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Sample records for reliable assay protocol

  1. Developing a yeast-based assay protocol to monitor total ...

    African Journals Online (AJOL)

    A yeast-based assay protocol developed for detecting oestrogenic activity in activated sludge (AS) supernatant is described. The protocol used Saccharomyces cerevisiae construct RMY/ER-ERE with human oestrogen receptor (ERα) and lacZ reporter genes, and was developed by modifying existing assays for use with AS ...

  2. A Hierarchical Energy Efficient Reliable Transport Protocol for Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    Prabhudutta Mohanty

    2014-12-01

    Full Text Available The two important requirements for many Wireless Senor Networks (WSNs are prolonged network lifetime and end-to-end reliability. The sensor nodes consume more energy during data transmission than the data sensing. In WSN, the redundant data increase the energy consumption, latency and reduce reliability during data transmission. Therefore, it is important to support energy efficient reliable data transport in WSNs. In this paper, we present a Hierarchical Energy Efficient Reliable Transport Protocol (HEERTP for the data transmission within the WSN. This protocol maximises the network lifetime by controlling the redundant data transmission with the co-ordination of Base Station (BS. The proposed protocol also achieves end-to-end reliability using a hop-by-hop acknowledgement scheme. We evaluate the performance of the proposed protocol through simulation. The simulation results reveal that our proposed protocol achieves better performance in terms of energy efficiency, latency and reliability than the existing protocols.

  3. ZyFISH: a simple, rapid and reliable zygosity assay for transgenic mice.

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    Donal McHugh

    Full Text Available Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH. The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations.

  4. Connectivity-Based Reliable Multicast MAC Protocol for IEEE 802.11 Wireless LANs

    Directory of Open Access Journals (Sweden)

    Woo-Yong Choi

    2009-01-01

    Full Text Available We propose the efficient reliable multicast MAC protocol based on the connectivity information among the recipients. Enhancing the BMMM (Batch Mode Multicast MAC protocol, the reliable multicast MAC protocol significantly reduces the RAK (Request for ACK frame transmissions in a reasonable computational time and enhances the MAC performance. By the analytical performance analysis, the throughputs of the BMMM protocol and our proposed MAC protocol are derived. Numerical examples show that our proposed MAC protocol increases the reliable multicast MAC performance for IEEE 802.11 wireless LANs.

  5. Evaluation of the reliability of maize reference assays for GMO quantification.

    Science.gov (United States)

    Papazova, Nina; Zhang, David; Gruden, Kristina; Vojvoda, Jana; Yang, Litao; Buh Gasparic, Meti; Blejec, Andrej; Fouilloux, Stephane; De Loose, Marc; Taverniers, Isabel

    2010-03-01

    A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb

  6. Design and Analysis of Transport Protocols for Reliable High-Speed Communications

    NARCIS (Netherlands)

    Oláh, A.

    1997-01-01

    The design and analysis of transport protocols for reliable communications constitutes the topic of this dissertation. These transport protocols guarantee the sequenced and complete delivery of user data over networks which may lose, duplicate and reorder packets. Reliable transport services are

  7. CPM Test-Retest Reliability: "Standard" vs "Single Test-Stimulus" Protocols.

    Science.gov (United States)

    Granovsky, Yelena; Miller-Barmak, Adi; Goldstein, Oren; Sprecher, Elliot; Yarnitsky, David

    2016-03-01

    Assessment of pain inhibitory mechanisms using conditioned pain modulation (CPM) is relevant clinically in prediction of pain and analgesic efficacy. Our objective is to provide necessary estimates of intersession CPM reliability, to enable transformation of the CPM paradigm into a clinical tool. Two cohorts of young healthy subjects (N = 65) participated in two dual-session studies. In Study I, a Bath-Thermode CPM protocol was used, with hot water immersion and contact heat as conditioning- and test-stimuli, respectively, in a classical parallel CPM design introducing test-stimulus first, and then the conditioning- and repeated test-stimuli in parallel. Study II consisted of two CPM protocols: 1) Two-Thermodes, one for each of the stimuli, in the same parallel design as above, and 2) single test-stimulus (STS) protocol with a single administration of a contact heat test-stimulus, partially overlapped in time by a remote shorter contact heat as conditioning stimulus. Test-retest reliability was assessed within 3-7 days. The STS-CPM had superior reliability intraclass correlation (ICC 2 ,: 1  = 0.59) over Bath-Thermode (ICC 2 ,: 1  = 0.34) or Two-Thermodes (ICC 2 ,: 1  = 0.21) protocols. The hand immersion conditioning pain had higher reliability than thermode pain (ICC 2 ,: 1  = 0.76 vs ICC 2 ,: 1  = 0.16). Conditioned test-stimulus pain scores were of good (ICC 2 ,: 1  = 0.62) or fair (ICC 2 ,: 1  = 0.43) reliability for the Bath-Thermode and the STS, respectively, but not for the Two-Thermodes protocol (ICC 2 ,: 1  = 0.20). The newly developed STS-CPM paradigm was more reliable than other CPM protocols tested here, and should be further investigated for its clinical relevance. It appears that large contact size of the conditioning-stimulus and use of single rather than dual test-stimulus pain contribute to augmentation of CPM reliability. © 2015 American Academy of Pain Medicine. All rights reserved. For permissions, please e

  8. An FEC Adaptive Multicast MAC Protocol for Providing Reliability in WLANs

    Science.gov (United States)

    Basalamah, Anas; Sato, Takuro

    For wireless multicast applications like multimedia conferencing, voice over IP and video/audio streaming, a reliable transmission of packets within short delivery delay is needed. Moreover, reliability is crucial to the performance of error intolerant applications like file transfer, distributed computing, chat and whiteboard sharing. Forward Error Correction (FEC) is frequently used in wireless multicast to enhance Packet Error Rate (PER) performance, but cannot assure full reliability unless coupled with Automatic Repeat Request forming what is knows as Hybrid-ARQ. While reliable FEC can be deployed at different levels of the protocol stack, it cannot be deployed on the MAC layer of the unreliable IEEE802.11 WLAN due to its inability to exchange ACKs with multiple recipients. In this paper, we propose a Multicast MAC protocol that enhances WLAN reliability by using Adaptive FEC and study it's performance through mathematical analysis and simulation. Our results show that our protocol can deliver high reliability and throughput performance.

  9. An Acetylcholinesterase-Based Chronoamperometric Biosensor for Fast and Reliable Assay of Nerve Agents

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    Rene Kizek

    2013-08-01

    Full Text Available The enzyme acetylcholinesterase (AChE is an important part of cholinergic nervous system, where it stops neurotransmission by hydrolysis of the neurotransmitter acetylcholine. It is sensitive to inhibition by organophosphate and carbamate insecticides, some Alzheimer disease drugs, secondary metabolites such as aflatoxins and nerve agents used in chemical warfare. When immobilized on a sensor (physico-chemical transducer, it can be used for assay of these inhibitors. In the experiments described herein, an AChE- based electrochemical biosensor using screen printed electrode systems was prepared. The biosensor was used for assay of nerve agents such as sarin, soman, tabun and VX. The limits of detection achieved in a measuring protocol lasting ten minutes were 7.41 × 10−12 mol/L for sarin, 6.31 × 10−12 mol /L for soman, 6.17 × 10−11 mol/L for tabun, and 2.19 × 10−11 mol/L for VX, respectively. The assay was reliable, with minor interferences caused by the organic solvents ethanol, methanol, isopropanol and acetonitrile. Isopropanol was chosen as suitable medium for processing lipophilic samples.

  10. An Energy-Efficient Link Layer Protocol for Reliable Transmission over Wireless Networks

    Directory of Open Access Journals (Sweden)

    Iqbal Adnan

    2009-01-01

    Full Text Available In multihop wireless networks, hop-by-hop reliability is generally achieved through positive acknowledgments at the MAC layer. However, positive acknowledgments introduce significant energy inefficiencies on battery-constrained devices. This inefficiency becomes particularly significant on high error rate channels. We propose to reduce the energy consumption during retransmissions using a novel protocol that localizes bit-errors at the MAC layer. The proposed protocol, referred to as Selective Retransmission using Virtual Fragmentation (SRVF, requires simple modifications to the positive-ACK-based reliability mechanism but provides substantial improvements in energy efficiency. The main premise of the protocol is to localize bit-errors by performing partial checksums on disjoint parts or virtual fragments of a packet. In case of error, only the corrupted virtual fragments are retransmitted. We develop stochastic models of the Simple Positive-ACK-based reliability, the previously-proposed Packet Length Optimization (PLO protocol, and the SRVF protocol operating over an arbitrary-order Markov wireless channel. Our analytical models show that SRVF provides significant theoretical improvements in energy efficiency over existing protocols. We then use bit-error traces collected over different real networks to empirically compare the proposed and existing protocols. These experimental results further substantiate that SRVF provides considerably better energy efficiency than Simple Positive-ACK and Packet Length Optimization protocols.

  11. Shoulder muscle endurance: the development of a standardized and reliable protocol

    Directory of Open Access Journals (Sweden)

    Roy Jean-Sébastien

    2011-01-01

    Full Text Available Abstract Background Shoulder muscle fatigue has been proposed as a possible link to explain the association between repetitive arm use and the development of rotator cuff disorders. To our knowledge, no standardized clinical endurance protocol has been developed to evaluate the effects of muscle fatigue on shoulder function. Such a test could improve clinical examination of individuals with shoulder disorders. Therefore, the purpose of this study was to establish a reliable protocol for objective assessment of shoulder muscle endurance. Methods An endurance protocol was developed on a stationary dynamometer (Biodex System 3. The endurance protocol was performed in isotonic mode with the resistance set at 50% of each subject's peak torque as measured for shoulder external (ER and internal rotation (IR. Each subject performed 60 continuous repetitions of IR/ER rotation. The endurance protocol was performed by 36 healthy individuals on two separate occasions at least two days apart. Maximal isometric shoulder strength tests were performed before and after the fatigue protocol to evaluate the effects of the endurance protocol and its reliability. Paired t-tests were used to evaluate the reduction in shoulder strength due to the protocol, while intraclass correlation coefficients (ICC and minimal detectable change (MDC were used to evaluate its reliability. Results Maximal isometric strength was significantly decreased after the endurance protocol (P 0.84. Conclusions Changes in muscular performance observed during and after the muscular endurance protocol suggests that the protocol did result in muscular fatigue. Furthermore, this study established that the resultant effects of fatigue of the proposed isotonic protocol were reproducible over time. The protocol was performed without difficulty by all volunteers and took less than 10 minutes to perform, suggesting that it might be feasible for clinical practice. This protocol could be used to induce

  12. Cacades: A reliable dissemination protocol for data collection sensor network

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    Peng, Y.; Song, W.; Huang, R.; Xu, M.; Shirazi, B.; LaHusen, R.; Pei, G.

    2009-01-01

    In this paper, we propose a fast and reliable data dissemination protocol Cascades to disseminate data from the sink(base station) to all or a subset of nodes in a data collection sensor network. Cascades makes use of the parentmonitor-children analogy to ensure reliable dissemination. Each node monitors whether or not its children have received the broadcast messages through snooping children's rebroadcasts or waiting for explicit ACKs. If a node detects a gap in its message sequences, it can fetch the missing messages from its neighbours reactively. Cascades also considers many practical issues for field deployment, such as dynamic topology, link/node failure, etc.. It therefore guarantees that a disseminated message from the sink will reach all intended receivers and the dissemination is terminated in a short time period. Notice that, all existing dissemination protocols either do not guarantee reliability or do not terminate [1, 2], which does not meet the requirement of real-time command control. We conducted experiment evaluations in both TOSSIM simulator and a sensor network testbed to compare Cascades with those existing dissemination protocols in TinyOS sensor networks, which show that Cascades achieves a higher degree of reliability, lower communication cost, and less delivery delay. ??2009 IEEE.

  13. Interoperability and Reliability of Multiplatform MPLS VPN: Comparison of Traffic Engineering with RSVP-TE Protocol and LDP Protocol

    Directory of Open Access Journals (Sweden)

    Nanang Ismail

    2017-10-01

    48% of packet loss per 100 sent packets while on RSVP packet loss percentage is 35.5% per 100 sent packets. Both protocols have interoperability on the third layer of multiplatform MPLS VPN, but on heavy loaded traffic condition, RSVP protocol has better reliability than the LDP protocol.

  14. Development of high-reliable real-time communication network protocol for SMART

    Energy Technology Data Exchange (ETDEWEB)

    Song, Ki Sang; Kim, Young Sik [Korea National University of Education, Chongwon (Korea); No, Hee Chon [Korea Advanced Institute of Science and Technology, Taejon (Korea)

    1999-04-01

    In this research, we first define protocol subsets for SMART(System-integrated Modular Advanced Reactor) communication network based on the requirement of SMART MMIS transmission delay and traffic requirements and OSI(Open System Interconnection) 7 layers' network protocol functions. Also, current industrial purpose LAN protocols are analyzed and the applicability of commercialized protocols are checked. For the suitability test, we have applied approximated SMART data traffic and maximum allowable transmission delay requirement. With the simulation results, we conclude that IEEE 802.5 and FDDI which is an ANSI standard, is the most suitable for SMART. We further analyzed the FDDI and token ring protocols for SMART and nuclear plant network environment including IEEE 802.4, IEEE 802.5, and ARCnet. The most suitable protocol for SMART is FDDI and FDDI MAC and RMT protocol specifications have been verified with LOTOS and the verification results show that FDDI MAC and RMT satisfy the reachability and liveness, but does not show deadlock and livelock. Therefore, we conclude that FDDI MAC and RMT is highly reliable protocol for SMART MMIS network. After that, we consider the stacking fault of IEEE 802.5 token ring protocol and propose a fault tolerant MAM(Modified Active Monitor) protocol. The simulation results show that the MAM protocol improves lower priority traffic service rate when stacking fault occurs. Therefore, proposed MAM protocol can be applied to SMART communication network for high reliability and hard real-time communication purpose in data acquisition and inter channel network. (author). 37 refs., 79 figs., 39 tabs.

  15. Is gait variability reliable in older adults and Parkinson's disease? Towards an optimal testing protocol.

    Science.gov (United States)

    Galna, Brook; Lord, Sue; Rochester, Lynn

    2013-04-01

    Despite the widespread use of gait variability in research and clinical studies, testing protocols designed to optimise its reliability have not been established. This study evaluates the impact of testing protocol and pathology on the reliability of gait variability. To (i) estimate the reliability of gait variability during continuous and intermittent walking protocols in older adults and people with Parkinson's disease (PD), (ii) determine optimal number of steps for acceptable levels of reliability of gait variability and (iii) provide sample size estimates for use in clinical trials. Gait variability was measured twice, one week apart, in 27 older adults and 25 PD participants. Participants walked at their preferred pace during: (i) a continuous 2 min walk and (ii) 3 intermittent walks over a 12 m walkway. Gait variability was calculated as the within-person standard deviation for step velocity, length and width, and step, stance and swing duration. Reliability of gait variability ranged from poor to excellent (intra class correlations .041-.860; relative limits of agreement 34-89%). Gait variability was more reliable during continuous walks. Control and PD participants demonstrated similar reliability. Increasing the number of steps improved reliability, with most improvement seen across the first 30 steps. In this study, we identified testing protocols that improve the reliability of measuring gait variability. We recommend using a continuous walking protocol and to collect no fewer than 30 steps. Early PD does not appear to impact negatively on the reliability of gait variability. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. The specification-based validation of reliable multicast protocol: Problem Report. M.S. Thesis

    Science.gov (United States)

    Wu, Yunqing

    1995-01-01

    Reliable Multicast Protocol (RMP) is a communication protocol that provides an atomic, totally ordered, reliable multicast service on top of unreliable IP multicasting. In this report, we develop formal models for RMP using existing automated verification systems, and perform validation on the formal RMP specifications. The validation analysis help identifies some minor specification and design problems. We also use the formal models of RMP to generate a test suite for conformance testing of the implementation. Throughout the process of RMP development, we follow an iterative, interactive approach that emphasizes concurrent and parallel progress of implementation and verification processes. Through this approach, we incorporate formal techniques into our development process, promote a common understanding for the protocol, increase the reliability of our software, and maintain high fidelity between the specifications of RMP and its implementation.

  17. Reliable Multicast MAC Protocol for IEEE 802.11 Wireless LANs with Extended Service Range

    Science.gov (United States)

    Choi, Woo-Yong

    2011-11-01

    In this paper, we propose the efficient reliable multicast MAC protocol by which the AP (Access Point) can transmit reliably its multicast data frames to the recipients in the AP's one-hop or two-hop transmission range. The AP uses the STAs (Stations) that are directly associated with itself as the relays for the data delivery to the remote recipients that cannot be reached directly from itself. Based on the connectivity information among the recipients, the reliable multicast MAC protocol optimizes the number of the RAK (Request for ACK) frame transmissions in a reasonable computational time. Numerical examples show that our proposed MAC protocol significantly enhances the MAC performance compared with the BMMM (Batch Mode Multicast MAC) protocol that is extended to support the recipients that are in the AP's one-hop or two-hop transmission range in IEEE 802.11 wireless LANs.

  18. Comparison of mRNA splicing assay protocols across multiple laboratories: recommendations for best practice in standardized clinical testing.

    Science.gov (United States)

    Whiley, Phillip J; de la Hoya, Miguel; Thomassen, Mads; Becker, Alexandra; Brandão, Rita; Pedersen, Inge Sokilde; Montagna, Marco; Menéndez, Mireia; Quiles, Francisco; Gutiérrez-Enríquez, Sara; De Leeneer, Kim; Tenés, Anna; Montalban, Gemma; Tserpelis, Demis; Yoshimatsu, Toshio; Tirapo, Carole; Raponi, Michela; Caldes, Trinidad; Blanco, Ana; Santamariña, Marta; Guidugli, Lucia; de Garibay, Gorka Ruiz; Wong, Ming; Tancredi, Mariella; Fachal, Laura; Ding, Yuan Chun; Kruse, Torben; Lattimore, Vanessa; Kwong, Ava; Chan, Tsun Leung; Colombo, Mara; De Vecchi, Giovanni; Caligo, Maria; Baralle, Diana; Lázaro, Conxi; Couch, Fergus; Radice, Paolo; Southey, Melissa C; Neuhausen, Susan; Houdayer, Claude; Fackenthal, Jim; Hansen, Thomas Van Overeem; Vega, Ana; Diez, Orland; Blok, Rien; Claes, Kathleen; Wappenschmidt, Barbara; Walker, Logan; Spurdle, Amanda B; Brown, Melissa A

    2014-02-01

    Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting. We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design. PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1G>T Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp). We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.

  19. Assessment of a robust model protocol with accelerated throughput for a human recombinant full length estrogen receptor-alpha binding assay: protocol optimization and intralaboratory assay performance as initial steps towards validation.

    Science.gov (United States)

    Freyberger, Alexius; Wilson, Vickie; Weimer, Marc; Tan, Shirlee; Tran, Hoai-Son; Ahr, Hans-Jürgen

    2010-08-01

    Despite about two decades of research in the field of endocrine active compounds, still no validated human recombinant (hr) estrogen receptor-alpha (ERalpha) binding assay is available, although hr-ERalpha is available from several sources. In a joint effort, US EPA and Bayer Schering Pharma with funding from the EU-sponsored 6th framework project, ReProTect, developed a model protocol for such a binding assay. Important features of this assay are the use of a full length hr-ERalpha and performance in a 96-well plate format. A full length hr-ERalpha was chosen, as it was considered to provide the most accurate and human-relevant results, whereas truncated receptors could perform differently. Besides three reference compounds [17beta-estradiol, norethynodrel, dibutylphthalate] nine test compounds with different affinities for the ERalpha [diethylstilbestrol (DES), ethynylestradiol, meso-hexestrol, equol, genistein, o,p'-DDT, nonylphenol, n-butylparaben, and corticosterone] were used to explore the performance of the assay. Three independent experiments per compound were performed on different days, and dilutions of test compounds from deep-frozen stocks, solutions of radiolabeled ligand and receptor preparation were freshly prepared for each experiment. The ERalpha binding properties of reference and test compounds were well detected. As expected dibutylphthalate and corticosterone were non-binders in this assay. In terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using a human recombinant ERalpha ligand binding domain. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.5. Our data demonstrate that the assay was robust and reliably ranked compounds with strong, weak, and no affinity for the ERalpha with high accuracy. It avoids the manipulation and use of animals, i.e., the preparation of uterine cytosol as

  20. Harmonization of radiobiological assays: why and how?

    International Nuclear Information System (INIS)

    Prasanna, Pataje G.

    2014-01-01

    The International Atomic Energy Agency has made available a technical manual for cytogenetic biodosimetry assays (dicentric chromosome aberration (DCA) and cytokinesis-block micronucleus (CBMN) assays) used for radiation dose assessment in radiation accidents. The International Standardization Organization, which develops standards and guidelines, also provides an avenue for laboratory accreditation, has developed guidelines and recommendations for performing cytogenetic biodosimetry assays. Harmonization of DCA and CBMN assays, has improved their accuracy. Double-blinded inter-laboratory comparison studies involving several networks have further validated DCA and CBMN assays and improved the confidence in their potential use for radiation dose assessment in mass casualties. This kind of international harmonization is lacking for pre-clinical radiobiology assays. The widely used pre-clinical assays that are relatively important to set stage for clinical trials include clonogenic assays, flow-cytometry assays, apoptotic assays, and tumor regression and growth delay assays. However, significant inter-laboratory variations occur with respect to data among laboratories. This raises concerns on the reliability and reproducibility of preclinical data that drives further development and translation. Lack of reproducibility may stem from a variety of factors such as poor scientist training, less than optimal experimental design, inadequate description of methodology, and impulse to publish only the positive data etc. Availability of technical manuals, standard operating procedures, accreditation avenues for laboratories performing such assays, inter-laboratory comparisons, and use of standardized protocols are necessary to enhance reliability and reproducibility. Thus, it is important that radiobiological assays are harmonized for laboratory protocols to ensure successful translation of pre-clinical research on radiation effect modulators to help design clinic trials with

  1. Fault recovery in the reliable multicast protocol

    Science.gov (United States)

    Callahan, John R.; Montgomery, Todd L.; Whetten, Brian

    1995-01-01

    The Reliable Multicast Protocol (RMP) provides a unique, group-based model for distributed programs that need to handle reconfiguration events at the application layer. This model, called membership views, provides an abstraction in which events such as site failures, network partitions, and normal join-leave events are viewed as group reformations. RMP provides access to this model through an application programming interface (API) that notifies an application when a group is reformed as the result of a some event. RMP provides applications with reliable delivery of messages using an underlying IP Multicast (12, 5) media to other group members in a distributed environment even in the case of reformations. A distributed application can use various Quality of Service (QoS) levels provided by RMP to tolerate group reformations. This paper explores the implementation details of the mechanisms in RMP that provide distributed applications with membership view information and fault recovery capabilities.

  2. An improved IEEE 802.11 protocol for reliable data transmission in power distribution fault diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Campoccia, F.; Di Silvestre, M.L.; Sanseverino, E.R.; Zizzo, G. [Palermo Univ., Palermo (Italy)

    2010-10-15

    In power systems, on-line transmission between local units and the central unit can be done by means of power line communications or wireless technology. During an electrical fault, the reliability of the distribution system depends on the security of the timely protective and restorative actions on the network. This paper focused on the WiFi system because of its economy and ease of installation. However, WiFi systems are typically managed by the IEEE 802.11 protocol, which is not reliable in terms of security in data communication. In WiFi networks, data is divided into packets and sent in succession to reduce errors within the radio channel. The IEEE 802.11 protocol has high probability for loss of packets or delay in their transmission. In order to ensure the reliability of data transmission times between two terminal units connected by WiFi stations, a new protocol was derived by modifying the IEEE 802.11. The improvements of the new protocol were highlighted and its capability for the diagnostic service was verified. The modified protocol eliminates the danger of collisions between packets and optimizes the transmission time for sending information. 6 refs., 7 tabs., 8 figs.

  3. An improved 96-well turbidity assay for T4 lysozyme activity.

    Science.gov (United States)

    Toro, Tasha B; Nguyen, Thao P; Watt, Terry J

    2015-01-01

    T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: •Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays;•Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and•Incorporates a simplified expression and purification protocol for T4 lysozyme.

  4. RELIABLE DYNAMIC SOURCE ROUTING PROTOCOL (RDSRP FOR ENERGY HARVESTING WIRELESS SENSOR NETWORKS

    Directory of Open Access Journals (Sweden)

    B. Narasimhan

    2015-03-01

    Full Text Available Wireless sensor networks (WSNs carry noteworthy pros over traditional communication. Though, unkind and composite environments fake great challenges in the reliability of WSN communications. It is more vital to develop a reliable unipath dynamic source routing protocol (RDSRPl for WSN to provide better quality of service (QoS in energy harvesting wireless sensor networks (EH-WSN. This paper proposes a dynamic source routing approach for attaining the most reliable route in EH-WSNs. Performance evaluation is carried out using NS-2 and throughput and packet delivery ratio are chosen as the metrics.

  5. Reliability and criterion validity of an observation protocol for working technique assessments in cash register work.

    Science.gov (United States)

    Palm, Peter; Josephson, Malin; Mathiassen, Svend Erik; Kjellberg, Katarina

    2016-06-01

    We evaluated the intra- and inter-observer reliability and criterion validity of an observation protocol, developed in an iterative process involving practicing ergonomists, for assessment of working technique during cash register work for the purpose of preventing upper extremity symptoms. Two ergonomists independently assessed 17 15-min videos of cash register work on two occasions each, as a basis for examining reliability. Criterion validity was assessed by comparing these assessments with meticulous video-based analyses by researchers. Intra-observer reliability was acceptable (i.e. proportional agreement >0.7 and kappa >0.4) for 10/10 questions. Inter-observer reliability was acceptable for only 3/10 questions. An acceptable inter-observer reliability combined with an acceptable criterion validity was obtained only for one working technique aspect, 'Quality of movements'. Thus, major elements of the cashiers' working technique could not be assessed with an acceptable accuracy from short periods of observations by one observer, such as often desired by practitioners. Practitioner Summary: We examined an observation protocol for assessing working technique in cash register work. It was feasible in use, but inter-observer reliability and criterion validity were generally not acceptable when working technique aspects were assessed from short periods of work. We recommend the protocol to be used for educational purposes only.

  6. Design, Implementation, and Verification of the Reliable Multicast Protocol. Thesis

    Science.gov (United States)

    Montgomery, Todd L.

    1995-01-01

    This document describes the Reliable Multicast Protocol (RMP) design, first implementation, and formal verification. RMP provides a totally ordered, reliable, atomic multicast service on top of an unreliable multicast datagram service. RMP is fully and symmetrically distributed so that no site bears an undue portion of the communications load. RMP provides a wide range of guarantees, from unreliable delivery to totally ordered delivery, to K-resilient, majority resilient, and totally resilient atomic delivery. These guarantees are selectable on a per message basis. RMP provides many communication options, including virtual synchrony, a publisher/subscriber model of message delivery, a client/server model of delivery, mutually exclusive handlers for messages, and mutually exclusive locks. It has been commonly believed that total ordering of messages can only be achieved at great performance expense. RMP discounts this. The first implementation of RMP has been shown to provide high throughput performance on Local Area Networks (LAN). For two or more destinations a single LAN, RMP provides higher throughput than any other protocol that does not use multicast or broadcast technology. The design, implementation, and verification activities of RMP have occurred concurrently. This has allowed the verification to maintain a high fidelity between design model, implementation model, and the verification model. The restrictions of implementation have influenced the design earlier than in normal sequential approaches. The protocol as a whole has matured smoother by the inclusion of several different perspectives into the product development.

  7. A Smart Collaborative Routing Protocol for Reliable Data Diffusion in IoT Scenarios.

    Science.gov (United States)

    Ai, Zheng-Yang; Zhou, Yu-Tong; Song, Fei

    2018-06-13

    It is knotty for current routing protocols to meet the needs of reliable data diffusion during the Internet of Things (IoT) deployments. Due to the random placement, limited resources and unattended features of existing sensor nodes, the wireless transmissions are easily exposed to unauthorized users, which becomes a vulnerable area for various malicious attacks, such as wormhole and Sybil attacks. However, the scheme based on geographic location is a suitable candidate to defend against them. This paper is inspired to propose a smart collaborative routing protocol, Geographic energy aware routing and Inspecting Node (GIN), for guaranteeing the reliability of data exchanging. The proposed protocol integrates the directed diffusion routing, Greedy Perimeter Stateless Routing (GPSR), and the inspecting node mechanism. We first discuss current wireless routing protocols from three diverse perspectives (improving transmission rate, shortening transmission range and reducing transmission consumption). Then, the details of GIN, including the model establishment and implementation processes, are presented by means of the theoretical analysis. Through leveraging the game theory, the inspecting node is elected to monitor the network behaviors. Thirdly, we evaluate the network performances, in terms of transmission delay, packet loss ratio, and throughput, between GIN and three traditional schemes (i.e., Flooding, GPSR, and GEAR). The simulation results illustrate that the proposed protocol is able to outperform the others.

  8. Characterization of perovskite solar cells: Towards a reliable measurement protocol

    Directory of Open Access Journals (Sweden)

    Eugen Zimmermann

    2016-09-01

    Full Text Available Lead halide perovskite solar cells have shown a tremendous rise in power conversion efficiency with reported record efficiencies of over 20% making this material very promising as a low cost alternative to conventional inorganic solar cells. However, due to a differently severe “hysteretic” behaviour during current density-voltage measurements, which strongly depends on scan rate, device and measurement history, preparation method, device architecture, etc., commonly used solar cell measurements do not give reliable or even reproducible results. For the aspect of commercialization and the possibility to compare results of different devices among different laboratories, it is necessary to establish a measurement protocol which gives reproducible results. Therefore, we compare device characteristics derived from standard current density-voltage measurements with stabilized values obtained from an adaptive tracking of the maximum power point and the open circuit voltage as well as characteristics extracted from time resolved current density-voltage measurements. Our results provide insight into the challenges of a correct determination of device performance and propose a measurement protocol for a reliable characterisation which is easy to implement and has been tested on varying perovskite solar cells fabricated in different laboratories.

  9. Test-retest reliability of jump execution variables using mechanography: a comparison of jump protocols.

    Science.gov (United States)

    Fitzgerald, John S; Johnson, LuAnn; Tomkinson, Grant; Stein, Jesse; Roemmich, James N

    2018-05-01

    Mechanography during the vertical jump may enhance screening and determining mechanistic causes underlying physical performance changes. Utility of jump mechanography for evaluation is limited by scant test-retest reliability data on force-time variables. This study examined the test-retest reliability of eight jump execution variables assessed from mechanography. Thirty-two women (mean±SD: age 20.8 ± 1.3 yr) and 16 men (age 22.1 ± 1.9 yr) attended a familiarization session and two testing sessions, all one week apart. Participants performed two variations of the squat jump with squat depth self-selected and controlled using a goniometer to 80º knee flexion. Test-retest reliability was quantified as the systematic error (using effect size between jumps), random error (using coefficients of variation), and test-retest correlations (using intra-class correlation coefficients). Overall, jump execution variables demonstrated acceptable reliability, evidenced by small systematic errors (mean±95%CI: 0.2 ± 0.07), moderate random errors (mean±95%CI: 17.8 ± 3.7%), and very strong test-retest correlations (range: 0.73-0.97). Differences in random errors between controlled and self-selected protocols were negligible (mean±95%CI: 1.3 ± 2.3%). Jump execution variables demonstrated acceptable reliability, with no meaningful differences between the controlled and self-selected jump protocols. To simplify testing, a self-selected jump protocol can be used to assess force-time variables with negligible impact on measurement error.

  10. A universal and reliable assay for molecular sex identification of three-spined sticklebacks (Gasterosteus aculeatus).

    Science.gov (United States)

    Toli, E-A; Calboli, F C F; Shikano, T; Merilä, J

    2016-11-01

    In heterogametic species, biological differences between the two sexes are ubiquitous, and hence, errors in sex identification can be a significant source of noise and bias in studies where sex-related sources of variation are of interest or need to be controlled for. We developed and validated a universal multimarker assay for reliable sex identification of three-spined sticklebacks (Gasterosteus aculeatus). The assay makes use of genotype scores from three sex-linked loci and utilizes Bayesian probabilistic inference to identify sex of the genotyped individuals. The results, validated with 286 phenotypically sexed individuals from six populations of sticklebacks representing all major genetic lineages (cf. Pacific, Atlantic and Japan Sea), indicate that in contrast to commonly used single-marker-based sex identification assays, the developed multimarker assay should be 100% accurate. As the markers in the assay can be scored from agarose gels, it provides a quick and cost-efficient tool for universal sex identification of three-spined sticklebacks. The general principle of combining information from multiple markers to improve the reliability of sex identification is transferable and can be utilized to develop and validate similar assays for other species. © 2016 John Wiley & Sons Ltd.

  11. A reliable transmission protocol for ZigBee-based wireless patient monitoring.

    Science.gov (United States)

    Chen, Shyr-Kuen; Kao, Tsair; Chan, Chia-Tai; Huang, Chih-Ning; Chiang, Chih-Yen; Lai, Chin-Yu; Tung, Tse-Hua; Wang, Pi-Chung

    2012-01-01

    Patient monitoring systems are gaining their importance as the fast-growing global elderly population increases demands for caretaking. These systems use wireless technologies to transmit vital signs for medical evaluation. In a multihop ZigBee network, the existing systems usually use broadcast or multicast schemes to increase the reliability of signals transmission; however, both the schemes lead to significantly higher network traffic and end-to-end transmission delay. In this paper, we present a reliable transmission protocol based on anycast routing for wireless patient monitoring. Our scheme automatically selects the closest data receiver in an anycast group as a destination to reduce the transmission latency as well as the control overhead. The new protocol also shortens the latency of path recovery by initiating route recovery from the intermediate routers of the original path. On the basis of a reliable transmission scheme, we implement a ZigBee device for fall monitoring, which integrates fall detection, indoor positioning, and ECG monitoring. When the triaxial accelerometer of the device detects a fall, the current position of the patient is transmitted to an emergency center through a ZigBee network. In order to clarify the situation of the fallen patient, 4-s ECG signals are also transmitted. Our transmission scheme ensures the successful transmission of these critical messages. The experimental results show that our scheme is fast and reliable. We also demonstrate that our devices can seamlessly integrate with the next generation technology of wireless wide area network, worldwide interoperability for microwave access, to achieve real-time patient monitoring.

  12. Overview of procalcitonin assays and procalcitonin-guided protocols for the management of patients with infections and sepsis.

    Science.gov (United States)

    Schuetz, Philipp; Bretscher, Celine; Bernasconi, Luca; Mueller, Beat

    2017-06-01

    Procalcitonin is a surrogate infection blood marker whose levels help estimate the likelihood of bacterial infections and correlate with their resolution. Recent trials have revealed the benefits of inclusion of procalcitonin in antibiotic stewardship protocols for initiation and discontinuation of antimicrobial therapy. Areas covered: Procalcitonin-guided antibiotic stewardship protocols have shown appreciable reductions in antibiotic use and duration of therapy in respiratory infections, sepsis, and other infections, with positive effects on clinical outcomes. Multiple fully automated and sensitive procalcitonin assays are routinely used in clinical practice. Utilization of these assays requires consideration of the clinical setting and knowledge of assay characteristics, particularly assay sensitivities, reproducibility, and performance across routinely used cut-off ranges. The authors provide an overview of the strengths and limitations of currently available procalcitonin assays and antibiotic therapy algorithms incorporating procalcitonin currently used in different clinical settings and in patients with different underlying infections. Expert commentary: Use of sensitive procalcitonin measurements in clinical algorithms can reduce antimicrobial overuse, decreasing the risk of side effects and controlling emerging bacterial multi-resistance. Before use in clinical practice, it is important to carefully assess the quality of novel PCT assays and rigorously evaluate them in target patient populations across clinically relevant cut-off ranges.

  13. Corrections to "Connectivity-Based Reliable Multicast MAC Protocol for IEEE 802.11 Wireless LANs"

    Directory of Open Access Journals (Sweden)

    Choi Woo-Yong

    2010-01-01

    Full Text Available We have found the errors in the throughput formulae presented in our paper "Connectivity-based reliable multicast MAC protocol for IEEE 802.11 wireless LANs". We provide the corrected formulae and numerical results.

  14. Establishment and intra-/inter-laboratory validation of a standard protocol of reactive oxygen species assay for chemical photosafety evaluation.

    Science.gov (United States)

    Onoue, Satomi; Hosoi, Kazuhiro; Wakuri, Shinobu; Iwase, Yumiko; Yamamoto, Toshinobu; Matsuoka, Naoko; Nakamura, Kazuichi; Toda, Tsuguto; Takagi, Hironori; Osaki, Naoto; Matsumoto, Yasuhiro; Kawakami, Satoru; Seto, Yoshiki; Kato, Masashi; Yamada, Shizuo; Ohno, Yasuo; Kojima, Hajime

    2013-11-01

    A reactive oxygen species (ROS) assay was previously developed for photosafety evaluation of pharmaceuticals, and the present multi-center study aimed to establish and validate a standard protocol for ROS assay. In three participating laboratories, two standards and 42 coded chemicals, including 23 phototoxins and 19 nonphototoxic drugs/chemicals, were assessed by the ROS assay according to the standardized protocol. Most phototoxins tended to generate singlet oxygen and/or superoxide under UV-vis exposure, but nonphototoxic chemicals were less photoreactive. In the ROS assay on quinine (200 µm), a typical phototoxic drug, the intra- and inter-day precisions (coefficient of variation; CV) were found to be 1.5-7.4% and 1.7-9.3%, respectively. The inter-laboratory CV for quinine averaged 15.4% for singlet oxygen and 17.0% for superoxide. The ROS assay on 42 coded chemicals (200 µm) provided no false negative predictions upon previously defined criteria as compared with the in vitro/in vivo phototoxicity, although several false positives appeared. Outcomes from the validation study were indicative of satisfactory transferability, intra- and inter-laboratory variability, and predictive capacity of the ROS assay. Copyright © 2012 John Wiley & Sons, Ltd.

  15. Test-retest reliability of a balance testing protocol with external perturbations in young healthy adults.

    Science.gov (United States)

    Robbins, Shawn M; Caplan, Ryan M; Aponte, Daniel I; St-Onge, Nancy

    2017-10-01

    External perturbations are utilized to challenge balance and mimic realistic balance threats in patient populations. The reliability of such protocols has not been established. The purpose was to examine test-retest reliability of balance testing with external perturbations. Healthy adults (n=34; mean age 23 years) underwent balance testing over two visits. Participants completed ten balance conditions in which the following parameters were combined: perturbation or non-perturbation, single or double leg, and eyes open or closed. Three trials were collected for each condition. Data were collected on a force plate and external perturbations were applied by translating the plate. Force plate center of pressure (CoP) data were summarized using 13 different CoP measures. Test-retest reliability was examined using intraclass correlation coefficients (ICC) and Bland-Altman plots. CoP measures of total speed and excursion in both anterior-posterior and medial-lateral directions generally had acceptable ICC values for perturbation conditions (ICC=0.46 to 0.87); however, many other CoP measures (e.g. range, area of ellipse) had unacceptable test-retest reliability (ICCbalance testing protocols that include external perturbations should be made to improve test-retest reliability and diminish learning including more extensive participant training and increasing the number of trials. CoP measures that consider all data points (e.g. total speed) are more reliable than those that only consider a few data points. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Dose-Response Assessment of Four Genotoxic Chemicals in a Combined Mouse and Rat Micronucleus and Comet Assay Protocol

    Science.gov (United States)

    Recio, Leslie; Hobbs, Cheryl; Caspary, William; Witt, Kristine L.

