF to stress by evaluating spaceborne SIF against in situ eddy covariance data. The response observed is as predicted by theory, and shows that SIF, used in conjunction with moderate resolution remote sensing, can detect and likely quantify stress by indexing the nonlinear part of the SIF-GPP relationship using the photochemical reflectance index and remotely observed light absorption. There are several exciting opportunities on the near horizon for the implementation of SIF, together with syngeristic measurements such as PRI and evapotranspiration that suggest the next few years will be a golden age for global ecology. Adancing the science and associated algorithms now is essential to fully exploiting the next wave of missions.

  12. Quantification of human upper extremity nerves and fascicular anatomy.

    Science.gov (United States)

    Brill, Natalie A; Tyler, Dustin J

    2017-09-01

    In this study we provide detailed quantification of upper extremity nerve and fascicular anatomy. The purpose is to provide values and trends in neural features useful for clinical applications and neural interface device design. Nerve cross-sections were taken from 4 ulnar, 4 median, and 3 radial nerves from 5 arms of 3 human cadavers. Quantified nerve features included cross-sectional area, minor diameter, and major diameter. Fascicular features analyzed included count, perimeter, area, and position. Mean fascicular diameters were 0.57 ± 0.39, 0.6 ± 0.3, 0.5 ± 0.26 mm in the upper arm and 0.38 ± 0.18, 0.47 ± 0.18, 0.4 ± 0.27 mm in the forearm of ulnar, median, and radial nerves, respectively. Mean fascicular diameters were inversely proportional to fascicle count. Detailed quantitative anatomy of upper extremity nerves is a resource for design of neural electrodes, guidance in extraneural procedures, and improved neurosurgical planning. Muscle Nerve 56: 463-471, 2017. © 2016 Wiley Periodicals, Inc.

  13. Quantification of Nociceptive Escape Response in C.elegans

    Science.gov (United States)

    Leung, Kawai; Mohammadi, Aylia; Ryu, William; Nemenman, Ilya

    2013-03-01

    Animals cannot rank and communicate their pain consciously. Thus in pain studies on animal models, one must infer the pain level from high precision experimental characterization of behavior. This is not trivial since behaviors are very complex and multidimensional. Here we explore the feasibility of C.elegans as a model for pain transduction. The nematode has a robust neurally mediated noxious escape response, which we show to be partially decoupled from other sensory behaviors. We develop a nociceptive behavioral response assay that allows us to apply controlled levels of pain by locally heating worms with an IR laser. The worms' motions are captured by machine vision programming with high spatiotemporal resolution. The resulting behavioral quantification allows us to build a statistical model for inference of the experienced pain level from the behavioral response. Based on the measured nociceptive escape of over 400 worms, we conclude that none of the simple characteristics of the response are reliable indicators of the laser pulse strength. Nonetheless, a more reliable statistical inference of the pain stimulus level from the measured behavior is possible based on a complexity-controlled regression model that takes into account the entire worm behavioral output. This work was partially supported by NSF grant No. IOS/1208126 and HFSP grant No. RGY0084/2011.

  14. Quantification of reactive oxygen species for photodynamic therapy

    Science.gov (United States)

    Tan, Zou; Zhang, Jinde; Lin, Lisheng; Li, Buhong

    2016-10-01

    Photodynamic therapy (PDT) is an effective therapeutic modality that uses a light source to activate light-sensitive photosensitizers to treat both oncologic and nononcological indications. Photosensitizers are excited to the long-lived triplet state, and they react with biomolecules via type I or II mechanism resulted in cell death and tumor necrosis. Free radicals and radical ions are formed by electron transfer reactions (type I), which rapidly react with oxygen leading to the production of reactive oxygen species (ROS), including superoxide ions, hydroxyl radicals and hydrogen peroxide. Singlet molecular oxygen is produced in a Type II reaction, in which the excited singlet state of the photosensitizer generated upon photon absorption by the ground-state photosensitizer molecule undergoes intersystem crossing to a long-lived triplet state. In this talk, the fundmental mechanisms and detection techniques for ROS generation in PDT will be introduced. In particular, the quantification of singlet oxygen generation for pre-clinical application will be highlighted, which plays an essential role in the establishment of robust singlet oxygen-mediated PDT dosimetry.

  15. Uncertainty Quantification for Monitoring of Civil Structures from Vibration Measurements

    Science.gov (United States)

    Döhler, Michael; Mevel, Laurent

    2014-05-01

    Health Monitoring of civil structures can be performed by detecting changes in the modal parameters of a structure, or more directly in the measured vibration signals. For a continuous monitoring the excitation of a structure is usually ambient, thus unknown and assumed to be noise. Hence, all estimates from the vibration measurements are realizations of random variables with inherent uncertainty due to (unknown) process and measurement noise and finite data length. In this talk, a strategy for quantifying the uncertainties of modal parameter estimates from a subspace-based system identification approach is presented and the importance of uncertainty quantification in monitoring approaches is shown. Furthermore, a damage detection method is presented, which is based on the direct comparison of the measured vibration signals without estimating modal parameters, while taking the statistical uncertainty in the signals correctly into account. The usefulness of both strategies is illustrated on data from a progressive damage action on a prestressed concrete bridge. References E. Carden and P. Fanning. Vibration based condition monitoring: a review. Structural Health Monitoring, 3(4):355-377, 2004. M. Döhler and L. Mevel. Efficient multi-order uncertainty computation for stochastic subspace identification. Mechanical Systems and Signal Processing, 38(2):346-366, 2013. M. Döhler, L. Mevel, and F. Hille. Subspace-based damage detection under changes in the ambient excitation statistics. Mechanical Systems and Signal Processing, 45(1):207-224, 2014.

  16. In vivo quantification of gingival inflammation using spectral imaging.

    Science.gov (United States)

    Zakian, Christian; Pretty, Iain; Ellwood, Roger; Hamlin, David

    2008-01-01

    Erythema is a reaction of the skin and oral soft tissues commonly associated with inflammation and an increase in blood flow. Diffuse reflection spectroscopy is a powerful tool for the assessment of skin inflammation where erythema has been linked to the relative concentration of oxygenated hemoglobin and blood perfusion. Here we demonstrate the applicability of a spectral imaging method for the quantification of gingival inflammation by looking at the gingival margin and papillary tip erythema. We present a longitudinal study on 22 healthy volunteers divided in two groups. The first was allowed to have normal oral hygiene and the second was subjected to an induced gingivitis for two weeks by cessation of oral hygiene. The spectral reflectance ratio at 615 and 460 nm, R(615)R(460), was proposed as a method to quantify and map the erythema spatial distribution. These wavelengths represent spectral absorption crossovers observed between oxygenated and deoxygenated hemoglobin. The spectral method presented shows a significant separation (pgingivitis was induced and correlates significantly (pgingival index scores. We believe that these investigations could contribute to the development of functional imaging methods for periodontal disease detection and monitoring.

  17. Atomic Resolution Imaging and Quantification of Chemical Functionality of Surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Schwarz, Udo D. [Yale Univ., New Haven, CT (United States). Dept. of Mechanical Engineering and Materials Science; Altman, Eric I. [Yale Univ., New Haven, CT (United States). Dept. of Chemical and Environmental Engineering

    2014-12-10

    The work carried out from 2006-2014 under DoE support was targeted at developing new approaches to the atomic-scale characterization of surfaces that include species-selective imaging and an ability to quantify chemical surface interactions with site-specific accuracy. The newly established methods were subsequently applied to gain insight into the local chemical interactions that govern the catalytic properties of model catalysts of interest to DoE. The foundation of our work was the development of three-dimensional atomic force microscopy (3DAFM), a new measurement mode that allows the mapping of the complete surface force and energy fields with picometer resolution in space (x, y, and z) and piconewton/millielectron volts in force/energy. From this experimental platform, we further expanded by adding the simultaneous recording of tunneling current (3D-AFM/STM) using chemically well-defined tips. Through comparison with simulations, we were able to achieve precise quantification and assignment of local chemical interactions to exact positions within the lattice. During the course of the project, the novel techniques were applied to surface-oxidized copper, titanium dioxide, and silicon oxide. On these materials, defect-induced changes to the chemical surface reactivity and electronic charge density were characterized with site-specific accuracy.

  18. Interactive image quantification tools in nuclear material forensics

    Energy Technology Data Exchange (ETDEWEB)

    Porter, Reid B [Los Alamos National Laboratory; Ruggiero, Christy [Los Alamos National Laboratory; Hush, Don [Los Alamos National Laboratory; Harvey, Neal [Los Alamos National Laboratory; Kelly, Pat [Los Alamos National Laboratory; Scoggins, Wayne [Los Alamos National Laboratory; Tandon, Lav [Los Alamos National Laboratory

    2011-01-03

    Morphological and microstructural features visible in microscopy images of nuclear materials can give information about the processing history of a nuclear material. Extraction of these attributes currently requires a subject matter expert in both microscopy and nuclear material production processes, and is a time consuming, and at least partially manual task, often involving multiple software applications. One of the primary goals of computer vision is to find ways to extract and encode domain knowledge associated with imagery so that parts of this process can be automated. In this paper we describe a user-in-the-loop approach to the problem which attempts to both improve the efficiency of domain experts during image quantification as well as capture their domain knowledge over time. This is accomplished through a sophisticated user-monitoring system that accumulates user-computer interactions as users exploit their imagery. We provide a detailed discussion of the interactive feature extraction and segmentation tools we have developed and describe our initial results in exploiting the recorded user-computer interactions to improve user productivity over time.