    2012-01-01

    The in vivo micronucleus (MN) assay has proven to be an effective measure of genotoxicity potential. However, sampling a single tissue (bone marrow) for a single indicator of genetic damage using the MN assay provides a limited genotoxicity profile. The in vivo alkaline (pH>13) Comet assay, which detects a broad spectrum of DNA damage, can be applied to a variety of rodent tissues following administration of test agents. To determine if the Comet assay is a useful supplement to the in vivo MN assay, a combined test protocol (MN/Comet assay) was conducted in male B6C3F1 mice and F344/N rats using four model genotoxicants: ethyl methanesulfonate (EMS), acrylamide (ACM), cyclophosphamide (CP), and vincristine sulfate (VS). Test compounds were administered on 4 consecutive days at 24-hour intervals (VS was administered to rats for 3 days); animals were euthanized 4 hours after the last administration. All compounds induced significant increases in micronucleated reticulocytes (MN-RET) in the peripheral blood of mice, and all but ACM induced MN-RET in rats. EMS and ACM induced significant increases in DNA damage, measured by the Comet assay, in multiple tissues of mice and rats. CP-induced DNA damage was detected in leukocytes and duodenum cells. VS, a spindle fiber disrupting agent, was negative in the Comet assay. Based on these results, the MN/Comet assay holds promise for providing more comprehensive assessments of potential genotoxicants, and the National Toxicology Program is presently using this combined protocol in its overall evaluation of the genotoxicity of substances of public health concern. PMID:20371966

  17. A Performance Evaluation of NACK-Oriented Protocols as the Foundation of Reliable Delay- Tolerant Networking Convergence Layers

    Science.gov (United States)

    Iannicca, Dennis; Hylton, Alan; Ishac, Joseph

    2012-01-01

    Delay-Tolerant Networking (DTN) is an active area of research in the space communications community. DTN uses a standard layered approach with the Bundle Protocol operating on top of transport layer protocols known as convergence layers that actually transmit the data between nodes. Several different common transport layer protocols have been implemented as convergence layers in DTN implementations including User Datagram Protocol (UDP), Transmission Control Protocol (TCP), and Licklider Transmission Protocol (LTP). The purpose of this paper is to evaluate several stand-alone implementations of negative-acknowledgment based transport layer protocols to determine how they perform in a variety of different link conditions. The transport protocols chosen for this evaluation include Consultative Committee for Space Data Systems (CCSDS) File Delivery Protocol (CFDP), Licklider Transmission Protocol (LTP), NACK-Oriented Reliable Multicast (NORM), and Saratoga. The test parameters that the protocols were subjected to are characteristic of common communications links ranging from terrestrial to cis-lunar and apply different levels of delay, line rate, and error.

  18. IRR (Inter-Rater Reliability) of a COP (Classroom Observation Protocol)--A Critical Appraisal

    Science.gov (United States)

    Rui, Ning; Feldman, Jill M.

    2012-01-01

    Notwithstanding broad utility of COPs (classroom observation protocols), there has been limited documentation of the psychometric properties of even the most popular COPs. This study attempted to fill this void by closely examining the item and domain-level IRR (inter-rater reliability) of a COP that was used in a federally funded striving readers…

  19. Integration of GC-MSD and ER-Calux® assay into a single protocol for determining steroid estrogens in environmental samples.

    Science.gov (United States)

    Avberšek, Miha; Žegura, Bojana; Filipič, Metka; Heath, Ester

    2011-11-01

    There are many published studies that use either chemical or biological methods to investigate steroid estrogens in the aquatic environment, but rarer are those that combine both. In this study, gas chromatography with mass selective detection (GC-MSD) and the ER-Calux(®) estrogenicity assay were integrated into a single protocol for simultaneous determination of natural (estrone--E1, 17β-estradiol--E2, estriol--E3) and synthetic (17α-ethinylestradiol--EE2) steroid estrogens concentrations and the total estrogenic potential of environmental samples. For integration purposes, several solvents were investigated and the commonly used dimethyl sulphoxide (DMSO) in the ER-Calux(®) assay was replaced by ethyl acetate, which is more compatible with gas chromatography and enables the same sample to be analysed by both GC-MSD and the ER-Calux(®) assay. The integrated protocol was initially tested using a standard mixture of estrogens. The results for pure standards showed that the estrogenicity calculated on the basis of GC-MSD and the ER-Calux(®) assay exhibited good correlation (r(2)=0.96; α=0.94). The result remained the same when spiked waste water extracts were tested (r(2)=0.92, α=1.02). When applied to real waste water influent and effluent samples the results proved (r(2)=0.93; α=0.99) the applicability of the protocol. The main advantages of this newly developed protocol are simple sample handling for both methods, and reduced material consumption and labour. In addition, it can be applied as either a complete or sequential analysis where the ER-Calux(®) assay is used as a pre-screening method prior to the chemical analysis. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Critical issues with the in vivo comet assay: A report of the comet assay working group in the 6th International Workshop on Genotoxicity Testing (IWGT).

    Science.gov (United States)

    Speit, Günter; Kojima, Hajime; Burlinson, Brian; Collins, Andrew R; Kasper, Peter; Plappert-Helbig, Ulla; Uno, Yoshifumi; Vasquez, Marie; Beevers, Carol; De Boeck, Marlies; Escobar, Patricia A; Kitamoto, Sachiko; Pant, Kamala; Pfuhler, Stefan; Tanaka, Jin; Levy, Dan D

    2015-05-01

    As a part of the 6th IWGT, an expert working group on the comet assay evaluated critical topics related to the use of the in vivo comet assay in regulatory genotoxicity testing. The areas covered were: identification of the domain of applicability and regulatory acceptance, identification of critical parameters of the protocol and attempts to standardize the assay, experience with combination and integration with other in vivo studies, demonstration of laboratory proficiency, sensitivity and power of the protocol used, use of different tissues, freezing of samples, and choice of appropriate measures of cytotoxicity. The standard protocol detects various types of DNA lesions but it does not detect all types of DNA damage. Modifications of the standard protocol may be used to detect additional types of specific DNA damage (e.g., cross-links, bulky adducts, oxidized bases). In addition, the working group identified critical parameters that should be carefully controlled and described in detail in every published study protocol. In vivo comet assay results are more reliable if they were obtained in laboratories that have demonstrated proficiency. This includes demonstration of adequate response to vehicle controls and an adequate response to a positive control for each tissue being examined. There was a general agreement that freezing of samples is an option but more data are needed in order to establish generally accepted protocols. With regard to tissue toxicity, the working group concluded that cytotoxicity could be a confounder of comet results. It is recommended to look at multiple parameters such as histopathological observations, organ-specific clinical chemistry as well as indicators of tissue inflammation to decide whether compound-specific toxicity might influence the result. The expert working group concluded that the alkaline in vivo comet assay is a mature test for the evaluation of genotoxicity and can be recommended to regulatory agencies for use

  1. Reassessing the reliability of the salivary cortisol assay for the diagnosis of Cushing syndrome.

    Science.gov (United States)

    Zhang, Qian; Dou, Jingtao; Gu, Weijun; Yang, Guoqing; Lu, Juming

    2013-10-01

    The cortisol concentration in saliva is 10-fold lower than total serum cortisol and accurately reflects the serum concentration, both levels being lowest around midnight. The salivary cortisol assay measures free cortisol and is unaffected by confounding factors. This study analysed published data on the sensitivity and specificity of salivary cortisol levels in the diagnosis of Cushing syndrome. Data from studies on the use of different salivary cortisol assay techniques in the diagnosis of Cushing syndrome, published between 1998 and 2012 and retrieved using Ovid MEDLINE®, were analysed for variance and correlation. For the 11 studies analysed, mean sensitivity and specificity of the salivary cortisol assay were both >90%. Repeated measurements were easily made with this assay, enabling improved diagnostic accuracy in comparison with total serum cortisol measurements. This analysis confirms the reliability of the saliva cortisol assay as pragmatic tool for the accurate diagnosis of Cushing syndrome. With many countries reporting a rising prevalence of metabolic syndrome, diabetes and obesity--in which there is often a high circulating cortisol level--salivary cortisol measurement will help distinguish these states from Cushing syndrome.

  2. Intra-rater and inter-rater reliability of the standardized ultrasound protocol for assessing subacromial structures

    DEFF Research Database (Denmark)

    Hougs Kjær, Birgitte; Ellegaard, Karen; Wieland, Ina

    2017-01-01

    BACKGROUND: US-examinations related to shoulder impingement (SI) often vary due to methodological differences, examiner positions, transducers, and recording parameters. Reliable US protocols for examination of different structures related to shoulder impingement are therefore needed. OBJECTIVES...... of the supraspinatus tendon (SUPRA) and subacromial subdeltoid (SASD) bursa in two imaging positions, and the acromial humeral distance (AHD) in one position. Additionally, agreement on dynamic impingement (DI) examination was performed. The intra- and inter-rater reliability was carried out on the same day...

  3. Nano-immunosafety: issues in assay validation

    International Nuclear Information System (INIS)

    Boraschi, Diana; Italiani, Paola; Oostingh, Gertie J; Duschl, Albert; Casals, Eudald; Puntes, Victor F; Nelissen, Inge

    2011-01-01

    Assessing the safety of engineered nanomaterials for human health must include a thorough evaluation of their effects on the immune system, which is responsible for defending the integrity of our body from damage and disease. An array of robust and representative assays should be set up and validated, which could be predictive of the effects of nanomaterials on immune responses. In a trans-European collaborative work, in vitro assays have been developed to this end. In vitro tests have been preferred for their suitability to standardisation and easier applicability. Adapting classical assays to testing the immunotoxicological effects of nanoparticulate materials has raised a series of issues that needed to be appropriately addressed in order to ensure reliability of results. Besides the exquisitely immunological problem of selecting representative endpoints predictive of the risk of developing disease, assay results turned out to be significantly biased by artefactual interference of the nanomaterials or contaminating agents with the assay protocol. Having addressed such problems, a series of robust and representative assays have been developed that describe the effects of engineered nanoparticles on professional and non-professional human defence cells. Two of such assays are described here, one based on primary human monocytes and the other employing human lung epithelial cells transfected with a reporter gene.

  4. Reliability of dipstick assay in predicting urinary tract infection

    Directory of Open Access Journals (Sweden)

    Anith Kumar Mambatta

    2015-01-01

    Full Text Available Aims: Urine dipstick analysis is a quick, cheap and a useful test in predicting Urinary Tract Infection (UTI in hospitalized patients. Our aim is to evaluate the reliability (sensitivity of urine dipstick analysis against urine culture in the diagnosis of UTI. Materials and Methods: Patients admitted to our hospital suspected of having UTI, with positive urine cultures were included in this study from a 2-year period (January 2011 to December 2012. Dipstick urinalysis was done using multistix 10 SG (Siemens and clinitek advantus analyzer. The sensitivity of dipstick nitrites, leukocyte esterase and blood in these culture-positive UTI patients was calculated retrospectively. Results: Urine dipstick analysis of 635 urine culture-positive patients was studied. The sensitivity of nitrite alone and leukocyte esterase alone were 23.31% and 48.5%, respectively. The sensitivity of blood alone in positive urine culture was 63.94%, which was the highest sensitivity for a single screening test. The presence of leukocyte esterase and/or blood increased the sensitivity to 72.28%. The sensitivity was found to be the highest when nitrite, leukocyte and blood were considered together. Conclusions: Nitrite test and leukocyte esterase test when used individually is not reliable to rule out UTI. Hence, symptomatic UTI patients with negative dipstick assay should be subjected to urine culture for a proper management.

  5. The reliability of a single protocol to determine endothelial, microvascular and autonomic functions in adolescents.

    Science.gov (United States)

    Bond, Bert; Williams, Craig A; Barker, Alan R

    2017-11-01

    Impairments in macrovascular, microvascular and autonomic function are present in asymptomatic youths with clustered cardiovascular disease risk factors. This study determines the within-day reliability and between-day reliability of a single protocol to non-invasively assess these outcomes in adolescents. Forty 12- to 15-year-old adolescents (20 boys) visited the laboratory in a fasted state on two occasions, approximately 1 week apart. One hour after a standardized cereal breakfast, macrovascular function was determined via flow-mediated dilation (FMD). Heart rate variability (root mean square of successive R-R intervals; RMSSD) was determined from the ECG-gated ultrasound images acquired during the FMD protocol prior to cuff occlusion. Microvascular function was simultaneously quantified as the peak (PRH) and total (TRH) hyperaemic response to occlusion in the cutaneous circulation of the forearm via laser Doppler imaging. To address within-day reliability, a subset of twenty adolescents (10 boys) repeated these measures 90 min afterwards on one occasion. The within-day typical error and between-day typical error expressed as a coefficient of variation of these outcomes are as follows: ratio-scaled FMD, 5·1% and 10·6%; allometrically scaled FMD, 4·4% and 9·4%; PRH, 11% and 13·3%; TRH, 29·9% and 23·1%; and RMSSD, 17·6% and 17·6%. The within- and between-day test-retest correlation coefficients for these outcomes were all significant (r > 0·54 for all). Macrovascular, microvascular and autonomic functions can be simultaneously and non-invasively determined in adolescents using a single protocol with an appropriate degree of reproducibility. Determining these outcomes may provide greater understanding of the progression of cardiovascular disease and aid early intervention. © 2016 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd.

  6. Reliability of single aliquot regenerative protocol (SAR) for dose estimation in quartz at different burial temperatures: A simulation study

    International Nuclear Information System (INIS)

    Koul, D.K.; Pagonis, V.; Patil, P.

    2016-01-01

    The single aliquot regenerative protocol (SAR) is a well-established technique for estimating naturally acquired radiation doses in quartz. This simulation work examines the reliability of SAR protocol for samples which experienced different ambient temperatures in nature in the range of −10 to 40 °C. The contribution of various experimental variables used in SAR protocols to the accuracy and precision of the method is simulated for different ambient temperatures. Specifically the effects of paleo-dose, test dose, pre-heating temperature and cut-heat temperature on the accuracy of equivalent dose (ED) estimation are simulated by using random combinations of the concentrations of traps and centers using a previously published comprehensive quartz model. The findings suggest that the ambient temperature has a significant bearing on the reliability of natural dose estimation using SAR protocol, especially for ambient temperatures above 0 °C. The main source of these inaccuracies seems to be thermal sensitization of the quartz samples caused by the well-known thermal transfer of holes between luminescence centers in quartz. The simulations suggest that most of this inaccuracy in the dose estimation can be removed by delivering the laboratory doses in pulses (pulsed irradiation procedures). - Highlights: • Ambient temperatures affect the reliability of SAR. • It overestimates the dose with increase in burial temperature and burial time periods. • Elevated temperature irradiation does not correct for these overestimations. • Inaccuracies in dose estimation can be removed by incorporating pulsed irradiation procedures.

  7. Modeling of coupled differential equations for cellular chemical signaling pathways: Implications for assay protocols utilized in cellular engineering.

    Science.gov (United States)

    O'Clock, George D

    2016-08-01

    Cellular engineering involves modification and control of cell properties, and requires an understanding of fundamentals and mechanisms of action for cellular derived product development. One of the keys to success in cellular engineering involves the quality and validity of results obtained from cell chemical signaling pathway assays. The accuracy of the assay data cannot be verified or assured if the effect of positive feedback, nonlinearities, and interrelationships between cell chemical signaling pathway elements are not understood, modeled, and simulated. Nonlinearities and positive feedback in the cell chemical signaling pathway can produce significant aberrations in assay data collection. Simulating the pathway can reveal potential instability problems that will affect assay results. A simulation, using an electrical analog for the coupled differential equations representing each segment of the pathway, provides an excellent tool for assay validation purposes. With this approach, voltages represent pathway enzyme concentrations and operational amplifier feedback resistance and input resistance values determine pathway gain and rate constants. The understanding provided by pathway modeling and simulation is strategically important in order to establish experimental controls for assay protocol structure, time frames specified between assays, and assay concentration variation limits; to ensure accuracy and reproducibility of results.

  8. Reliability and fatigue characteristics of a standing hip isometric endurance protocol.

    Science.gov (United States)

    Mutchler, Jessica A; Weinhandl, Joshua T; Hoch, Matthew C; Van Lunen, Bonnie L

    2015-08-01

    Muscle fatigue is a common consideration when evaluating and rehabilitating athletic injuries. The presence of muscular fatigue has been previously determined by quantifying median frequency (MF) through a power spectral analysis on EMG signals collected throughout an endurance task. Research has not yet determined if a prolonged isometric test in a standing position generates muscular fatigue of the hip. The purpose of this study was to determine the reliability and fatigue characteristics of a standing hip isometric endurance test. Twenty healthy participants completed one 60-s Maximum Voluntary Isometric Contraction of standing hip flexion, extension, adduction, and abduction. MF of the participants' dominant limb rectus femoris (RF), biceps femoris (BF), gluteus maximus (GMax), gluteus medius (GMed) and adductor longus (ADD) was determined via surface electromyography during two sessions, 30-min apart. Reliability values (ICC2,1) were moderate-to-excellent for all time intervals of each action (FlexionRF: >0.80; ExtensionBF: >0.89; ExtensionGMax: >0.60; AdductionADD: >0.78; AbductionGMed: >0.60) and MF significantly decreased over time for all actions. Results suggest the endurance test is a reliable technique to generate muscular fatigue for hip flexion, extension, adduction and abduction. It can be used as a time efficient fatigue protocol specific to the RF, BF, GMax, ADD and GMed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Inter-observer reliability of animal-based welfare indicators included in the Animal Welfare Indicators welfare assessment protocol for dairy goats.

    Science.gov (United States)

    Vieira, A; Battini, M; Can, E; Mattiello, S; Stilwell, G

    2018-01-08

    This study was conducted within the context of the Animal Welfare Indicators (AWIN) project and the underlying scientific motivation for the development of the study was the scarcity of data regarding inter-observer reliability (IOR) of welfare indicators, particularly given the importance of reliability as a further step for developing on-farm welfare assessment protocols. The objective of this study is therefore to evaluate IOR of animal-based indicators (at group and individual-level) of the AWIN welfare assessment protocol (prototype) for dairy goats. In the design of the study, two pairs of observers, one in Portugal and another in Italy, visited 10 farms each and applied the AWIN prototype protocol. Farms in both countries were visited between January and March 2014, and all the observers received the same training before the farm visits were initiated. Data collected during farm visits, and analysed in this study, include group-level and individual-level observations. The results of our study allow us to conclude that most of the group-level indicators presented the highest IOR level ('substantial', 0.85 to 0.99) in both field studies, pointing to a usable set of animal-based welfare indicators that were therefore included in the first level of the final AWIN welfare assessment protocol for dairy goats. Inter-observer reliability of individual-level indicators was lower, but the majority of them still reached 'fair to good' (0.41 to 0.75) and 'excellent' (0.76 to 1) levels. In the paper we explore reasons for the differences found in IOR between the group and individual-level indicators, including how the number of individual-level indicators to be assessed on each animal and the restraining method may have affected the results. Furthermore, we discuss the differences found in the IOR of individual-level indicators in both countries: the Portuguese pair of observers reached a higher level of IOR, when compared with the Italian observers. We argue how the

  10. Specification and Design of a Fault Recovery Model for the Reliable Multicast Protocol

    Science.gov (United States)

    Montgomery, Todd; Callahan, John R.; Whetten, Brian

    1996-01-01

    The Reliable Multicast Protocol (RMP) provides a unique, group-based model for distributed programs that need to handle reconfiguration events at the application layer. This model, called membership views, provides an abstraction in which events such as site failures, network partitions, and normal join-leave events are viewed as group reformations. RMP provides access to this model through an application programming interface (API) that notifies an application when a group is reformed as the result of a some event. RMP provides applications with reliable delivery of messages using an underlying IP Multicast media to other group members in a distributed environment even in the case of reformations. A distributed application can use various Quality of Service (QoS) levels provided by RMP to tolerate group reformations. This paper explores the implementation details of the mechanisms in RMP that provide distributed applications with membership view information and fault recovery capabilities.

  11. Reliability of plant root comet assay in comparison with human leukocyte comet assay for assessment environmental genotoxic agents.

    Science.gov (United States)

    Reis, Gabriela Barreto Dos; Andrade-Vieira, Larissa Fonseca; Moraes, Isabella de Campos; César, Pedro Henrique Souza; Marcussi, Silvana; Davide, Lisete Chamma

    2017-08-01

    Comet assay is an efficient test to detect genotoxic compounds based on observation of DNA damage. The aim of this work was to compare the results obtained from the comet assay in two different type of cells extracted from the root tips from Lactuca sativa L. and human blood. For this, Spent Pot Liner (SPL), and its components (aluminum and fluoride) were applied as toxic agents. SPL is a solid waste generated in industry from the aluminum mining and processing with known toxicity. Three concentrations of all tested solutions were applied and the damages observed were compared to negative and positive controls. It was observed an increase in the frequency of DNA damage for human leukocytes and plant cells, in all treatments. On human leukocytes, SPL induced the highest percentage of damage, with an average of 87.68%. For root tips cells of L. sativa the highest percentage of damage was detected for aluminum (93.89%). Considering the arbitrary units (AU), the average of nuclei with high levels of DNA fragmentation was significant for both cells type evaluated. The tested cells demonstrated equal effectiveness for detection of the genotoxicity induced by the SPL and its chemical components, aluminum and fluoride. Further, using a unique method, the comet assay, we proved that cells from root tips of Lactuca sativa represent a reliable model to detect DNA damage induced by genotoxic pollutants is in agreement of those observed in human leukocytes as model. So far, plant cells may be suggested as important system to assess the toxicological risk of environmental agents. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Inter-rater and intra-rater reliability of a clinical protocol for measuring turnout in collegiate dancers.

    Science.gov (United States)

    Greene, Amanda; Lasner, Andrea; Deu, Rajwinder; Oliphant, Seth; Johnson, Kenneth

    2018-02-02

    Reliable methods of measuring turnout in dancers and comparing active turnout (used in class) with functional (uncompensated) turnout are needed. Authors have suggested measurement techniques but there is no clinically useful, easily reproducible technique with established inter-rater and intra-rater reliability. We adapted a technique based on previous research, which is easily reproducible. We hypothesized excellent inter-rater and intra-rater reliability between experienced physical therapists (PTs) and a briefly trained faculty member from a university's department of dance. Thirty-two participants were recruited from the same dance department. Dancers' active and functional turnout was measured by each rater. We found that our technique for measuring active and functional turnout has excellent inter-rater and intra-rater reliability when performed by two experienced PTs and by one briefly trained university-level dance faculty member. For active turnout, inter-rater reliability was 0.78 among all raters and 0.82 among only the PT raters; intra-rater reliability was 0.82 among all raters and 0.85 among only the PT raters. For functional turnout, inter-rater reliability was 0.86 among all raters and 0.88 among only the PT raters; intra-rater reliability was 0.87 among all raters and 0.88 among only the PT raters. The measurement technique described provides a standardized protocol with excellent inter-rater and intra-rater reliability when performed by experienced PTs or by a briefly trained university-level dance faculty member.

  13. Determining Reliability of a Dual-Task Functional Mobility Protocol for Individuals With Lower Extremity Amputation.

    Science.gov (United States)

    Hunter, Susan W; Frengopoulos, Courtney; Holmes, Jeff; Viana, Ricardo; Payne, Michael W

    2018-04-01

    To determine the relative and absolute reliability of a dual-task functional mobility assessment. Cross-sectional study. Academic rehabilitation hospital. Individuals (N=60) with lower extremity amputation attending an outpatient amputee clinic (mean age, 58.21±12.59y; 18, 80% male) who were stratified into 3 groups: (1) transtibial amputation of vascular etiology (n=20); (2) transtibial amputation of nonvascular etiology (n=20); and (3) transfemoral or bilateral amputation of any etiology (n=20). Not applicable. Time to complete the L Test measured functional mobility under single- and dual-task conditions. The addition of a cognitive task (serial subtractions by 3's) created dual-task conditions. Single-task performance on the cognitive task was also reported. Intraclass correlation coefficients (ICCs) measured relative reliability; SEM and minimal detectable change with a 95% confidence interval (MDC 95 ) measured absolute reliability. Bland-Altman plots measured agreement between assessments. Relative reliability results were excellent for all 3 groups. Values for the dual-task L Test for those with transtibial amputation of vascular etiology (n=20; mean age, 60.36±7.84y; 19, 90% men) were ICC=.98 (95% confidence interval [CI], .94-.99), SEM=1.36 seconds, and MDC 95 =3.76 seconds; for those with transtibial amputation of nonvascular etiology (n=20; mean age, 55.85±14.08y; 17, 85% men), values were ICC=.93 (95% CI, .80-.98), SEM=1.34 seconds, and MDC 95 =3.71 seconds; and for those with transfemoral or bilateral amputation (n=20; mean age, 58.21±14.88y; 13, 65% men), values were ICC=.998 (95% CI, .996-.999), SEM=1.03 seconds, and MDC 95 =2.85 seconds. Bland-Altman plots indicated that assessments did not vary systematically for each group. This dual-task assessment protocol achieved approved levels of relative reliability values for the 3 groups tested. This protocol may be used clinically or in research settings to assess the interaction between cognition

  14. Automation of a Nile red staining assay enables high throughput quantification of microalgal lipid production.

    Science.gov (United States)

    Morschett, Holger; Wiechert, Wolfgang; Oldiges, Marco

    2016-02-09

    Within the context of microalgal lipid production for biofuels and bulk chemical applications, specialized higher throughput devices for small scale parallelized cultivation are expected to boost the time efficiency of phototrophic bioprocess development. However, the increasing number of possible experiments is directly coupled to the demand for lipid quantification protocols that enable reliably measuring large sets of samples within short time and that can deal with the reduced sample volume typically generated at screening scale. To meet these demands, a dye based assay was established using a liquid handling robot to provide reproducible high throughput quantification of lipids with minimized hands-on-time. Lipid production was monitored using the fluorescent dye Nile red with dimethyl sulfoxide as solvent facilitating dye permeation. The staining kinetics of cells at different concentrations and physiological states were investigated to successfully down-scale the assay to 96 well microtiter plates. Gravimetric calibration against a well-established extractive protocol enabled absolute quantification of intracellular lipids improving precision from ±8 to ±2 % on average. Implementation into an automated liquid handling platform allows for measuring up to 48 samples within 6.5 h, reducing hands-on-time to a third compared to manual operation. Moreover, it was shown that automation enhances accuracy and precision compared to manual preparation. It was revealed that established protocols relying on optical density or cell number for biomass adjustion prior to staining may suffer from errors due to significant changes of the cells' optical and physiological properties during cultivation. Alternatively, the biovolume was used as a measure for biomass concentration so that errors from morphological changes can be excluded. The newly established assay proved to be applicable for absolute quantification of algal lipids avoiding limitations of currently established

  15. The Vitotox and ToxTracker assays: A two-test combination for quick and reliable assessment of genotoxic hazards.

    Science.gov (United States)

    Ates, Gamze; Favyts, Dorien; Hendriks, Giel; Derr, Remco; Mertens, Birgit; Verschaeve, Luc; Rogiers, Vera; Y Doktorova, Tatyana

    2016-11-01

    To ensure safety for humans, it is essential to characterize the genotoxic potential of new chemical entities, such as pharmaceutical and cosmetic substances. In a first tier, a battery of in vitro tests is recommended by international regulatory agencies. However, these tests suffer from inadequate specificity: compounds may be wrongly categorized as genotoxic, resulting in unnecessary, time-consuming, and expensive in vivo follow-up testing. In the last decade, novel assays (notably, reporter-based assays) have been developed in an attempt to overcome these drawbacks. Here, we have investigated the performance of two in vitro reporter-based assays, Vitotox and ToxTracker. A set of reference compounds was selected to span a variety of mechanisms of genotoxic action and applicability domains (e.g., pharmaceutical and cosmetic ingredients). Combining the performance of the two assays, we achieved 93% sensitivity and 79% specificity for prediction of gentoxicity for this set of compounds. Both assays permit quick high-throughput analysis of drug candidates, while requiring only small quantities of the test substances. Our study shows that these two assays, when combined, can be a reliable method for assessment of genotoxicity hazard. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. The reliability of a maximal isometric hip strength and simultaneous surface EMG screening protocol in elite, junior rugby league athletes.

    Science.gov (United States)

    Charlton, Paula C; Mentiplay, Benjamin F; Grimaldi, Alison; Pua, Yong-Hao; Clark, Ross A

    2017-02-01

    Firstly to describe the reliability of assessing maximal isometric strength of the hip abductor and adductor musculature using a hand held dynamometry (HHD) protocol with simultaneous wireless surface electromyographic (sEMG) evaluation of the gluteus medius (GM) and adductor longus (AL). Secondly, to describe the correlation between isometric strength recorded with the HHD protocol and a laboratory standard isokinetic device. Reliability and correlational study. A sample of 24 elite, male, junior, rugby league athletes, age 16-20 years participated in repeated HHD and isometric Kin-Com (KC) strength testing with simultaneous sEMG assessment, on average (range) 6 (5-7) days apart by a single assessor. Strength tests included; unilateral hip abduction (ABD) and adduction (ADD) and bilateral ADD assessed with squeeze (SQ) tests in 0 and 45° of hip flexion. HHD demonstrated good to excellent inter-session reliability for all outcome measures (ICC (2,1) =0.76-0.91) and good to excellent association with the laboratory reference KC (ICC (2,1) =0.80-0.88). Whilst intra-session, inter-trial reliability of EMG activation and co-activation outcome measures ranged from moderate to excellent (ICC (2,1) =0.70-0.94), inter-session reliability was poor (all ICC (2,1) Isometric strength testing of the hip ABD and ADD musculature using HHD may be measured reliably in elite, junior rugby league athletes. Due to the poor inter-session reliability of sEMG measures, it is not recommended for athlete screening purposes if using the techniques implemented in this study. Copyright © 2016 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

  17. MQARR-AODV: A NOVEL MULTIPATH QOS AWARE RELIABLE REVERSE ON-DEMAND DISTANCE VECTOR ROUTING PROTOCOL FOR MOBILE AD-HOC NETWORKS

    Directory of Open Access Journals (Sweden)

    K.G. Santhiya

    2012-12-01

    Full Text Available MANET (Mobile Ad-hoc Network is an infra structure less wireless ad-hoc network that does not require any basic central control. The topology of the network changes drastically due to very fast mobility of nodes. So an adaptive routing protocol is needed for routing in MANET. AODV (Ad-hoc On-demand Distance Vector routing is the effective and prominent on-demand Ad-hoc routing protocols. During route establishment phase in traditional AODV, only one route reply message will be sent in the reverse path to establish routing path. The high mobility of nodes may affect the reply messages which lead to the retransmission of route request message by the sender which in turn leads to higher communication delay, power consumption and the reduction in the ratio of packets delivered. Sending multiple route reply messages and establishing multiple paths in a single path discovery will reduce the routing overhead involved in maintaining the connection between source and destination nodes. Multipath routing can render high scalability, end-to-end throughput and provide load balancing in MANET. The new proposed novel Multipath QoS aware reliable routing protocol establishes two routes of maximum node disjoint paths and the data transfer is carried out in the two paths simultaneously. To select best paths, the new proposed protocol uses three parameters Link Eminence, MAC overhead and node residual energy. The experimental values prove that the MQARR-AODV protocol achieves high reliability, stability, low latency and outperforms AODV by the less energy consumption, overhead and delay.

  18. Field experience with a mobile tomographic nondestructive assay system

    International Nuclear Information System (INIS)

    Prettyman, T.H.; Betts, S.E.; Taggart, D.P.; Estep, R.J.; Nicholas, N.J.; Lucas, M.C.; Harlan, R.A.

    1995-01-01

    A mobile tomographic gamma-ray scanner (TGS) developed by Los Alamos National Laboratory was recently demonstrated at the Rocky Flats Environmental Technology Site and is currently in use at Los Alamos waste storage areas. The scanner was developed to assay radionuclides in low-level, transuranic, and mixed waste in containers ranging in size from 2 ft 3 boxes to 83-gallon overpacks. The tomographic imaging capability provides a complete correction for source distribution and matrix attenuation effects, enabling accurate assays of Pu-239 and other gamma-ray emitting isotopes. In addition, the system can reliably detect self-absorbing material such as plutonium metal shot, and can correct for bias caused by self-absorption. The system can be quickly configured to execute far-field scans, segmented gamma-ray scans, and a host of intermediate scanning protocols, enabling higher throughput (up to 20 drums per 8-hour shift). In this paper, we will report on the results of field trials of the mobile system at Rocky Flats and Los Alamos. Assay accuracy is confirmed for cases in which TGS assays can be compared with assays (e.g. with calorimetry) of individual packages within the drums. The mobile tomographic technology is expected to considerably reduce characterization costs at DOE production and environmental technology sites

  19. Reliability of soluble IL-2 receptor measurements obtained with enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    Akiyama, Mitoshi; Takaishi, Masatoshi; Murakami, Yoshie; Ueda, Ryuzo; Yamakido, Michio; Tsubokura, Tokuo.

    1989-09-01

    Using an enzyme-linked immunosorbent assay (ELISA), human soluble interleukin-2 receptors (IL-2R) were measured in the serum of patients with various autoimmune system diseases. To study the sensitivity and specificity of the assay, soluble IL-2Rs were measured in the culture supernatants and in the cell extracts of peripheral blood mononuclear cells activated with phytohemagglutinin (PHA), purified protein derivative of tuberculin, and allogeneic lymphocytes, as well as in the serum of patients with various collagen diseases. The results correlated well with reports from other laboratories. For example, when stimulated by PHA, the greatest amount of soluble IL-2Rs was produced at the fastest rate. In addition, soluble IL-2R levels in the serum of collagen disease patients were significantly higher than those in healthy persons, who themselves exhibited low levels of detectable soluble IL-2Rs. It is hoped that reliable ELISA measurements of soluble IL-2Rs in the serum of atomic bomb survivors will assist in the interpretation of data collected during the work described in RP 2-87, a study of autoimmunity and autoimmune diseases in the Adult Health Study. (author)

  20. Development and utilization of an ex vivo bromodeoxyuridine local lymph node assay protocol for assessing potential chemical sensitizers.

    Science.gov (United States)

    Williams, W C; Copeland, C; Boykin, E; Quell, S J; Lehmann, D M

    2015-01-01

    The murine local lymph node assay (LLNA) is widely used to identify chemicals that may cause allergic contact dermatitis. Exposure to a dermal sensitizer results in proliferation of local lymph node T cells, which has traditionally been measured by in vivo incorporation of [(3) H]methyl thymidine. A more recent non-isotopic variation of the assay utilizes bromodeoxyuridine (BrdU) incorporation in vivo. To further improve the utility of this assay, we developed an ex vivo BrdU labeling procedure eliminating the need for in vivo injections. The results of this assay correctly identified a strong sensitizer (i.e., trimellitic anhydride) as well as weak/moderate sensitizers (i.e., eugenol, cinnamaldehyde and hexylcinnaminic aldehyde). As anticipated, neither non-sensitizers isopropanol and lactic acid nor the false negative chemical nickel II sulfate hexahydrate induced a positive threshold response in the assay. The results of this assay are in close agreement with those of the in vivo LLNA:BrdU-enzyme-linked immunosorbent assay labeling procedure. We also used the ex vivo BrdU LLNA procedure to evaluate ammonium hexachloroplatinate, ammonium tetrachloroplatinate and cis-diamminedichloroplatinum(II) and the assay correctly identified them as sensitizers based on the calculation of EC2 values. We conclude that this ex vivo BrdU labeling method offers predictive capacity comparable to previously established LLNA protocols while eliminating animal injections and the use of radioisotope. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

  1. A Functional Assay for GPR55: Envision Protocol.

    Science.gov (United States)

    Anavi-Goffer, Sharon; Ross, Ruth A

    2016-01-01

    AlphaScreen(®) SureFire(®) assay is a novel technology that combines luminescent oxygen channeling technology, nano-beads, and monocloncal antibodies to detect the level of a selected protein in a volume lower than 5 μl. This method is more sensitive compared with the traditional enzyme-linked immunosorbent assays (ELISA), and can detect an increasing number of new targets. Here, we described a method for AlphaScreen(®) SureFire(®) assay that targets ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys activation of GPR55 by L-α-lysophosphatidylinositol (LPI) and certain cannabinoids.

  2. A shortened protocol for assessing cognitive bias in rats.

    Science.gov (United States)

    Brydges, Nichola M; Hall, Lynsey

    2017-07-15

    Reliable measurement of affective state in animals is a significant goal of animal welfare. Such measurements would also improve the validity of pre-clinical mental health research which relies on animal models. However, at present, affective states in animals are inaccessible to direct measurement. In humans, changes in cognitive processing can give reliable indications of emotional state. Therefore, similar techniques are increasingly being used to gain proxy measures of affective states in animals. In particular, the 'cognitive bias' assay has gained popularity in recent years. Major disadvantages of this technique include length of time taken for animals to acquire the task (typically several weeks), negative experiences associated with task training, and issues of motivation. Here we present a shortened cognitive bias protocol using only positive reinforcers which must actively be responded to. The protocol took an average of 4days to complete, and produced similar results to previous, longer methods (minimum 30days). Specifically, rats housed in standard laboratory conditions demonstrated negative cognitive biases when presented with ambiguous stimuli, and took longer to make a decision when faced with an ambiguous stimulus. Compared to previous methods, this protocol is significantly shorter (average 4days vs. minimum 30days), utilises only positive reinforcers to avoid inducing negative affective states, and requires active responses to all cues, avoiding potential confounds of motivational state. We have successfully developed a shortened cognitive bias protocol, suitable for use with laboratory rats. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  3. Interface Assignment-Based AODV Routing Protocol to Improve Reliability in Multi-Interface Multichannel Wireless Mesh Networks

    Directory of Open Access Journals (Sweden)

    Won-Suk Kim

    2015-01-01

    Full Text Available The utilization of wireless mesh networks (WMNs has greatly increased, and the multi-interface multichannel (MIMC technic has been widely used for the backbone network. Unfortunately, the ad hoc on-demand distance vector (AODV routing protocol defined in the IEEE 802.11s standard was designed for WMNs using the single-interface single-channel technic. So, we define a problem that happens when the legacy AODV is used in MIMC WMNs and propose an interface assignment-based AODV (IA-AODV in order to resolve that problem. IA-AODV, which is based on multitarget path request, consists of the PREQ prediction scheme, the PREQ loss recovery scheme, and the PREQ sender assignment scheme. A detailed operation according to various network conditions and services is introduced, and the routing efficiency and network reliability of a network using IA-AODV are analyzed over the presented system model. Finally, after a real-world test-bed for MIMC WMNs using the IA-AODV routing protocol is implemented, the various indicators of the network are evaluated through experiments. When the proposed routing protocol is compared with the existing AODV routing protocol, it performs the path update using only 14.33% of the management frames, completely removes the routing malfunction, and reduces the UDP packet loss ratio by 0.0012%.