  19. Quantification of sea ice production in Weddell Sea polynyas

    Science.gov (United States)

    Zentek, Rolf; Heinemann, Günther; Paul, Stephan; Stulic, Lukrecia; Timmermann, Ralph

    2017-04-01

    The regional climate model COSMO-CLM was used to perform simulations the Weddell Sea region in Antarctica for the time period 2002-2015 with the focus on atmosphere-ocean-sea ice interactions. The original model was adapted to polar regions by the use of a thermodynamic sea ice module with snow cover and an temperature-dependent albedo scheme for sea ice. The recently published topography RTopo2 was used. The model was run with nesting in ERA-Interim data in a forecast mode. Sea ice concentrations were taken from satellite measurements (AMSR-E, SSMI/S, AMSR2) and were updated daily to allow for a close-to-reality hindcast. Simulations were done with 15 km resolution for the whole period 2002-2015 with the goal to force the sea-ice ocean model FESOM. In a second step a 5 km simulation was one-way nested for the winter period (April - September) 2002-2015 to allow for a better quantification of sea ice production in the Weddell Sea. Estimates of sea ice production and comparisons of the results to remote sensing data will be presented.

  20. Quantification of Folding in Sheet-like Nanostructures

    Science.gov (United States)

    Rai, Durgesh; Beaucage, Gregory; Ramachandran, Ramanth; Pradhan, Siddharth

    2012-02-01

    Two-dimensional nanostructures are of interest to a wide range of scientists from biologists interested in membranes to polymer scientists producing grapheme reinforced nanocomposites. Considering two--dimensional structures as a class of nanomaterials, we could use the sheet thickness and lateral size as structural description. In addition to these parameters, two-dimensional structures are, at times, capable of folding or crumpling, largely depending on the interfacial chemistry and to some extent on thermodynamics. We have studied this crumpling behavior using small-angle neutron and x-ray scattering in a range of nano-materials, specifically, membrane bilayers, graphene oxide, as well as exfoliated nano-sheets of molybdenum sulfide, boron nitride, and tungsten sulfide. A new parameterization of crumpling in these two-dimensional nanostructures will be described with indications of how this quantification can lead to general categories of crumpling behavior that differ in the systems mentioned above. (A helpful discussion with Fyl Pincus assisted with this work.)

  1. Micelle Mediated Trace Level Sulfide Quantification through Cloud Point Extraction

    Directory of Open Access Journals (Sweden)

    Samrat Devaramani

    2012-01-01

    Full Text Available A simple cloud point extraction protocol has been proposed for the quantification of sulfide at trace level. The method is based on the reduction of iron (III to iron (II by the sulfide and the subsequent complexation of metal ion with nitroso-R salt in alkaline medium. The resulting green-colored complex was extracted through cloud point formation using cationic surfactant, that is, cetylpyridinium chloride, and the obtained surfactant phase was homogenized by ethanol before its absorbance measurement at 710 nm. The reaction variables like metal ion, ligand, surfactant concentration, and medium pH on the cloud point extraction of the metal-ligand complex have been optimized. The interference effect of the common anions and cations was studied. The proposed method has been successfully applied to quantify the trace level sulfide in the leachate samples of the landfill and water samples from bore wells and ponds. The validity of the proposed method has been studied by spiking the samples with known quantities of sulfide as well as comparing with the results obtained by the standard method.

  2. Quantification of the degree of reaction of fly ash

    International Nuclear Information System (INIS)

    Ben Haha, M.; De Weerdt, K.; Lothenbach, B.

    2010-01-01

    The quantification of the fly ash (FA) in FA blended cements is an important parameter to understand the effect of the fly ash on the hydration of OPC and on the microstructural development. The FA reaction in two different blended OPC-FA systems was studied using a selective dissolution technique based on EDTA/NaOH, diluted NaOH solution, the portlandite content and by backscattered electron image analysis. The amount of FA determined by selective dissolution using EDTA/NaOH is found to be associated with a significant possible error as different assumptions lead to large differences in the estimate of FA reacted. In addition, at longer hydration times, the reaction of the FA is underestimated by this method due to the presence of non-dissolved hydrates and MgO rich particles. The dissolution of FA in diluted NaOH solution agreed during the first days well with the dissolution as observed by image analysis. At 28 days and longer, the formation of hydrates in the diluted solutions leads to an underestimation. Image analysis appears to give consistent results and to be most reliable technique studied.

  3. Current analytical methods for plant auxin quantification--A review.

    Science.gov (United States)

    Porfírio, Sara; Gomes da Silva, Marco D R; Peixe, Augusto; Cabrita, Maria J; Azadi, Parastoo

    2016-01-01

    Plant hormones, and especially auxins, are low molecular weight compounds highly involved in the control of plant growth and development. Auxins are also broadly used in horticulture, as part of vegetative plant propagation protocols, allowing the cloning of genotypes of interest. Over the years, large efforts have been put in the development of more sensitive and precise methods of analysis and quantification of plant hormone levels in plant tissues. Although analytical techniques have evolved, and new methods have been implemented, sample preparation is still the limiting step of auxin analysis. In this review, the current methods of auxin analysis are discussed. Sample preparation procedures, including extraction, purification and derivatization, are reviewed and compared. The different analytical techniques, ranging from chromatographic and mass spectrometry methods to immunoassays and electrokinetic methods, as well as other types of detection are also discussed. Considering that auxin analysis mirrors the evolution in analytical chemistry, the number of publications describing new and/or improved methods is always increasing and we considered appropriate to update the available information. For that reason, this article aims to review the current advances in auxin analysis, and thus only reports from the past 15 years will be covered. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Absolute Quantification of Toxicological Biomarkers via Mass Spectrometry.

    Science.gov (United States)

    Lau, Thomas Y K; Collins, Ben C; Stone, Peter; Tang, Ning; Gallagher, William M; Pennington, Stephen R

    2017-01-01

    With the advent of "-omics" technologies there has been an explosion of data generation in the field of toxicology, as well as many others. As new candidate biomarkers of toxicity are being regularly discovered, the next challenge is to validate these observations in a targeted manner. Traditionally, these validation experiments have been conducted using antibody-based technologies such as Western blotting, ELISA, and immunohistochemistry. However, this often produces a significant bottleneck as the time, cost, and development of successful antibodies are often far outpaced by the generation of targets of interest. In response to this, there recently have been several developments in the use of triple quadrupole (QQQ) mass spectrometry (MS) as a platform to provide quantification of proteins. This technology does not require antibodies; it is typically less expensive and quicker to develop assays and has the opportunity for more accessible multiplexing. The speed of these experiments combined with their flexibility and ability to multiplex assays makes the technique a valuable strategy to validate biomarker discovery.

  5. Mesh refinement for uncertainty quantification through model reduction

    International Nuclear Information System (INIS)

    Li, Jing; Stinis, Panos

    2015-01-01

    We present a novel way of deciding when and where to refine a mesh in probability space in order to facilitate uncertainty quantification in the presence of discontinuities in random space. A discontinuity in random space makes the application of generalized polynomial chaos expansion techniques prohibitively expensive. The reason is that for discontinuous problems, the expansion converges very slowly. An alternative to using higher terms in the expansion is to divide the random space in smaller elements where a lower degree polynomial is adequate to describe the randomness. In general, the partition of the random space is a dynamic process since some areas of the random space, particularly around the discontinuity, need more refinement than others as time evolves. In the current work we propose a way to decide when and where to refine the random space mesh based on the use of a reduced model. The idea is that a good reduced model can monitor accurately, within a random space element, the cascade of activity to higher degree terms in the chaos expansion. In turn, this facilitates the efficient allocation of computational sources to the areas of random space where they are more needed. For the Kraichnan–Orszag system, the prototypical system to study discontinuities in random space, we present theoretical results which show why the proposed method is sound and numerical results which corroborate the theory

  6. Ultrasonography findings and tumour quantification in patients with pseudomyxoma peritonei

    International Nuclear Information System (INIS)

    Krause, J.; Bergman, A.; Graf, W.; Nilsson, A.; Mahteme, H.

    2012-01-01

    Pseudomyxoma peritonei (PMP) is a disease with various clinical presentations and the diagnostic value of ultrasonography (US) is under investigated. The purpose of this study was to identify the most common US finding in PMP and to investigate US sensitivity, specificity, positive and negative predictive value in quantifying tumour burden in different abdomino-pelvic regions in PMP patients. Between February 2006 and December 2008, 54 patients were treated with cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) due to PMP. The results from preoperative US examination with and without intravenously administrated contrast (SonoVue) were compared to surgical findings. The mean US peritoneal cancer index (PCI) was 6 (range 0–25) and the surgical PCI was 18 (range 3–27) p < 0.0001. The histo-pathological subtypes did not influence the US findings. Ascites, bowel loops adhesions and omental cake were mostly visualised correctly by US. The sensitivity of US in quantification of tumour nodules was 91.5% (range 74–100%) and specificity was 33.8% (range 18–55%). The positive predictive value of US examination in PMP was 22% (range 11–44%) and the negative predictive value was 93% (range 77–100%). US can detect the most common PMP findings (ascites and omental cake). The sensitivity of US to quantify PMP tumour burden in different abdominio-pelvic region was relatively high, however, this imaging tool had low specificity.