  4. Calf-raise senior: a new test for assessment of plantar flexor muscle strength in older adults: protocol, validity, and reliability.

    Science.gov (United States)

    André, Helô-Isa; Carnide, Filomena; Borja, Edgar; Ramalho, Fátima; Santos-Rocha, Rita; Veloso, António P

    2016-01-01

    This study aimed to develop a new field test protocol with a standardized measurement of strength and power in plantar flexor muscles targeted to functionally independent older adults, the calf-raise senior (CRS) test, and also evaluate its reliability and validity. Forty-one subjects aged 65 years and older of both sexes participated in five different cross-sectional studies: 1) pilot (n=12); 2) inter- and intrarater agreement (n=12); 3) construct (n=41); 4) criterion validity (n=33); and 5) test-retest reliability (n=41). Different motion parameters were compared in order to define a specifically designed protocol for seniors. Two raters evaluated each participant twice, and the results of the same individual were compared between raters and participants to assess the interrater and intrarater agreement. The validity and reliability studies involved three testing sessions that lasted 2 weeks, including a battery of functional fitness tests, CRS test in two occasions, accelerometry, and strength assessments in an isokinetic dynamometer. The CRS test presented an excellent test-retest reliability (intraclass correlation coefficient [ICC] =0.90, standard error of measurement =2.0) and interrater reliability (ICC =0.93-0.96), as well as a good intrarater agreement (ICC =0.79-0.84). Participants with better results in the CRS test were younger and presented higher levels of physical activity and functional fitness. A significant association between test results and all strength parameters (isometric, r =0.87, r 2 =0.75; isokinetic, r =0.86, r 2 =0.74; and rate of force development, r =0.77, r 2 =0.59) was shown. This study was successful in demonstrating that the CRS test can meet the scientific criteria of validity and reliability. The test can be a good indicator of ankle strength in older adults and proved to discriminate significantly between individuals with improved functionality and levels of physical activity.

  5. Mac protocols for cyber-physical systems

    CERN Document Server

    Xia, Feng

    2015-01-01

    This book provides a literature review of various wireless MAC protocols and techniques for achieving real-time and reliable communications in the context of cyber-physical systems (CPS). The evaluation analysis of IEEE 802.15.4 for CPS therein will give insights into configuration and optimization of critical design parameters of MAC protocols. In addition, this book also presents the design and evaluation of an adaptive MAC protocol for medical CPS, which exemplifies how to facilitate real-time and reliable communications in CPS by exploiting IEEE 802.15.4 based MAC protocols. This book wil

  6. Assessment of a recombinant androgen receptor binding assay: initial steps towards validation.

    Science.gov (United States)

    Freyberger, Alexius; Weimer, Marc; Tran, Hoai-Son; Ahr, Hans-Jürgen

    2010-08-01

    Despite more than a decade of research in the field of endocrine active compounds with affinity for the androgen receptor (AR), still no validated recombinant AR binding assay is available, although recombinant AR can be obtained from several sources. With funding from the European Union (EU)-sponsored 6th framework project, ReProTect, we developed a model protocol for such an assay based on a simple AR binding assay recently developed at our institution. Important features of the protocol were the use of a rat recombinant fusion protein to thioredoxin containing both the hinge region and ligand binding domain (LBD) of the rat AR (which is identical to the human AR-LBD) and performance in a 96-well plate format. Besides two reference compounds [dihydrotestosterone (DHT), androstenedione] ten test compounds with different affinities for the AR [levonorgestrel, progesterone, prochloraz, 17alpha-methyltestosterone, flutamide, norethynodrel, o,p'-DDT, dibutylphthalate, vinclozolin, linuron] were used to explore the performance of the assay. At least three independent experiments per compound were performed. The AR binding properties of reference and test compounds were well detected, in terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using recombinant AR preparations. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.6. Our data demonstrate that the assay reliably ranked compounds with strong, weak, and no/marginal affinity for the AR with high accuracy. It avoids the manipulation and use of animals, as a recombinant protein is used and thus contributes to the 3R concept. On the whole, this assay is a promising candidate for further validation. Copyright 2009 Elsevier Inc. All rights reserved.

  7. Assaying Cellular Viability Using the Neutral Red Uptake Assay.

    Science.gov (United States)

    Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

    2017-01-01

    The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

  8. A reliable protocol for the isolation of viable, chondrogenically differentiated human mesenchymal stem cells from high-density pellet cultures.

    Science.gov (United States)

    Ullah, Mujib; Hamouda, Houda; Stich, Stefan; Sittinger, Michael; Ringe, Jochen

    2012-12-01

    Administration of chondrogenically differentiated mesenchymal stem cells (MSC) is discussed as a promising approach for the regenerative treatment of injured or diseased cartilage. The high-density pellet culture is the standard culture for chondrogenic differentiation, but cells in pellets secrete extracellular matrix (ECM) that they become entrapped in. Protocols for cell isolation from pellets often result in cell damage and dedifferentiation towards less differentiated MSC. Therefore, our aim was to develop a reliable protocol for the isolation of viable, chondrogenically differentiated MSC from high-density pellet cultures. Human bone marrow MSC were chondrogenically stimulated with transforming growth factor-β3, and the cartilaginous structure of the pellets was verified by alcian blue staining of cartilage proteoglycans, antibody staining of cartilage collagen type II, and quantitative real-time reverse-transcription polymerase chain reaction of the marker genes COL2A1 and SOX9. Trypsin and collagenases II and P were tested alone or in combination, and for different concentrations and times, to find a protocol for optimized pellet digestion. Whereas trypsin was not able to release viable cells, 90-min digestion with 300 U of collagenase II, 20 U of collagenase P, and 2 mM CaCl2 worked quite well and resulted in about 2.5×10(5) cells/pellet. The protocol was further optimized for the separation of released cells and ECM from each other. Cells were alcian blue and collagen type II positive and expressed COL2A1 and SOX9, verifying a chondrogenic character. However, they had different morphological shapes. The ECM was also uniformly alcian blue and collagen type II positive but showed different organizational and structural forms. To conclude, our protocol allows the reliable isolation of a defined number of viable, chondrogenically differentiated MSC from high-density pellet cultures. Such cells, as well as the ECM components, are of interest as

  9. A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl

    Science.gov (United States)

    Smith, Matthew M.; Schmutz, Joel A.; Apelgren, Chloe; Ramey, Andy M.

    2015-01-01

    Microscopic examination of blood smears can be effective at diagnosing and quantifying hematozoa infections. However, this method requires highly trained observers, is time consuming, and may be inaccurate for detection of infections at low levels of parasitemia. To develop a molecular methodology for identifying and quantifying Leucocytozoon parasite infection in wild waterfowl (Anseriformes), we designed a real-time, quantitative PCR protocol to amplify Leucocytozoon mitochondrial DNA using TaqMan fluorogenic probes and validated our methodology using blood samples collected from waterfowl in interior Alaska during late summer and autumn (n = 105). By comparing our qPCR results to those derived from a widely used nested PCR protocol, we determined that our assay showed high levels of sensitivity (91%) and specificity (100%) in detecting Leucocytozoon DNA from host blood samples. Additionally, results of a linear regression revealed significant correlation between the raw measure of parasitemia produced by our qPCR assay (Ct values) and numbers of parasites observed on blood smears (R2 = 0.694, P = 0.003), indicating that our assay can reliably determine the relative parasitemia levels among samples. This methodology provides a powerful new tool for studies assessing effects of haemosporidian infection in wild avian species.

  10. A Comprehensive Review on Clinical Applications of Comet Assay

    Science.gov (United States)

    Gunasekarana, Vidya; Chand, Parkash

    2015-01-01

    Increased levels of DNA damage and ineffective repair mechanisms are the underlying bio-molecular events in the pathogenesis of most of the life-threatening diseases like cancer and degenerative diseases. The sources of DNA damage can be either exogenous or endogenous in origin. Imbalance between the oxidants and antioxidants resulting in increased reactive oxygen species mostly accounts for the endogenously derived attacks on DNA. Among the various methods employed in the estimation of DNA damage, alkaline comet assay is proven to be a relatively simple and versatile tool in the assessment of DNA damage and also in determining the efficacy of DNA repair mechanism. The aim of this article is to review the application of comet assay in the field of medicine towards human biomonitoring, understanding the pathogenesis of cancer and progression of chronic and degenerative diseases, prediction of tumour radio & chemosensitivity and in male infertility. A standardized protocol and analysis system of various variants of comet assay in different types of cells, across the labs will be of useful and reliable clinical tool in the field of Medicine for the estimation of levels of DNA damage and repair mechanisms. PMID:25954633

  11. Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening.

    Science.gov (United States)

    Ng, Khong Y; Machida, Kazuya

    2017-01-01

    With a growing number of high-throughput studies, structural analyses, and availability of protein-protein interaction databases, it is now possible to apply web-based prediction tools to SH2 domain-interactions. However, in silico prediction is not always reliable and requires experimental validation. Rosette assay is a dot blot-based reverse-phase assay developed for the assessment of binding between SH2 domains and their ligands. It is conveniently customizable, allowing for low- to high-throughput analysis of interactions between various numbers of SH2 domains and their ligands, e.g., short peptides, purified proteins, and cell lysates. The binding assay is performed in a 96-well plate (MBA or MWA apparatus) in which a sample spotted membrane is incubated with up to 96 labeled SH2 domains. Bound domains are detected and quantified using a chemiluminescence or near-infrared fluorescence (IR) imaging system. In this chapter, we describe a practical protocol for rosette assay to assess interactions between synthesized tyrosine phosphorylated peptides and a library of GST-tagged SH2 domains. Since the methodology is not confined to assessment of SH2-pTyr interactions, rosette assay can be broadly utilized for ligand and drug screening using different protein interaction domains or antibodies.

  12. Towards Reliable Integrated Services for Dependable Systems

    DEFF Research Database (Denmark)

    Schiøler, Henrik; Ravn, Anders Peter; Izadi-Zamanabadi, Roozbeh

    Reliability issues for various technical systems are discussed and focus is directed towards distributed systems, where communication facilities are vital to maintain system functionality. Reliability in communication subsystems is considered as a resource to be shared among a number of logical c...... applications residing on alternative routes. Details are provided for the operation of RRRSVP based on reliability slack calculus. Conclusions summarize the considerations and give directions for future research....... connections and a reliability management framework is suggested. We suggest a network layer level reliability management protocol RRSVP (Reliability Resource Reservation Protocol) as a counterpart of the RSVP for bandwidth and time resource management. Active and passive standby redundancy by background...

  13. Towards Reliable Integrated Services for Dependable Systems

    DEFF Research Database (Denmark)

    Schiøler, Henrik; Ravn, Anders Peter; Izadi-Zamanabadi, Roozbeh

    2003-01-01

    Reliability issues for various technical systems are discussed and focus is directed towards distributed systems, where communication facilities are vital to maintain system functionality. Reliability in communication subsystems is considered as a resource to be shared among a number of logical c...... applications residing on alternative routes. Details are provided for the operation of RRRSVP based on reliability slack calculus. Conclusions summarize the considerations and give directions for future research....... connections and a reliability management framework is suggested. We suggest a network layer level reliability management protocol RRSVP (Reliability Resource Reservation Protocol) as a counterpart of the RSVP for bandwidth and time resource management. Active and passive standby redundancy by background...

  14. Magnetic particle separation technique: a reliable and simple tool for RIA/IRMA and quantitative PCR assay

    International Nuclear Information System (INIS)

    Shen Rongsen; Shen Decun

    1998-01-01

    Five types of magnetic particles without or with aldehyde, amino and carboxyl functional groups, respectively were used to immobilize first or second antibody by three models, i. e. physical adsorption, chemical coupling and immuno-affinity, forming four types of magnetic particle antibodies. The second antibody immobilized on polyacrolein magnetic particles through aldehyde functional groups and the first antibodies immobilized on carboxylic polystyrene magnetic particles through carboxyl functional groups were recommended to apply to RIAs and/or IRMAs. Streptavidin immobilized on commercial magnetic particles through amino functional groups was successfully applied to separating specific PCR product for quantification of human cytomegalovirus. In the paper typical data on reliability of these magnetic particle ligands were reported and simplicity of the magnetic particle separation technique was discussed. The results showed that the technique was a reliable and simple tool for RIA/IRMA and quantitative PCR assay. (author)

  15. Design and Implementation of a MAC Protocol for Timely and Reliable Delivery of Command and Data in Dynamic Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    Phan Van Vinh

    2013-09-01

    Full Text Available This paper proposes and implements a new TDMA-based MAC protocol for providing timely and reliable delivery of data and command for monitoring and control networks. In this kind of network, sensor nodes are required to sense data from the monitoring environment periodically and then send the data to a sink. The sink determines whether the environment is safe or not by analyzing the acquired data. Sometimes, a command or control message is sent from the sink to a particular node or a group of nodes to execute the services or request further interested data. The proposed MAC protocol enables bidirectional communication, controls active and sleep modes of a sensor node to conserve energy, and addresses the problem of load unbalancing between the nodes near a sink and the other nodes. It can improve reliability of communication significantly while extending network lifetime. These claims are supported by the experimental results.

  16. A reliable, practical, and economical protocol for inducing diarrhea and severe dehydration in the neonatal calf.

    OpenAIRE

    Walker, P G; Constable, P D; Morin, D E; Drackley, J K; Foreman, J H; Thurmon, J C

    1998-01-01

    Fifteen healthy, colostrum-fed, male dairy calves, aged 2 to 7 d were used in a study to develop a diarrhea protocol for neonatal calves that is reliable, practical, and economical. After instrumentation and recording baseline data, diarrhea and dehydration were induced by administering milk replacer [16.5 mL/kg of body weight (BW), PO], sucrose (2 g/kg in a 20% aqueous solution, p.o.), spironolactone and hydrochlorothiazide (1 mg/kg, PO) every 8 h, and furosemide (2 mg/kg, i.m., q6h). Calves...

  17. New Application of the Comet Assay

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I.; Dávila-Rodríguez, Martha I.; Fernández, José Luís; López-Fernández, Carmen; Gosálbez, Altea; Gosálvez, Jaime

    2011-01-01

    The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains. PMID:21540337

  18. Reliability of the MODS assay decentralisation process in three health regions in Peru

    Science.gov (United States)

    Mendoza, A.; Castillo, E.; Gamarra, N.; Huamán, T.; Perea, M.; Monroi, Y.; Salazar, R.; Coronel, J.; Acurio, M.; Obregón, G.; Roper, M.; Bonilla, C.; Asencios, L.; Moore, D. A. J.

    2011-01-01

    OBJECTIVE To deliver rapid isoniazid (INH) and rifampicin (RMP) drug susceptibility testing (DST) close to the patient, we designed a decentralisation process for the microscopic observation drug susceptibility (MODS) assay in Peru and evaluated its reliability. METHODS After 2 weeks of training, laboratory staff processed ≥120 consecutive sputum samples each in three regional laboratories. Samples were processed in parallel with MODS testing at an expert laboratory. Blinded paired results were independently analysed by the Instituto Nacional de Salud (INS) according to predetermined criteria: concordance for culture, DST against INH and RMP and diagnosis of multidrug-resistant t uberculosis (MDR-TB) ≥ 95%, McNemar's P > 0.05, kappa index (κ) ≥ 0.75 and contamination 1–4%. Sensitivity and specificity for MDR-TB were calculated. RESULTS The accreditation process for Callao (126 samples, 79.4% smear-positive), Lima Sur (n = 130, 84%) and Arequipa (n = 126, 80%) took respectively 94, 97 and 173 days. Pre-determined criteria in all regional laboratories were above expected values. The sensitivity and specificity for detecting MDR-TB in regional laboratories were >95%, except for sensitivity in Lima Sur, which was 91.7%. Contamination was 1.0–2.3%. Mean delay to positive MODS results was 9.9–12.9 days. CONCLUSION Technology transfer of MODS was reliable, effective and fast, enabling the INS to accredit regional laboratories swiftly. PMID:21219684

  19. Use of a standardized JaCVAM in vivo rat comet assay protocol to assess the genotoxicity of three coded test compounds; ampicillin trihydrate, 1,2-dimethylhydrazine dihydrochloride, and N-nitrosodimethylamine.

    Science.gov (United States)

    McNamee, J P; Bellier, P V

    2015-07-01

    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), our laboratory examined ampicillin trihydrate (AMP), 1,2-dimethylhydrazine dihydrochloride (DMH), and N-nitrosodimethylamine (NDA) using a standard comet assay validation protocol (v14.2) developed by the JaCVAM validation management team (VMT). Coded samples were received by our laboratory along with basic MSDS information. Solubility analysis and range-finding experiments of the coded test compounds were conducted for dose selection. Animal dosing schedules, the comet assay processing and analysis, and statistical analysis were conducted in accordance with the standard protocol. Based upon our blinded evaluation, AMP was not found to exhibit evidence of genotoxicity in either the rat liver or stomach. However, both NDA and DMH were observed to cause a significant increase in % tail DNA in the rat liver at all dose levels tested. While acute hepatoxicity was observed for these compounds in the high dose group, in the investigators opinion there were a sufficient number of consistently damaged/measurable cells at the medium and low dose groups to judge these compounds as genotoxic. There was no evidence of genotoxicity from either NDA or DMH in the rat stomach. In conclusion, our laboratory observed increased DNA damage from two blinded test compounds in rat liver (later identified as genotoxic carcinogens), while no evidence of genotoxicity was observed for the third blinded test compound (later identified as a non-genotoxic, non-carcinogen). This data supports the use of a standardized protocol of the in vivo comet assay as a cost-effective alternative genotoxicity assay for regulatory testing purposes. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

  20. Cost-optimization of the IPv4 zeroconf protocol

    NARCIS (Netherlands)

    Bohnenkamp, H.C.; van der Stok, Peter; Hermanns, H.; Vaandrager, Frits

    2003-01-01

    This paper investigates the tradeoff between reliability and effectiveness for the IPv4 Zeroconf protocol, proposed by Cheshire/Adoba/Guttman in 2002, dedicated to the selfconfiguration of IP network interfaces. We develop a simple stochastic cost model of the protocol, where reliability is measured

  1. Integrated cryptosporidium assay to determine oocyst density, infectivity, and genotype for risk assessment of source and reuse water.

    Science.gov (United States)

    King, Brendon; Fanok, Stella; Phillips, Renae; Swaffer, Brooke; Monis, Paul

    2015-05-15

    Cryptosporidium continues to be problematic for the water industry, with risk assessments often indicating that treatment barriers may fail under extreme conditions. However, risk analyses have historically used oocyst densities and not considered either oocyst infectivity or species/genotype, which can result in an overestimation of risk if the oocysts are not human infective. We describe an integrated assay for determining oocyst density, infectivity, and genotype from a single-sample concentrate, an important advance that overcomes the need for processing multiple-grab samples or splitting sample concentrates for separate analyses. The assay incorporates an oocyst recovery control and is compatible with standard primary concentration techniques. Oocysts were purified from primary concentrates using immunomagnetic separation prior to processing by an infectivity assay. Plate-based cell culture was used to detect infectious foci, with a monolayer washing protocol developed to allow recovery and enumeration of oocysts. A simple DNA extraction protocol was developed to allow typing of any wells containing infectious Cryptosporidium. Water samples from a variety of source water and wastewater matrices, including a semirural catchment, wastewater, an aquifer recharge site, and storm water, were analyzed using the assay. Results demonstrate that the assay can reliably determine oocyst densities, infectivity, and genotype from single-grab samples for a variety of water matrices and emphasize the varying nature of Cryptosporidium risk extant throughout source waters and wastewaters. This assay should therefore enable a more comprehensive understanding of Cryptosporidium risk for different water sources, assisting in the selection of appropriate risk mitigation measures. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. Chromogenic in situ hybridization is a reliable assay for detection of ALK rearrangements in adenocarcinomas of the lung.

    Science.gov (United States)

    Schildhaus, Hans-Ulrich; Deml, Karl-Friedrich; Schmitz, Katja; Meiboom, Maren; Binot, Elke; Hauke, Sven; Merkelbach-Bruse, Sabine; Büttner, Reinhard

    2013-11-01

    Reliable detection of anaplastic lymphoma kinase (ALK) rearrangements is a prerequisite for personalized treatment of lung cancer patients, as ALK rearrangements represent a predictive biomarker for the therapy with specific tyrosine kinase inhibitors. Currently, fluorescent in situ hybridization (FISH) is considered to be the standard method for assessing formalin-fixed and paraffin-embedded tissue for ALK inversions and translocations. However, FISH requires a specialized equipment, the signals fade rapidly and it is difficult to detect overall morphology and tumor heterogeneity. Chromogenic in situ hybridization (CISH) has been successfully introduced as an alternative test for the detection of several genetic aberrations. This study validates a newly developed ALK CISH assay by comparing FISH and CISH signal patterns in lung cancer samples with and without ALK rearrangements. One hundred adenocarcinomas of the lung were included in this study, among them 17 with known ALK rearrangement. FISH and CISH were carried out and evaluated according to the manufacturers' recommendations. For both assays, tumors were considered positive if ≥15% of tumor cells showed either isolated 3' signals or break-apart patterns or a combination of both. A subset of tumors was exemplarily examined by using a novel EML4 (echinoderm microtubule-associated protein-like 4) CISH probe. Red, green and fusion CISH signals were clearcut and different signal patterns were easily recognized. The percentage of aberrant tumor cells was statistically highly correlated (PCISH. On the basis of 86 samples that were evaluable by ALK CISH, we found a 100% sensitivity and 100% specificity of this assay. Furthermore, EML4 rearrangements could be recognized by CISH. CISH is a highly reliable, sensitive and specific method for the detection of ALK gene rearrangements in pulmonary adenocarcinomas. Our results suggest that CISH might serve as a suitable alternative to FISH, which is the current gold

  3. Revaluation of biological variation of glycated hemoglobin (HbA(1c)) using an accurately designed protocol and an assay traceable to the IFCC reference system.

    Science.gov (United States)

    Braga, Federica; Dolci, Alberto; Montagnana, Martina; Pagani, Franca; Paleari, Renata; Guidi, Gian Cesare; Mosca, Andrea; Panteghini, Mauro

    2011-07-15

    Glycated hemoglobin (HbA(1c)) has a key role for diagnosing diabetes and monitoring glycemic state. As recently reviewed, available data on HbA(1c) biological variation show marked heterogeneity. Here we experimentally revaluated these data using a well designed protocol. We took five EDTA whole blood specimens from 18 apparently healthy subjects on the same day, every two weeks for two months. Samples were stored at -80°C until analysis and assayed in duplicate in a single run by Roche Tina-quant® Gen.2 immunoassay. Data were analyzed by the ANOVA. To assess the assay traceability to the IFCC reference method, we preliminarily carried out a correlation experiment. The bias (mean±SD) of the Roche immunoassay was 0.3%±0.7%, confirming the traceability of the employed assay. No difference was found in HbA(1c) values between men and women. Within- and between-subject CV were 2.5% and 7.1%, respectively. Derived desirable analytical goals for imprecision, bias, and total error resulted 1.3%, 1.9%, and 3.9%, respectively. HbA(1c) had marked individuality, limiting the use of population-based reference limits for test interpretation. The estimated critical difference was ~10%. For the first time we defined biological variation and derived indices for the clinical application of HbA(1c) measurements using an accurately designed protocol and an assay standardized according to the IFCC. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Optimisation of the microplate resazurin assay for screening and bioassay-guided fractionation of phytochemical extracts against Mycobacterium tuberculosis.

    Science.gov (United States)

    O'Neill, Taryn E; Li, Haoxin; Colquhoun, Caitlyn D; Johnson, John A; Webster, Duncan; Gray, Christopher A

    2014-01-01

    Because of increased resistance to current drugs, there is an urgent need to discover new anti-mycobacterial compounds for the development of novel anti-tuberculosis drugs. The microplate resazurin assay (MRA) is commonly used to evaluate natural products and synthetic compounds for anti-mycobacterial activity. However, the assay can be problematic and unreliable when screening methanolic phytochemical extracts. To optimise the MRA for the screening and bioassay-guided fractionation of phytochemical extracts using Mycobacterium tuberculosis H37Ra. The effects of varying assay duration, resazurin solution composition, solvent (dimethyl sulphoxide - DMSO) concentration and type of microtitre plate used on the results and reliability of the MRA were investigated. The optimal bioassay protocol was applied to methanolic extracts of medicinal plants that have been reported to possess anti-mycobacterial activity. The variables investigated were found to have significant effects on the results obtained with the MRA. A standardised procedure that can reliably quantify anti-mycobacterial activity of phytochemical extracts in as little as 48 h was identified. The optimised MRA uses 2% aqueous DMSO, with an indicator solution of 62.5 µg/mL resazurin in 5% aqueous Tween 80 over 96 h incubation. The study has identified an optimal procedure for the MRA when used with M. tuberculosis H37Ra that gives rapid, reliable and consistent results. The assay procedure has been used successfully for the screening and bioassay-guided fractionation of anti-mycobacterial compounds from methanol extracts of Canadian medicinal plants. Copyright © 2014 John Wiley & Sons, Ltd.

  5. Interobserver reliability of the 'Welfare Quality(®) Animal Welfare Assessment Protocol for Growing Pigs'.

    Science.gov (United States)

    Czycholl, I; Kniese, C; Büttner, K; Beilage, E Grosse; Schrader, L; Krieter, J

    2016-01-01

    The present paper focuses on evaluating the interobserver reliability of the 'Welfare Quality(®) Animal Welfare Assessment Protocol for Growing Pigs'. The protocol for growing pigs mainly consists of a Qualitative Behaviour Assessment (QBA), direct behaviour observations (BO) carried out by instantaneous scan sampling and checks for different individual parameters (IP), e.g. presence of tail biting, wounds and bursitis. Three trained observers collected the data by performing 29 combined assessments, which were done at the same time and on the same animals; but they were carried out completely independent of each other. The findings were compared by the calculation of Spearman Rank Correlation Coefficients (RS), Intraclass Correlation Coefficients (ICC), Smallest Detectable Changes (SDC) and Limits of Agreements (LoA). There was no agreement found concerning the adjectives belonging to the QBA (e.g. active: RS: 0.50, ICC: 0.30, SDC: 0.38, LoA: -0.05 to 0.45; fearful: RS: 0.06, ICC: 0.0, SDC: 0.26, LoA: -0.20 to 0.30). In contrast, the BO showed good agreement (e.g. social behaviour: RS: 0.45, ICC: 0.50, SDC: 0.09, LoA: -0.09 to 0.03 use of enrichment material: RS: 0.75, ICC: 0.68, SDC: 0.06, LoA: -0.03 to 0.03). Overall, observers agreed well in the IP, e.g. tail biting (RS: 0.52, ICC: 0.88; SDC: 0.05, LoA: -0.01 to 0.02) and wounds (RS: 0.43, ICC: 0.59, SDC: 0.10, LoA: -0.09 to 0.10). The parameter bursitis showed great differences (RS: 0.10, ICC: 0.0, SDC: 0.35, LoA: -0.37 to 0.40), which can be explained by difficulties in the assessment when the animals moved around quickly or their legs were soiled. In conclusion, the interobserver reliability was good in the BO and most IP, but not for the parameter bursitis and the QBA.

  6. Complete validation of a unique digestion assay to detect Trichinella larvae in horse meat demonstrates the reliability of this assay for meeting food safety and trade requirements.

    Science.gov (United States)

    Forbes, L B; Hill, D E; Parker, S; Tessaro, S V; Gamble, H R; Gajadhar, A A

    2008-03-01

    A tissue digestion assay using a double separatory funnel procedure for the detection of Trichinella larvae in horse meat was validated for application in food safety programs and trade. The assay consisted of a pepsin-HCl digestion step to release larvae from muscle tissue and two sequential sedimentation steps in separatory funnels to recover and concentrate larvae for detection with a stereomicroscope. With defined critical control points, the assay was conducted within a quality assurance system compliant with International Organization for Standardization-International Electrotechnical Commission (ISO/IEC) 17025 guidelines. Samples used in the validation were obtained from horses experimentally infected with Trichinella spiralis to obtain a range of muscle larvae densities. One-, 5-, and 10-g samples of infected tissue were combined with 99, 95, and 90 g, respectively, of known negative horse tissue to create a 100-g sample for testing. Samples of 5 and 10 g were more likely to be positive than were 1-g samples when larval densities were less than three larvae per gram (lpg). This difference is important because ingested meat with 1 lpg is considered the threshold for clinical disease in humans. Using a 5-g sample size, all samples containing 1.3 to 2 lpg were detected, and 60 to 100% of samples with infected horse meat containing 0.1 to 0.7 lpg were detected. In this study, the double separatory funnel digestion assay was efficient and reliable for its intended use in food safety and trade. This procedure is the only digestion assay for Trichinella in horse meat that has been validated as consistent and effective at critical levels of sensitivity.

  7. Reproducibility of microbial mutagenicity assays. I. Tests with Salmonella typhimurium and Escherichia coli using a standardized protocol

    International Nuclear Information System (INIS)

    Dunkel, V.C.; Zeiger, E.; Brusick, D.; McCoy, E.; McGregor, D.; Mortelmans, K.; Rosenkranz, H.S.; Simmon, V.F.

    1984-01-01

    The Salmonella/microsome test developed by Ames and his coworkers has been widely used in the evaluation of chemicals for genotoxic potential. Although the value of this assay is well recognized, there have been no comprehensive studies on the interlaboratory reproducibility of the method using a standardized protocol. A program was therefore initiated to compare the results obtained in four laboratories from testing a series of coded mutagens and nonmutagens using a standardized protocol. Additional objectives of this study were to compare male Fisher 344 rat, B6C3F1 mouse, and Syrian hamster liver S-9 preparations for the activation of chemicals; to compare Aroclor 1254-induced liver S-9 from all three species with the corresponding non-induced liver S-9's; and to compare the response of Escherichia coli WP-2 uvrA with the Salmonella typhimurium tester strains recommended by Ames. Since a primary use of in vitro microbial mutagenesis tests is the identification of potential carcinogens by their mutagenicity, the authors decided to compare the animal species and strains used by the National Cancer Institute/National Toxicology Program (NCI/NTP) for animal carcinogenicity studies

  8. Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay.

    Science.gov (United States)

    Cui, Heying; Loftus, Kyle M; Noell, Crystal R; Solmaz, Sozanne R

    2018-05-03

    Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer. Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin α. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

  9. A reliable, practical, and economical protocol for inducing diarrhea and severe dehydration in the neonatal calf.

    Science.gov (United States)

    Walker, P G; Constable, P D; Morin, D E; Drackley, J K; Foreman, J H; Thurmon, J C

    1998-07-01

    Fifteen healthy, colostrum-fed, male dairy calves, aged 2 to 7 d were used in a study to develop a diarrhea protocol for neonatal calves that is reliable, practical, and economical. After instrumentation and recording baseline data, diarrhea and dehydration were induced by administering milk replacer [16.5 mL/kg of body weight (BW), PO], sucrose (2 g/kg in a 20% aqueous solution, p.o.), spironolactone and hydrochlorothiazide (1 mg/kg, PO) every 8 h, and furosemide (2 mg/kg, i.m., q6h). Calves were administered sucrose and diuretic agents for 48 h to induce diarrhea and severe dehydration. Clinical changes after 48 h were severe watery diarrhea, severe depression, and marked dehydration (mean, 14% BW loss). Cardiac output, stroke volume, mean central venous pressure, plasma volume, thiocyanate space, blood pH and bicarbonate concentration, base excess, serum chloride concentration, and fetlock temperature were decreased. Plasma lactate concentration, hematocrit, and serum potassium, creatinine, phosphorus, total protein and albumin concentrations were increased. This non-infectious calf diarrhea protocol has a 100% response rate, while providing a consistent and predictable hypovolemic state with diarrhea that reflects most of the clinicopathologic changes observed in osmotic/maldigestive diarrhea caused by infection with rotavirus, coronavirus or cryptosporidia. Limitations of the protocol, when compared to infectious diarrhea models, include failure to induce a severe metabolic acidosis, absence of hyponatremia, renal instead of enteric loss of chloride, renal as well as enteric loss of free water, absence of profound clinical depression and suspected differences in the morphologic and functional effect on intestinal epithelium. Despite these differences, the sucrose/diuretic protocol should be useful in the initial screening of new treatment modalities for calf diarrhea. To confirm their efficacy, the most effective treatment methods should then be examined in

  10. Overview of the InterGroup protocols

    Energy Technology Data Exchange (ETDEWEB)

    Berket, Karlo [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Agarwal, Deborah A. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Melliar-Smith, P. Michael [Univ. of California, Santa Barbara, CA (United States); Moser, Louise E. [Univ. of California, Santa Barbara, CA (United States)

    2001-03-01

    Existing reliable ordered group communication protocols have been developed for local-area networks and do not, in general, scale well to large numbers of nodes and wide-area networks. The InterGroup suite of protocols is a scalable group communication system that introduces a novel approach to handling group membership, and supports a receiver-oriented selection of service. The protocols are intended for a wide-area network, with a large number of nodes, that has highly variable delays and a high message loss rate, such as the Internet. The levels of the message delivery service range from unreliable unordered to reliable group timestamp ordered.

  11. Comparison of mRNA Splicing Assay Protocols across Multiple Laboratories

    DEFF Research Database (Denmark)

    Whiley, Phillip J; de la Hoya, Miguel; Thomassen, Mads

    2014-01-01

    Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data...

  12. Arabidopsis seedling flood-inoculation technique: a rapid and reliable assay for studying plant-bacterial interactions

    Directory of Open Access Journals (Sweden)

    Uppalapati Srinivasa R

    2011-10-01

    Full Text Available Abstract Background The Arabidopsis thaliana-Pseudomonas syringae model pathosystem is one of the most widely used systems to understand the mechanisms of microbial pathogenesis and plant innate immunity. Several inoculation methods have been used to study plant-pathogen interactions in this model system. However, none of the methods reported to date are similar to those occurring in nature and amicable to large-scale mutant screens. Results In this study, we developed a rapid and reliable seedling flood-inoculation method based on young Arabidopsis seedlings grown on MS medium. This method has several advantages over conventional soil-grown plant inoculation assays, including a shorter growth and incubation period, ease of inoculation and handling, uniform infection and disease development, requires less growth chamber space and is suitable for high-throughput screens. In this study we demonstrated the efficacy of the Arabidopsis seedling assay to study 1 the virulence factors of P. syringae pv. tomato DC3000, including type III protein secretion system (TTSS and phytotoxin coronatine (COR; 2 the effector-triggered immunity; and 3 Arabidopsis mutants affected in salicylic acid (SA- and pathogen-associated molecular pattern (PAMPs-mediated pathways. Furthermore, we applied this technique to study nonhost resistance (NHR responses in Arabidopsis using nonhost pathogens, such as P. syringae pv. tabaci, pv. glycinea and pv. tomato T1, and confirmed the functional role of FLAGELLIN-SENSING 2 (FLS2 in NHR. Conclusions The Arabidopsis seedling flood-inoculation assay provides a rapid, efficient and economical method for studying Arabidopsis-Pseudomonas interactions with minimal growth chamber space and time. This assay could also provide an excellent system for investigating the virulence mechanisms of P. syringae. Using this method, we demonstrated that FLS2 plays a critical role in conferring NHR against nonhost pathovars of P. syringae, but not to

  13. A safe and reliable neutralization assay based on pseudovirus to measure neutralizing antibody titer against poliovirus.

    Science.gov (United States)

    Liu, Shaohua; Song, Dongmei; Bai, Han; Lu, Weiwei; Dai, Xinxian; Hao, Chunsheng; Zhang, Zhongyang; Guo, Huijie; Zhang, Yue; Li, Xiuling

    2017-12-01

    With the promotion of inactivated poliomyelitis vaccine (IPV) and live attenuated oral poliomyelitis vaccine (OPV), the global reported cases of poliomyelitis have reduced sharply from 0.35 million in 1988 to 74 in 2015. The Polio Eradication & Endgame Strategic Plan published by WHO in 2013 included the strategy of implementation of poliovirus safe handling and containment measures to minimize the risks of facility-associated reintroduction of virus into the polio-free community to prevent the re-import of poliovirus. Toward this strategy, we produced replication-incompetent pseudovirus of poliovirus type 1, 2, 3 attenuated strains by constructing poliovirus capsid expression vectors and poliovirus replicon then transfecting HEK293T cells and developed a pseudovirus-based neutralization assay (pNA) to determine neutralizing antibody titer which is more secure, time-saving and reliable than conventional neutralization assay (cNA). By using anti-poliovirus rat serum, we demonstrated excellent correlation between neutralizing antibody titers measured by cNA and pNA. It was concluded that pNA can be a potential alternative to replace cNA as a safe and time-saving system for titer determination after live poliovirus's safekeeping. © 2017 Wiley Periodicals, Inc.

  14. Reliability of IP Tunnels in Military Networks

    Directory of Open Access Journals (Sweden)

    Pólkowski Marcin

    2016-10-01

    Full Text Available The military networks, contrary to commercial ones, require standards which provide the highest level of security and reliability. The process to assuring redundancy of the main connections through applying various protocols and transmission media causes problem with time needed to re-establish virtual tunnels between different locations in case of damaged link. This article compares reliability of different IP (Internet Protocol tunnels, which were implemented on military network devices.

  15. Reliable Multihop Broadcast Protocol with a Low-Overhead Link Quality Assessment for ITS Based on VANETs in Highway Scenarios

    Directory of Open Access Journals (Sweden)

    Alejandro Galaviz-Mosqueda

    2014-01-01

    Full Text Available Vehicular ad hoc networks (VANETs have been identified as a key technology to enable intelligent transport systems (ITS, which are aimed to radically improve the safety, comfort, and greenness of the vehicles in the road. However, in order to fully exploit VANETs potential, several issues must be addressed. Because of the high dynamic of VANETs and the impairments in the wireless channel, one key issue arising when working with VANETs is the multihop dissemination of broadcast packets for safety and infotainment applications. In this paper a reliable low-overhead multihop broadcast (RLMB protocol is proposed to address the well-known broadcast storm problem. The proposed RLMB takes advantage of the hello messages exchanged between the vehicles and it processes such information to intelligently select a relay set and reduce the redundant broadcast. Additionally, to reduce the hello messages rate dependency, RLMB uses a point-to-zone link evaluation approach. RLMB performance is compared with one of the leading multihop broadcast protocols existing to date. Performance metrics show that our RLMB solution outperforms the leading protocol in terms of important metrics such as packet dissemination ratio, overhead, and delay.

  16. Can the comet assay be used reliably to detect nanoparticle-induced genotoxicity?

    Science.gov (United States)

    Karlsson, Hanna L; Di Bucchianico, Sebastiano; Collins, Andrew R; Dusinska, Maria

    2015-03-01

    The comet assay is a sensitive method to detect DNA strand breaks as well as oxidatively damaged DNA at the level of single cells. Today the assay is commonly used in nano-genotoxicology. In this review we critically discuss possible interactions between nanoparticles (NPs) and the comet assay. Concerns for such interactions have arisen from the occasional observation of NPs in the "comet head", which implies that NPs may be present while the assay is being performed. This could give rise to false positive or false negative results, depending on the type of comet assay endpoint and NP. For most NPs, an interaction that substantially impacts the comet assay results is unlikely. For photocatalytically active NPs such as TiO2 , on the other hand, exposure to light containing UV can lead to increased DNA damage. Samples should therefore not be exposed to such light. By comparing studies in which both the comet assay and the micronucleus assay have been used, a good consistency between the assays was found in general (69%); consistency was even higher when excluding studies on TiO2 NPs (81%). The strong consistency between the comet and micronucleus assays for a range of different NPs-even though the two tests measure different endpoints-implies that both can be trusted in assessing the genotoxicity of NPs, and that both could be useful in a standard battery of test methods. © 2014 Wiley Periodicals, Inc.

  17. Introducing MINA--The Molecularly Imprinted Nanoparticle Assay.

    Science.gov (United States)

    Shutov, Roman V; Guerreiro, Antonio; Moczko, Ewa; de Vargas-Sansalvador, Isabel Perez; Chianella, Iva; Whitcombe, Michael J; Piletsky, Sergey A

    2014-03-26

    A new ELISA- (enzyme-linked immunosorbent assay)-like assay is demonstrated in which no elements of biological origin are used for molecular recognition or signaling. Composite imprinted nanoparticles that contain a catalytic core and which are synthesized by using a solid-phase approach can simultaneously act as recognition/signaling elements, and be used with minimal modifications to standard assay protocols. This assay provides a new route towards replacement of unstable biomolecules in immunoassays. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. A Protocol for Advanced Psychometric Assessment of Surveys

    Science.gov (United States)

    Squires, Janet E.; Hayduk, Leslie; Hutchinson, Alison M.; Cranley, Lisa A.; Gierl, Mark; Cummings, Greta G.; Norton, Peter G.; Estabrooks, Carole A.

    2013-01-01

    Background and Purpose. In this paper, we present a protocol for advanced psychometric assessments of surveys based on the Standards for Educational and Psychological Testing. We use the Alberta Context Tool (ACT) as an exemplar survey to which this protocol can be applied. Methods. Data mapping, acceptability, reliability, and validity are addressed. Acceptability is assessed with missing data frequencies and the time required to complete the survey. Reliability is assessed with internal consistency coefficients and information functions. A unitary approach to validity consisting of accumulating evidence based on instrument content, response processes, internal structure, and relations to other variables is taken. We also address assessing performance of survey data when aggregated to higher levels (e.g., nursing unit). Discussion. In this paper we present a protocol for advanced psychometric assessment of survey data using the Alberta Context Tool (ACT) as an exemplar survey; application of the protocol to the ACT survey is underway. Psychometric assessment of any survey is essential to obtaining reliable and valid research findings. This protocol can be adapted for use with any nursing survey. PMID:23401759

  19. Adaptation of a MR imaging protocol into a real-time clinical biometric ultrasound protocol for persons with spinal cord injury at risk for deep tissue injury: A reliability study.

    Science.gov (United States)

    Swaine, Jillian M; Moe, Andrew; Breidahl, William; Bader, Daniel L; Oomens, Cees W J; Lester, Leanne; O'Loughlin, Edmond; Santamaria, Nick; Stacey, Michael C

    2018-02-01

    High strain in soft tissues that overly bony prominences are considered a risk factor for pressure ulcers (PUs) following spinal cord impairment (SCI) and have been computed using Finite Element methods (FEM). The aim of this study was to translate a MRI protocol into ultrasound (US) and determine between-operator reliability of expert sonographers measuring diameter of the inferior curvature of the ischial tuberosity (IT) and the thickness of the overlying soft tissue layers on able-bodied (AB) and SCI using real-time ultrasound. Part 1: Fourteen AB participants with a mean age of 36.7 ± 12.09 years with 7 males and 7 females had their 3 soft tissue layers in loaded and unloaded sitting measured independently by 2 sonographers: tendon/muscle, skin/fat and total soft tissue and the diameter of the IT in its short and long axis. Part 2: Nineteen participants with SCI were screened, three were excluded due to abnormal skin signs, and eight participants (42%) were excluded for abnormal US signs with normal skin. Eight SCI participants with a mean age of 31.6 ± 13.6 years and all male with 4 paraplegics and 4 tetraplegics were measured by the same sonographers for skin, fat, tendon, muscle and total. Skin/fat and tendon/muscle were computed. AB between-operator reliability was good (ICC = 0.81-0.90) for 3 soft tissues layers in unloaded and loaded sitting and poor for both IT short and long axis (ICC = -0.028 and -0.01). SCI between-operator reliability was good in unloaded and loaded for total, muscle, fat, skin/fat, tendon/muscle (ICC = 0.75-0.97) and poor for tendon (ICC = 0.26 unloaded and ICC = -0.71 loaded) and skin (ICC = 0.37 unloaded and ICC = 0.10). A MRI protocol was successfully adapted for a reliable 3 soft tissue layer model and could be used in a 2-D FEM model designed to estimate soft tissue strain as a novel risk factor for the development of a PU. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. An Indication of Reliability of the Two-Level Approach of the AWIN Welfare Assessment Protocol for Horses

    Directory of Open Access Journals (Sweden)

    Irena Czycholl

    2018-01-01

    Full Text Available To enhance feasibility, the Animal Welfare Indicators (AWIN assessment protocol for horses consists of two levels: the first is a visual inspection of a sample of horses performed from a distance, the second a close-up inspection of all horses. The aim was to analyse whether information would be lost if only the first level were performed. In this study, 112 first and 112 second level assessments carried out on a subsequent day by one observer were compared by calculating the Spearman’s Rank Correlation Coefficient (RS, Intraclass Correlation Coefficients (ICC, Smallest Detectable Changes (SDC and Limits of Agreements (LoA. Most indicators demonstrated sufficient reliability between the two levels. Exceptions were the Horse Grimace Scale, the Avoidance Distance Test and the Voluntary Human Approach Test (e.g., Voluntary Human Approach Test: RS: 0.38, ICC: 0.38, SDC: 0.21, LoA: −0.25–0.17, which could, however, be also interpreted as a lack of test-retest reliability. Further disagreement was found for the indicator consistency of manure (RS: 0.31, ICC: 0.38, SDC: 0.36, LoA: −0.38–0.36. For these indicators, an adaptation of the first level would be beneficial. Overall, in this study, the division into two levels was reliable and might therewith have the potential to enhance feasibility in other welfare assessment schemes.

  1. Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis.

    Science.gov (United States)

    Li, Wenbin; Teixeira, Diva C; Hartung, John S; Huang, Qi; Duan, Yongping; Zhou, Lijuan; Chen, Jianchi; Lin, Hong; Lopes, Silvio; Ayres, A Juliano; Levy, Laurene

    2013-01-01

    The xylem-limited, Gram-negative, fastidious plant bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease affecting approximately half of the citrus plantations in the State of São Paulo, Brazil. The disease was recently found in Central America and is threatening the multi-billion U.S. citrus industry. Many strains of X. fastidiosa are pathogens or endophytes in various plants growing in the U.S., and some strains cross infect several host plants. In this study, a TaqMan-based assay targeting the 16S rDNA signature region was developed for the identification of X. fastidiosa at the species level. Another TaqMan-based assay was developed for the specific identification of the CVC strains. Both new assays have been systematically validated in comparison with the primer/probe sets from four previously published assays on one platform and under similar PCR conditions, and shown to be superior. The species specific assay detected all X. fastidiosa strains and did not amplify any other citrus pathogen or endophyte tested. The CVC-specific assay detected all CVC strains but did not amplify any non-CVC X. fastidiosa nor any other citrus pathogen or endophyte evaluated. Both sets were multiplexed with a reliable internal control assay targeting host plant DNA, and their diagnostic specificity and sensitivity remained unchanged. This internal control provides quality assurance for DNA extraction, performance of PCR reagents, platforms and operators. The limit of detection for both assays was equivalent to 2 to 10 cells of X. fastidiosa per reaction for field citrus samples. Petioles and midribs of symptomatic leaves of sweet orange harbored the highest populations of X. fastidiosa, providing the best materials for detection of the pathogen. These new species specific assay will be invaluable for molecular identification of X. fastidiosa at the species level, and the CVC specific assay will be very powerful for the

  2. In silico toxicology protocols.

    Science.gov (United States)

    Myatt, Glenn J; Ahlberg, Ernst; Akahori, Yumi; Allen, David; Amberg, Alexander; Anger, Lennart T; Aptula, Aynur; Auerbach, Scott; Beilke, Lisa; Bellion, Phillip; Benigni, Romualdo; Bercu, Joel; Booth, Ewan D; Bower, Dave; Brigo, Alessandro; Burden, Natalie; Cammerer, Zoryana; Cronin, Mark T D; Cross, Kevin P; Custer, Laura; Dettwiler, Magdalena; Dobo, Krista; Ford, Kevin A; Fortin, Marie C; Gad-McDonald, Samantha E; Gellatly, Nichola; Gervais, Véronique; Glover, Kyle P; Glowienke, Susanne; Van Gompel, Jacky; Gutsell, Steve; Hardy, Barry; Harvey, James S; Hillegass, Jedd; Honma, Masamitsu; Hsieh, Jui-Hua; Hsu, Chia-Wen; Hughes, Kathy; Johnson, Candice; Jolly, Robert; Jones, David; Kemper, Ray; Kenyon, Michelle O; Kim, Marlene T; Kruhlak, Naomi L; Kulkarni, Sunil A; Kümmerer, Klaus; Leavitt, Penny; Majer, Bernhard; Masten, Scott; Miller, Scott; Moser, Janet; Mumtaz, Moiz; Muster, Wolfgang; Neilson, Louise; Oprea, Tudor I; Patlewicz, Grace; Paulino, Alexandre; Lo Piparo, Elena; Powley, Mark; Quigley, Donald P; Reddy, M Vijayaraj; Richarz, Andrea-Nicole; Ruiz, Patricia; Schilter, Benoit; Serafimova, Rositsa; Simpson, Wendy; Stavitskaya, Lidiya; Stidl, Reinhard; Suarez-Rodriguez, Diana; Szabo, David T; Teasdale, Andrew; Trejo-Martin, Alejandra; Valentin, Jean-Pierre; Vuorinen, Anna; Wall, Brian A; Watts, Pete; White, Angela T; Wichard, Joerg; Witt, Kristine L; Woolley, Adam; Woolley, David; Zwickl, Craig; Hasselgren, Catrin

    2018-04-17

    The present publication surveys several applications of in silico (i.e., computational) toxicology approaches across different industries and institutions. It highlights the need to develop standardized protocols when conducting toxicity-related predictions. This contribution articulates the information needed for protocols to support in silico predictions for major toxicological endpoints of concern (e.g., genetic toxicity, carcinogenicity, acute toxicity, reproductive toxicity, developmental toxicity) across several industries and regulatory bodies. Such novel in silico toxicology (IST) protocols, when fully developed and implemented, will ensure in silico toxicological assessments are performed and evaluated in a consistent, reproducible, and well-documented manner across industries and regulatory bodies to support wider uptake and acceptance of the approaches. The development of IST protocols is an initiative developed through a collaboration among an international consortium to reflect the state-of-the-art in in silico toxicology for hazard identification and characterization. A general outline for describing the development of such protocols is included and it is based on in silico predictions and/or available experimental data for a defined series of relevant toxicological effects or mechanisms. The publication presents a novel approach for determining the reliability of in silico predictions alongside experimental data. In addition, we discuss how to determine the level of confidence in the assessment based on the relevance and reliability of the information. Copyright © 2018. Published by Elsevier Inc.

  3. The fluorometric microculture cytotoxicity assay.

    Science.gov (United States)

    Lindhagen, Elin; Nygren, Peter; Larsson, Rolf

    2008-01-01

    The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.

  4. Using generalizability theory to develop clinical assessment protocols.

    Science.gov (United States)

    Preuss, Richard A

    2013-04-01

    Clinical assessment protocols must produce data that are reliable, with a clinically attainable minimal detectable change (MDC). In a reliability study, generalizability theory has 2 advantages over classical test theory. These advantages provide information that allows assessment protocols to be adjusted to match individual patient profiles. First, generalizability theory allows the user to simultaneously consider multiple sources of measurement error variance (facets). Second, it allows the user to generalize the findings of the main study across the different study facets and to recalculate the reliability and MDC based on different combinations of facet conditions. In doing so, clinical assessment protocols can be chosen based on minimizing the number of measures that must be taken to achieve a realistic MDC, using repeated measures to minimize the MDC, or simply based on the combination that best allows the clinician to monitor an individual patient's progress over a specified period of time.

  5. Immunochemical protocols

    National Research Council Canada - National Science Library

    Pound, John D

    1998-01-01

    ... easy and important refinements often are not published. This much anticipated 2nd edition of Immunochemzcal Protocols therefore aims to provide a user-friendly up-to-date handbook of reliable techniques selected to suit the needs of molecular biologists. It covers the full breadth of the relevant established immunochemical methods, from protein blotting and immunoa...

  6. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Design of Bus Protocol Intelligent Initiation System Based On RS485

    Directory of Open Access Journals (Sweden)

    Li Liming

    2017-01-01

    Full Text Available In order to design an effective and reliable RS485 bus protocol based on RS485 bus, this paper introduces the structure and transmission mode of the command frame and the response frame, and also introduce four control measures and the communication in order to process quality of this system. The communication protocol is open, tolerant, reliable and fast, and can realize ignition more reliable and accurate in the intelligent initiation system.

  8. Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material.

    Science.gov (United States)

    Cheng, Yo Ching; Hannaoui, Samia; John, Theodore Ralph; Dudas, Sandor; Czub, Stefanie; Gilch, Sabine

    2017-09-29

    The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with ᵦ-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrP Sc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrP Sc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

  9. Reliability of the Star Excursion Balance Test and Two New Similar Protocols to Measure Trunk Postural Control.

    Science.gov (United States)

    López-Plaza, Diego; Juan-Recio, Casto; Barbado, David; Ruiz-Pérez, Iñaki; Vera-Garcia, Francisco J

    2018-05-18

    Although the Star Excursion Balance test (SEBT) has shown a good intrasession reliability, the intersession reliability of this test has not been deeply studied. Furthermore, there is an evident high influence of the lower limbs in the performance of the SEBT, so even if it has been used to measure core stability, it is possibly not the most suitable measurement. The aims of this study were to (1) to assess the absolute and relative between-session reliability of the SEBT and 2 novel variations of this test to assess trunk postural control while sitting, ie, the Star Excursion Sitting Test (SEST) and the Star Excursion Timing Test (SETT); and (2) to analyze the relationships between these 3 test scores. Correlational and reliability test-retest study. Controlled laboratory environment. Twenty-seven physically active men (age: 24.54 ± 3.05 years). Relative and absolute reliability of the SEBT, SEST, and SETT were calculated through the intraclass correlation coefficient (ICC) and standard error of measurement (SEM), respectively. A Pearson correlation analysis was carried out between the variables of the 3 tests. Maximum normalized reach distances were assessed for different SEBT and SEST directions. In addition, composite indexes were calculated for SEBT, SEST, and SETT. The SEBT (dominant leg: ICC = 0.87 [0.73-0.94], SEM = 2.12 [1.66-2.93]; nondominant leg: ICC = 0.74 [0.50-0.87], SEM = 3.23 [2.54-4.45]), SEST (ICC = 0.85 [0.68-0.92], SEM = 1.27 [1.03-1.80]), and SETT (ICC = 0.61 [0.30-0.80], SEM = 2.31 [1.82-3.17]) composite indexes showed moderate-to-high 1-month reliability. A learning effect was detected for some SEBT and SEST directions and for SEST and SETT composite indexes. No significant correlations were found between SEBT and its 2 variations (r ≤ .366; P > .05). A significant correlation was found between the SEST and SETT composite indexes (r = .520; P > .01). SEBT, SEST, and SETT are reliable field protocols to measure postural control. However

  10. Network Coding Protocols for Smart Grid Communications

    DEFF Research Database (Denmark)

    Prior, Rui; Roetter, Daniel Enrique Lucani; Phulpin, Yannick

    2014-01-01

    We propose a robust network coding protocol for enhancing the reliability and speed of data gathering in smart grids. At the heart of our protocol lies the idea of tunable sparse network coding, which adopts the transmission of sparsely coded packets at the beginning of the transmission process b...

  11. Design Protocols and Analytical Strategies that Incorporate Structural Reliability Models

    Science.gov (United States)

    Duffy, Stephen F.

    1997-01-01

    Al single crystal turbine blade material; map a simplistic failure strength envelope of the material; develop a statistically based reliability computer algorithm, verify the reliability model and computer algorithm, and model stator vanes for rig tests. Thus establishing design protocols that enable the engineer to analyze and predict the mechanical behavior of ceramic composites and intermetallics would mitigate the prototype (trial and error) approach currently used by the engineering community. The primary objective of the research effort supported by this short term grant is the continued creation of enabling technologies for the macroanalysis of components fabricated from ceramic composites and intermetallic material systems. The creation of enabling technologies aids in shortening the product development cycle of components fabricated from the new high technology materials.

  12. Improving the communication reliability of body sensor networks based on the IEEE 802.15.4 protocol.

    Science.gov (United States)

    Gomes, Diogo; Afonso, José A

    2014-03-01

    Body sensor networks (BSNs) enable continuous monitoring of patients anywhere, with minimum constraints to daily life activities. Although the IEEE 802.15.4 and ZigBee(®) (ZigBee Alliance, San Ramon, CA) standards were mainly developed for use in wireless sensors network (WSN) applications, they are also widely used in BSN applications because of device characteristics such as low power, low cost, and small form factor. However, compared with WSNs, BSNs present some very distinctive characteristics in terms of traffic and mobility patterns, heterogeneity of the nodes, and quality of service requirements. This article evaluates the suitability of the carrier sense multiple access-collision avoidance protocol, used by the IEEE 802.15.4 and ZigBee standards, for data-intensive BSN applications, through the execution of experimental tests in different evaluation scenarios, in order to take into account the effects of contention, clock drift, and hidden nodes on the communication reliability. Results show that the delivery ratio may decrease substantially during transitory periods, which can last for several minutes, to a minimum of 90% with retransmissions and 13% without retransmissions. This article also proposes and evaluates the performance of the BSN contention avoidance mechanism, which was designed to solve the identified reliability problems. This mechanism was able to restore the delivery ratio to 100% even in the scenario without retransmissions.

  13. Temperature Switch PCR (TSP: Robust assay design for reliable amplification and genotyping of SNPs

    Directory of Open Access Journals (Sweden)

    Mather Diane E

    2009-12-01

    Full Text Available Abstract Background Many research and diagnostic applications rely upon the assay of individual single nucleotide polymorphisms (SNPs. Thus, methods to improve the speed and efficiency for single-marker SNP genotyping are highly desirable. Here, we describe the method of temperature-switch PCR (TSP, a biphasic four-primer PCR system with a universal primer design that permits amplification of the target locus in the first phase of thermal cycling before switching to the detection of the alleles. TSP can simplify assay design for a range of commonly used single-marker SNP genotyping methods, and reduce the requirement for individual assay optimization and operator expertise in the deployment of SNP assays. Results We demonstrate the utility of TSP for the rapid construction of robust and convenient endpoint SNP genotyping assays based on allele-specific PCR and high resolution melt analysis by generating a total of 11,232 data points. The TSP assays were performed under standardised reaction conditions, requiring minimal optimization of individual assays. High genotyping accuracy was verified by 100% concordance of TSP genotypes in a blinded study with an independent genotyping method. Conclusion Theoretically, TSP can be directly incorporated into the design of assays for most current single-marker SNP genotyping methods. TSP provides several technological advances for single-marker SNP genotyping including simplified assay design and development, increased assay specificity and genotyping accuracy, and opportunities for assay automation. By reducing the requirement for operator expertise, TSP provides opportunities to deploy a wider range of single-marker SNP genotyping methods in the laboratory. TSP has broad applications and can be deployed in any animal and plant species.

  14. Biomonitoring of genotoxic risk in radar facility workers: comparison of the comet assay with micronucleus assay and chromatid breakage assay

    International Nuclear Information System (INIS)

    Garaj-Vrhovac, V.; Kopjar, N.

    2003-01-01

    Genotoxic risks of occupational exposure in a radar facility were evaluated by using alkaline comet assay, micronucleus assay and chromatid breakage assay on peripheral blood leukocytes in exposed subjects and corresponding controls. Results show that occupational exposure to microwave radiation correlates with an increase of genome damage in somatic cells. The levels of DNA damage in exposed subjects determined by using alkaline comet assay were increased compared to control and showed interindividual variations. Incidence of micronuclei was also significantly increased compared to baseline control values. After short exposure of cultured lymphocytes to bleomycin, cells of occupationally exposed subjects responded with high numbers of chromatid breaks. Although the level of chromosome damage generated by bleomycin varied greatly between individuals, in exposed subjects a significantly elevated number of chromatid breaks was observed. Our results support data reported in literature indicating that microwave radiation represents a potential DNA-damaging hazard. Alkaline comet assay is confirmed as a sensitive and highly reproducible technique for detection of primary DNA damage inflicted in somatic cells. Micronucleus assay was confirmed as reliable bio-markers of effect and chromatid breakage assay as sensitive bio-marker of individual cancer susceptibility. The results obtained also confirm the necessity to improve measures and to perform accurate health surveillance of individuals occupationally exposed to microwave radiation

  15. Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I.; López-Fernández, Carmen; Fernández, José Luis; Dávila-Rodríguez, Martha I.; Johnston, Stephen D.; Gosálvez, Jaime

    2014-01-01

    Key Concepts The two-dimensional Two-Tailed Comet assay (TT-comet) protocol is a valuable technique to differentiate between single-stranded (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell.Protein lysis inherent with the TT-comet protocol accounts for differences in sperm protamine composition at a species-specific level to produce reliable visualization of sperm DNA damage.Alkaline treatment may break the sugar–phosphate backbone in abasic sites or at sites with deoxyribose damage, transforming these lesions into DNA breaks that are also converted into ssDNA. These lesions are known as Alkali Labile Sites “ALSs.”DBD–FISH permits the in situ visualization of DNA breaks, abasic sites or alkaline-sensitive DNA regions.The alkaline comet single assay reveals that all mammalian species display constitutive ALS related with the requirement of the sperm to undergo transient changes in DNA structure linked with chromatin packing.Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome.The TT is a valuable tool for identifying SSBs or DSBs in sperm cells with DNA fragmentation and can be therefore used for the purposes of fertility assessment. Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. A series of methodologies to assess DNA damage in spermatozoa have been developed but most are unable to differentiate between single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. The two-dimensional Two-Tailed Comet assay (TT-comet) protocol highlighted in this review overcomes this limitation and emphasizes the importance in accounting for the difference in sperm protamine composition at a species-specific level for the appropriate preparation of the assay. The TT-comet is a modification of the original comet assay that uses a two dimensional electrophoresis to

  16. Molecular recognition and self-assembly special feature: A general protocol for creating high-throughput screening assays for reaction yield and enantiomeric excess applied to hydrobenzoin.

    Science.gov (United States)

    Shabbir, Shagufta H; Regan, Clinton J; Anslyn, Eric V

    2009-06-30

    A general approach to high-throughput screening of enantiomeric excess (ee) and concentration was developed by using indicator displacement assays (IDAs), and the protocol was then applied to the vicinal diol hydrobenzoin. The method involves the sequential utilization of what we define herein as screening, training, and analysis plates. Several enantioselective boronic acid-based receptors were screened by using 96-well plates, both for their ability to discriminate the enantiomers of hydrobenzoin and to find their optimal pairing with indicators resulting in the largest optical responses. The best receptor/indicator combination was then used to train an artificial neural network to determine concentration and ee. To prove the practicality of the developed protocol, analysis plates were created containing true unknown samples of hydrobenzoin generated by established Sharpless asymmetric dihydroxylation reactions, and the best ligand was correctly identified.

  17. Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.

    Science.gov (United States)

    Nikolova, Teodora; Marini, Federico; Kaina, Bernd

    2017-10-01

    Genotoxicity testing relies on the quantitative measurement of adverse effects, such as chromosome aberrations, micronuclei, and mutations, resulting from primary DNA damage. Ideally, assays will detect DNA damage and cellular responses with high sensitivity, reliability, and throughput. Several novel genotoxicity assays may fulfill these requirements, including the comet assay and the more recently developed γH2AX assay. Although they are thought to be specific for genotoxicants, a systematic comparison of the assays has not yet been undertaken. In the present study, we compare the γH2AX focus assay with the alkaline and neutral versions of the comet assay, as to their sensitivities and limitations for detection of genetic damage. We investigated the dose-response relationships of γH2AX foci and comet tail intensities at various times following treatment with four prototypical genotoxicants, methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide (H 2 O 2 ) and we tested whether there is a correlation between the endpoints, i.e., alkali-labile sites and DNA strand breaks on the one hand and the cell's response to DNA double-strand breaks and blocked replication forks on the other. Induction of γH2AX foci gave a linear dose response and all agents tested were positive in the assay. The increase in comet tail intensity was also a function of dose; however, mitomycin C was almost completely ineffective in the comet assay, and the doses needed to achieve a significant effect were somewhat higher for some treatments in the comet assay than in the γH2AX foci assay, which was confirmed by threshold analysis. There was high correlation between tail intensity and γH2AX foci for MMS and H 2 O 2 , less for MNNG, and none for mitomycin C. From this we infer that the γH2AX foci assay is more reliable, sensitive, and robust than the comet assay for detecting genotoxicant-induced DNA damage. Copyright © 2017 Elsevier

  18. MDP: Reliable File Transfer for Space Missions

    Science.gov (United States)

    Rash, James; Criscuolo, Ed; Hogie, Keith; Parise, Ron; Hennessy, Joseph F. (Technical Monitor)

    2002-01-01

    This paper presents work being done at NASA/GSFC by the Operating Missions as Nodes on the Internet (OMNI) project to demonstrate the application of the Multicast Dissemination Protocol (MDP) to space missions to reliably transfer files. This work builds on previous work by the OMNI project to apply Internet communication technologies to space communication. The goal of this effort is to provide an inexpensive, reliable, standard, and interoperable mechanism for transferring files in the space communication environment. Limited bandwidth, noise, delay, intermittent connectivity, link asymmetry, and one-way links are all possible issues for space missions. Although these are link-layer issues, they can have a profound effect on the performance of transport and application level protocols. MDP, a UDP-based reliable file transfer protocol, was designed for multicast environments which have to address these same issues, and it has done so successfully. Developed by the Naval Research Lab in the mid 1990's, MDP is now in daily use by both the US Post Office and the DoD. This paper describes the use of MDP to provide automated end-to-end data flow for space missions. It examines the results of a parametric study of MDP in a simulated space link environment and discusses the results in terms of their implications for space missions. Lessons learned are addressed, which suggest minor enhancements to the MDP user interface to add specific features for space mission requirements, such as dynamic control of data rate, and a checkpoint/resume capability. These are features that are provided for in the protocol, but are not implemented in the sample MDP application that was provided. A brief look is also taken at the status of standardization. A version of MDP known as NORM (Neck Oriented Reliable Multicast) is in the process of becoming an IETF standard.

  19. Assessment of Quality of Assay Data on Drill Samples from Golden ...

    African Journals Online (AJOL)

    The success of a mining company is dependent on the integrity of the resource database. The quality of assay data and thus the validity of the database can only be guaranteed when appropriate sampling and assaying protocols have been implemented. It is necessary to convince investors and project financiers that ...

  20. The reliability of knee joint position testing using electrogoniometry

    Directory of Open Access Journals (Sweden)

    Winter Adele

    2008-01-01

    Full Text Available Abstract Background The current investigation examined the inter- and intra-tester reliability of knee joint angle measurements using a flexible Penny and Giles Biometric® electrogoniometer. The clinical utility of electrogoniometry was also addressed. Methods The first study examined the inter- and intra-tester reliability of measurements of knee joint angles in supine, sitting and standing in 35 healthy adults. The second study evaluated inter-tester and intra-tester reliability of knee joint angle measurements in standing and after walking 10 metres in 20 healthy adults, using an enhanced measurement protocol with a more detailed electrogoniometer attachment procedure. Both inter-tester reliability studies involved two testers. Results In the first study, inter-tester reliability (ICC[2,10] ranged from 0.58–0.71 in supine, 0.68–0.79 in sitting and 0.57–0.80 in standing. The standard error of measurement between testers was less than 3.55° and the limits of agreement ranged from -12.51° to 12.21°. Reliability coefficients for intra-tester reliability (ICC[3,10] ranged from 0.75–0.76 in supine, 0.86–0.87 in sitting and 0.87–0.88 in standing. The standard error of measurement for repeated measures by the same tester was less than 1.7° and the limits of agreement ranged from -8.13° to 7.90°. The second study showed that using a more detailed electrogoniometer attachment protocol reduced the error of measurement between testers to 0.5°. Conclusion Using a standardised protocol, reliable measures of knee joint angles can be gained in standing, supine and sitting by using a flexible goniometer.

  1. Inter-laboratory variation in DNA damage using a standard comet assay protocol

    DEFF Research Database (Denmark)

    Forchhammer, Lykke; Ersson, Clara; Loft, Steffen

    2012-01-01

    determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol...... analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained...

  2. Integrated bioassays in microfluidic devices: botulinum toxin assays.

    Science.gov (United States)

    Mangru, Shakuntala; Bentz, Bryan L; Davis, Timothy J; Desai, Nitin; Stabile, Paul J; Schmidt, James J; Millard, Charles B; Bavari, Sina; Kodukula, Krishna

    2005-12-01

    A microfluidic assay was developed for screening botulinum neurotoxin serotype A (BoNT-A) by using a fluorescent resonance energy transfer (FRET) assay. Molded silicone microdevices with integral valves, pumps, and reagent reservoirs were designed and fabricated. Electrical and pneumatic control hardware were constructed, and software was written to automate the assay protocol and data acquisition. Detection was accomplished by fluorescence microscopy. The system was validated with a peptide inhibitor, running 2 parallel assays, as a feasibility demonstration. The small footprint of each bioreactor cell (0.5 cm2) and scalable fluidic architecture enabled many parallel assays on a single chip. The chip is programmable to run a dilution series in each lane, generating concentration-response data for multiple inhibitors. The assay results showed good agreement with the corresponding experiments done at a macroscale level. Although the system has been developed for BoNT-A screening, a wide variety of assays can be performed on the microfluidic chip with little or no modification.

  3. Evaluation of a manual DNA extraction protocol and an isothermal amplification assay for detecting HIV-1 DNA from dried blood spots for use in resource-limited settings.

    Science.gov (United States)

    Jordan, Jeanne A; Ibe, Christine O; Moore, Miranda S; Host, Christel; Simon, Gary L

    2012-05-01

    In resource-limited settings (RLS) dried blood spots (DBS) are collected on infants and transported through provincial laboratories to a central facility where HIV-1 DNA PCR testing is performed using specialized equipment. Implementing a simpler approach not requiring such equipment or skilled personnel could allow the more numerous provincial laboratories to offer testing, improving turn-around-time to identify and treat infected infants sooner. Assess performances of a manual DNA extraction method and helicase-dependent amplification (HDA) assay for detecting HIV-1 DNA from DBS. 60 HIV-1 infected adults were enrolled, blood samples taken and DBS made. DBS extracts were assessed for DNA concentration and beta globin amplification using PCR and melt-curve analysis. These same extracts were then tested for HIV-1 DNA using HDA and compared to results generated by PCR and pyrosequencing. Finally, HDA limit of detection (LOD) studies were performed using DBS extracts prepared with known numbers of 8E5 cells. The manual extraction protocol consistently yielded high concentrations of amplifiable DNA from DBS. LOD assessment demonstrated HDA detected ∼470 copies/ml of HIV-1 DNA extracts in 4/4 replicates. No statistical difference was found using the McNemar's test when comparing HDA to PCR for detecting HIV-1 DNA from DBS. Using just a magnet, heat block and pipettes, the manual extraction protocol and HDA assay detected HIV-1 DNA from DBS at levels that would be useful for early infant diagnosis. Next steps will include assessing HDA for non-B HIV-1 subtypes recognition and comparison to Roche HIV-1 DNA v1.5 PCR assay. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. The Americleft Speech Project: A Training and Reliability Study.

    Science.gov (United States)

    Chapman, Kathy L; Baylis, Adriane; Trost-Cardamone, Judith; Cordero, Kelly Nett; Dixon, Angela; Dobbelsteyn, Cindy; Thurmes, Anna; Wilson, Kristina; Harding-Bell, Anne; Sweeney, Triona; Stoddard, Gregory; Sell, Debbie

    2016-01-01

    To describe the results of two reliability studies and to assess the effect of training on interrater reliability scores. The first study (1) examined interrater and intrarater reliability scores (weighted and unweighted kappas) and (2) compared interrater reliability scores before and after training on the use of the Cleft Audit Protocol for Speech-Augmented (CAPS-A) with British English-speaking children. The second study examined interrater and intrarater reliability on a modified version of the CAPS-A (CAPS-A Americleft Modification) with American and Canadian English-speaking children. Finally, comparisons were made between the interrater and intrarater reliability scores obtained for Study 1 and Study 2. The participants were speech-language pathologists from the Americleft Speech Project. In Study 1, interrater reliability scores improved for 6 of the 13 parameters following training on the CAPS-A protocol. Comparison of the reliability results for the two studies indicated lower scores for Study 2 compared with Study 1. However, this appeared to be an artifact of the kappa statistic that occurred due to insufficient variability in the reliability samples for Study 2. When percent agreement scores were also calculated, the ratings appeared similar across Study 1 and Study 2. The findings of this study suggested that improvements in interrater reliability could be obtained following a program of systematic training. However, improvements were not uniform across all parameters. Acceptable levels of reliability were achieved for those parameters most important for evaluation of velopharyngeal function.

  5. Targeted resequencing and variant validation using pxlence PCR assays

    Directory of Open Access Journals (Sweden)

    Frauke Coppieters

    2016-01-01

    Full Text Available The advent of next-generation sequencing technologies had a profound impact on molecular diagnostics. PCR is a popular method for target enrichment of disease gene panels. Using our proprietary primer-design pipeline, primerXL, we have created almost one million assays covering over 98% of the human exome. Here we describe the assay specification and both in silico and wet-lab validation of a selected set of 2294 assays using both next-generation sequencing and Sanger sequencing. Using a universal PCR protocol without optimization, these assays result in high coverage uniformity and limited non-specific coverage. In addition, data indicates a positive correlation between the predictive in silico specificity score and the amount of assay non-specific coverage.

  6. Assessment and reduction of comet assay variation in relation to DNA damage: studies from the European Comet Assay Validation Group

    DEFF Research Database (Denmark)

    Møller, Peter; Möller, Lennart; Godschalk, Roger W L

    2010-01-01

    The alkaline single cell gel electrophoresis (comet) assay has become a widely used method for the detection of DNA damage and repair in cells and tissues. Still, it has been difficult to compare results from different investigators because of differences in assay conditions and because the data...... are reported in different units. The European Comet Assay Validation Group (ECVAG) was established for the purpose of validation of the comet assay with respect to measures of DNA damage formation and its repair. The results from this inter-laboratory validation trail showed a large variation in measured level...... reliability for the measurement of DNA damage by the comet assay but there is still a need for further validation to reduce both assay and inter-laboratory variation....

  7. A reliable, delay bounded and less complex communication protocol for multicluster FANETs

    Directory of Open Access Journals (Sweden)

    Wajiya Zafar

    2017-02-01

    Full Text Available Recently, Flying Ad-hoc Networks (FANETs, enabling ad-hoc networking between Unmanned Aerial Vehicles (UAVs is gaining importance in several military and civilian applications. The sensitivity of the applications requires adaptive; efficient; delay bounded and scalable communication network among UAVs for data transmission. Due to communication protocol complexity; rigidity; cost of commercial-off-the-shelf (COT components; limited radio bandwidth; high mobility and computational resources; maintaining the desired level of Quality of Service (QoS becomes a daunting task. For the first time in this research we propose multicluster FANETs for efficient network management; the proposed scheme considerably reduces communication cost and optimizes network performance as well as exploit low power; less complex and low cost IEEE 802.15.4 (MAC protocol for intercluster and intracluster communication. In this research both beacon enabled mode and beaconless modes have been investigated with Guaranteed Time Slots (GTS and virtual Time Division Multiple Access (TDMA respectively. The methodology plays a key role towards reserving bandwidth for latency critical applications; eliminate collisions and medium access delays. Moreover analysis ad-hoc routing protocols including two proactive (OLSR, DSDV and one reactive (AODV is also presented. The results shows that the proposed scheme guarantees high packet delivery ratios while maintaining acceptable levels of latency requirements comparable with more complex and dedicatedly designed protocols in literature.

  8. Linearization of the bradford protein assay.

    Science.gov (United States)

    Ernst, Orna; Zor, Tsaffrir

    2010-04-12

    Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

  9. [Evaluation of serum PIVKA-II by Lumipulse PrestoII assay].

    Science.gov (United States)

    Hiramatsu, Kumiko; Tanaka, Yasuhito; Takagi, Kazumi; Kani, Satomi; Goto, Takaaki; Takasaka, Yoshimitsu; Matsuura, Kentaro; Sugauchi, Fuminaka; Moriyama, Kazushige; Murakami, Hiroshi; Kitajima, Sachiko; Mizokami, Masashi

    2009-03-01

    Measurements of serum concentrations of Des-gamma-carboxy Prothrombin (PIVKA-II) are widely used for diagnosing hepatocellular carcinoma (HCC). Recently, in Lumipulsef assay, it was reported that antibodies against alkaline phosphatase (ALP) derived from anti bleeding sheets led false high values of PIVKA-II in the patients with HCC resection. To improve the previous issue, newly developed Lumipulse PrestoII assay was examined. (1) The assay was reliable and positively correlated with the previous assays (Lumipulse f and Picolumi, R = 0.997 and 0.994 (n=115), respectively). (2) Eleven cases, which had false high values of PIVKA-II by the Lumipulsef assay, were examined by the PrestoII assay with excess of inactive ALP. The false high values of 10 cases were improved, but only one was still high. False reactivity of this case was stronger than other cases, more effective adsorption was required. (3) Comparing the absorbent activity of inactive ALP among 6 different kinds, we found inactive ALP with much higher adsorbent activity. When this inactive ALP was applied to assay, false high values of PIVKA-II were improved in all 11 cases. In conclusion, the PrestoII assay, which applies the inactive ALP with high activity, is reliable and useful for clinical screening.