  7. Stochastic modeling for magnetic resonance quantification of myocardial blood flow

    Science.gov (United States)

    Seethamraju, Ravi T.; Muehling, Olaf; Panse, Prasad M.; Wilke, Norbert M.; Jerosch-Herold, Michael

    2000-10-01

    Quantification of myocardial blood flow is useful for determining the functional severity of coronary artery lesions. With advances in MR imaging it has become possible to assess myocardial perfusion and blood flow in a non-invasive manner by rapid serial imaging following injection of contrast agent. To date most approaches reported in the literature relied mostly on deriving relative indices of myocardial perfusion directly from the measured signal intensity curves. The central volume principle on the other hand states that it is possible to derive absolute myocardial blood flow from the tissue impulse response. Because of the sensitivity involved in deconvolution due to noise in measured data, conventional methods are sub-optimal, hence, we propose to use stochastic time series modeling techniques like ARMA to obtain a robust impulse response estimate. It is shown that these methods when applied for the optical estimation of the transfer function give accurate estimates of myocardial blood flow. The most significant advantage of this approach, compared with compartmental tracer kinetic models, is the use of a minimum set of prior assumptions on data. The bottleneck in assessing myocardial blood flow, does not lie in the MRI acquisition, but rather in the effort or time for post processing. It is anticipated that the very limited requirements for user input and interaction will be of significant advantage for the clinical application of these methods. The proposed methods are validated by comparison with mean blood flow measurements obtained from radio-isotope labeled microspheres.

  8. The Method of Manufactured Universes for validating uncertainty quantification methods

    KAUST Repository

    Stripling, H.F.

    2011-09-01

    The Method of Manufactured Universes is presented as a validation framework for uncertainty quantification (UQ) methodologies and as a tool for exploring the effects of statistical and modeling assumptions embedded in these methods. The framework calls for a manufactured reality from which experimental data are created (possibly with experimental error), an imperfect model (with uncertain inputs) from which simulation results are created (possibly with numerical error), the application of a system for quantifying uncertainties in model predictions, and an assessment of how accurately those uncertainties are quantified. The application presented in this paper manufactures a particle-transport universe, models it using diffusion theory with uncertain material parameters, and applies both Gaussian process and Bayesian MARS algorithms to make quantitative predictions about new experiments within the manufactured reality. The results of this preliminary study indicate that, even in a simple problem, the improper application of a specific UQ method or unrealized effects of a modeling assumption may produce inaccurate predictions. We conclude that the validation framework presented in this paper is a powerful and flexible tool for the investigation and understanding of UQ methodologies. © 2011 Elsevier Ltd. All rights reserved.

  9. Forensic Uncertainty Quantification of Explosive Dispersal of Particles

    Science.gov (United States)

    Hughes, Kyle; Park, Chanyoung; Haftka, Raphael; Kim, Nam-Ho

    2017-06-01

    In addition to the numerical challenges of simulating the explosive dispersal of particles, validation of the simulation is often plagued with poor knowledge of the experimental conditions. The level of experimental detail required for validation is beyond what is usually included in the literature. This presentation proposes the use of forensic uncertainty quantification (UQ) to investigate validation-quality experiments to discover possible sources of uncertainty that may have been missed in initial design of experiments or under-reported. The current experience of the authors has found that by making an analogy to crime scene investigation when looking at validation experiments, valuable insights may be gained. One examines all the data and documentation provided by the validation experimentalists, corroborates evidence, and quantifies large sources of uncertainty a posteriori with empirical measurements. In addition, it is proposed that forensic UQ may benefit from an independent investigator to help remove possible implicit biases and increases the likelihood of discovering unrecognized uncertainty. Forensic UQ concepts will be discussed and then applied to a set of validation experiments performed at Eglin Air Force Base. This work was supported in part by the U.S. Department of Energy, National Nuclear Security Administration, Advanced Simulation and Computing Program.

  10. A Quantification Index for Power Systems Transient Stability

    Directory of Open Access Journals (Sweden)

    Shengen Chen

    2017-07-01

    Full Text Available In order to assess the reliability of power systems, transient stability simulations must be conducted in addition to steady state study. The transient stability component of reliability studies usually involves extensive simulations generating large amounts of data to be analyzed. Conventional stability analysis relies on a visual examination of selected simulation data plots to classify the severity of disturbances. This conventional examination, which aims to compare the simulations results to established performance criteria, is not comprehensive, is time consuming and prone to subjective interpretation. This paper presents a quantification method for power system performance evaluation. It applies a range of criteria such as rotor angle separation, loss of source, damping, and voltage sag directly to the simulation data files to achieve a more efficient and objective stability assessment. By using stability modules, the proposed method evaluates the performance of every fault location, numerically, by providing a local stability index, as well as an overall global stability index. The method also provides an evaluation of dispatches and their impacts on system stability. The IEEE 39-bus test system and the Northeast Interconnection Power System were used to show the results of this method. This method will free engineers from tedious, time-consuming and error-susceptible offline visual analysis and yield significantly quantified results.

  11. In vivo cell tracking and quantification method in adult zebrafish

    Science.gov (United States)

    Zhang, Li; Alt, Clemens; Li, Pulin; White, Richard M.; Zon, Leonard I.; Wei, Xunbin; Lin, Charles P.

    2012-03-01

    Zebrafish have become a powerful vertebrate model organism for drug discovery, cancer and stem cell research. A recently developed transparent adult zebrafish using double pigmentation mutant, called casper, provide unparalleled imaging power in in vivo longitudinal analysis of biological processes at an anatomic resolution not readily achievable in murine or other systems. In this paper we introduce an optical method for simultaneous visualization and cell quantification, which combines the laser scanning confocal microscopy (LSCM) and the in vivo flow cytometry (IVFC). The system is designed specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish casper, under physiological conditions in the same fish over time. The confocal imaging part in this system serves the dual purposes of imaging fish tissue microstructure and a 3D navigation tool to locate a suitable vessel for circulating cell counting. The multi-color, multi-channel instrument allows the detection of multiple cell populations or different tissues or organs simultaneously. We demonstrate initial testing of this novel instrument by imaging vasculature and tracking circulating cells in CD41: GFP/Gata1: DsRed transgenic casper fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. Circulating fluorescent cell incidents were recorded and counted repeatedly over time and in different types of vessels. Great application opportunities in cancer and stem cell researches are discussed.

  12. Impact Induced Delamination Detection and Quantification With Guided Wavefield Analysis

    Science.gov (United States)

    Tian, Zhenhua; Leckey, Cara A. C.; Yu, Lingyu; Seebo, Jeffrey P.

    2015-01-01

    This paper studies impact induced delamination detection and quantification by using guided wavefield data and spatial wavenumber imaging. The complex geometry impact-like delamination is created through a quasi-static indentation on a CFRP plate. To detect and quantify the impact delamination in the CFRP plate, PZT-SLDV sensing and spatial wavenumber imaging are performed. In the PZT-SLDV sensing, the guided waves are generated from the PZT, and the high spatial resolution guided wavefields are measured by the SLDV. The guided wavefield data acquired from the PZT-SLDV sensing represent guided wave propagation in the composite laminate and include guided wave interaction with the delamination damage. The measured guided wavefields are analyzed through the spatial wavenumber imaging method, which generates an image containing the dominant local wavenumber at each spatial location. The spatial wavenumber imaging result for the simple single layer Teflon insert delamination provided quantitative information on delamination damage size and location. The location of delamination damage is indicated by the area with larger wavenumbers in the spatial wavenumber image. The impact-like delamination results only partially agreed with the damage size and shape. The results also demonstrated the dependence on excitation frequency. Future work will further investigate the accuracy of the wavenumber imaging method for real composite damage and the dependence on frequency of excitation.

  13. Quantification of Safety-Critical Software Test Uncertainty

    International Nuclear Information System (INIS)

    Khalaquzzaman, M.; Cho, Jaehyun; Lee, Seung Jun; Jung, Wondea

    2015-01-01

    The method, conservatively assumes that the failure probability of a software for the untested inputs is 1, and the failure probability turns in 0 for successful testing of all test cases. However, in reality the chance of failure exists due to the test uncertainty. Some studies have been carried out to identify the test attributes that affect the test quality. Cao discussed the testing effort, testing coverage, and testing environment. Management of the test uncertainties was discussed in. In this study, the test uncertainty has been considered to estimate the software failure probability because the software testing process is considered to be inherently uncertain. A reliability estimation of software is very important for a probabilistic safety analysis of a digital safety critical system of NPPs. This study focused on the estimation of the probability of a software failure that considers the uncertainty in software testing. In our study, BBN has been employed as an example model for software test uncertainty quantification. Although it can be argued that the direct expert elicitation of test uncertainty is much simpler than BBN estimation, however the BBN approach provides more insights and a basis for uncertainty estimation

  14. Quantification of motility of carabid beetles in farmland.

    Science.gov (United States)

    Allema, A B; van der Werf, W; Groot, J C J; Hemerik, L; Gort, G; Rossing, W A H; van Lenteren, J C