  10. Hippocampal MRI volumetry at 3 Tesla: reliability and practical guidance.

    Science.gov (United States)

    Jeukens, Cécile R L P N; Vlooswijk, Mariëlle C G; Majoie, H J Marian; de Krom, Marc C T F M; Aldenkamp, Albert P; Hofman, Paul A M; Jansen, Jacobus F A; Backes, Walter H

    2009-09-01

    Although volumetry of the hippocampus is considered to be an established technique, protocols reported in literature are not described in great detail. This article provides a complete and detailed protocol for hippocampal volumetry applicable to T1-weighted magnetic resonance (MR) images acquired at 3 Tesla, which has become the standard for structural brain research. The protocol encompasses T1-weighted image acquisition at 3 Tesla, anatomic guidelines for manual hippocampus delineation, requirements of delineation software, reliability measures, and criteria to assess and ensure sufficient reliability. Moreover, the validity of the correction for total intracranial volume size was critically assessed. The protocol was applied by 2 readers to the MR images of 36 patients with cryptogenic localization-related epilepsy, 4 patients with unilateral hippocampal sclerosis, and 20 healthy control subjects. The uncorrected hippocampal volumes were 2923 +/- 500 mm3 (mean +/- SD) (left) and 3120 +/- 416 mm3 (right) for the patient group and 3185 +/- 411 mm3 (left) and 3302 +/- 411 mm3 (right) for the healthy control group. The volume of the 4 pathologic hippocampi of the patients with unilateral hippocampal sclerosis was 2980 +/- 422 mm3. The inter-reader reliability values were determined: intraclass-correlation-coefficient (ICC) = 0.87 (left) and 0.86 (right), percentage volume difference (VD) = 7.0 +/- 4.7% (left) and 6.0 +/- 3.8% (right), and overlap ratio (OR) = 0.82 +/- 0.04 (left) and 0.82 +/- 0.03 (right). The positive Pearson correlation between hippocampal volume and total intracranial volume was found to be low: r = 0.48 (P = 0.03, left) and r = 0.62 (P = 0.004, right) and did not significantly reduce the volumetric variances, showing the limited benefit of the brain size correction. A protocol was described to determine hippocampal volumes based on 3 Tesla MR images with high inter-reader reliability. Although the reliability of hippocampal volumetry at 3 Tesla

  11. A Mechanism for Reliable Mobility Management for Internet of Things Using CoAP

    Directory of Open Access Journals (Sweden)

    Seung-Man Chun

    2017-01-01

    Full Text Available Under unreliable constrained wireless networks for Internet of Things (IoT environments, the loss of the signaling message may frequently occur. Mobile Internet Protocol version 6 (MIPv6 and its variants do not consider this situation. Consequently, as a constrained device moves around different wireless networks, its Internet Protocol (IP connectivity may be frequently disrupted and power can be drained rapidly. This can result in the loss of important sensing data or a large delay for time-critical IoT services such as healthcare monitoring and disaster management. This paper presents a reliable mobility management mechanism in Internet of Things environments with lossy low-power constrained device and network characteristics. The idea is to use the Internet Engineering Task Force (IETF Constrained Application Protocol (CoAP retransmission mechanism to achieve both reliability and simplicity for reliable IoT mobility management. Detailed architecture, algorithms, and message extensions for reliable mobility management are presented. Finally, performance is evaluated using both mathematical analysis and simulation.

  12. A Mechanism for Reliable Mobility Management for Internet of Things Using CoAP.

    Science.gov (United States)

    Chun, Seung-Man; Park, Jong-Tae

    2017-01-12

    Under unreliable constrained wireless networks for Internet of Things (IoT) environments, the loss of the signaling message may frequently occur. Mobile Internet Protocol version 6 (MIPv6) and its variants do not consider this situation. Consequently, as a constrained device moves around different wireless networks, its Internet Protocol (IP) connectivity may be frequently disrupted and power can be drained rapidly. This can result in the loss of important sensing data or a large delay for time-critical IoT services such as healthcare monitoring and disaster management. This paper presents a reliable mobility management mechanism in Internet of Things environments with lossy low-power constrained device and network characteristics. The idea is to use the Internet Engineering Task Force (IETF) Constrained Application Protocol (CoAP) retransmission mechanism to achieve both reliability and simplicity for reliable IoT mobility management. Detailed architecture, algorithms, and message extensions for reliable mobility management are presented. Finally, performance is evaluated using both mathematical analysis and simulation.

  13. Validation and modification of dried blood spot-based glycosylated hemoglobin assay for the longitudinal aging study in India.

    Science.gov (United States)

    Hu, Peifeng; Edenfield, Michael; Potter, Alan; Kale, Varsha; Risbud, Arun; Williams, Sharon; Lee, Jinkook; Bloom, David E; Crimmins, Eileen; Seeman, Teresa

    2015-01-01

    This study aims to validate a modified dried blood spot (DBS)-based glycosylated hemoglobin (HbA1c) assay protocol, after a pretest in India showed poor correlation between the original DBS-based protocol and venous results. The original protocol was tested on different chemistry analyzers and then simplified at the University of Washington (UW). A second pretest was conducted in India to validate the modified assay protocol, using 44 quality control specimens. Data from UW indicated that, using the original protocol, the correlation coefficients between DBS and venous results were above 0.98 on both Bio-Rad and Olympus chemistry analyzers. The protocol worked equally well on filter paper, with or without pre-treatment, and when the recommended amount of blood spot material, or less, was used. A second pretest of the modified protocol confirmed that DBS-based levels from both Olympus and Roche chemistry analyzers were well correlated with DBS results from UW (correlation coefficients were above 0.96), as well as with venous values (correlation coefficients were above 0.94). The DBS-based HbA1c values are highly correlated with venous results. The pre-treatment of filter paper does not appear to be necessary. The poor results from the first pretest are probably due to factors unrelated to the protocol, such as problems with the chemistry analyzer or assay reagents. © 2015 Wiley Periodicals, Inc.

  14. Mutagenicity of the potent rat hepatocarcinogen 6BT to the liver of transgenic (lacI) rats: consideration of a reduced mutation assay protocol.

    Science.gov (United States)

    Lefevre, P A; Tinwell, H; Ashby, J

    1997-01-01

    6-(p-dimethylaminophenylazo)benzothiazole (6BT) is an unusually potent rat hepatocarcinogen, producing large malignant liver tumours after only 2-3 months of dietary administration in a riboflavin-deficient diet. This azocarcinogen has been evaluated in a Big Blue F344 transgenic rat (lacI) gene mutation assay. In a reproduction of the early stages of the carcinogenesis bioassay of this agent, rats were maintained on a riboflavin-deficient diet and were given 10 consecutive daily doses of 6BT (10 mg/kg) by oral gavage. The animals were killed and the livers examined 11 days after the final dose. The livers of 6BT-treated rats showed evidence of hepatocellular hypertrophy in centrolobular areas, with some indication of an increased incidence of mitotic figures. An approximately 10-fold increase in the mutation frequency of DNA isolated from an aliquot of the combined liver homogenates of 6BT-treated rats was observed over that obtained from an equivalent aliquot from control animals. Examination of DNA samples isolated from the livers of individual animals confirmed that 6BT was mutagenic in Big Blue rat livers. These data extend the sensitivity of this transgenic assay to include azo hepatocarcinogens. The determination of mutation frequencies using pooled tissue samples represented a major resource-saving adaptation of the assay protocol in the present study; the general advantages and disadvantages of this practice are discussed.

  15. A reliable radiochromatographic assay technique for hepatic microsomal 16α-hydroxylase activity towards oestrone 3-sulphate

    International Nuclear Information System (INIS)

    Tsoutsoulis, C.J.; Hobkirk, R.

    1980-01-01

    A reliable procedure for the assay of liver microsomal 16α-hydroxylation of oestrone 3-sulphate has been developed for the guinea pig. It is based on the rapid, quantitative separation of oestradiol and oestriol by Sephadex LH-20 columns after the chemical reduction and enzymic hydrolysis of the incubation products. Microsomal preparations and incubation conditions that optimized 16α-hydroxylation of oestrone 3-sulphate were employed. Under these circumstances, reduction of the substrate at C-17 and hydrolysis of the sulphate were minimized. Conditions were established that yielded reaction linearity with respect to time and microsomal concentration. This hydroxylation had an absolute requirement for NADPH, which could not be satisfied by NADH. Apparent Ksub(m) values for oestrone 3-sulphate and NADPH, under the conditions used, were 14μM and 0.17mM respectively. 16α-hydroxylase activity was present in the liver microsomal fraction from heavily pigmented, female English Shorthaired guinea pigs. Much lower activity was detected in mature pigmented males and albino females. No activity could be demonstrated in mature, albino males. (author)

  16. A SURVEY ON MULTICAST ROUTING PROTOCOLS FOR PERFORMANCE EVALUATION IN WIRELESS SENSOR NETWORK

    Directory of Open Access Journals (Sweden)

    A. Suruliandi

    2015-03-01

    Full Text Available Multicast is a process used to transfer same message to multiple receivers at the same time. This paper presents the simulation and analysis of the performance of six different multicast routing protocols for Wireless Sensor Network (WSN. They are On Demand Multicast Routing Protocol (ODMRP, Protocol for Unified Multicasting through Announcement (PUMA, Multicast Adhoc On demand Distance Vector Protocol (MAODV, Overlay Boruvka-based Adhoc Multicast Protocol (OBAMP, Application Layer Multicast Algorithm (ALMA and enhanced version of ALMA (ALMA-H for WSN. Among them, ODMRP, MAODV and PUMA are reactive protocols while OBAMP, ALMA and ALMA-H are proactive protocols. This paper compares the performance of these protocols with common parameters such as Throughput, Reliability, End-to-End delay and Packet Delivery Ratio (PDR with increasing the numbers of nodes and increasing the speed of the nodes. The main objective of this work is to select the efficient multicast routing protocol for WSN among six multicast routing protocol based on relative strength and weakness of each protocol. The summary of above six multicast routing protocols is presented with a table of different performance characteristics. Experimental result shows that ODMRP attains higher throughput, reliability and higher packet delivery ratio than other multicast routing protocol, while incurring far less end-to-end delay.

  17. Relationship between the radioisotopic footpad assay and other immunological assays in tumor bearing rats

    International Nuclear Information System (INIS)

    Mizushima, Yutaka; Takeichi, Noritoshi; Minami, Akio; Kasai, Masaharu; Itaya, Toshiyuki

    1981-01-01

    KMT-17, a fibrosarcoma induced by 3-methylcholanthrene in a WKA rat, is a sensitive tumor to various kinds of immunological assays and is a suitable model tumor for the study of the immune status in tumor bearing hosts. The antitumor immune response of KMT-17 bearing rats was studied by a radioisotopic footpad assay (FPA) in comparison with other in vivo and in vitro assays. Delayed hypersensitivity to tumor antigens measured by the FPA was observed from the 8th day after transplantation of KMT-17 cells, reached a peak on the 12 - 15th day, and then declined in the late stage on the 17th day. The kinetics of the FPA correlated well with those of an in vivo Winn assay and of an in vitro lymphocyte cytotoxicity assay ( 51 Cr-release assay). The appearance of an antitumor antibody detected by a complement dependent cytotoxicity test also correlated well with the kinetics of the FPA. A growth inhibition assay (GIA) for non-specific cell-mediated immunity also showed similar kinetics to that of the FPA. The delayed hypersensitivity footpad reaction to tumor cell extracts measured by this FPA was tumor-specific. These results suggest that the FPA is a simple and reliable in vivo assay for evaluating antitumor immunity in tumor bearing hosts. (author)

  18. Detection and identification of Toscana and other phleboviruses by RT-nested-PCR assays with degenerated primers.

    Science.gov (United States)

    Sánchez-Seco, María-Paz; Echevarría, José-Manuel; Hernández, Lourdes; Estévez, Domingo; Navarro-Marí, José-María; Tenorio, Antonio

    2003-09-01

    Phleboviruses are a large and widespread group of viruses that are transmitted by arthropods. Toscana virus is one of the principal agents that causes meningitis in humans during the summer in Italy and, possibly, in other Mediterranean countries. Rift Valley Fever virus can cause serious illness in both animals and humans, leading to high morbidity and mortality, and is considered to be a potential agent for epizootics and human epidemics. Since information on this group of viruses is still scant, reliable laboratory tools for diagnosis and epidemiological surveillance must be developed, in order to ascertain their real impact on Public Health. Sequence data obtained from Spanish isolates of Toscana virus and other phleboviruses confirmed that natural genome variability may hamper the diagnosis of these agents by molecular methods, so this must be borne in mind when developing reliable assays. In view of the above, a novel and useful protocol has been developed for the detection and specific identification of every member of the phlebovirus genus present in a sample, including Toscana virus, based on a generic RT-nested-PCR, followed by sequencing of the amplified fragment. A change in this method also allowed specific direct detection and identification of wild isolates of Toscana virus of different geographical origin, using newly designed primers. Testing clinical samples with these assays confirmed the role of Toscana virus as an agent that causes acute aseptic meningitis in the central region of Spain. Copyright 2003 Wiley-Liss, Inc.

  19. JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: I. Summary of pre-validation study results.

    Science.gov (United States)

    Uno, Yoshifumi; Kojima, Hajime; Omori, Takashi; Corvi, Raffaella; Honma, Masamistu; Schechtman, Leonard M; Tice, Raymond R; Burlinson, Brian; Escobar, Patricia A; Kraynak, Andrew R; Nakagawa, Yuzuki; Nakajima, Madoka; Pant, Kamala; Asano, Norihide; Lovell, David; Morita, Takeshi; Ohno, Yasuo; Hayashi, Makoto

    2015-07-01

    The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this validation effort was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The purpose of the pre-validation studies (i.e., Phase 1 through 3), conducted in four or five laboratories with extensive comet assay experience, was to optimize the protocol to be used during the definitive validation study. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. The electrophoretic mobility shift assay (EMSA)

    OpenAIRE

    sprotocols

    2015-01-01

    The electrophoretic mobility shift assay (EMSA), also known as “gel shift assay”, is used to examine the binding parameters and relative affinities of protein and DNA interactions. We produced recombinant CCA1 protein and tested its binding affinity for the promoter fragments that contain CBS (AAAAATCT) or evening element (EE, AAAATATCT) (1) using a modified procedure adopted from published protocols (2,3).

  1. BTP: a Block Transfer Protocol for Delay Tolerant Wireless Sensor Networks

    DEFF Research Database (Denmark)

    Hansen, Morten Tranberg; Biagioni, Edoardo S.

    2010-01-01

    Wireless sensor networks that are energy-constrained must transmit and receive data as efficiently as possible.  If the transmission is delay tolerant, transferring blocks of accumulated data can be more efficient than transferring each sensed measurement as soon as it is available.  This paper...... proposes a Block Transfer Protocol (BTP) designed for efficient and reliable transmission in wireless sensor networks.  BTP reduces the time it takes to reliably transfer a block of packets compared to conventional link layer protocols, by piggybacking in data packets information about the transfer......, minimizing the number of acknowledgements needed for reliable transmission, and reducing the need for timeouts, which can substantially slow down communication when transmission is unreliable.  In addition, BTP improves reliability by handling false positive acknowledgements and by letting the receivers...

  2. The intergroup protocols: Scalable group communication for the internet

    Energy Technology Data Exchange (ETDEWEB)

    Berket, Karlo [Univ. of California, Santa Barbara, CA (United States)

    2000-12-04

    Reliable group ordered delivery of multicast messages in a distributed system is a useful service that simplifies the programming of distributed applications. Such a service helps to maintain the consistency of replicated information and to coordinate the activities of the various processes. With the increasing popularity of the Internet, there is an increasing interest in scaling the protocols that provide this service to the environment of the Internet. The InterGroup protocol suite, described in this dissertation, provides such a service, and is intended for the environment of the Internet with scalability to large numbers of nodes and high latency links. The InterGroup protocols approach the scalability problem from various directions. They redefine the meaning of group membership, allow voluntary membership changes, add a receiver-oriented selection of delivery guarantees that permits heterogeneity of the receiver set, and provide a scalable reliability service. The InterGroup system comprises several components, executing at various sites within the system. Each component provides part of the services necessary to implement a group communication system for the wide-area. The components can be categorized as: (1) control hierarchy, (2) reliable multicast, (3) message distribution and delivery, and (4) process group membership. We have implemented a prototype of the InterGroup protocols in Java, and have tested the system performance in both local-area and wide-area networks.

  3. Coordination Protocols for a Reliable Sensor, Actuator, and Device Network (SADN

    Directory of Open Access Journals (Sweden)

    Keiji Ozaki

    2008-01-01

    Full Text Available A sensor, actuator, and device network (SADN is composed of three types of nodes, which are sensor, actuator, and actuation device nodes. Sensor nodes and actuator nodes are interconnected in wireless networks as discussed in wireless sensor and actuator networks (WSANs. Actuator nodes and device nodes are interconnected in types of networks, i.e. wireless and wired network. Sensor nodes sense an physical event and send sensed values of the event to actuator nodes. An actuator node makes a decision on proper actions on receipt of sensed values and then issue the action requests to the device nodes. A device node really acts to the physical world. For example, moves a robot arms by performing the action on receipt of the action request. Messages may be lost and nodes may be faulty. Especially, messages are lost due to noise and collision in a wireless network. We propose a fully redundant model for an SADN where each of sensor, actuator, and device functions is replicated in multiple nodes and each of sensor-actuator and actuator-device communication is realized in many-to-many type of communication protocols. Even if some number of nodes are faulty, the other nodes can perform requested tasks. Here, each sensor node sends sensed values to multiple actuator nodes and each actuator node receives sensed values from multiple sensor nodes. While multiple actuator nodes communicate with multiple replica nodes of a device. Even if messages are lost and some number of nodes are faulty, device nodes can surely receive action requests required for sensed values and the actions are performed. In this paper, we discuss a type of semi-passive coordination (SPC protocol of multiple actuator nodes for multiple sensor nodes. We discuss a type of active coordination protocol for multiple actuator nodes and multiple actuation device nodes. We evaluate the SPC protocol for the sensor-actuator coordination in terms of the number of messages exchanged among

  4. Generation of a reliable full-length cDNA of infectiousTembusu virus using a PCR-based protocol.

    Science.gov (United States)

    Liang, Te; Liu, Xiaoxiao; Cui, Shulin; Qu, Shenghua; Wang, Dan; Liu, Ning; Wang, Fumin; Ning, Kang; Zhang, Bing; Zhang, Dabing

    2016-02-02

    Full-length cDNA of Tembusu virus (TMUV) cloned in a plasmid has been found instable in bacterial hosts. Using a PCR-based protocol, we generated a stable full-length cDNA of TMUV. Different cDNA fragments of TMUV were amplified by reverse transcription (RT)-PCR, and cloned into plasmids. Fragmented cDNAs were amplified and assembled by fusion PCR to produce a full-length cDNA using the recombinant plasmids as templates. Subsequently, a full-length RNA was transcribed from the full-length cDNA in vitro and transfected into BHK-21 cells; infectious viral particles were rescued successfully. Following several passages in BKH-21 cells, the rescued virus was compared with the parental virus by genetic marker checks, growth curve determinations and animal experiments. These assays clearly demonstrated the genetic and biological stabilities of the rescued virus. The present work will be useful for future investigations on the molecular mechanisms involved in replication and pathogenesis of TMUV. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Robust against route failure using power proficient reliable routing in MANET

    Directory of Open Access Journals (Sweden)

    M. Malathi

    2018-03-01

    Full Text Available The aim of this paper was to propose a novel routing protocol for Mobile Adhoc Network communication which reduces the route failure during transmission. The proposed routing protocol uses 3 salient parameters to discover the path which ensure the reliable communication. The quality of the channel, link quality and energy level of the node are the major reasons for unintentional node failure in mobile Adhoc network. So the proposed routing protocol considers these three parameters to select the best forwarder node in the path. The reliable data communication is achieved by transmitting data via path selected by the proposed routing scheme has been proven using network simulator (NS2. Keywords: Channel quality, Link quality, Mobile Adhoc Network (MANET, Residual energy

  6. Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

    OpenAIRE

    Zhu, Qinchang; Yu, Zhiqiang; Kabashima, Tsutomu; Yin, Sheng; Dragusha, Shpend; El-Mahdy, Ahmed F. M.; Ejupi, Valon; Shibata, Takayuki; Kai, Masaaki

    2015-01-01

    Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates f...

  7. Bacteriophage amplification assay for detection of Listeria spp. using virucidal laser treatment

    Directory of Open Access Journals (Sweden)

    I.C. Oliveira

    2012-09-01

    Full Text Available A protocol for the bacteriophage amplification technique was developed for quantitative detection of viable Listeria monocytogenes cells using the A511 listeriophage with plaque formation as the end-point assay. Laser and toluidine blue O (TBO were employed as selective virucidal treatment for destruction of exogenous bacteriophage. Laser and TBO can bring a total reduction in titer phage (ca. 10(8 pfu/mL without affecting the viability of L. monocytogenes cells. Artificially inoculated skimmed milk revealed mean populations of the bacteria as low as between 13 cfu/mL (1.11 log cfu/mL, after a 10-h assay duration. Virucidal laser treatment demonstrated better protection of Listeria cells than the other agents previously tested. The protocol was faster and easier to perform than standard procedures. This protocol constitutes an alternative for rapid, sensitive and quantitative detection of L. monocytogenes.

  8. Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

    International Nuclear Information System (INIS)

    Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei; Loo, Say Chye Joachim

    2015-01-01

    Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein–particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges

  9. Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei, E-mail: kwng@ntu.edu.sg; Loo, Say Chye Joachim, E-mail: joachimloo@ntu.edu.sg [Nanyang Technological University, School of Materials Science and Engineering (Singapore)

    2015-01-15

    Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein–particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges.

  10. Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

    Science.gov (United States)

    Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei; Loo, Say Chye Joachim

    2015-01-01

    Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein-particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges.

  11. Advanced flooding-based routing protocols for underwater sensor networks

    OpenAIRE

    Isufi, E.; Dol, H.; Leus, G.J.T.

    2016-01-01

    Flooding-based protocols are a reliable solution to deliver packets in underwater sensor networks. However, these protocols potentially involve all the nodes in the forwarding process. Thus, the performance and energy efficiency are not optimal. In this work, we propose some advances of a flooding-based protocol with the goal to improve the performance and the energy efficiency. The first idea considers the node position information in order to reduce the number of relays that may apply flood...

  12. Chromosome aberration assays in Allium

    Energy Technology Data Exchange (ETDEWEB)

    Grant, W.F.

    1982-01-01

    The common onion (Allium cepa) is an excellent plant for the assay of chromosome aberrations after chemical treatment. Other species of Allium (A. cepa var. proliferum, A. carinatum, A. fistulosum and A. sativum) have also been used but to a much lesser extent. Protocols have been given for using root tips from either bulbs or seeds of Allium cepa to study the cytological end-points, such as chromosome breaks and exchanges, which follow the testing of chemicals in somatic cells. It is considered that both mitotic and meiotic end-points should be used to a greater extent in assaying the cytogenetic effects of a chemical. From a literature survey, 148 chemicals are tabulated that have been assayed in 164 Allium tests for their clastogenic effect. Of the 164 assays which have been carried out, 75 are reported as giving a positive reaction, 49 positive and with a dose response, 1 positive and temperature-related, 9 borderline positive, and 30 negative; 76% of the chemicals gave a definite positive response. It is proposed that the Allium test be included among those tests routinely used for assessing chromosomal damage induced by chemicals.

  13. WEAMR — A Weighted Energy Aware Multipath Reliable Routing Mechanism for Hotline-Based WSNs

    Directory of Open Access Journals (Sweden)

    Ki-Hyung Kim

    2013-05-01

    Full Text Available Reliable source to sink communication is the most important factor for an efficient routing protocol especially in domains of military, healthcare and disaster recovery applications. We present weighted energy aware multipath reliable routing (WEAMR, a novel energy aware multipath routing protocol which utilizes hotline-assisted routing to meet such requirements for mission critical applications. The protocol reduces the number of average hops from source to destination and provides unmatched reliability as compared to well known reactive ad hoc protocols i.e., AODV and AOMDV. Our protocol makes efficient use of network paths based on weighted cost calculation and intelligently selects the best possible paths for data transmissions. The path cost calculation considers end to end number of hops, latency and minimum energy node value in the path. In case of path failure path recalculation is done efficiently with minimum latency and control packets overhead. Our evaluation shows that our proposal provides better end-to-end delivery with less routing overhead and higher packet delivery success ratio compared to AODV and AOMDV. The use of multipath also increases overall life time of WSN network using optimum energy available paths between sender and receiver in WDNs.

  14. Reliability of diagnostic imaging techniques in suspected acute appendicitis: proposed diagnostic protocol

    International Nuclear Information System (INIS)

    Cura del, J. L.; Oleaga, L.; Grande, D.; Vela, A. C.; Ibanez, A. M.

    2001-01-01

    To study the utility of ultrasound and computed tomography (CT) in case of suspected appendicitis. To determine the diagnostic yield in terms of different clinical contexts and patient characteristics. to assess the costs and benefits of introducing these techniques and propose a protocol for their use. Negative appendectomies, complications and length of hospital stay in a group of 152 patients with suspected appendicitis who underwent ultrasound and CT were compared with those of 180 patients who underwent appendectomy during the same time period, but had not been selected for the first group: these patients costs for each group were calculated. In the first group, the diagnostic value of the clinical signs was also evaluated. The reliability of the clinical signs was limited, while the results with ultrasound and CT were excellent. The incidence of negative appendectomy was 9.6% in the study group and 12.2% in the control group. Moreover, there were fewer complications and a shorter hospital stay in the first group. Among men, however, the rate of negative appendectomy was lower in the control group. The cost of using ultrasound and CT in the management of appendicitis was only slightly higher than that of the control group. Although ultrasound and CT are not necessary in cases in which the probability of appendicitis is low or in men presenting clear clinical evidence, the use of these techniques is indicated in the remaining cases in which appendicitis is suspected. In children, ultrasound is the technique of choice. In all other patients, if negative results are obtained with one of the two techniques, the other should be performed. (Author) 49 refs

  15. Addressing the need for biomarker liquid chromatography/mass spectrometry assays: a protocol for effective method development for the bioanalysis of endogenous compounds in cerebrospinal fluid.

    Science.gov (United States)

    Benitex, Yulia; McNaney, Colleen A; Luchetti, David; Schaeffer, Eric; Olah, Timothy V; Morgan, Daniel G; Drexler, Dieter M

    2013-08-30

    Research on disorders of the central nervous system (CNS) has shown that an imbalance in the levels of specific endogenous neurotransmitters may underlie certain CNS diseases. These alterations in neurotransmitter levels may provide insight into pathophysiology, but can also serve as disease and pharmacodynamic biomarkers. To measure these potential biomarkers in vivo, the relevant sample matrix is cerebrospinal fluid (CSF), which is in equilibrium with the brain's interstitial fluid and circulates through the ventricular system of the brain and spinal cord. Accurate analysis of these potential biomarkers can be challenging due to low CSF sample volume, low analyte levels, and potential interferences from other endogenous compounds. A protocol has been established for effective method development of bioanalytical assays for endogenous compounds in CSF. Database searches and standard-addition experiments are employed to qualify sample preparation and specificity of the detection thus evaluating accuracy and precision. This protocol was applied to the study of the histaminergic neurotransmitter system and the analysis of histamine and its metabolite 1-methylhistamine in rat CSF. The protocol resulted in a specific and sensitive novel method utilizing pre-column derivatization ultra high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS), which is also capable of separating an endogenous interfering compound, identified as taurine, from the analytes of interest. Copyright © 2013 John Wiley & Sons, Ltd.

  16. Validation of IT-based Data Communication Protocol for Nuclear Power Plant

    International Nuclear Information System (INIS)

    Jeong, K. I.; Kim, D. H.; Lee, J. C.

    2009-12-01

    The communication network designed to transmit control and processing signals in digital Instrument and Control (I and C) systems in Nuclear Power Plant (NPP), should provide a high level of safety and reliability. There are different features between the communication networks of NPPs and other commercial communication networks. Safety and reliability are the most important factors in the communication networks of an NPP rather than efficiency which are important factors of a commercial communication network design. To develop Data Communication Protocol for Nuclear Power Plant, We analyze the design criteria and performance requirements of existing commercial communication protocols based on Information Technology(IT). And also, we examine the adaptability to the communication protocol of an NPP. Based on these results, we developed our own protocol(Nuclear power plant Safety Communication Protocol : NSCP) for NPP I and C, which meet the required specifications through design overall protocol architecture and data frame format, definition of functional requirements and specifications. NSCP is the communication protocol designed for a safety-grade control network in the nuclear power plant. In this report, we had specified NSCP protocol by FDT(Formal Description Technique) and established validation procedures based on the validation methodology. It was confirmed specification error, major function's validity and reachability of NSCP by performing simulation and the validation process using Telelogic Tau tool

  17. Evaluation of PCR and DNA hybridization protocols for detection of viable enterotoxigenic Clostridium perfringens in irradiated beef

    International Nuclear Information System (INIS)

    Baez, L.A.; Juneja, V.K.; Thayer, D.W.; Sackitey, S.

    1997-01-01

    The sensitivity of DNA hybridization and polymerase chain reaction (PCR), was evaluated in irradiated cooked and raw beef samples. A membrane-based colony hybridization assay and a PCR protocol, both with specificity for the enterotoxin A gene of Clostridium perfringens, were compared with viable plate counts. The results of the colony hybridization procedure were in agreement with viable plate counts for detection and enumeration of enterotoxigenic C. perfringens. The PCR procedure combined a 4 h enrichment followed by a nucleic acid extraction step and assessed the amplification of 183 and 750 base pair enterotoxin gene targets. Detection of C. perfringens by PCR did not show a reliable correlation with viable plate counts or the colony hybridization assay. C. perfringens killed by irradiation were not detected by the plate count or colony hybridization methods; however, killed cells were detected with the PCR technique. By relying on the growth of viable cells for detection and/or enumeration, the colony hybridization and plate count methods provided a direct correlation with the presence of viable bacteria

  18. DOE assay methods used for characterization of contact-handled transuranic waste

    Energy Technology Data Exchange (ETDEWEB)

    Schultz, F.J. (Oak Ridge National Lab., TN (United States)); Caldwell, J.T. (Pajarito Scientific Corp., Los Alamos, NM (United States))

    1991-08-01

    US Department of Energy methods used for characterization of contact-handled transuranic (CH-TRU) waste prior to shipment to the Waste Isolation Pilot Plant (WIPP) are described and listed by contractor site. The methods described are part of the certification process. All CH-TRU waste must be assayed for determination of fissile material content and decay heat values prior to shipment and prior to storage on-site. Both nondestructive assay (NDA) and destructive assay methods are discussed, and new NDA developments such as passive-action neutron (PAN) crate counter improvements and neutron imaging are detailed. Specifically addressed are assay method physics; applicability to CH-TRU wastes; calibration standards and implementation; operator training requirements and practices; assay procedures; assay precision, bias, and limit of detection; and assay limitation. While PAN is a new technique and does not yet have established American Society for Testing and Materials. American National Standards Institute, or Nuclear Regulatory Commission guidelines or methods describing proper calibration procedures, equipment setup, etc., comparisons of PAN data with the more established assay methods (e.g., segmented gamma scanning) have demonstrated its reliability and accuracy. Assay methods employed by DOE have been shown to reliable and accurate in determining fissile, radionuclide, alpha-curie content, and decay heat values of CH-TRU wastes. These parameters are therefore used to characterize packaged waste for use in certification programs such as that used in shipment of CH-TRU waste to the WIPP. 36 refs., 10 figs., 7 tabs.

  19. DOE assay methods used for characterization of contact-handled transuranic waste

    International Nuclear Information System (INIS)

    Schultz, F.J.; Caldwell, J.T.

    1991-08-01

    US Department of Energy methods used for characterization of contact-handled transuranic (CH-TRU) waste prior to shipment to the Waste Isolation Pilot Plant (WIPP) are described and listed by contractor site. The methods described are part of the certification process. All CH-TRU waste must be assayed for determination of fissile material content and decay heat values prior to shipment and prior to storage on-site. Both nondestructive assay (NDA) and destructive assay methods are discussed, and new NDA developments such as passive-action neutron (PAN) crate counter improvements and neutron imaging are detailed. Specifically addressed are assay method physics; applicability to CH-TRU wastes; calibration standards and implementation; operator training requirements and practices; assay procedures; assay precision, bias, and limit of detection; and assay limitation. While PAN is a new technique and does not yet have established American Society for Testing and Materials. American National Standards Institute, or Nuclear Regulatory Commission guidelines or methods describing proper calibration procedures, equipment setup, etc., comparisons of PAN data with the more established assay methods (e.g., segmented gamma scanning) have demonstrated its reliability and accuracy. Assay methods employed by DOE have been shown to reliable and accurate in determining fissile, radionuclide, alpha-curie content, and decay heat values of CH-TRU wastes. These parameters are therefore used to characterize packaged waste for use in certification programs such as that used in shipment of CH-TRU waste to the WIPP. 36 refs., 10 figs., 7 tabs

  20. Detection of proteins using a colorimetric bio-barcode assay.

    Science.gov (United States)

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

    2007-01-01

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

  1. Rapid 2,2'-bicinchoninic-based xylanase assay compatible with high throughput screening

    Science.gov (United States)

    William R. Kenealy; Thomas W. Jeffries

    2003-01-01

    High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2'-bicinchoninic-based protein reagent. Endo-1,4-â-D-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml−1. The assay is linear for sugar...

  2. BAVP: Blockchain-Based Access Verification Protocol in LEO Constellation Using IBE Keys

    Directory of Open Access Journals (Sweden)

    Songjie Wei

    2018-01-01

    Full Text Available LEO constellation has received intensive research attention in the field of satellite communication. The existing centralized authentication protocols traditionally used for MEO/GEO satellite networks cannot accommodate LEO satellites with frequent user connection switching. This paper proposes a fast and efficient access verification protocol named BAVP by combining identity-based encryption and blockchain technology. Two different key management schemes with IBE and blockchain, respectively, are investigated, which further enhance the authentication reliability and efficiency in LEO constellation. Experiments on OPNET simulation platform evaluate and demonstrate the effectiveness, reliability, and fast-switching efficiency of the proposed protocol. For LEO networks, BAVP surpasses the well-known existing solutions with significant advantages in both performance and scalability which are supported by theoretical analysis and simulation results.

  3. International Network for Comparison of HIV Neutralization Assays: The NeutNet Report

    NARCIS (Netherlands)

    Fenyö, Eva Maria; Heath, Alan; Dispinseri, Stefania; Holmes, Harvey; Lusso, Paolo; Zolla-Pazner, Susan; Donners, Helen; Heyndrickx, Leo; Alcami, Jose; Bongertz, Vera; Jassoy, Christian; Malnati, Mauro; Montefiori, David; Moog, Christiane; Morris, Lynn; Osmanov, Saladin; Polonis, Victoria; Sattentau, Quentin; Schuitemaker, Hanneke; Sutthent, Ruengpung; Wrin, Terri; Scarlatti, Gabriella

    2009-01-01

    BACKGROUND: Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration

  4. Protocol: A simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis

    Directory of Open Access Journals (Sweden)

    Coustham Vincent

    2011-03-01

    Full Text Available Abstract Background Many experiments in modern plant molecular biology require the processing of large numbers of samples for a variety of applications from mutant screens to the analysis of natural variants. A severe bottleneck to many such analyses is the acquisition of good yields of high quality RNA suitable for use in sensitive downstream applications such as real time quantitative reverse-transcription-polymerase chain reaction (real time qRT-PCR. Although several commercial kits are available for high-throughput RNA extraction in 96-well format, only one non-kit method has been described in the literature using the commercial reagent TRIZOL. Results We describe an unusual phenomenon when using TRIZOL reagent with young Arabidopsis seedlings. This prompted us to develop a high-throughput RNA extraction protocol (HTP96 adapted from a well established phenol:chloroform-LiCl method (P:C-L that is cheap, reliable and requires no specialist equipment. With this protocol 192 high quality RNA samples can be prepared in 96-well format in three hours (less than 1 minute per sample with less than 1% loss of samples. We demonstrate that the RNA derived from this protocol is of high quality and suitable for use in real time qRT-PCR assays. Conclusion The development of the HTP96 protocol has vastly increased our sample throughput, allowing us to fully exploit the large sample capacity of modern real time qRT-PCR thermocyclers, now commonplace in many labs, and develop an effective high-throughput gene expression platform. We propose that the HTP96 protocol will significantly benefit any plant scientist with the task of obtaining hundreds of high quality RNA extractions.

  5. Assays of D-Amino Acid Oxidase Activity

    Directory of Open Access Journals (Sweden)

    Elena Rosini

    2018-01-01

    Full Text Available D-amino acid oxidase (DAAO is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs. Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples.

  6. Serotype determination of Salmonella by xTAG assay.

    Science.gov (United States)

    Zheng, Zhibei; Zheng, Wei; Wang, Haoqiu; Pan, Jincao; Pu, Xiaoying

    2017-10-01

    Currently, no protocols or commercial kits are available to determine the serotypes of Salmonella by using Luminex MAGPIX®. In this study, an xTAG assay for serotype determination of Salmonella suitable for Luminex MAGPIX® is described and 228 Salmonella isolates were serotype determined by this xTAG assay. The xTAG assay consists of two steps: 1) Multiplex PCR to amplify simultaneously O, H and Vi antigen genes of Salmonella, and 2) Magplex-TAG™ microsphere hybridization to identify accurately the specific PCR products of different antigens. Compared with the serotyping results of traditional serum agglutination test, the sensitivity and specificity of the xTAG assay were 95.1% and 100%, respectively. The agreement rate of these two assays was 95.2%. Compared with Luminex xMAP® Salmonella Serotyping Assay (SSA) kit, the advantages of this xTAG assay are: First, the magnetic beads make it applicable to both the Luminex®100/200™ and MAGPIX® systems. Second, only primers rather than both primers and probes are needed in the xTAG assay, and the process of coupling antigen-specific oligonucleotide probes to beads is circumvented, which make the xTAG assay convenient to be utilized by other laboratories. The xTAG assay may serve as a rapid alternative or complementary method for traditional Salmonella serotyping tests, especially for laboratories that utilize the MAGPIX® systems. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. A Field-Based Testing Protocol for Assessing Gross Motor Skills in Preschool Children: The Children's Activity and Movement in Preschool Study Motor Skills Protocol

    Science.gov (United States)

    Williams, Harriet G.; Pfeiffer, Karin A.; Dowda, Marsha; Jeter, Chevy; Jones, Shaverra; Pate, Russell R.

    2009-01-01

    The purpose of this study was to develop a valid and reliable tool for use in assessing motor skills in preschool children in field-based settings. The development of the Children's Activity and Movement in Preschool Study Motor Skills Protocol included evidence of its reliability and validity for use in field-based environments as part of large…

  8. The best of both worlds: Building on the COPUS and RTOP observation protocols to easily and reliably measure various levels of reformed instructional practice.