    2015-04-01

    Quantification of the movement of insects at field and landscape levels helps us to understand their ecology and ecological functions. We conducted a meta-analysis on movement of carabid beetles (Coleoptera: Carabidae), to identify key factors affecting movement and population redistribution. We characterize the rate of redistribution using motility μ (L2 T-1), which is a measure for diffusion of a population in space and time that is consistent with ecological diffusion theory and which can be used for upscaling short-term data to longer time frames. Formulas are provided to calculate motility from literature data on movement distances. A field experiment was conducted to measure the redistribution of mass-released carabid, Pterostichus melanarius in a crop field, and derive motility by fitting a Fokker-Planck diffusion model using inverse modelling. Bias in estimates of motility from literature data is elucidated using the data from the field experiment as a case study. The meta-analysis showed that motility is 5.6 times as high in farmland as in woody habitat. Species associated with forested habitats had greater motility than species associated with open field habitats, both in arable land and woody habitat. The meta-analysis did not identify consistent differences in motility at the species level, or between clusters of larger and smaller beetles. The results presented here provide a basis for calculating time-varying distribution patterns of carabids in farmland and woody habitat. The formulas for calculating motility can be used for other taxa.

  15. Quantification of deep medullary veins at 7 T brain MRI

    Energy Technology Data Exchange (ETDEWEB)

    Kuijf, Hugo J.; Viergever, Max A.; Vincken, Koen L. [University Medical Center Utrecht, Image Sciences Institute, Utrecht (Netherlands); Bouvy, Willem H.; Razoux Schultz, Tom B.; Biessels, Geert Jan [University Medical Center Utrecht, Department of Neurology, Brain Center Rudolf Magnus, Utrecht (Netherlands); Zwanenburg, Jaco J.M. [University Medical Center Utrecht, Image Sciences Institute, Utrecht (Netherlands); University Medical Center Utrecht, Department of Radiology, Utrecht (Netherlands)

    2016-10-15

    Deep medullary veins support the venous drainage of the brain and may display abnormalities in the context of different cerebrovascular diseases. We present and evaluate a method to automatically detect and quantify deep medullary veins at 7 T. Five participants were scanned twice, to assess the robustness and reproducibility of manual and automated vein detection. Additionally, the method was evaluated on 24 participants to demonstrate its application. Deep medullary veins were assessed within an automatically created region-of-interest around the lateral ventricles, defined such that all veins must intersect it. A combination of vesselness, tubular tracking, and hysteresis thresholding located individual veins, which were quantified by counting and computing (3-D) density maps. Visual assessment was time-consuming (2 h/scan), with an intra-/inter-observer agreement on absolute vein count of ICC = 0.76 and 0.60, respectively. The automated vein detection showed excellent inter-scan reproducibility before (ICC = 0.79) and after (ICC = 0.88) visually censoring false positives. It had a positive predictive value of 71.6 %. Imaging at 7 T allows visualization and quantification of deep medullary veins. The presented method offers fast and reliable automated assessment of deep medullary veins. (orig.)

  16. Independent code assessment: Sandia-proposed accuracy quantification methodology

    International Nuclear Information System (INIS)

    Kmetyk, L.N.; Elrick, M.G.; Byers, R.K.; Buxton, L.D.

    1986-08-01

    Code assessment is performed to obtain a judgement of the accuracy and validity of any given code over its range of applicability. To achieve this, the codes are exercised against a matrix of experiments, resulting in comparisons between code predictions and measured data. This far, conclusions on code accuracy drawn from previous independent assessment studies have appeared to be mostly phenomenological and/or qualitative. Therefore, there is an increasing emphasis within the NRC code assessment effort to formulate more coherent, quantitative conclusions on the capabilities and accuracies of the codes. This can be done by using statistically based methods to evaluate the overall accuracy of the codes with respect to particular key phenomena, based on the same code analyses of individual experiments. One possibility for a statistically-based code accuracy quantification methodology is presented in this report. This method yields three nested accuracy estimates for any given time period: the code accuracy bias, the average variation in accuracy for the overall behavior. Quantifying assessment results using a common method will allow the results of a number of independent code assessors to be combined to provide broad-based information on code accuracy for applications to regulatory needs and other power plant studies and to define further code development needs and priorities

  17. Quantification of habitat fragmentation reveals extinction risk in terrestrial mammals.

    Science.gov (United States)

    Crooks, Kevin R; Burdett, Christopher L; Theobald, David M; King, Sarah R B; Di Marco, Moreno; Rondinini, Carlo; Boitani, Luigi

    2017-07-18

    Although habitat fragmentation is often assumed to be a primary driver of extinction, global patterns of fragmentation and its relationship to extinction risk have not been consistently quantified for any major animal taxon. We developed high-resolution habitat fragmentation models and used phylogenetic comparative methods to quantify the effects of habitat fragmentation on the world's terrestrial mammals, including 4,018 species across 26 taxonomic Orders. Results demonstrate that species with more fragmentation are at greater risk of extinction, even after accounting for the effects of key macroecological predictors, such as body size and geographic range size. Species with higher fragmentation had smaller ranges and a lower proportion of high-suitability habitat within their range, and most high-suitability habitat occurred outside of protected areas, further elevating extinction risk. Our models provide a quantitative evaluation of extinction risk assessments for species, allow for identification of emerging threats in species not classified as threatened, and provide maps of global hotspots of fragmentation for the world's terrestrial mammals. Quantification of habitat fragmentation will help guide threat assessment and strategic priorities for global mammal conservation.

  18. Uncertainty quantification for large-scale ocean circulation predictions.

    Energy Technology Data Exchange (ETDEWEB)

    Safta, Cosmin; Debusschere, Bert J.; Najm, Habib N.; Sargsyan, Khachik

    2010-09-01

    Uncertainty quantificatio in climate models is challenged by the sparsity of the available climate data due to the high computational cost of the model runs. Another feature that prevents classical uncertainty analyses from being easily applicable is the bifurcative behavior in the climate data with respect to certain parameters. A typical example is the Meridional Overturning Circulation in the Atlantic Ocean. The maximum overturning stream function exhibits discontinuity across a curve in the space of two uncertain parameters, namely climate sensitivity and CO{sub 2} forcing. We develop a methodology that performs uncertainty quantificatio in the presence of limited data that have discontinuous character. Our approach is two-fold. First we detect the discontinuity location with a Bayesian inference, thus obtaining a probabilistic representation of the discontinuity curve location in presence of arbitrarily distributed input parameter values. Furthermore, we developed a spectral approach that relies on Polynomial Chaos (PC) expansions on each sides of the discontinuity curve leading to an averaged-PC representation of the forward model that allows efficient uncertainty quantification and propagation. The methodology is tested on synthetic examples of discontinuous data with adjustable sharpness and structure.

  19. Uranium quantification in semen by inductively coupled plasma mass spectrometry

    Science.gov (United States)

    Todorov, Todor; Ejnik, John W.; Guandalini, Gustavo S.; Xu, Hanna; Hoover, Dennis; Anderson, Larry W.; Squibb, Katherine; McDiarmid, Melissa A.; Centeno, Jose A.

    2013-01-01

    In this study we report uranium analysis for human semen samples. Uranium quantification was performed by inductively coupled plasma mass spectrometry. No additives, such as chymotrypsin or bovine serum albumin, were used for semen liquefaction, as they showed significant uranium content. For method validation we spiked 2 g aliquots of pooled control semen at three different levels of uranium: low at 5 pg/g, medium at 50 pg/g, and high at 1000 pg/g. The detection limit was determined to be 0.8 pg/g uranium in human semen. The data reproduced within 1.4–7% RSD and spike recoveries were 97–100%. The uranium level of the unspiked, pooled control semen was 2.9 pg/g of semen (n = 10). In addition six semen samples from a cohort of Veterans exposed to depleted uranium (DU) in the 1991 Gulf War were analyzed with no knowledge of their exposure history. Uranium levels in the Veterans’ semen samples ranged from undetectable (<0.8 pg/g) to 3350 pg/g. This wide concentration range for uranium in semen is consistent with known differences in current DU body burdens in these individuals, some of whom have retained embedded DU fragments.

  20. Crowdsourced Quantification and Visualization of Urban Mobility Space Inequality

    Directory of Open Access Journals (Sweden)

    Michael Szell

    2018-03-01

    Full Text Available Most cities are car-centric, allocating a privileged amount of urban space to cars at the expense of sustainable mobility like cycling. Simultaneously, privately owned vehicles are vastly underused, wasting valuable opportunities for accommodating more people in a livable urban environment by occupying spacious parking areas. Since a data-driven quantification and visualization of such urban mobility space inequality is lacking, here we explore how crowdsourced data can help to advance its understanding. In particular, we describe how the open-source online platform What the Street!? uses massive user-generated data from OpenStreetMap for the interactive exploration of city-wide mobility spaces. Using polygon packing and graph algorithms, the platform rearranges all parking and mobility spaces of cars, rails, and bicycles of a city to be directly comparable, making mobility space inequality accessible to a broad public. This crowdsourced method confirms a prevalent imbalance between modal share and space allocation in 23 cities worldwide, typically discriminating bicycles. Analyzing the guesses of the platform’s visitors about mobility space distributions, we find that this discrimination is consistently underestimated in the public opinion. Finally, we discuss a visualized scenario in which extensive parking areas are regained through fleets of shared, autonomous vehicles. We outline how such accessible visualization platforms can facilitate urban planners and policy makers to reclaim road and parking space for pushing forward sustainable transport solutions.