    Science.gov (United States)

    Lund, Travis J; Pilarz, Matthew; Velasco, Jonathan B; Chakraverty, Devasmita; Rosploch, Kaitlyn; Undersander, Molly; Stains, Marilyne

    2015-01-01

    Researchers, university administrators, and faculty members are increasingly interested in measuring and describing instructional practices provided in science, technology, engineering, and mathematics (STEM) courses at the college level. Specifically, there is keen interest in comparing instructional practices between courses, monitoring changes over time, and mapping observed practices to research-based teaching. While increasingly common observation protocols (Reformed Teaching Observation Protocol [RTOP] and Classroom Observation Protocol in Undergraduate STEM [COPUS]) at the postsecondary level help achieve some of these goals, they also suffer from weaknesses that limit their applicability. In this study, we leverage the strengths of these protocols to provide an easy method that enables the reliable and valid characterization of instructional practices. This method was developed empirically via a cluster analysis using observations of 269 individual class periods, corresponding to 73 different faculty members, 28 different research-intensive institutions, and various STEM disciplines. Ten clusters, called COPUS profiles, emerged from this analysis; they represent the most common types of instructional practices enacted in the classrooms observed for this study. RTOP scores were used to validate the alignment of the 10 COPUS profiles with reformed teaching. Herein, we present a detailed description of the cluster analysis method, the COPUS profiles, and the distribution of the COPUS profiles across various STEM courses at research-intensive universities. © 2015 T. J. Lund et al. CBE—Life Sciences Education © 2015 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  9. Analysis of Security Protocols by Annotations

    DEFF Research Database (Denmark)

    Gao, Han

    . The development of formal techniques, e.g. control flow analyses, that can check various security properties, is an important tool to meet this challenge. This dissertation contributes to the development of such techniques. In this dissertation, security protocols are modelled in the process calculus LYSA......The trend in Information Technology is that distributed systems and networks are becoming increasingly important, as most of the services and opportunities that characterise the modern society are based on these technologies. Communication among agents over networks has therefore acquired a great...... deal of research interest. In order to provide effective and reliable means of communication, more and more communication protocols are invented, and for most of them, security is a significant goal. It has long been a challenge to determine conclusively whether a given protocol is secure or not...

  10. Multivariate performance reliability prediction in real-time

    International Nuclear Information System (INIS)

    Lu, S.; Lu, H.; Kolarik, W.J.

    2001-01-01

    This paper presents a technique for predicting system performance reliability in real-time considering multiple failure modes. The technique includes on-line multivariate monitoring and forecasting of selected performance measures and conditional performance reliability estimates. The performance measures across time are treated as a multivariate time series. A state-space approach is used to model the multivariate time series. Recursive forecasting is performed by adopting Kalman filtering. The predicted mean vectors and covariance matrix of performance measures are used for the assessment of system survival/reliability with respect to the conditional performance reliability. The technique and modeling protocol discussed in this paper provide a means to forecast and evaluate the performance of an individual system in a dynamic environment in real-time. The paper also presents an example to demonstrate the technique

  11. The reliability and validity of fatigue measures during multiple-sprint work: an issue revisited.

    Science.gov (United States)

    Glaister, Mark; Howatson, Glyn; Pattison, John R; McInnes, Gill

    2008-09-01

    The ability to repeatedly produce a high-power output or sprint speed is a key fitness component of most field and court sports. The aim of this study was to evaluate the validity and reliability of eight different approaches to quantify this parameter in tests of multiple-sprint performance. Ten physically active men completed two trials of each of two multiple-sprint running protocols with contrasting recovery periods. Protocol 1 consisted of 12 x 30-m sprints repeated every 35 seconds; protocol 2 consisted of 12 x 30-m sprints repeated every 65 seconds. All testing was performed in an indoor sports facility, and sprint times were recorded using twin-beam photocells. All but one of the formulae showed good construct validity, as evidenced by similar within-protocol fatigue scores. However, the assumptions on which many of the formulae were based, combined with poor or inconsistent test-retest reliability (coefficient of variation range: 0.8-145.7%; intraclass correlation coefficient range: 0.09-0.75), suggested many problems regarding logical validity. In line with previous research, the results support the percentage decrement calculation as the most valid and reliable method of quantifying fatigue in tests of multiple-sprint performance.

  12. Reliable Communication in Wireless Meshed Networks using Network Coding

    DEFF Research Database (Denmark)

    Pahlevani, Peyman; Paramanathan, Achuthan; Hundebøll, Martin

    2012-01-01

    The advantages of network coding have been extensively studied in the field of wireless networks. Integrating network coding with existing IEEE 802.11 MAC layer is a challenging problem. The IEEE 802.11 MAC does not provide any reliability mechanisms for overheard packets. This paper addresses...... this problem and suggests different mechanisms to support reliability as part of the MAC protocol. Analytical expressions to this problem are given to qualify the performance of the modified network coding. These expressions are confirmed by numerical result. While the suggested reliability mechanisms...

  13. Results of in vitro chemosensitivity assays

    International Nuclear Information System (INIS)

    Tanigawa, Nobuhiko; Morimoto, Hideki; Akita, Toshiaki; Inoue, Hiroshi; Tanaka, Takeo.

    1986-01-01

    The authors reviewed their experiences to date with chemosensitivity testing of 629 tumors by human tumor clonogenic assay (HTCA) and of 199 tumors by scintillation assay (SA). HTCA and SA were both performed using a double-layer-soft-agar system with continuous exposure of cells to one concentration of standard anticancer drugs. Overall, 60 % of specimens in HTCA and 58 % in SA produced significant growth in vitro. HTCA was 52 % (13/25) reliable for predicting in vivo sensitivity, and 95 % (36/38) reliable for in vivo resistance, whereas SA was 40 % (8/20) reliable for in vivo sensitivity and 88 % (21/24) for in vivo resistance. In vitro success rates were variable, depending on the tumor histology. In vitro growth of gastric cancer specimens was characteristically lower than that of colon cancer specimens (48 % and 60 % in HTCA, and 46 % and 68 % in SA, respectively). (p < 0.005). Optimal in vitro-in vivo drug concentrations and culture conditions are still being defined. Correlation studies of in vitro-in vivo responses of gastrointestinal cancers suggested that in vitro concentrations of 5-fluorouracil and mitomycin C used in this study were considerably higher than their optimal doses. Tumor cell heterogeneity poses significant problems in the clinical use of chemosensitivity assays. In this last study, we sought evidence of tumor heterogeneity by comparing chemosensitivity responses between : 1) different portions of a single tumor, 2) a primary and a metastatic biopsy taken from a patient on the same day, and 3) different metastases from a patient taken on the same day. The results demonstrated the presence of considerable heterogeneity of response to chemotherapy among different tumors from the same patient, and even within the same tumor. The reported discrepancies of in vitro and in vivo sensitivity may be due to such therapeutic heterogeneity among tumors. (J.P.N.)

  14. A comparative study of PD-L1 immunohistochemical assays with four reliable antibodies in thymic carcinoma.

    Science.gov (United States)

    Sakane, Tadashi; Murase, Takayuki; Okuda, Katsuhiro; Takino, Hisashi; Masaki, Ayako; Oda, Risa; Watanabe, Takuya; Kawano, Osamu; Haneda, Hiroshi; Moriyama, Satoru; Saito, Yushi; Yamada, Takeshi; Nakanishi, Ryoichi; Inagaki, Hiroshi

    2018-01-23

    Currently, four immunohistochemical assays are registered with the US Food and Drug Administration to detect the expression of PD-L1. We investigated the PD-L1 expression in thymic carcinomas using these four diagnostic assays. The cases of 53 patients were reviewed and their specimens were subjected to four PD-L1 assays with different antibodies (SP142, SP263, 22C3, and 28-8). The PD-L1 expression in tumor cells (TCs) and immune cells (ICs) was evaluated. In TCs, the four assays showed similar scores in each case. Histopathologically, high TC scores were observed in squamous cell carcinomas (SqCCs). Meanwhile, there were no significant relationships among the IC scores in the four assays. In SqCCs, the high expression of PD-L1 (defined as ≥50% TC score) in TCs tended to be associated with early stage cancer. The patients with high expression levels of PD-L1 tended to show longer overall survival in the 22C3 assays (p=0.0200). In thymic carcinomas, the staining pattern showed high concordance among the four assays when TCs - rather than ICs - were stained. High PD-L1 positivity in TCs, especially in SqCCs, indicated that PD-1/PD-L1 targeted therapy may be a promising therapeutic approach.

  15. Network protocol 'EPAP'; Network protokoru 'EPAP'

    Energy Technology Data Exchange (ETDEWEB)

    Kobori, T.; Fujita, F.; Iwamoto, S. [Fuji Electric Co. Ltd., Toyo (Japan)

    2000-10-10

    The Ethernet, a standard of information networks, has begun to be applied to the control local area network (LAN). To apply the Ethernet to the field level, Fuji Electric has newly developed the communication protocol 'Ethernet precision access protocol (EPAP)' in which a command/response method is structured on the user datagram protocol (UDP) to realize real time and high reliability. Further, we have implemented the EPAP on the bus interface module of the open PIO. This paper outlines the EPAP and its implementation. (author)

  16. Rapid screening method for male DNA by using the loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Kitamura, Masashi; Kubo, Seiji; Tanaka, Jin; Adachi, Tatsushi

    2017-08-12

    Screening for male-derived biological material from collected samples plays an important role in criminal investigations, especially those involving sexual assaults. We have developed a loop-mediated isothermal amplification (LAMP) assay targeting multi-repeat sequences of the Y chromosome for detecting male DNA. Successful amplification occurred with 0.5 ng of male DNA under isothermal conditions of 61 to 67 °C, but no amplification occurred with up to 10 ng of female DNA. Under the optimized conditions, the LAMP reaction initiated amplification within 10 min and amplified for 20 min. The LAMP reaction was sensitive at levels as low as 1-pg male DNA, and a quantitative LAMP assay could be developed because of the strong correlation between the reaction time and the amount of template DNA in the range of 10 pg to 10 ng. Furthermore, to apply the LAMP assay to on-site screening for male-derived samples, we evaluated a protocol using a simple DNA extraction method and a colorimetric intercalating dye that allows detection of the LAMP reaction by evaluating the change in color of the solution. Using this protocol, samples of male-derived blood and saliva stains were processed in approximately 30 min from DNA extraction to detection. Because our protocol does not require much hands-on time or special equipment, this LAMP assay promises to become a rapid and simple screening method for male-derived samples in forensic investigations.

  17. High-throughput screening of carbohydrate-degrading enzymes using novel insoluble chromogenic substrate assay kits

    DEFF Research Database (Denmark)

    Schückel, Julia; Kracun, Stjepan Kresimir; Willats, William George Tycho

    2016-01-01

    for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay...... CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used...... for screening the activities of enzymes, enzyme cocktails, and broths....

  18. Implementing voice over Internet protocol in mobile ad hoc network – analysing its features regarding efficiency, reliability and security

    Directory of Open Access Journals (Sweden)

    Naveed Ahmed Sheikh

    2014-05-01

    Full Text Available Providing secure and efficient real-time voice communication in mobile ad hoc network (MANET environment is a challenging problem. Voice over Internet protocol (VoIP has originally been developed over the past two decades for infrastructure-based networks. There are strict timing constraints for acceptable quality VoIP services, in addition to registration and discovery issues in VoIP end-points. In MANETs, ad hoc nature of networks and multi-hop wireless environment with significant packet loss and delays present formidable challenges to the implementation. Providing a secure real-time VoIP service on MANET is the main design objective of this paper. The authors have successfully developed a prototype system that establishes reliable and efficient VoIP communication and provides an extremely flexible method for voice communication in MANETs. The authors’ cooperative mesh-based MANET implementation can be used for rapidly deployable VoIP communication with survivable and efficient dynamic networking using open source software.

  19. Comparison of Four PD-L1 Immunohistochemical Assays in Lung Cancer.

    Science.gov (United States)

    Hendry, Shona; Byrne, David J; Wright, Gavin M; Young, Richard J; Sturrock, Sue; Cooper, Wendy A; Fox, Stephen B

    2018-03-01

    Four different programmed death ligand 1 immunohistochemical assays are approved or in development as companion or complementary diagnostics to different immunotherapeutic agents in lung carcinoma. We sought to determine whether these assays are technically equivalent and whether one antibody can be used on an alternate staining platform. Serial sections of tissue microarrays constructed from 368 cases of resected lung cancer were stained for 22C3 and 28-8 on the Dako Link 48 platform (Dako, Carpinteria, Ca) and for SP142 and SP263 on the Ventana Benchmark Ultra platform (Ventana Medical Systems, Tucson, AZ) strictly as per product insert. A protocol was developed to use the 22C3 antibody on the Ventana Benchmark Ultra platform. Differences in mean tumor cell and immune cell staining were observed between the four assays (p Link 48 platform and the alternate Ventana Benchmark Ultra platform (ICC = 0.921, κ = 0.897). Concordance between the four programmed death ligand 1 immunohistochemical assays when performed and scored as intended show that apart from 28-8 and 22C3, they cannot be used interchangeably in clinical practice. A protocol was successfully developed to use 22C3 on an alternate platform, which may help to overcome some barriers to implementation. Copyright © 2017 International Association for the Study of Lung Cancer. All rights reserved.

  20. Principles of validation of diagnostic assays for infectious diseases

    International Nuclear Information System (INIS)

    Jacobson, R.H.

    1998-01-01

    Assay validation requires a series of inter-related processes. Assay validation is an experimental process: reagents and protocols are optimized by experimentation to detect the analyte with accuracy and precision. Assay validation is a relative process: its diagnostic sensitivity and diagnostic specificity are calculated relative to test results obtained from reference animal populations of known infection/exposure status. Assay validation is a conditional process: classification of animals in the target population as infected or uninfected is conditional upon how well the reference animal population used to validate the assay represents the target population; accurate predictions of the infection status of animals from test results (PV+ and PV-) are conditional upon the estimated prevalence of disease/infection in the target population. Assay validation is an incremental process: confidence in the validity of an assay increases over time when use confirms that it is robust as demonstrated by accurate and precise results; the assay may also achieve increasing levels of validity as it is upgraded and extended by adding reference populations of known infection status. Assay validation is a continuous process: the assay remains valid only insofar as it continues to provide accurate and precise results as proven through statistical verification. Therefore, the work required for validation of diagnostic assays for infectious diseases does not end with a time-limited series of experiments based on a few reference samples rather, to assure valid test results from an assay requires constant vigilance and maintenance of the assay, along with reassessment of its performance characteristics for each unique population of animals to which it is applied. (author)

  1. Controlling variation in the comet assay

    Directory of Open Access Journals (Sweden)

    Andrew Richard Collins

    2014-10-01

    Full Text Available Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardising the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature and voltage gradient are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e. cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay, or photosensitiser plus light to oxidise guanine (positive control for Fpg- or OGG1-sensitive sites. Reference standards are especially valuable when performing a series of experiments over a long period - for example, analysing samples of white blood cells from a large human biomonitoring trial - to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation.

  2. Infrared microspectroscopy of live cells in microfluidic devices (MD-IRMS): toward a powerful label-free cell-based assay.

    Science.gov (United States)

    Vaccari, L; Birarda, G; Businaro, L; Pacor, S; Grenci, G

    2012-06-05

    Until nowadays most infrared microspectroscopy (IRMS) experiments on biological specimens (i.e., tissues or cells) have been routinely carried out on fixed or dried samples in order to circumvent water absorption problems. In this paper, we demonstrate the possibility to widen the range of in-vitro IRMS experiments to vibrational analysis of live cellular samples, thanks to the development of novel biocompatible IR-visible transparent microfluidic devices (MD). In order to highlight the biological relevance of IRMS in MD (MD-IRMS), we performed a systematic exploration of the biochemical alterations induced by different fixation protocols, ethanol 70% and formaldehyde solution 4%, as well as air-drying on U937 leukemic monocytes by comparing their IR vibrational features with the live U937 counterpart. Both fixation and air-drying procedures affected lipid composition and order as well as protein structure at a different extent while they both induced structural alterations in nucleic acids. Therefore, only IRMS of live cells can provide reliable information on both DNA and RNA structure and on their cellular dynamic. In summary, we show that MD-IRMS of live cells is feasible, reliable, and biologically relevant to be recognized as a label-free cell-based assay.

  3. L2-LBMT: A Layered Load Balance Routing Protocol for underwater multimedia data transmission

    Science.gov (United States)

    Lv, Ze; Tang, Ruichun; Tao, Ye; Sun, Xin; Xu, Xiaowei

    2017-12-01

    Providing highly efficient underwater transmission of mass multimedia data is challenging due to the particularities of the underwater environment. Although there are many schemes proposed to optimize the underwater acoustic network communication protocols, from physical layer, data link layer, network layer to transport layer, the existing routing protocols for underwater wireless sensor network (UWSN) still cannot well deal with the problems in transmitting multimedia data because of the difficulties involved in high energy consumption, low transmission reliability or high transmission delay. It prevents us from applying underwater multimedia data to real-time monitoring of marine environment in practical application, especially in emergency search, rescue operation and military field. Therefore, the inefficient transmission of marine multimedia data has become a serious problem that needs to be solved urgently. In this paper, A Layered Load Balance Routing Protocol (L2-LBMT) is proposed for underwater multimedia data transmission. In L2-LBMT, we use layered and load-balance Ad Hoc Network to transmit data, and adopt segmented data reliable transfer (SDRT) protocol to improve the data transport reliability. And a 3-node variant of tornado (3-VT) code is also combined with the Ad Hoc Network to transmit little emergency data more quickly. The simulation results show that the proposed protocol can balance energy consumption of each node, effectively prolong the network lifetime and reduce transmission delay of marine multimedia data.

  4. Field Programmable Gate Array Reliability Analysis Guidelines for Launch Vehicle Reliability Block Diagrams

    Science.gov (United States)

    Al Hassan, Mohammad; Britton, Paul; Hatfield, Glen Spencer; Novack, Steven D.

    2017-01-01

    Field Programmable Gate Arrays (FPGAs) integrated circuits (IC) are one of the key electronic components in today's sophisticated launch and space vehicle complex avionic systems, largely due to their superb reprogrammable and reconfigurable capabilities combined with relatively low non-recurring engineering costs (NRE) and short design cycle. Consequently, FPGAs are prevalent ICs in communication protocols and control signal commands. This paper will identify reliability concerns and high level guidelines to estimate FPGA total failure rates in a launch vehicle application. The paper will discuss hardware, hardware description language, and radiation induced failures. The hardware contribution of the approach accounts for physical failures of the IC. The hardware description language portion will discuss the high level FPGA programming languages and software/code reliability growth. The radiation portion will discuss FPGA susceptibility to space environment radiation.

  5. Radioreceptor assay: theory and applications to pharmacology

    International Nuclear Information System (INIS)

    Perret, G.; Simon, P.

    1984-01-01

    The aim of the first part of this work is to present the theory of the radioreceptor assay and to compare it to the other techniques of radioanalysis (radioimmunoassay, competitive protein binding assays). The technology of the radioreceptor assay is then presented and its components (preparation of the receptors, radioligand, incubation medium) are described. The analytical characteristics of the radioreceptor assay (specificity, sensitivity, reproductibility, accuracy) and the pharmacological significance of the results are discussed. The second part is devoted to the description of the radioreceptor assays of some pharmacological classes (neuroleptics, tricyclic antidepressants, benzodiazepines, β-blockers, anticholinergic drugs) and to their use in therapeutic drug monitoring. In conclusion, by their nature, radioreceptor assays are highly sensitive, reliable, precise, accurate and simple to perform. Their chief disadvantage relates to specificity, since any substance having an appreciable affinity to the receptor site will displace the specifically bound radioligand. Paradoxically in some cases, this lack of specificity may be advantageous in that it allows for the detection of not only the apparent compound but of active metabolites and endogenous receptor agonists as well and in that radioreceptors assays can be devised for a whole pharmacological class and not only for one drug as it is the case for classical physico-chemical techniques. For all these reasons future of radioreceptor assay in pharmacology appears promising [fr

  6. Design and performance testing of a DNA extraction assay for sensitive and reliable quantification of acetic acid bacteria directly in red wine using real time PCR

    Directory of Open Access Journals (Sweden)

    Cédric eLONGIN

    2016-06-01

    Full Text Available Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence there is a real need for a rapid, specific, sensitive and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR. Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP at 1% (v/v during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 mL to 10 mL. Thus the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage.

  7. Development and performance assessment of a luminex xMAP® direct hybridization assay for the detection and identification of indoor air fungal contamination.

    Science.gov (United States)

    Libert, Xavier; Packeu, Ann; Bureau, Fabrice; Roosens, Nancy H; De Keersmaecker, Sigrid C J

    2017-01-01

    Considered as a public health problem, indoor fungal contamination is generally monitored using classical protocols based on culturing. However, this culture dependency could influence the representativeness of the fungal population detected in an analyzed sample as this includes the dead and uncultivable fraction. Moreover, culture-based protocols are often time-consuming. In this context, molecular tools are a powerful alternative, especially those allowing multiplexing. In this study a Luminex xMAP® assay was developed for the simultaneous detection of 10 fungal species which are most frequently in indoor air and that may cause health problems. This xMAP® assay was found to be sensitive, i.e. its limit of detection is ranging between 0.05 and 0.01 ng of gDNA. The assay was subsequently tested with environmental air samples which were also analyzed with a classical protocol. All the species identified with the classical method were also detected with the xMAP® assay, however in a shorter time frame. These results demonstrate that the Luminex xMAP® fungal assay developed in this study could contribute to the improvement of public health and specifically to the indoor fungal contamination treatment.

  8. Development and performance assessment of a luminex xMAP® direct hybridization assay for the detection and identification of indoor air fungal contamination.

    Directory of Open Access Journals (Sweden)

    Xavier Libert

    Full Text Available Considered as a public health problem, indoor fungal contamination is generally monitored using classical protocols based on culturing. However, this culture dependency could influence the representativeness of the fungal population detected in an analyzed sample as this includes the dead and uncultivable fraction. Moreover, culture-based protocols are often time-consuming. In this context, molecular tools are a powerful alternative, especially those allowing multiplexing. In this study a Luminex xMAP® assay was developed for the simultaneous detection of 10 fungal species which are most frequently in indoor air and that may cause health problems. This xMAP® assay was found to be sensitive, i.e. its limit of detection is ranging between 0.05 and 0.01 ng of gDNA. The assay was subsequently tested with environmental air samples which were also analyzed with a classical protocol. All the species identified with the classical method were also detected with the xMAP® assay, however in a shorter time frame. These results demonstrate that the Luminex xMAP® fungal assay developed in this study could contribute to the improvement of public health and specifically to the indoor fungal contamination treatment.

  9. The Design of Finite State Machine for Asynchronous Replication Protocol

    Science.gov (United States)

    Wang, Yanlong; Li, Zhanhuai; Lin, Wei; Hei, Minglei; Hao, Jianhua

    Data replication is a key way to design a disaster tolerance system and to achieve reliability and availability. It is difficult for a replication protocol to deal with the diverse and complex environment. This means that data is less well replicated than it ought to be. To reduce data loss and to optimize replication protocols, we (1) present a finite state machine, (2) run it to manage an asynchronous replication protocol and (3) report a simple evaluation of the asynchronous replication protocol based on our state machine. It's proved that our state machine is applicable to guarantee the asynchronous replication protocol running in the proper state to the largest extent in the event of various possible events. It also can helpful to build up replication-based disaster tolerance systems to ensure the business continuity.

  10. Validation of a standard forensic anthropology examination protocol by measurement of applicability and reliability on exhumed and archive samples of known biological attribution.

    Science.gov (United States)

    Francisco, Raffaela Arrabaça; Evison, Martin Paul; Costa Junior, Moacyr Lobo da; Silveira, Teresa Cristina Pantozzi; Secchieri, José Marcelo; Guimarães, Marco Aurelio

    2017-10-01

    Forensic anthropology makes an important contribution to human identification and assessment of the causes and mechanisms of death and body disposal in criminal and civil investigations, including those related to atrocity, disaster and trafficking victim identification. The methods used are comparative, relying on assignment of questioned material to categories observed in standard reference material of known attribution. Reference collections typically originate in Europe and North America, and are not necessarily representative of contemporary global populations. Methods based on them must be validated when applied to novel populations. This study describes the validation of a standardized forensic anthropology examination protocol by application to two contemporary Brazilian skeletal samples of known attribution. One sample (n=90) was collected from exhumations following 7-35 years of burial and the second (n=30) was collected following successful investigations following routine case work. The study presents measurement of (1) the applicability of each of the methods: used and (2) the reliability with which the biographic parameters were assigned in each case. The results are discussed with reference to published assessments of methodological reliability regarding sex, age and-in particular-ancestry estimation. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Improvement in QA protocol for TLD based personnel monitoring laboratory in last five year

    International Nuclear Information System (INIS)

    Rakesh, R.B.

    2018-01-01

    The Quality Assurance (QA) in Personnel monitoring (PM) is a tool to assess the performance of PM laboratories and reliability of dose estimation with respect to standards laid down by international agencies such as IAEA (ISO trumpet curve), IEC, ANSI etc. Reliable personal dose estimation is a basic requirement for radiation protection planning as well as decision making continuous improvement in radiation protection is inherent in radiation protection practices which is highly dependent on accuracy and reliability of the monitoring data. Experience based evolution of Quality control (QC) measures as well as Quality assurance (QA) protocol are two important aspects towards continuous improvement in accuracy and reliability of personnel monitoring results. The paper describes improvement in QC measures and QA protocols initiated during the last five years which led to improvement in the quality of PM services

  12. Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

    Science.gov (United States)

    Zhu, Qinchang; Yu, Zhiqiang; Kabashima, Tsutomu; Yin, Sheng; Dragusha, Shpend; El-Mahdy, Ahmed F. M.; Ejupi, Valon; Shibata, Takayuki; Kai, Masaaki

    2015-01-01

    Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates for the enzymatic reaction. In this study, susceptibility of HIV mutations to drugs was evaluated by selective formation of three FL products after the enzymatic HIV-PR reaction. This proof-of-concept study indicates that the present HPLC-FL method could be an alternative to current phenotypic assays for the evaluation of HIV drug resistance. PMID:25988960

  13. Human neuron-astrocyte 3D co-culture-based assay for evaluation of neuroprotective compounds.

    Science.gov (United States)

    Terrasso, Ana Paula; Silva, Ana Carina; Filipe, Augusto; Pedroso, Pedro; Ferreira, Ana Lúcia; Alves, Paula Marques; Brito, Catarina

    Central nervous system drug development has registered high attrition rates, mainly due to the lack of efficacy of drug candidates, highlighting the low reliability of the models used in early-stage drug development and the need for new in vitro human cell-based models and assays to accurately identify and validate drug candidates. 3D human cell models can include different tissue cell types and represent the spatiotemporal context of the original tissue (co-cultures), allowing the establishment of biologically-relevant cell-cell and cell-extracellular matrix interactions. Nevertheless, exploitation of these 3D models for neuroprotection assessment has been limited due to the lack of data to validate such 3D co-culture approaches. In this work we combined a 3D human neuron-astrocyte co-culture with a cell viability endpoint for the implementation of a novel in vitro neuroprotection assay, over an oxidative insult. Neuroprotection assay robustness and specificity, and the applicability of Presto Blue, MTT and CytoTox-Glo viability assays to the 3D co-culture were evaluated. Presto Blue was the adequate endpoint as it is non-destructive and is a simpler and reliable assay. Semi-automation of the cell viability endpoint was performed, indicating that the assay setup is amenable to be transferred to automated screening platforms. Finally, the neuroprotection assay setup was applied to a series of 36 test compounds and several candidates with higher neuroprotective effect than the positive control, Idebenone, were identified. The robustness and simplicity of the implemented neuroprotection assay with the cell viability endpoint enables the use of more complex and reliable 3D in vitro cell models to identify and validate drug candidates. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. An eDNA Assay to Monitor a Globally Invasive Fish Species from Flowing Freshwater.

    Science.gov (United States)

    Adrian-Kalchhauser, Irene; Burkhardt-Holm, Patricia

    2016-01-01

    Ponto-Caspian gobies are a flock of five invasive fish species that have colonized freshwaters and brackish waters in Europe and North America. One of them, the round goby Neogobius melanostomus, figures among the 100 worst invaders in Europe. Current methods to detect the presence of Ponto-Caspian gobies involve catching or sighting the fish. These approaches are labor intense and not very sensitive. Consequently, populations are usually detected only when they have reached high densities and when management or containment efforts are futile. To improve monitoring, we developed an assay based on the detection of DNA traces (environmental DNA, or eDNA) of Ponto-Caspian gobies in river water. The assay specifically detects invasive goby DNA and does not react to any native fish species. We apply the assay to environmental samples and demonstrate that parameters such as sampling depth, sampling location, extraction protocol, PCR protocol and PCR inhibition greatly impact detection. We further successfully outline the invasion front of Ponto-Caspian gobies in a large river, the High Rhine in Switzerland, and thus demonstrate the applicability of the assay to lotic environments. The eDNA assay requires less time, equipment, manpower, skills, and financial resources than the conventional monitoring methods such as electrofishing, angling or diving. Samples can be taken by untrained individuals, and the assay can be performed by any molecular biologist on a conventional PCR machine. Therefore, this assay enables environment managers to map invaded areas independently of fishermen's' reports and fish community monitorings.

  15. Individual response to ionising radiation: What predictive assay(s) to choose?

    International Nuclear Information System (INIS)

    Granzotto, A.; Viau, M.; Devic, C.; Maalouf, M.; Thomas, Ch.; Vogin, G.; Foray, N.; Granzotto, A.; Vogin, G.; Balosso, J.; Joubert, A.; Maalouf, M.; Vogin, G.; Colin, C.; Malek, K.; Balosso, J.; Colin, C.

    2011-01-01

    Individual response to ionizing radiation is an important information required to apply an efficient radiotherapy treatment against tumour and to avoid any adverse effects in normal tissues. In 1981, Fertil and Malaise have demonstrated that the post-irradiation local tumor control determined in vivo is correlated with clonogenic cell survival assessed in vitro. Furthermore, these authors have reminded the relevance of the concept of intrinsic radiosensitivity that is specific to each individual organ (Fertil and Malaise, 1981) [1]. To date, since clonogenicity assays are too time-consuming and do not provide any other molecular information, a plethora of research groups have attempted to determine the molecular bases of intrinsic radiosensitivity in order to propose reliable and faster predictive assays. To this aim, several approaches have been developed. Notably, the recent revolution in genomic and proteomics technologies is providing a considerable number of data but their link with radiosensitivity still remains to be elucidated. On another hand, the systematic screening of some candidate genes potentially involved in the radiation response is highlighting the complexity of the molecular and cellular mechanisms of DNA damage sensing and signalling and shows that an abnormal radiation response is not necessarily due to the impairment of one single protein. Finally, more modest approaches consisting in focusing some specific functions of DNA repair seem to provide more reliable clues to predict over-acute reactions caused by radiotherapy. In this review, we endeavored to analyse the contributions of these major approaches to predict human radiosensitivity. (authors)

  16. Isokinetic Strength and Endurance Tests used Pre- and Post-Spaceflight: Test-Retest Reliability

    Science.gov (United States)

    Laughlin, Mitzi S.; Lee, Stuart M. C.; Loehr, James A.; Amonette, William E.

    2009-01-01

    To assess changes in muscular strength and endurance after microgravity exposure, NASA measures isokinetic strength and endurance across multiple sessions before and after long-duration space flight. Accurate interpretation of pre- and post-flight measures depends upon the reliability of each measure. The purpose of this study was to evaluate the test-retest reliability of the NASA International Space Station (ISS) isokinetic protocol. Twenty-four healthy subjects (12 M/12 F, 32.0 +/- 5.6 years) volunteered to participate. Isokinetic knee, ankle, and trunk flexion and extension strength as well as endurance of the knee flexors and extensors were measured using a Cybex NORM isokinetic dynamometer. The first weekly session was considered a familiarization session. Data were collected and analyzed for weeks 2-4. Repeated measures analysis of variance (alpha=0.05) was used to identify weekly differences in isokinetic measures. Test-retest reliability was evaluated by intraclass correlation coefficients (ICC) (3,1). No significant differences were found between weeks in any of the strength measures and the reliability of the strength measures were all considered excellent (ICC greater than 0.9), except for concentric ankle dorsi-flexion (ICC=0.67). Although a significant difference was noted in weekly endurance measures of knee extension (p less than 0.01), the reliability of endurance measure by week were considered excellent for knee flexion (ICC=0.97) and knee extension (ICC=0.96). Except for concentric ankle dorsi-flexion, the isokinetic strength and endurance measures are highly reliable when following the NASA ISS protocol. This protocol should allow accurate interpretation isokinetic data even with a small number of crew members.

  17. A duplex PCR assay for the detection of Ralstonia solanacearum phylotype II strains in Musa spp.

    Directory of Open Access Journals (Sweden)

    Gilles Cellier

    Full Text Available Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato, no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae.

  18. MS transport assays for γ-aminobutyric acid transporters--an efficient alternative for radiometric assays.

    Science.gov (United States)

    Schmitt, Sebastian; Höfner, Georg; Wanner, Klaus T

    2014-08-05

    Transport assays for neurotransmitters based on radiolabeled substrates are widely spread and often indispensable in basic research and the drug development process, although the use of radioisotopes is inherently coupled to issues concerning radioactive waste and safety precautions. To overcome these disadvantages, we developed mass spectrometry (MS)-based transport assays for γ-aminobutyric acid (GABA), which is the major inhibitory neurotransmitter in the central nervous system (CNS). These "MS Transport Assays" provide all capabilities of [(3)H]GABA transport assays and therefore represent the first substitute for the latter. The performance of our approach is demonstrated for GAT1, the most important GABA transporter (GAT) subtype. As GABA is endogenously present in COS-7 cells employed as hGAT1 expression system, ((2)H6)GABA was used as a substrate to differentiate transported from endogenous GABA. To record transported ((2)H6)GABA, a highly sensitive, short, robust, and reliable HILIC-ESI-MS/MS quantification method using ((2)H2)GABA as an internal standard was developed and validated according to the Center for Drug Evaluation and Research (CDER) guidelines. Based on this LC-MS quantification, a setup to characterize hGAT1 mediated ((2)H6)GABA transport in a 96-well format was established, that enables automated processing and avoids any sample preparation. The K(m) value for ((2)H6)GABA determined for hGAT1 is in excellent agreement with results obtained from [(3)H]GABA uptake assays. In addition, the established assay format enables efficient determination of the inhibitory potency of GAT1 inhibitors, is capable of identifying those inhibitors transported as substrates, and furthermore allows characterization of efflux. The approach described here combines the strengths of LC-MS/MS with the high efficiency of transport assays based on radiolabeled substrates and is applicable to all GABA transporter subtypes.

  19. Data transmission protocol for Pi-of-the-Sky cameras

    Science.gov (United States)

    Uzycki, J.; Kasprowicz, G.; Mankiewicz, M.; Nawrocki, K.; Sitek, P.; Sokolowski, M.; Sulej, R.; Tlaczala, W.

    2006-10-01

    The large amount of data collected by the automatic astronomical cameras has to be transferred to the fast computers in a reliable way. The method chosen should ensure data streaming in both directions but in nonsymmetrical way. The Ethernet interface is very good choice because of its popularity and proven performance. However it requires TCP/IP stack implementation in devices like cameras for full compliance with existing network and operating systems. This paper describes NUDP protocol, which was made as supplement to standard UDP protocol and can be used as a simple-network protocol. The NUDP does not need TCP protocol implementation and makes it possible to run the Ethernet network with simple devices based on microcontroller and/or FPGA chips. The data transmission idea was created especially for the "Pi of the Sky" project.

  20. Rapid identification of tomato Sw-5 resistance-breaking isolates of Tomato spotted wilt virus using high resolution melting and TaqMan SNP Genotyping assays as allelic discrimination techniques.

    Directory of Open Access Journals (Sweden)

    Valentina di Rienzo

    Full Text Available In tomato, resistance to Tomato spotted wilt virus (TSWV is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan™ SNP Genotyping and high-resolution melting (HRM assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50-70 TSWV RNA copies and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke and summer (tomato crops, in the same cultivated areas of Southern Italy.

  1. A novel approach for reliable detection of cathepsin S activities in mouse antigen presenting cells.

    Science.gov (United States)

    Steimle, Alex; Kalbacher, Hubert; Maurer, Andreas; Beifuss, Brigitte; Bender, Annika; Schäfer, Andrea; Müller, Ricarda; Autenrieth, Ingo B; Frick, Julia-Stefanie

    2016-05-01

    Cathepsin S (CTSS) is a eukaryotic protease mostly expressed in professional antigen presenting cells (APCs). Since CTSS activity regulation plays a role in the pathogenesis of various autoimmune diseases like multiple sclerosis, atherosclerosis, Sjögren's syndrome and psoriasis as well as in cancer progression, there is an ongoing interest in the reliable detection of cathepsin S activity. Various applications have been invented for specific detection of this enzyme. However, most of them have only been shown to be suitable for human samples, do not deliver quantitative results or the experimental procedure requires technical equipment that is not commonly available in a standard laboratory. We have tested a fluorogen substrate, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2, that has been described to specifically detect CTSS activities in human APCs for its potential use for mouse samples. We have modified the protocol and thereby offer a cheap, easy, reproducible and quick activity assay to detect CTSS activities in mouse APCs. Since most of basic research on CTSS is performed in mice, this method closes a gap and offers a possibility for reliable and quantitative CTSS activity detection that can be performed in almost every laboratory. Copyright © 2016. Published by Elsevier B.V.

  2. BioAssay templates for the semantic web

    Directory of Open Access Journals (Sweden)

    Alex M. Clark

    2016-05-01

    Full Text Available Annotation of bioassay protocols using semantic web vocabulary is a way to make experiment descriptions machine-readable. Protocols are communicated using concise scientific English, which precludes most kinds of analysis by software algorithms. Given the availability of a sufficiently expressive ontology, some or all of the pertinent information can be captured by asserting a series of facts, expressed as semantic web triples (subject, predicate, object. With appropriate annotation, assays can be searched, clustered, tagged and evaluated in a multitude of ways, analogous to other segments of drug discovery informatics. The BioAssay Ontology (BAO has been previously designed for this express purpose, and provides a layered hierarchy of meaningful terms which can be linked to. Currently the biggest challenge is the issue of content creation: scientists cannot be expected to use the BAO effectively without having access to software tools that make it straightforward to use the vocabulary in a canonical way. We have sought to remove this barrier by: (1 defining a BioAssay Template (BAT data model; (2 creating a software tool for experts to create or modify templates to suit their needs; and (3 designing a common assay template (CAT to leverage the most value from the BAO terms. The CAT was carefully assembled by biologists in order to find a balance between the maximum amount of information captured vs. low degrees of freedom in order to keep the user experience as simple as possible. The data format that we use for describing templates and corresponding annotations is the native format of the semantic web (RDF triples, and we demonstrate some of the ways that generated content can be meaningfully queried using the SPARQL language. We have made all of these materials available as open source (http://github.com/cdd/bioassay-template, in order to encourage community input and use within diverse projects, including but not limited to our own

  3. Comparative study of ß-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.)

    DEFF Research Database (Denmark)

    Jiménez, Natalia Ivonne Vera; Pietretti, D.; Wiegertjes, G. F.

    2013-01-01

    kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS......-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head...... on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head...

  4. Receiver-Based Ad Hoc On Demand Multipath Routing Protocol for Mobile Ad Hoc Networks.

    Science.gov (United States)

    Al-Nahari, Abdulaziz; Mohamad, Mohd Murtadha

    2016-01-01

    Decreasing the route rediscovery time process in reactive routing protocols is challenging in mobile ad hoc networks. Links between nodes are continuously established and broken because of the characteristics of the network. Finding multiple routes to increase the reliability is also important but requires a fast update, especially in high traffic load and high mobility where paths can be broken as well. The sender node keeps re-establishing path discovery to find new paths, which makes for long time delay. In this paper we propose an improved multipath routing protocol, called Receiver-based ad hoc on demand multipath routing protocol (RB-AOMDV), which takes advantage of the reliability of the state of the art ad hoc on demand multipath distance vector (AOMDV) protocol with less re-established discovery time. The receiver node assumes the role of discovering paths when finding data packets that have not been received after a period of time. Simulation results show the delay and delivery ratio performances are improved compared with AOMDV.

  5. Development of simple immunoradiometric assays using avidin coupled to polystyrene beads as a common solid phase

    International Nuclear Information System (INIS)

    Jyotsna, N.; Singh, Y.; Chouthkanthiwar, V.; Paradkar, S.; Sivaprasad, N.

    1998-01-01

    In this paper, we describe the preparation and application of avidin coupled polystyrene beads as a common solid phase for use in immunoradiometric assays (IRMAs). The assay system is based on two matched commercial monoclonal antibodies, of which, the capture antibody is biotinylated using biotinamidocaproate N-hydroxysuccinimide ester and the detection antibody is radiolabeled with 125 I by conventional Chloramine-T method. Avidin was immobilized on the polystyrene beads through a primary coat of bovine serum albumin using glutaraldhyde activation method. Various factors, such as concentration of reagents, incubation time, etc. were optimised to obtain a simple assay protocol consisting of only two pipetting steps, namely, that of a mixture of the two labelled antibodies (radiolabelled and biotinylated) and of the standard or sample. The advantage of the Avidin-Biotin system is the improved sensitivity, economy of antibody and the possibility to use a common solid phase in assays for different analytes. Using the polystyrene beads along with the novel decanting device, it has been possible to achieve the convenience of the 'coated-tube' technology without the expensive automation necessary for large scale preparation of antibody coated tubes. This protocol has been successfully applied to Prolactin, LH and FSH assays. The sensitivity of the Prolactin assay is 8μIU/mL (0.3 ng/mL), that of the FSH assay is 1mIU/mL and that of the LH assay is 0.9 mIU/mL. The intra-assay and inter-assay variations were <10%. Shelf life of the avidin coupled beads was found to be about 8 months and that of the biotin labelled antibodies up to 18 months. (author)

  6. Comparative study of β-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.).

    Science.gov (United States)

    Vera-Jimenez, N I; Pietretti, D; Wiegertjes, G F; Nielsen, M E

    2013-05-01

    The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Privacy-Preserving Meter Report Protocol of Isolated Smart Grid Devices

    Directory of Open Access Journals (Sweden)

    Zhiwei Wang

    2017-01-01

    Full Text Available Smart grid aims to improve the reliability, efficiency, and security of the traditional grid, which allows two-way transmission and efficiency-driven response. However, a main concern of this new technique is that the fine-grained metering data may leak the personal privacy information of the customers. Thus, the data aggregation mechanism for privacy protection is required for the meter report protocol in smart grid. In this paper, we propose an efficient privacy-preserving meter report protocol for the isolated smart grid devices. Our protocol consists of an encryption scheme with additively homomorphic property and a linearly homomorphic signature scheme, where the linearly homomorphic signature scheme is suitable for privacy-preserving data aggregation. We also provide security analysis of our protocol in the context of some typical attacks in smart grid. The implementation of our protocol on the Intel Edison platform shows that our protocol is efficient enough for the physical constrained devices, like smart meters.

  8. IoT real time data acquisition using MQTT protocol

    Science.gov (United States)

    Atmoko, R. A.; Riantini, R.; Hasin, M. K.

    2017-05-01

    The Internet of Things (IoT) provides ease to monitor and to gain sensor data through the Internet [1]. The need of high quality data is increasing to the extent that data monitoring and acquisition system in real time is required, such as smart city or telediagnostic in medical areas [2]. Therefore, an appropriate communication protocol is required to resolve these problems. Lately, researchers have developed a lot of communication protocols for IoT, of which each has advantages and disadvantages. This study proposes the utilization of MQTT as a communication protocol, which is one of data communication protocols for IoT. This study used temperature and humidity sensors because the physical parameters are often needed as parameters of environment condition [3]. Data acquisition was done in real-time and stored in MySQL database. This study is also completed by interface web-based and mobile for online monitoring. This result of this study is the enhancement of data quality and reliability using MQTT protocol.

  9. A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

    Science.gov (United States)

    Su, Mengna; Venkatachalam, Mahesh; Liu, Changqi; Zhang, Ying; Roux, Kenneth H; Sathe, Shridhar K

    2013-11-13

    A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (assay variability assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

  10. Dissolvable fluidic time delays for programming multi-step assays in instrument-free paper diagnostics.

    Science.gov (United States)

    Lutz, Barry; Liang, Tinny; Fu, Elain; Ramachandran, Sujatha; Kauffman, Peter; Yager, Paul

    2013-07-21

    Lateral flow tests (LFTs) are an ingenious format for rapid and easy-to-use diagnostics, but they are fundamentally limited to assay chemistries that can be reduced to a single chemical step. In contrast, most laboratory diagnostic assays rely on multiple timed steps carried out by a human or a machine. Here, we use dissolvable sugar applied to paper to create programmable flow delays and present a paper network topology that uses these time delays to program automated multi-step fluidic protocols. Solutions of sucrose at different concentrations (10-70% of saturation) were added to paper strips and dried to create fluidic time delays spanning minutes to nearly an hour. A simple folding card format employing sugar delays was shown to automate a four-step fluidic process initiated by a single user activation step (folding the card); this device was used to perform a signal-amplified sandwich immunoassay for a diagnostic biomarker for malaria. The cards are capable of automating multi-step assay protocols normally used in laboratories, but in a rapid, low-cost, and easy-to-use format.

  11. Link reliability based hybrid routing for tactical mobile ad hoc network

    Institute of Scientific and Technical Information of China (English)

    Xie Xiaochuan; Wei Gang; Wu Keping; Wang Gang; Jia Shilou

    2008-01-01

    Tactical mobile ad hoc network (MANET) is a collection of mobile nodes forming a temporary network,without the aid of pre-established network infrastructure. The routing protocol has a crucial impact on the networkperformance in battlefields. Link reliability based hybrid routing (LRHR) is proposed, which is a novel hybrid routing protocol, for tactical MANET. Contrary to the traditional single path routing strategy, multiple paths are established between a pair of source-destination nodes. In the hybrid routing strategy, the rate of topological change provides a natural mechanism for switching dynamically between table-driven and on-demand routing. The simulation results indicate that the performances of the protocol in packet delivery ratio, routing overhead, and average end-to-end delay are better than the conventional routing protocol.

  12. Study and development of a remote biometric authentication protocol

    OpenAIRE

    Bistarelli, Stefano; Claudio, Viti

    2003-01-01

    This paper reports the phases of study and implementation of a remote biometric authentication protocol developed during my internship at the I.i.t. of the C.n.r. in Pisa. Starting from the study of authentication history we had a look from the first system used since the 60ies to the latest technology; this helped us understand how we could realize a demonstration working protocol that could achieve a web remote authentication granting good reliability: to do this we choosed to modify the SS...

  13. Genotyping assay for differentiation of wild-type and vaccine viruses in subjects immunized with live attenuated influenza vaccine.

    Directory of Open Access Journals (Sweden)

    Victoria Matyushenko

    Full Text Available Live attenuated influenza vaccines (LAIVs are considered as safe and effective tool to control influenza in different age groups, especially in young children. An important part of the LAIV safety evaluation is the detection of vaccine virus replication in the nasopharynx of the vaccinees, with special attention to a potential virus transmission to the unvaccinated close contacts. Conducting LAIV clinical trials in some geographical regions with year-round circulation of influenza viruses warrants the development of robust and reliable tools for differentiating vaccine viruses from wild-type influenza viruses in nasal pharyngeal wash (NPW specimens of vaccinated subjects. Here we report the development of genotyping assay for the detection of wild-type and vaccine-type influenza virus genes in NPW specimens of young children immunized with Russian-backbone seasonal trivalent LAIV using Sanger sequencing from newly designed universal primers. The new primer set allowed amplification and sequencing of short fragments of viral genes in NPW specimens and appeared to be more sensitive than conventional real-time RT-PCR protocols routinely used for the detection and typing/subtyping of influenza virus in humans. Furthermore, the new assay is capable of defining the origin of wild-type influenza virus through BLAST search with the generated sequences of viral genes fragments.

  14. Radioligand assay for biotin in liver tissues

    International Nuclear Information System (INIS)

    Rettenmaier, R.

    1979-01-01

    A radioligand assay for biotin in liver tissue is described. 3 H-biotin is used as tracer and avidin as binder. The biotin-loaded avidin is separated from free biotin on dextran-coated charcoal, which leaves the avidin-biotin complex in the supernatant liquid. Thus, the avidin-biotin complex can easily be utilized for determination of the radioactivity. Calibration with known additions of biotin in the range 0.25-8.0 ng per assay sample yields a linear logit-log plot. The biotin is extracted from liver tissues by enzymatic proteolysis with papain. This treatment is optimized to liberate the bound forms of the vitamin. Microbiological parallel assays with Lactobacillus plantarum were in good agreement with the radioligand assay giving a regression coefficient of 0.974(n=44). The coefficient of variation was found to be 4.2% in the range 500-1200 ng of biotin per g of liver tissue (n=46). The method is simple and reliable and allows the simultaneous analysis of a considerable number of samples. (Auth.)

  15. Comet Assay on Daphnia magna in eco-genotoxicity testing.

    Science.gov (United States)

    Pellegri, Valerio; Gorbi, Gessica; Buschini, Annamaria

    2014-10-01

    Detection of potentially hazardous compounds in water bodies is a priority in environmental risk assessment. For the evaluation and monitoring of water quality, a series of methodologies may be applied. Among them, the worldwide used toxicity tests with organisms of the genus Daphnia is one of the most powerful. In recent years, some attempts were made to utilize Daphnia magna in genotoxicity testing as many of the new environmental contaminants are described as DNA-damaging agents in aquatic organisms. The aim of this research was to develop a highly standardized protocol of the Comet Assay adapted for D. magna, especially regarding the isolation of cells derived from the same tissue (haemolymph) from newborn organisms exposed in vivo. Several methods for haemolymph extraction and different Comet Assay parameters were compared. Electrophoretic conditions were adapted in order to obtain minimum DNA migration in cells derived from untreated organisms and, at the same time, maximum sensitivity in specimens treated with known genotoxicants (CdCl2 and H2O2). Additional tests were performed to investigate if life-history traits of the cladoceran (such as the age of adult organisms that provide newborns, the clutch size of origin, the number of generations reared in standard conditions) and the water composition as well, might influence the response of the assay. This study confirms the potential application of the Comet Assay in D. magna for assessing genotoxic loads in aqueous solution. The newly developed protocol could integrate the acute toxicity bioassay, thus expanding the possibility of using this model species in freshwater monitoring (waters, sediment and soil elutriates) and is in line with the spirit of the EU Water Framework Directive in reducing the number of bioassays that involve medium-sized species. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. QoS-aware self-adaptation of communication protocols in a pervasive service middleware

    DEFF Research Database (Denmark)

    Zhang, Weishan; Hansen, Klaus Marius; Fernandes, João

    2010-01-01

    Pervasive computing is characterized by heterogeneous devices that usually have scarce resources requiring optimized usage. These devices may use different communication protocols which can be switched at runtime. As different communication protocols have different quality of service (Qo......S) properties, this motivates optimized self-adaption of protocols for devices, e.g., considering power consumption and other QoS requirements, e.g. round trip time (RTT) for service invocations, throughput, and reliability. In this paper, we present an extensible approach for self-adaptation of communication...... protocols for pervasive web services, where protocols are designed as reusable connectors and our middleware infrastructure can hide the complexity of using different communication protocols to upper layers. We also propose to use Genetic Algorithms (GAs) to find optimized configurations at runtime...

  17. New simple spectrophotometric assay of total carotenes in margarines

    NARCIS (Netherlands)

    Luterotti, S.; Bicanic, D.D.; Pozgaj, R.

    2006-01-01

    Direct and reliable spectrophotometric method for assaying total carotenes (TC) in margarines with the minimum of sample manipulation is proposed. For the first time saponification step used in determination of carotenes in margarines was omitted leading to a substantial cost saving and reduction of

  18. The reliability and validity of fatigue measures during short-duration maximal-intensity intermittent cycling.

    Science.gov (United States)

    Glaister, Mark; Stone, Michael H; Stewart, Andrew M; Hughes, Michael; Moir, Gavin L

    2004-08-01

    The purpose of the present study was to assess the reliability and validity of fatigue measures, as derived from 4 separate formulae, during tests of repeat sprint ability. On separate days over a 3-week period, 2 groups of 7 recreationally active men completed 6 trials of 1 of 2 maximal (20 x 5 seconds) intermittent cycling tests with contrasting recovery periods (10 or 30 seconds). All trials were conducted on a friction-braked cycle ergometer, and fatigue scores were derived from measures of mean power output for each sprint. Apart from formula 1, which calculated fatigue from the percentage difference in mean power output between the first and last sprint, all remaining formulae produced fatigue scores that showed a reasonably good level of test-retest reliability in both intermittent test protocols (intraclass correlation range: 0.78-0.86; 95% likely range of true values: 0.54-0.97). Although between-protocol differences in the magnitude of the fatigue scores suggested good construct validity, within-protocol differences highlighted limitations with each formula. Overall, the results support the use of the percentage decrement score as the most valid and reliable measure of fatigue during brief maximal intermittent work.

  19. Implementation of two-party protocols in the noisy-storage model

    International Nuclear Information System (INIS)

    Wehner, Stephanie; Curty, Marcos; Schaffner, Christian; Lo, Hoi-Kwong

    2010-01-01

    The noisy-storage model allows the implementation of secure two-party protocols under the sole assumption that no large-scale reliable quantum storage is available to the cheating party. No quantum storage is thereby required for the honest parties. Examples of such protocols include bit commitment, oblivious transfer, and secure identification. Here, we provide a guideline for the practical implementation of such protocols. In particular, we analyze security in a practical setting where the honest parties themselves are unable to perform perfect operations and need to deal with practical problems such as errors during transmission and detector inefficiencies. We provide explicit security parameters for two different experimental setups using weak coherent, and parametric down-conversion sources. In addition, we analyze a modification of the protocols based on decoy states.

  20. Energy efficient routing protocols for wireless sensor networks: comparison and future directions

    Directory of Open Access Journals (Sweden)

    Loganathan Murukesan

    2017-01-01

    Full Text Available Wireless sensor network consists of nodes with limited resources. Hence, it is important to design protocols or algorithms which increases energy efficiency in order to improve the network lifetime. In this paper, techniques used in the network layer (routing of the internet protocol stack to achieve energy efficiency are reviewed. Usually, the routing protocols are classified into four main schemes: (1 Network Structure, (2 Communication Model, (3 Topology Based, and (4 Reliable Routing. In this work, only network structure based routing protocols are reviewed due to the page constraint. Besides, this type of protocols are much popular among the researchers since they are fairly simple to implement and produce good results as presented in this paper. Also, the pros and cons of each protocols are presented. Finally, the paper concludes with possible further research directions.

  1. A substrate-optimized electrophoretic mobility shift assay for ADAM12

    DEFF Research Database (Denmark)

    Kotzsch, Alexander; Skovgaard, Tine; Buus, Uwe

    2014-01-01

    long been investigated as pharmaceutical drug targets; however, due to lack of potency and in vivo side effects, none of the small-molecule inhibitors discovered so far has made it beyond clinical testing. Ongoing research on novel selective inhibitors of ADAMs requires reliable biochemical assays...... to validate molecular probes from large-scale screening efforts. Here we describe an electrophoretic mobility shift assay for ADAM12 based on the identification of an optimized peptide substrate that is characterized by excellent performance and reproducibility....

  2. Analytical performances of the Diazyme ADA assay on the Cobas® 6000 system.

    Science.gov (United States)

    Delacour, Hervé; Sauvanet, Christophe; Ceppa, Franck; Burnat, Pascal

    2010-12-01

    To evaluate the analytical performance of the Diazyme ADA assay on the Cobas® 6000 system for pleural fluid samples analysis. Imprecision, linearity, calibration curve stability, interference, and correlation studies were completed. The Diazyme ADA assay demonstrated excellent precision (CVADA assay correlated well with the Giusti method (r(2)=0.93) but exhibited a negative bias (~ -30%). The Diazyme ADA assay on the Cobas® 6000 system represents a rapid, accurate, precise and reliable method for determination of ADA activity in pleural fluid samples. Copyright © 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  3. Analytical validation of a flow cytometric protocol for quantification of platelet microparticles in dogs.

    Science.gov (United States)

    Cremer, Signe E; Krogh, Anne K H; Hedström, Matilda E K; Christiansen, Liselotte B; Tarnow, Inge; Kristensen, Annemarie T

    2018-06-01

    Platelet microparticles (PMPs) are subcellular procoagulant vesicles released upon platelet activation. In people with clinical diseases, alterations in PMP concentrations have been extensively investigated, but few canine studies exist. This study aims to validate a canine flow cytometric protocol for PMP quantification and to assess the influence of calcium on PMP concentrations. Microparticles (MP) were quantified in citrated whole blood (WB) and platelet-poor plasma (PPP) using flow cytometry. Anti-CD61 antibody and Annexin V (AnV) were used to detect platelets and phosphatidylserine, respectively. In 13 healthy dogs, CD61 + /AnV - concentrations were analyzed with/without a calcium buffer. CD61 + /AnV - , CD61 + /AnV + , and CD61 - /AnV + MP quantification were validated in 10 healthy dogs. The coefficient of variation (CV) for duplicate (intra-assay) and parallel (inter-assay) analyses and detection limits (DLs) were calculated. CD61 + /AnV - concentrations were higher in calcium buffer; 841,800 MP/μL (526,000-1,666,200) vs without; 474,200 MP/μL (278,800-997,500), P < .05. In WB, PMP were above DLs and demonstrated acceptable (<20%) intra-assay and inter-assay CVs in 9/10 dogs: 1.7% (0.5-8.9) and 9.0% (0.9-11.9), respectively, for CD61 + /AnV - and 2.4% (0.2-8.7) and 7.8% (0.0-12.8), respectively, for CD61 + /AnV + . Acceptable CVs were not seen for the CD61 - /AnV + MP. In PPP, quantifications were challenged by high inter-assay CV, overlapping DLs and hemolysis and lipemia interfered with quantification in 5/10 dogs. Calcium induced higher in vitro PMP concentrations, likely due to platelet activation. PMP concentrations were reliably quantified in WB, indicating the potential for clinical applications. PPP analyses were unreliable due to high inter-CV and DL overlap, and not obtainable due to hemolysis and lipemia interference. © 2018 American Society for Veterinary Clinical Pathology.

  4. The Assessment of Parameters Affecting the Quality of Cord Blood by the Appliance of the Annexin V Staining Method and Correlation with CFU Assays

    Directory of Open Access Journals (Sweden)

    Teja Falk Radke

    2013-01-01

    Full Text Available The assessment of nonviable haematopoietic cells by Annexin V staining method in flow cytometry has recently been published by Duggleby et al. Resulting in a better correlation with the observed colony formation in methylcellulose assays than the standard ISHAGE protocol, it presents a promising method to predict cord blood potency. Herein, we applied this method for examining the parameters during processing which potentially could affect cord blood viability. We could verify that the current standards regarding time and temperature are sufficient, since no significant difference was observed within 48 hours or in storage at 4°C up to 26°C. However, the addition of DMSO for cryopreservation alone leads to an inevitable increase in nonviable haematopoietic stem cells from initially 14.8% ± 4.3% to at least 30.6% ± 5.5%. Furthermore, CFU-assays with varied seeding density were performed in order to evaluate the applicability as a quantitative method. The results revealed that only in a narrow range reproducible clonogenic efficiency (ClonE could be assessed, giving at least a semiquantitative estimation. We conclude that both Annexin V staining method and CFU-assays with defined seeding density are reliable means leading to a better prediction of the final potency. Especially Annexin V, due to its fast readout, is a practical tool for examining and optimising specific steps in processing, while CFU-assays add a functional confirmation.

  5. A Lightweight Protocol for Secure Video Streaming.

    Science.gov (United States)

    Venčkauskas, Algimantas; Morkevicius, Nerijus; Bagdonas, Kazimieras; Damaševičius, Robertas; Maskeliūnas, Rytis

    2018-05-14

    The Internet of Things (IoT) introduces many new challenges which cannot be solved using traditional cloud and host computing models. A new architecture known as fog computing is emerging to address these technological and security gaps. Traditional security paradigms focused on providing perimeter-based protections and client/server point to point protocols (e.g., Transport Layer Security (TLS)) are no longer the best choices for addressing new security challenges in fog computing end devices, where energy and computational resources are limited. In this paper, we present a lightweight secure streaming protocol for the fog computing "Fog Node-End Device" layer. This protocol is lightweight, connectionless, supports broadcast and multicast operations, and is able to provide data source authentication, data integrity, and confidentiality. The protocol is based on simple and energy efficient cryptographic methods, such as Hash Message Authentication Codes (HMAC) and symmetrical ciphers, and uses modified User Datagram Protocol (UDP) packets to embed authentication data into streaming data. Data redundancy could be added to improve reliability in lossy networks. The experimental results summarized in this paper confirm that the proposed method efficiently uses energy and computational resources and at the same time provides security properties on par with the Datagram TLS (DTLS) standard.

  6. The reliability-quality relationship for quality systems and quality risk management.

    Science.gov (United States)

    Claycamp, H Gregg; Rahaman, Faiad; Urban, Jason M

    2012-01-01

    Engineering reliability typically refers to the probability that a system, or any of its components, will perform a required function for a stated period of time and under specified operating conditions. As such, reliability is inextricably linked with time-dependent quality concepts, such as maintaining a state of control and predicting the chances of losses from failures for quality risk management. Two popular current good manufacturing practice (cGMP) and quality risk management tools, failure mode and effects analysis (FMEA) and root cause analysis (RCA) are examples of engineering reliability evaluations that link reliability with quality and risk. Current concepts in pharmaceutical quality and quality management systems call for more predictive systems for maintaining quality; yet, the current pharmaceutical manufacturing literature and guidelines are curiously silent on engineering quality. This commentary discusses the meaning of engineering reliability while linking the concept to quality systems and quality risk management. The essay also discusses the difference between engineering reliability and statistical (assay) reliability. The assurance of quality in a pharmaceutical product is no longer measured only "after the fact" of manufacturing. Rather, concepts of quality systems and quality risk management call for designing quality assurance into all stages of the pharmaceutical product life cycle. Interestingly, most assays for quality are essentially static and inform product quality over the life cycle only by being repeated over time. Engineering process reliability is the fundamental concept that is meant to anticipate quality failures over the life cycle of the product. Reliability is a well-developed theory and practice for other types of manufactured products and manufacturing processes. Thus, it is well known to be an appropriate index of manufactured product quality. This essay discusses the meaning of reliability and its linkages with quality

  7. How reliable are Functional Movement Screening scores? A systematic review of rater reliability.

    Science.gov (United States)

    Moran, Robert W; Schneiders, Anthony G; Major, Katherine M; Sullivan, S John

    2016-05-01

    Several physical assessment protocols to identify intrinsic risk factors for injury aetiology related to movement quality have been described. The Functional Movement Screen (FMS) is a standardised, field-expedient test battery intended to assess movement quality and has been used clinically in preparticipation screening and in sports injury research. To critically appraise and summarise research investigating the reliability of scores obtained using the FMS battery. Systematic literature review. Systematic search of Google Scholar, Scopus (including ScienceDirect and PubMed), EBSCO (including Academic Search Complete, AMED, CINAHL, Health Source: Nursing/Academic Edition), MEDLINE and SPORTDiscus. Studies meeting eligibility criteria were assessed by 2 reviewers for risk of bias using the Quality Appraisal of Reliability Studies checklist. Overall quality of evidence was determined using van Tulder's levels of evidence approach. 12 studies were appraised. Overall, there was a 'moderate' level of evidence in favour of 'acceptable' (intraclass correlation coefficient ≥0.6) inter-rater and intra-rater reliability for composite scores derived from live scoring. For inter-rater reliability of composite scores derived from video recordings there was 'conflicting' evidence, and 'limited' evidence for intra-rater reliability. For inter-rater reliability based on live scoring of individual subtests there was 'moderate' evidence of 'acceptable' reliability (κ≥0.4) for 4 subtests (Deep Squat, Shoulder Mobility, Active Straight-leg Raise, Trunk Stability Push-up) and 'conflicting' evidence for the remaining 3 (Hurdle Step, In-line Lunge, Rotary Stability). This review found 'moderate' evidence that raters can achieve acceptable levels of inter-rater and intra-rater reliability of composite FMS scores when using live ratings. Overall, there were few high-quality studies, and the quality of several studies was impacted by poor study reporting particularly in relation to

  8. Validity and Reliability of Baseline Testing in a Standardized Environment.

    Science.gov (United States)

    Higgins, Kathryn L; Caze, Todd; Maerlender, Arthur

    2017-08-11

    The Immediate Postconcussion Assessment and Cognitive Testing (ImPACT) is a computerized neuropsychological test battery commonly used to determine cognitive recovery from concussion based on comparing post-injury scores to baseline scores. This model is based on the premise that ImPACT baseline test scores are a valid and reliable measure of optimal cognitive function at baseline. Growing evidence suggests that this premise may not be accurate and a large contributor to invalid and unreliable baseline test scores may be the protocol and environment in which baseline tests are administered. This study examined the effects of a standardized environment and administration protocol on the reliability and performance validity of athletes' baseline test scores on ImPACT by comparing scores obtained in two different group-testing settings. Three hundred-sixty one Division 1 cohort-matched collegiate athletes' baseline data were assessed using a variety of indicators of potential performance invalidity; internal reliability was also examined. Thirty-one to thirty-nine percent of the baseline cases had at least one indicator of low performance validity, but there were no significant differences in validity indicators based on environment in which the testing was conducted. Internal consistency reliability scores were in the acceptable to good range, with no significant differences between administration conditions. These results suggest that athletes may be reliably performing at levels lower than their best effort would produce. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. EVA Human Health and Performance Benchmarking Study Overview and Development of a Microgravity Protocol

    Science.gov (United States)

    Norcross, Jason; Jarvis, Sarah; Bekdash, Omar; Cupples, Scott; Abercromby, Andrew

    2017-01-01

    The primary objective of this study is to develop a protocol to reliably characterize human health and performance metrics for individuals working inside various EVA suits under realistic spaceflight conditions. Expected results and methodologies developed during this study will provide the baseline benchmarking data and protocols with which future EVA suits and suit configurations (e.g., varied pressure, mass, center of gravity [CG]) and different test subject populations (e.g., deconditioned crewmembers) may be reliably assessed and compared. Results may also be used, in conjunction with subsequent testing, to inform fitness-for-duty standards, as well as design requirements and operations concepts for future EVA suits and other exploration systems.

  10. Highly parallel and short-acting amplification with locus-specific primers to detect single nucleotide polymorphisms by the DigiTag2 assay.

    Directory of Open Access Journals (Sweden)

    Nao Nishida

    Full Text Available The DigiTag2 assay enables analysis of a set of 96 SNPs using Kapa 2GFast HotStart DNA polymerase with a new protocol that has a total running time of about 7 hours, which is 6 hours shorter than the previous protocol. Quality parameters (conversion rate, call rate, reproducibility and concordance were at the same levels as when genotype calls were acquired using the previous protocol. Multiplex PCR with 192 pairs of locus-specific primers was available for target preparation in the DigiTag2 assay without the optimization of reaction conditions, and quality parameters had the same levels as those acquired with 96-plex PCR. The locus-specific primers were able to achieve sufficient (concentration of target amplicon ≥5 nM and specific (concentration of unexpected amplicons <2 nM amplification within 2 hours, were also able to achieve detectable amplifications even when working in a 96-plex or 192-plex form. The improved DigiTag2 assay will be an efficient platform for screening an intermediate number of SNPs (tens to hundreds of sites in the replication analysis after genome-wide association study. Moreover, highly parallel and short-acting amplification with locus-specific primers may thus facilitate widespread application to other PCR-based assays.

  11. Teleportation protocol with non-ideal conditional local operations

    Energy Technology Data Exchange (ETDEWEB)

    Di Franco, C., E-mail: cdifranco@caesar.ucc.i [Department of Physics, University College Cork, Cork (Ireland); Ballester, D. [School of Mathematics and Physics, Queen' s University, Belfast BT7 1NN (United Kingdom)

    2010-07-12

    We analyze teleportation protocol when some of receiver's conditional operations are more reliable than others and a non-maximally entangled channel is shared by the two parts. We show that the average fidelity of teleportation can be maximized by choosing properly the basis in which the sender performs her two-qubit measurement.

  12. A quantitative assay for lysosomal acidification rates in human osteoclasts

    DEFF Research Database (Denmark)

    Jensen, Vicki Kaiser; Nosjean, Olivier; Dziegiel, Morten Hanefeld

    2011-01-01

    The osteoclast initiates resorption by creating a resorption lacuna. The ruffled border surrounding the lacunae arises from exocytosis of lysosomes. To dissolve the inorganic phase of the bone, the vacuolar adenosine triphosphatase, located in the ruffled border, pumps protons into the resorption...... assay with respect to lysosomal acidification and assess whether it is a reliable test of a compound's ability to inhibit acidification. Investigated were the expression levels of the lysosomal acidification machinery, the activation of the assay by adenosine triphosphate, H(+) and Cl(-) dependency...

  13. A fast and easy real-time PCR genotyping method for the HLA-G 14-bp insertion/deletion polymorphism in the 3' untranslated region

    DEFF Research Database (Denmark)

    Djurisic, S; Sørensen, A E; Hviid, T V F

    2012-01-01

    and reliable method to screen for the HLA-G 14-bp insertion/deletion polymorphism using an optimized real-time polymerase chain reaction protocol. The genotyping assay has been validated by comparison with conventional methods. As results can be obtained within a few hours, the assay will have a potential...

  14. Nested PCR Assay for Detection of Leishmania donovani in Slit Aspirates from Post-Kala-Azar Dermal Leishmaniasis Lesions

    Science.gov (United States)

    Sreenivas, Gannavaram; Ansari, N. A.; Kataria, Joginder; Salotra, Poonam

    2004-01-01

    A nested PCR assay to detect parasite DNA in slit aspirates from skin lesions of patients with post-kala-azar dermal lesihmaniasis (PKDL) is described. PCR results were positive in 27 of 29 (93%) samples by nested PCR assay, while only 20 of 29 (69%) were positive in a primary PCR assay. The nested PCR assay allowed reliable diagnosis of PKDL in a noninvasive manner. PMID:15071047

  15. Nested PCR Assay for Detection of Leishmania donovani in Slit Aspirates from Post-Kala-Azar Dermal Leishmaniasis Lesions

    OpenAIRE

    Sreenivas, Gannavaram; Ansari, N. A.; Kataria, Joginder; Salotra, Poonam

    2004-01-01

    A nested PCR assay to detect parasite DNA in slit aspirates from skin lesions of patients with post-kala-azar dermal lesihmaniasis (PKDL) is described. PCR results were positive in 27 of 29 (93%) samples by nested PCR assay, while only 20 of 29 (69%) were positive in a primary PCR assay. The nested PCR assay allowed reliable diagnosis of PKDL in a noninvasive manner.

  16. Performance standard-based validation study for local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method.

    Science.gov (United States)

    Ahn, Ilyoung; Kim, Tae-Sung; Jung, Eun-Sun; Yi, Jung-Sun; Jang, Won-Hee; Jung, Kyoung-Mi; Park, Miyoung; Jung, Mi-Sook; Jeon, Eun-Young; Yeo, Kyeong-Uk; Jo, Ji-Hoon; Park, Jung-Eun; Kim, Chang-Yul; Park, Yeong-Chul; Seong, Won-Keun; Lee, Ai-Young; Chun, Young Jin; Jeong, Tae Cheon; Jeung, Eui Bae; Lim, Kyung-Min; Bae, SeungJin; Sohn, Soojung; Heo, Yong

    2016-10-01

    Local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method (LLNA: BrdU-FCM) is a modified non-radioisotopic technique with the additional advantages of accommodating multiple endpoints with the introduction of FCM, and refinement and reduction of animal use by using a sophisticated prescreening scheme. Reliability and accuracy of the LLNA: BrdU-FCM was determined according to OECD Test Guideline (TG) No. 429 (Skin Sensitization: Local Lymph Node Assay) performance standards (PS), with the participation of four laboratories. Transferability was demonstrated through successfully producing stimulation index (SI) values for 25% hexyl cinnamic aldehyde (HCA) consistently greater than 3, a predetermined threshold, by all participating laboratories. Within- and between-laboratory reproducibility was shown using HCA and 2,4-dinitrochlorobenzene, in which EC2.7 values (the estimated concentrations eliciting an SI of 2.7, the threshold for LLNA: BrdU-FCM) fell consistently within the acceptance ranges, 0.025-0.1% and 5-20%, respectively. Predictive capacity was tested using the final protocol version 1.3 for the 18 reference chemicals listed in OECD TG 429, of which results showed 84.6% sensitivity, 100% specificity, and 88.9% accuracy compared with the original LLNA. The data presented are considered to meet the performance criteria for the PS, and its predictive capacity was also sufficiently validated. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Stochastic Petri nets for the reliability analysis of communication network applications with alternate-routing

    International Nuclear Information System (INIS)

    Balakrishnan, Meera; Trivedi, Kishor S.

    1996-01-01

    In this paper, we present a comparative reliability analysis of an application on a corporate B-ISDN network under various alternate-routing protocols. For simple cases, the reliability problem can be cast into fault-tree models and solved rapidly by means of known methods. For more complex scenarios, state space (Markov) models are required. However, generation of large state space models can get very labor intensive and error prone. We advocate the use of stochastic reward nets (a variant of stochastic Petri nets) for the concise specification, automated generation and solution of alternate-routing protocols in networks. This paper is written in a tutorial style so as to make it accessible to a large audience

  18. A high performance totally ordered multicast protocol

    Science.gov (United States)

    Montgomery, Todd; Whetten, Brian; Kaplan, Simon

    1995-01-01

    This paper presents the Reliable Multicast Protocol (RMP). RMP provides a totally ordered, reliable, atomic multicast service on top of an unreliable multicast datagram service such as IP Multicasting. RMP is fully and symmetrically distributed so that no site bears un undue portion of the communication load. RMP provides a wide range of guarantees, from unreliable delivery to totally ordered delivery, to K-resilient, majority resilient, and totally resilient atomic delivery. These QoS guarantees are selectable on a per packet basis. RMP provides many communication options, including virtual synchrony, a publisher/subscriber model of message delivery, an implicit naming service, mutually exclusive handlers for messages, and mutually exclusive locks. It has commonly been held that a large performance penalty must be paid in order to implement total ordering -- RMP discounts this. On SparcStation 10's on a 1250 KB/sec Ethernet, RMP provides totally ordered packet delivery to one destination at 842 KB/sec throughput and with 3.1 ms packet latency. The performance stays roughly constant independent of the number of destinations. For two or more destinations on a LAN, RMP provides higher throughput than any protocol that does not use multicast or broadcast.

  19. Real‑time, fast neutron detection for stimulated safeguards assay

    International Nuclear Information System (INIS)

    Joyce, Malcolm J.; Adamczyk, Justyna; Plenteda, Romano; Aspinall, Michael D.; Cave, Francis D.

    2015-01-01

    The advent of low‑hazard organic liquid scintillation detectors and real‑time pulse‑shape discrimination (PSD) processing has suggested a variety of modalities by which fast neutrons, as opposed to neutrons moderated prior to detection, can be used directly to benefit safeguards needs. In this paper we describe a development of a fast‑neutron based safeguards assay system designed for the assessment of 235 U content in fresh fuel. The system benefits from real‑time pulse‑shape discrimination processing and auto‑calibration of the detector system parameters to ensure a rapid and effective set‑up protocol. These requirements are essential in optimising the speed and limit of detection of the fast neutron technique, whilst minimising the intervention needed to perform the assay.

  20. Telomerase Repeated Amplification Protocol (TRAP).

    Science.gov (United States)

    Mender, Ilgen; Shay, Jerry W

    2015-11-20

    Telomeres are found at the end of eukaryotic linear chromosomes, and proteins that bind to telomeres protect DNA from being recognized as double-strand breaks thus preventing end-to-end fusions (Griffith et al. , 1999). However, due to the end replication problem and other factors such as oxidative damage, the limited life span of cultured cells (Hayflick limit) results in progressive shortening of these protective structures (Hayflick and Moorhead, 1961; Olovnikov, 1973). The ribonucleoprotein enzyme complex telomerase-consisting of a protein catalytic component hTERT and a functional RNA component hTR or hTERC - counteracts telomere shortening by adding telomeric repeats to the end of chromosomes in ~90% of primary human tumors and in some transiently proliferating stem-like cells (Shay and Wright, 1996; Shay and Wright, 2001). This results in continuous proliferation of cells which is a hallmark of cancer. Therefore, telomere biology has a central role in aging, cancer progression/metastasis as well as targeted cancer therapies. There are commonly used methods in telomere biology such as Telomere Restriction Fragment (TRF) (Mender and Shay, 2015b), Telomere Repeat Amplification Protocol (TRAP) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this detailed protocol we describe Telomere Repeat Amplification Protocol (TRAP). The TRAP assay is a popular method to determine telomerase activity in mammalian cells and tissue samples (Kim et al. , 1994). The TRAP assay includes three steps: extension, amplification, and detection of telomerase products. In the extension step, telomeric repeats are added to the telomerase substrate (which is actually a non telomeric oligonucleotide, TS) by telomerase. In the amplification step, the extension products are amplified by the polymerase chain reaction (PCR) using specific primers (TS upstream primer and ACX downstream primer) and in the detection step, the presence or absence of telomerase is

  1. Validation of the Thermo Scientific SureTect Escherichia coli O157:H7 Real-Time PCR Assay for Raw Beef and Produce Matrixes.

    Science.gov (United States)

    Cloke, Jonathan; Crowley, Erin; Bird, Patrick; Bastin, Ben; Flannery, Jonathan; Agin, James; Goins, David; Clark, Dorn; Radcliff, Roy; Wickstrand, Nina; Kauppinen, Mikko

    2015-01-01

    . coli O157:NM isolate. Nonmotile isolates of E. coli O157 have been demonstrated to still contain the H7 gene; therefore, this result is not unexpected. Robustness testing was conducted to evaluate the performance of the SureTect assay with specific deviations to the assay protocol, which were outside the recommended parameters and which are open to variation. This study demonstrated that the SureTect assay gave reliable performance. A final study to verify the shelf life of the product, under accelerated conditions was also conducted.

  2. Materials and Reliability Handbook for Semiconductor Optical and Electron Devices

    CERN Document Server

    Pearton, Stephen

    2013-01-01

    Materials and Reliability Handbook for Semiconductor Optical and Electron Devices provides comprehensive coverage of reliability procedures and approaches for electron and photonic devices. These include lasers and high speed electronics used in cell phones, satellites, data transmission systems and displays. Lifetime predictions for compound semiconductor devices are notoriously inaccurate due to the absence of standard protocols. Manufacturers have relied on extrapolation back to room temperature of accelerated testing at elevated temperature. This technique fails for scaled, high current density devices. Device failure is driven by electric field or current mechanisms or low activation energy processes that are masked by other mechanisms at high temperature. The Handbook addresses reliability engineering for III-V devices, including materials and electrical characterization, reliability testing, and electronic characterization. These are used to develop new simulation technologies for device operation and ...

  3. Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization.

    Science.gov (United States)

    Machida, Kazuya; Liu, Bernard

    2017-01-01

    Recognition of phosphotyrosine-containing sequences by SH2 domains confers specificity in tyrosine kinase pathways. By assessing interactions between isolated SH2 domains and their binding proteins, it is possible to gain insight into otherwise inaccessible complex cellular systems. Far-Western, pull-down, and fluorescence polarization (FP) have been frequently used for characterization of phosphotyrosine signaling. Here, we outline standard protocols for these established assays using recombinant SH2 domain, emphasizing the importance of appropriate sample preparation and assay controls.

  4. A precise, efficient radiometric assay for bacterial growth

    International Nuclear Information System (INIS)

    Boonkitticharoen, V.; Ehrhardt, C.; Kirchner, P.T.

    1984-01-01

    The two-compartment radiometric assay for bacterial growth promised major advantages over systems in clinical use, but poor reproducibility and counting efficiency limited its application. In this method, 14-CO/sub 2/ produced by bacterial metabolism of C-14-glucose is trapped and counted on filter paper impregnated with NaOH and fluors. The authors sought to improve assay efficiency and precision through a systematic study of relevant physical and chemical factors. Improvements in efficiency (88% vs. 10%) and in precision (relative S.D. 5% vs. 40%) were produced by a) reversing growth medium and scintillator chambers to permit vigorous agitation, b) increasing NaOH quantity and using a supersaturated PPO solution and c) adding detergent to improve uniformity of NaOH-PPO mixture. Inoculum size, substrate concentration and O/sub 2/ transfer rate affected assay sensitivity but not bacterial growth rate. The authors' assay reliably detects bacterial growth for inocula of 10,000 organisms in 1 hour and for 25 organisms within 4 1/2 hours, thus surpassing other existing clinical and research methods

  5. An indicator cell assay for blood-based diagnostics.

    Directory of Open Access Journals (Sweden)

    Samuel A Danziger

    Full Text Available We have established proof of principle for the Indicator Cell Assay Platform™ (iCAP™, a broadly applicable tool for blood-based diagnostics that uses specifically-selected, standardized cells as biosensors, relying on their innate ability to integrate and respond to diverse signals present in patients' blood. To develop an assay, indicator cells are exposed in vitro to serum from case or control subjects and their global differential response patterns are used to train reliable, disease classifiers based on a small number of features. In a feasibility study, the iCAP detected pre-symptomatic disease in a murine model of amyotrophic lateral sclerosis (ALS with 94% accuracy (p-Value = 3.81E-6 and correctly identified samples from a murine Huntington's disease model as non-carriers of ALS. Beyond the mouse model, in a preliminary human disease study, the iCAP detected early stage Alzheimer's disease with 72% cross-validated accuracy (p-Value = 3.10E-3. For both assays, iCAP features were enriched for disease-related genes, supporting the assay's relevance for disease research.

  6. The effect of different methods and image analyzers on the results of the in vivo comet assay.

    Science.gov (United States)

    Kyoya, Takahiro; Iwamoto, Rika; Shimanura, Yuko; Terada, Megumi; Masuda, Shuichi

    2018-01-01

    The in vivo comet assay is a widely used genotoxicity test that can detect DNA damage in a range of organs. It is included in the Organisation for Economic Co-operation and Development Guidelines for the Testing of Chemicals. However, various protocols are still used for this assay, and several different image analyzers are used routinely to evaluate the results. Here, we verified a protocol that largely contributes to the equivalence of results, and we assessed the effect on the results when slides made from the same sample were analyzed using two different image analyzers (Comet Assay IV vs Comet Analyzer). Standardizing the agarose concentrations and DNA unwinding and electrophoresis times had a large impact on the equivalence of the results between the different methods used for the in vivo comet assay. In addition, there was some variation in the sensitivity of the two different image analyzers tested; however this variation was considered to be minor and became negligible when the test conditions were standardized between the two different methods. By standardizing the concentrations of low melting agarose and DNA unwinding and electrophoresis times between both methods used in the current study, the sensitivity to detect the genotoxicity of a positive control substance in the in vivo comet assay became generally comparable, independently of the image analyzer used. However, there may still be the possibility that other conditions, except for the three described here, could affect the reproducibility of the in vivo comet assay.

  7. Non destructive assay techniques applied to nuclear materials

    International Nuclear Information System (INIS)

    Gavron, A.

    2001-01-01

    Nondestructive assay is a suite of techniques that has matured and become precise, easily implementable, and remotely usable. These techniques provide elaborate safeguards of nuclear material by providing the necessary information for materials accounting. NDA techniques are ubiquitous, reliable, essentially tamper proof, and simple to use. They make the world a safer place to live in, and they make nuclear energy viable. (author)

  8. An efficient and reliable multi-hop geographical broadcast protocol in vehicular ad-hoc networks

    NARCIS (Netherlands)

    Rajendran, R.; Jongh, J. de

    2013-01-01

    In Intelligent Transportation Systems (ITS), disseminating warning messages in a timely and efficient way through wireless short-range communications can save many lives and reduce traffic congestion. A geographical broadcast protocol provides data delivery to specified geographical areas, using

  9. Protocol converter for serial communication between digital rectifier controllers and a power plant SCADA system

    Directory of Open Access Journals (Sweden)

    Vukić Vladimir Đ.

    2016-01-01

    Full Text Available The paper describes the protocol converter INT-485-MBRTU, developed for serial communication between the thyristor rectifier (based on the proprietary protocol "INT-CPD-05", according to standard RS-485 and the SCADA system (based on protocol "Modbus RTU", of the same standard in the thermal power plant "Nikola Tesla B1". Elementary data on industrial communication protocols and communication gateways were provided. The basic technical characteristics of the "Omron" programmable logic controller CJ series were described, as well as the developed device INT-485-MBRTU. Protocol converters with two versions of communication software were tested, differing only in one control word, intended for a forced successive change of communication sequences, in opposite to automatic sequence relieve. The device iNT-485-MBRTU, with the program for forced successive change of communication sequences, demonstrated the reliability of data transfer of 100 %, in a sample of approximately 480 messages. For nearly the same sample, the same protocol converter, with a version of the program without any type of message identifiers, transferred less than 60 % of the foreseen data. During multiple sixty-hour tests, the reliability of data transfer of at least 99.9979% was recorded, in 100% of the analysed cases, and for a sample of nearly 96,000 pairs of the send and receive messages. We analysed the results and estimated the additional possibilities for application of the INT-485-MBRTU protocol converter.

  10. Towards Reliable and Energy-Efficient Incremental Cooperative Communication for Wireless Body Area Networks.

    Science.gov (United States)

    Yousaf, Sidrah; Javaid, Nadeem; Qasim, Umar; Alrajeh, Nabil; Khan, Zahoor Ali; Ahmed, Mansoor

    2016-02-24

    In this study, we analyse incremental cooperative communication for wireless body area networks (WBANs) with different numbers of relays. Energy efficiency (EE) and the packet error rate (PER) are investigated for different schemes. We propose a new cooperative communication scheme with three-stage relaying and compare it to existing schemes. Our proposed scheme provides reliable communication with less PER at the cost of surplus energy consumption. Analytical expressions for the EE of the proposed three-stage cooperative communication scheme are also derived, taking into account the effect of PER. Later on, the proposed three-stage incremental cooperation is implemented in a network layer protocol; enhanced incremental cooperative critical data transmission in emergencies for static WBANs (EInCo-CEStat). Extensive simulations are conducted to validate the proposed scheme. Results of incremental relay-based cooperative communication protocols are compared to two existing cooperative routing protocols: cooperative critical data transmission in emergencies for static WBANs (Co-CEStat) and InCo-CEStat. It is observed from the simulation results that incremental relay-based cooperation is more energy efficient than the existing conventional cooperation protocol, Co-CEStat. The results also reveal that EInCo-CEStat proves to be more reliable with less PER and higher throughput than both of the counterpart protocols. However, InCo-CEStat has less throughput with a greater stability period and network lifetime. Due to the availability of more redundant links, EInCo-CEStat achieves a reduced packet drop rate at the cost of increased energy consumption.

  11. A lightweight neighbor-info-based routing protocol for no-base-station taxi-call system.

    Science.gov (United States)

    Zhu, Xudong; Wang, Jinhang; Chen, Yunchao

    2014-01-01

    Since the quick topology change and short connection duration, the VANET has had unstable routing and wireless signal quality. This paper proposes a kind of lightweight routing protocol-LNIB for call system without base station, which is applicable to the urban taxis. LNIB maintains and predicts neighbor information dynamically, thus finding the reliable path between the source and the target. This paper describes the protocol in detail and evaluates the performance of this protocol by simulating under different nodes density and speed. The result of evaluation shows that the performance of LNIB is better than AODV which is a classic protocol in taxi-call scene.

  12. Reliability and accuracy of a video analysis protocol to assess core ability.

    Science.gov (United States)

    McDonald, Dawn A; Delgadillo, James Q; Fredericson, Michael; McConnell, Jennifer; Hodgins, Melissa; Besier, Thor F

    2011-03-01

    To develop and test a method to measure core ability in healthy athletes with 2-dimensional video analysis software (SiliconCOACH). Specific objectives were to: (1) develop a standardized exercise battery with progressions of increasing difficulty to evaluate areas of core ability in elite athletes; (2) develop an objective and quantitative grading rubric with the use of video analysis software; (3) assess the test-retest reliability of the exercise battery; (4) assess the interrater and intrarater reliability of the video analysis system; and (5) assess the accuracy of the assessment. Test-retest repeatability and accuracy. Testing was conducted in the Stanford Human Performance Laboratory, Stanford University, Stanford, CA. Nine female gymnasts currently training with the Stanford Varsity Women's Gymnastics Team participated in testing. Participants completed a test battery composed of planks, side planks, and leg bridges of increasing difficulty. Subjects completed two 20-minute testing sessions within a 4- to 10-day period. Two-dimensional sagittal-plane video was captured simultaneously with 3-dimensional motion capture. The main outcome measures were pelvic displacement and time that elapsed until failure occurred, as measured with SiliconCOACH video analysis software. Test-retest and interrater and intrarater reliability of the video analysis measures was assessed. Accuracy as compared with 3-dimensional motion capture also was assessed. Levels reached during the side planks and leg bridges had an excellent test-retest correlation (r(2) = 0.84, r(2) = 0.95). Pelvis displacements measured by examiner 1 and examiner 2 had an excellent correlation (r(2) = 0.86, intraclass correlation coefficient = 0.92). Pelvis displacements measured by examiner 1 during independent grading sessions had an excellent correlation (r(2) = 0.92). Pelvis displacements from the plank and from a set of combined plank and side plank exercises both had an excellent correlation with 3

  13. Performance Evaluation of AODV Routing Protocol in VANET with NS2

    Directory of Open Access Journals (Sweden)

    Divya Rathi

    2017-03-01

    Full Text Available In intelligent transportation systems, the collaboration between vehicles and the road side units is essential to bring these systems to realization. The emerging Vehicular Ad Hoc Network (VANET is becoming more and more important as it provides intelligent transportation application, comfort, safety, entertainment for people in vehicles. In order to provide stable routes and to get good performance in VANET, there is a need of proper routing protocols must be designed. In this paper, we are working with the very well-known ad-hoc on-demand distance vector (AODV routing protocol. The existing Routing protocol AODV-L which is based on the Link expiration time is extended to propose a more reliable AODV-AD which is based on multichannel MAC protocol. For the performance evaluation of routing protocols, a simulation tool ‘NS2’ has been used. Simulation results show that the proposed AODV-AD protocol can achieves better performances in forms of high Route stability, Packet Delivery ratio and packet loss rate than traditional AODV-L and traditional AODV.

  14. Performance of a commercial assay for the diagnosis of influenza A (H1N1 infection in comparison to the Centers for Disease Control and Prevention protocol of real time RT-PCR

    Directory of Open Access Journals (Sweden)

    María G Barbás

    2012-03-01

    Full Text Available At the time of influenza A (H1N1 emergency, the WHO responded with remarkable speed by releasing guidelines and a protocol for a real-time RT-PCR assay (rRT-PCR. The aim of the present study was to evalúate the performance of the "Real Time Ready Influenza A/H1N1 Detection Set" (June 2009-Roche kit in comparison to the CDC reference rRT-PCR protocol. The overall sensitivity of the Roche assay for detection of the Inf A gene in the presence or absence of the H1 gene was 74.5 %. The sensitivity for detecting samples that were only positive for the Inf A gene (absence of the H1 gene was 53.3 % whereas the sensitivity for H1N1-positive samples (presence of the Inf A gene and any other swine gene was 76.4 %. The specificity of the assay was 97.1 %. A new version of the kit (November 2009 is now available, and a recent evaluation of its performance showed good sensitivity to detect pandemic H1N1 compared to other molecular assays.Durante la pandemia de influenza A (H1N1, la OMS recomendó algoritmos y protocolos de detección del virus mediante RT-PCR en tiempo real. El objetivo del presente estudio fue evaluar el desempeño del equipo que comercializa la empresa Roche, Real Time Ready Influenza A/H1N1 Detection Set (junio de 2009, en comparación con el protocolo de RT-PCR en tiempo real de los CDC. La sensibilidad global del ensayo de Roche para la detección del gen Inf A en presencia o ausencia del gen H1 fue 74,5 %. La sensibilidad para la detección de muestras positivas solo para el gen Inf A (ausencia del gen H1 fue 53,3 % y la sensibilidad para la detección de muestras positivas para H1N1 (presencia del gen Inf A y cualquier otro gen porcino fue 76,4 %. La especificidad fue 97,1 %. Existe una nueva versión del equipo (noviembre 2009 que, según se ha descrito, presenta buena sensibilidad en comparación con otros ensayos moleculares para detectar H1N1 pandémica.

  15. Intra-laboratory validation of a human cell based in vitro angiogenesis assay for testing angiogenesis modulators

    Directory of Open Access Journals (Sweden)

    Jertta-Riina Sarkanen

    2011-01-01

    Full Text Available The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis e.g. pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using 6 reference chemicals, which are widely used pharmaceuticals that inhibit angiogenesis: acetyl salicylic acid, erlotinib, 2-methoxyestradiol, levamisole, thalidomide, and anti-vascular endothelial growth factor. In the intra-laboratory validation, the sensitivity of the assay (upper and lower limits of detection and linearity of response in tubule formation, batch to batch variation in tubule formation between different Master cell bank batches, and precision as well as the reliability of the assay (reproducibility and repeatability were tested. The pre-set acceptance criteria for the intra-laboratory validation study were met. The relevance of the assay in man was investigated by comparing the effects of reference chemicals and their concentrations to the published human data. The comparison showed a good concordance, which indicates that this human cell based angiogenesis model predicts well the effects in man and has the potential to be used to supplement and/or replace of animal tests.

  16. 4-Meter Gait Speed Test in Chronic Obstructive Pulmonary Disease: INTERRATER RELIABILITY USING A STOPWATCH.

    Science.gov (United States)

    Bisca, Gianna Waldrich; Fava, Lucas Rodrigues; Morita, Andrea Akemi; Machado, Felipe Vilaça Cavallari; Pitta, Fabio; Hernandes, Nidia Aparecida

    2017-12-14

    4-meter gait speed (4MGS) is increasingly used to assess functional performance in patients with chronic obstructive pulmonary disease. However, the current literature lacks information regarding some technical standards for this test. Therefore, the purpose of this study was to compare and to evaluate the interrater reliability between a stopwatch and video recording used as timing systems for the 4MGS in patients with chronic obstructive pulmonary disease, as well as to verify the interrater reliability between 2 observers measuring the 4MGS time using a manual stopwatch. Fifty-one patients performed the 4MGS using 4 different protocols (random order): walking at the usual and maximum speed in a 4-meter course and walking at the same 2 speeds on an 8-m course using a 2-m acceleration zone, a 4-meter timing area, and a 2-m deceleration zone. Gait speed was measured simultaneously using a stopwatch and a video recording. In a subanalysis (n = 24), 2 independent observers timed the 4MGS using a stopwatch. There was no significant difference in comparison between the 2 timing methods (P > .05 for all), and the reliability between video recording and stopwatch was excellent in all 4MGS studied protocols (intraclass correlation coefficient ≥ 0.91). Moreover, when comparing gait speed measured by 2 observers using a stopwatch, no significant difference was found among all proposed protocols (P > .05 for all), and there was also excellent reliability between the 2 independent observers (intraclass correlation coefficient ≥ 0.94). The stopwatch, a low-cost and feasible tool, is reliable as a timing device for the 4MGS in patients with chronic obstructive pulmonary disease.

  17. Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays.

    Science.gov (United States)

    Glais, Laurent; Jacquot, Emmanuel

    2015-01-01

    Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay.

  18. Simplified PCR protocols for INNO-LiPA HBV Genotyping and INNO-LiPA HBV PreCore assays

    NARCIS (Netherlands)

    Qutub, Mohammed O.; Germer, Jeffrey J.; Rebers, Sjoerd P. H.; Mandrekar, Jayawant N.; Beld, Marcel G. H. M.; Yao, Joseph D. C.

    2006-01-01

    INNO-LiPA HBV Genotyping (LiPA HBV GT) and INNO-LiPA HBV PreCore (LiPA HBV PC) are commercially available assays for hepatitis B virus (HBV) characterization. These assays are labor-intensive and may be prone to exogenous DNA contamination due to their use of nested PCR amplification procedures and

  19. Code verification and Y2K testing, calibration, testing, and installation of the radionuclide assay system-photon (RAS-P) at multiple sites for the Savannah River Site

    International Nuclear Information System (INIS)

    Hodge, C.A.

    2000-01-01

    The Radionuclide Assay System - Photon (RAS-P) is a near-field, transmission-corrected assay system developed for measurement of the actinide content of relatively homogeneous waste generated by facility operations. It is intended for use by facility operations personnel, and has features to enhance its usefulness and efficiency. These include multinuclide assay capability, automatic (off-shift) collection of background and straight-through transmission source data, enforcement of measurement control requirements, Go-NoGo or Assay modes, password protection, and reporting of total fissile gram equivalent values. System hardware consists of a shielded high-resolution germanium detector, a turntable, a shielded transmission source and shutter assembly, and a desktop computer and laser printer mounted on a compact frame. RAS-P was designed to assay the contents of cylindrical containers up to 30 inches diameter by 32 inches high, boxes up to 30 inches diagonal by 32 inches high, and HE PA filters up to 2 x 2 x 1 feet. Prior to installation at the Savannah River Site (SRS), code validation, system performance, and assurance against Y2K effects all were confirmed. Code validation was accomplished using spreadsheet calculations that were independent of the original code to calculate intermediate and final result produced by RAS-P. System testing was performed by repeated operation of the instrument under all required circumstances. Y2K testing was performed simultaneously with code validation following a protocol prescribed by the SRS Y2K subcommittee that required assays with dates varying throughout the expected useful life of the RAS-P, particularly those bracketing Y2K boundaries. Performance history has been compiled demonstrating reliability (system availability), diversability (the ability to alter assay parameters and obtain results quickly), and measurement control characteristics

  20. Impact of Transport Layer Protocols on Reliable Information Access in Smart Grids

    DEFF Research Database (Denmark)

    Shahid, Kamal; Saeed, Aamir; Kristensen, Thomas le Fevre

    2017-01-01

    Time is critical for certain types of dynamic information (e.g. frequency control) in a smart grid scenario. The usefulness of such information depends upon the arrival within a specific frame of time, which in other case may not serve the purpose and effect controller’s performance....... The question is addressed by analyzing the performance of UDP and TCP over imperfect network conditions to show how the selection of transport layer protocol can dramatically affect controller’s performance. This analysis is based on a quality metric called mismatch probability that considers occurrence...

  1. Evaluation of the highly sensitive Roche thyroglobulin II assay and establishment of a reference limit for thyroglobulin-negative patient samples.

    Science.gov (United States)

    Rotteveel-de Groot, Dorien M; Ross, H Alec; Janssen, Marcel J R; Netea-Maier, Romana T; Oosting, Janine D; Sweep, Fred C G J; van Herwaarden, Antonius E

    2016-08-01

    Thyroglobulin (Tg) measurements are used to monitor for residual thyroid tissue in patients with differentiated thyroid cancer (DTC) after thyroidectomy and radioiodine ablative therapy. In recent years highly sensitive Tg assays have been developed. In this study the analytical performance of the new Roche Elecsys Tg II assay was evaluated and compared with the well documented Access2 Tg assay (Beckman-Coulter). Analytical performance was examined using various Clinical and Laboratory Standards Institute (CLSI) evaluation protocols. Tg negative patient sera were used to establish an upper reference limit (URL) for the Elecsys Tg II assay. Non-linearity, drift and carry-over according to CLSI EP10 and EP6 in a measuring range of 0.04-500 ng/mL were non-significant. Total precision according to CLSI EP5 was 10% at a Tg concentration of 0.08 ng/mL. A patient serum comparison performed according to a modified CLSI EP9 protocol showed a significant difference of a factor of approximately 1.4, despite using an identical CRM calibrator. The Elecsys Tg II assay measured Tg with a two-fold higher sensitivity than the Access2 assay. Finally, using human sera without Tg, an URL of 0.05 ng/mL was determined. In our hands the highly sensitive Elecsys Tg II assay shows a good analytical performance and a higher sensitivity compared to the Access2 Tg assay. An URL of 0.05 ng/mL for the Elecsys Tg II assay was determined which may improve the clinical utility of the assay for the detection of residual DTC or disease recurrence.

  2. Analytical performances of a new enzymatic assay for hemoglobin A1c.

    Science.gov (United States)

    Jaisson, Stéphane; Desmons, Aurore; Renard, Benoît; Chevelle, Benjamin; Leroy, Nathalie; Gillery, Philippe

    2014-07-01

    HbA1c is considered the gold standard for the follow-up of diabetic patients and a new diagnostic tool for diabetes mellitus, which implies the availability of reliable assay methods. We have evaluated a new assay developed by Abbott Laboratories, based on the enzymatic quantification of HbA1c by a fructosyl dipeptide oxidase using Architect analyzers. Precision, linearity, correlation with a HPLC method, accuracy and potential impact interferences on HbA1c measurement have been evaluated. Intra-day and between-day CVs were lower than 1.2% and linearity was excellent from 19 mmol/mol (3.9%) to 163 mmol/mol (17.1%). The results were well correlated with those obtained by the HPLC (Variant II device, kit NU - BioRad): HbA1c [Architect, mmol/mol]=0.986×HbA1c [Variant II, mmol/mol]+0.713 (r=0.998, n=109). This method provided consistent results with IFCC titrated quality control samples. Classical interferences in HbA1c assays (i.e. labile HbA1c, carbamylated hemoglobin, triglycerides or bilirubin) did not have an impact on HbA1c quantification by this method. This new enzymatic assay proved to be a robust and reliable method for HbA1c measurement suitable for routine practice in clinical chemistry laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. A Study on IP Network Recovery through Routing Protocols

    Directory of Open Access Journals (Sweden)

    K. Karthik

    2016-09-01

    Full Text Available Internet has taken major role in our communication infrastructure. Such that requirement of internet availability and reliability has increasing accordingly. The major network failure reasons are failure of node and failure of link among the nodes. This can reduce the performance of major applications in an IP networks. The network recovery should be fast enough so that service interruption of link or node failure. The new path taken by the diverted traffic can be computed either at the time of failures or before failures. These mechanisms are known as Reactive and Proactive protocols respectively. In this paper, we surveyed reactive and proactive protocols mechanisms for IP network recovery.

  4. Validation of the 3D Skin Comet assay using full thickness skin models: Transferability and reproducibility.

    Science.gov (United States)

    Reisinger, Kerstin; Blatz, Veronika; Brinkmann, Joep; Downs, Thomas R; Fischer, Anja; Henkler, Frank; Hoffmann, Sebastian; Krul, Cyrille; Liebsch, Manfred; Luch, Andreas; Pirow, Ralph; Reus, Astrid A; Schulz, Markus; Pfuhler, Stefan

    2018-03-01

    Recently revised OECD Testing Guidelines highlight the importance of considering the first site-of-contact when investigating the genotoxic hazard. Thus far, only in vivo approaches are available to address the dermal route of exposure. The 3D Skin Comet and Reconstructed Skin Micronucleus (RSMN) assays intend to close this gap in the in vitro genotoxicity toolbox by investigating DNA damage after topical application. This represents the most relevant route of exposure for a variety of compounds found in household products, cosmetics, and industrial chemicals. The comet assay methodology is able to detect both chromosomal damage and DNA lesions that may give rise to gene mutations, thereby complementing the RSMN which detects only chromosomal damage. Here, the comet assay was adapted to two reconstructed full thickness human skin models: the EpiDerm™- and Phenion ® Full-Thickness Skin Models. First, tissue-specific protocols for the isolation of single cells and the general comet assay were transferred to European and US-American laboratories. After establishment of the assay, the protocol was then further optimized with appropriate cytotoxicity measurements and the use of aphidicolin, a DNA repair inhibitor, to improve the assay's sensitivity. In the first phase of an ongoing validation study eight chemicals were tested in three laboratories each using the Phenion ® Full-Thickness Skin Model, informing several validation modules. Ultimately, the 3D Skin Comet assay demonstrated a high predictive capacity and good intra- and inter-laboratory reproducibility with four laboratories reaching a 100% predictivity and the fifth yielding 70%. The data are intended to demonstrate the use of the 3D Skin Comet assay as a new in vitro tool for following up on positive findings from the standard in vitro genotoxicity test battery for dermally applied chemicals, ultimately helping to drive the regulatory acceptance of the assay. To expand the database, the validation will

  5. The PRECIS-2 tool has good interrater reliability and modest discriminant validity.

    Science.gov (United States)

    Loudon, Kirsty; Zwarenstein, Merrick; Sullivan, Frank M; Donnan, Peter T; Gágyor, Ildikó; Hobbelen, Hans J S M; Althabe, Fernando; Krishnan, Jerry A; Treweek, Shaun

    2017-08-01

    PRagmatic Explanatory Continuum Indicator Summary (PRECIS)-2 is a tool that could improve design insight for trialists. Our aim was to validate the PRECIS-2 tool, unlike its predecessor, testing the discriminant validity and interrater reliability. Over 80 international trialists, methodologists, clinicians, and policymakers created PRECIS-2 helping to ensure face validity and content validity. The interrater reliability of PRECIS-2 was measured using 19 experienced trialists who used PRECIS-2 to score a diverse sample of 15 randomized controlled trial protocols. Discriminant validity was tested with two raters to independently determine if the trial protocols were more pragmatic or more explanatory, with scores from the 19 raters for the 15 trials as predictors of pragmatism. Interrater reliability was generally good, with seven of nine domains having an intraclass correlation coefficient over 0.65. Flexibility (adherence) and recruitment had wide confidence intervals, but raters found these difficult to rate and wanted more information. Each of the nine PRECIS-2 domains could be used to differentiate between trials taking more pragmatic or more explanatory approaches with better than chance discrimination for all domains. We have assessed the validity and reliability of PRECIS-2. An elaboration study and web site provide guidance to help future users of the tool which is continuing to be tested by trial teams, systematic reviewers, and funders. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Comparison of human septal nuclei MRI measurements using automated segmentation and a new manual protocol based on histology

    Science.gov (United States)

    Butler, Tracy; Zaborszky, Laszlo; Pirraglia, Elizabeth; Li, Jinyu; Wang, Xiuyuan Hugh; Li, Yi; Tsui, Wai; Talos, Delia; Devinsky, Orrin; Kuchna, Izabela; Nowicki, Krzysztof; French, Jacqueline; Kuzniecky, Rubin; Wegiel, Jerzy; Glodzik, Lidia; Rusinek, Henry; DeLeon, Mony J.; Thesen, Thomas

    2014-01-01

    Septal nuclei, located in basal forebrain, are strongly connected with hippocampi and important in learning and memory, but have received limited research attention in human MRI studies. While probabilistic maps for estimating septal volume on MRI are now available, they have not been independently validated against manual tracing of MRI, typically considered the gold standard for delineating brain structures. We developed a protocol for manual tracing of the human septal region on MRI based on examination of neuroanatomical specimens. We applied this tracing protocol to T1 MRI scans (n=86) from subjects with temporal epilepsy and healthy controls to measure septal volume. To assess the inter-rater reliability of the protocol, a second tracer used the same protocol on 20 scans that were randomly selected from the 72 healthy controls. In addition to measuring septal volume, maximum septal thickness between the ventricles was measured and recorded. The same scans (n=86) were also analysed using septal probabilistic maps and Dartel toolbox in SPM. Results show that our manual tracing algorithm is reliable, and that septal volume measurements obtained via manual and automated methods correlate significantly with each other (pautomated methods detected significantly enlarged septal nuclei in patients with temporal lobe epilepsy in accord with a proposed compensatory neuroplastic process related to the strong connections between septal nuclei and hippocampi. Septal thickness, which was simple to measure with excellent inter-rater reliability, correlated well with both manual and automated septal volume, suggesting it could serve as an easy-to-measure surrogate for septal volume in future studies. Our results call attention to the important though understudied human septal region, confirm its enlargement in temporal lobe epilepsy, and provide a reliable new manual delineation protocol that will facilitate continued study of this critical region. PMID:24736183

  7. A novel hematoxylin and eosin stain assay for detection of the parasitic dinoflagellate Amoebophrya.

    Science.gov (United States)

    Li, Caiwen; Chen, Tiantian

    2017-02-01

    The parasitic dinoflagellate Amoebophrya infects broad range of marine organisms. Particularly, Amoebophrya infections in planktonic dinoflagellates can prevent or delay the formation of algal blooms, and recycle undergrazed planktonic dinoflagellates back to the microbial loop by disrupting host cells. Its ecological significance was gradually recognized along with the discovery of its enormous molecular diversity in oceanic and coastal ecosystems. Thus, we developed a reliable, easily accessible and less time-consuming assay, to detect and assess Amoebophrya infections in planktonic dinoflagellates. The modified hematoxylin and eosin staining assay provided reliable diagnosis of Amoebophrya infection by identifying the characteristic "beehive" of the multinucleate trophonts. After staining, the typical multinucleate "beehive" is evidently distinguishable from the compact nuclei of uninfected host cells. The modified hematoxylin and eosin (H & E) staining assay is easy to use, that can be routinely performed within 3h (up to 20 samples/batch) using general laboratory equipment, supplies and chemical reagents. The produced slides with agar-embedded dinoflagellate cells can be stored for several months or even years in a dry place without noticeable loss in quality of staining. With suitable calculation, the modified H & E assay can be applied to assess the prevalence of Amoebophrya infection in planktonic dinoflagellates. This efficient and powerful assay will facilitate the investigation on the ecological roles of Amoebophryidae in coastal and oceanic ecosystem. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Continuous-variable protocol for oblivious transfer in the noisy-storage model

    DEFF Research Database (Denmark)

    Furrer, Fabian; Gehring, Tobias; Schaffner, Christian

    2018-01-01

    for oblivious transfer for optical continuous-variable systems, and prove its security in the noisy-storage model. This model allows us to establish security by sending more quantum signals than an attacker can reliably store during the protocol. The security proof is based on uncertainty relations which we...... derive for continuous-variable systems, that differ from the ones used in quantum key distribution. We experimentally demonstrate in a proof-of-principle experiment the proposed oblivious transfer protocol for various channel losses by using entangled two-mode squeezed states measured with balanced...

  9. Magnetic resonance imaging protocols for examination of the neurocranium at 3 T.

    Science.gov (United States)

    Schwindt, W; Kugel, H; Bachmann, R; Kloska, S; Allkemper, T; Maintz, D; Pfleiderer, B; Tombach, B; Heindel, W

    2003-09-01

    The increasing availability of high-field (3 T) MR scanners requires adapting and optimizing clinical imaging protocols to exploit the theoretically higher signal-to-noise ratio (SNR) of the higher field strength. Our aim was to establish reliable and stable protocols meeting the clinical demands for imaging the neurocranium at 3 T. Two hundred patients with a broad range of indications received an examination of the neurocranium with an appropriate assortment of imaging techniques at 3 T. Several imaging parameters were optimized. Keeping scan times comparable to those at 1.5 T we increased spatial resolution. Contrast-enhanced and non-enhanced T1-weighted imaging was best applying gradient-echo and inversion recovery (rather than spin-echo) techniques, respectively. For fluid-attenuated inversion recovery (FLAIR) imaging a TE of 120 ms yielded optimum contrast-to-noise ratio (CNR). High-resolution isotropic 3D data sets were acquired within reasonable scan times. Some artifacts were pronounced, but generally imaging profited from the higher SNR. We present a set of optimized examination protocols for neuroimaging at 3 T, which proved to be reliable in a clinical routine setting.

  10. Magnetic resonance imaging protocols for examination of the neurocranium at 3 T

    Energy Technology Data Exchange (ETDEWEB)

    Schwindt, W.; Kugel, H.; Bachmann, R.; Kloska, S.; Allkemper, T.; Maintz, D.; Pfleiderer, B.; Tombach, B.; Heindel, W. [Institut fuer Klinische Radiologie, Universitaetsklinikum Muenster, Albert-Schweitzer-Strasse 33, 48129, Muenster (Germany)

    2003-09-01

    The increasing availability of high-field (3 T) MR scanners requires adapting and optimizing clinical imaging protocols to exploit the theoretically higher signal-to-noise ratio (SNR) of the higher field strength. Our aim was to establish reliable and stable protocols meeting the clinical demands for imaging the neurocranium at 3 T. Two hundred patients with a broad range of indications received an examination of the neurocranium with an appropriate assortment of imaging techniques at 3 T. Several imaging parameters were optimized. Keeping scan times comparable to those at 1.5 T we increased spatial resolution. Contrast-enhanced and non-enhanced T1-weighted imaging was best applying gradient-echo and inversion recovery (rather than spin-echo) techniques, respectively. For fluid-attenuated inversion recovery (FLAIR) imaging a TE of 120 ms yielded optimum contrast-to-noise ratio (CNR). High-resolution isotropic 3D data sets were acquired within reasonable scan times. Some artifacts were pronounced, but generally imaging profited from the higher SNR. We present a set of optimized examination protocols for neuroimaging at 3 T, which proved to be reliable in a clinical routine setting. (orig.)

  11. Magnetic resonance imaging protocols for examination of the neurocranium at 3 T

    International Nuclear Information System (INIS)

    Schwindt, W.; Kugel, H.; Bachmann, R.; Kloska, S.; Allkemper, T.; Maintz, D.; Pfleiderer, B.; Tombach, B.; Heindel, W.

    2003-01-01

    The increasing availability of high-field (3 T) MR scanners requires adapting and optimizing clinical imaging protocols to exploit the theoretically higher signal-to-noise ratio (SNR) of the higher field strength. Our aim was to establish reliable and stable protocols meeting the clinical demands for imaging the neurocranium at 3 T. Two hundred patients with a broad range of indications received an examination of the neurocranium with an appropriate assortment of imaging techniques at 3 T. Several imaging parameters were optimized. Keeping scan times comparable to those at 1.5 T we increased spatial resolution. Contrast-enhanced and non-enhanced T1-weighted imaging was best applying gradient-echo and inversion recovery (rather than spin-echo) techniques, respectively. For fluid-attenuated inversion recovery (FLAIR) imaging a TE of 120 ms yielded optimum contrast-to-noise ratio (CNR). High-resolution isotropic 3D data sets were acquired within reasonable scan times. Some artifacts were pronounced, but generally imaging profited from the higher SNR. We present a set of optimized examination protocols for neuroimaging at 3 T, which proved to be reliable in a clinical routine setting. (orig.)

  12. Model-Checking Driven Design of QoS-Based Routing Protocol for Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    Zhi Chen

    2015-01-01

    Full Text Available Accurate and reliable routing protocols with Quality of Service (QoS support determine the mission-critical application efficiency in WSNs. This paper proposes a model-checking design driven framework for designing the QoS-based routing protocols of WSNs, which involves the light-weight design process, the timed automata model, and the alternative QoS verification properties. The accurate feedback of continually model checking in the iterative design process effectively stimulates the parameter tuning of the protocols. We demonstrate the straightforward and modular characteristics of the proposed framework in designing a prototype QoS-based routing protocol. The prototype study shows that the model-checking design framework may complement other design methods and ensure the QoS implementation of the QoS-based routing protocol design for WSNs.

  13. Appraisal of the sensitising potential of orally and dermally administered mercaptobenzothiazole by a biphasic protocol of the local lymph node assay.

    Science.gov (United States)

    Ahuja, Varun; Wanner, Reinhard; Platzek, Thomas; Stahlmann, Ralf

    2009-10-01

    Mercaptobenzothiazole (MBT) is used while manufacturing natural rubber products. Our study deals with assessing its allergenic potential following dermal and oral routes of exposure, using a biphasic local lymph node assay (LLNA). Female Balb/c mice were treated with MBT (dermally 3, 10, 30% concentrations in DMSO; orally 1, 10, 100 mg/kg doses in corn oil) on the back (dermal study) or through oral administration (oral study) on days 1-3 followed by auricular application of 3, 10 and 30% concentrations, respectively, on days 15-17. End points determined on day 19 included ear thickness, ear punch weight, lymph node weight, lymph node cell count, and lymphocyte subpopulations (CD4+, CD8+, CD45+). After dermal application of 3% or 10% solution, a significant increase in cell count and lymph node weight along with significant decrease in CD8+ cells was observed. After initial oral administration of 1 mg/kg, we noticed a significant amplification in cell count. Following oral administration of 10 mg/kg, we observed a similar increase in cell count and lymph node weight. The results of our study show that the modified biphasic LLNA protocol can be used to study the sensitising potential of a compound also following the oral route of exposure.

  14. Quantification of methanogenic biomass by enzyme-linked immunosorbent assay and by analysis of specific methanogenic cofactors

    Energy Technology Data Exchange (ETDEWEB)

    Gorris, L G.M.; Kemp, H A; Archer, D B

    1987-01-01

    The reliability and accuracy with which enzyme-linked immunosorbent assay (ELISA) and an assay of methanogenic cofactors detect and quantify methanogenic species were investigated. Both assays required standardization with laboratory cultures of methanogenic bacteria and were applied to mixtures of pure cultures and samples from anaerobic digesters. ELISA was shown to be a simple method for detecting and quantifying individual methanogenic species. The range of species which can be assayed is limited by the range of antisera available but, potentially, ELISA can be applied to all methanogens. Although the cofactor assay is not species-specific it can distinguish hydrogenotrophic and acetotrophic methanogens and is quantitative.

  15. Evaluation of the highly sensitive Roche thyroglobulin II assay and establishment of a reference limit for thyroglobulin-negative patient samples

    Directory of Open Access Journals (Sweden)

    Dorien M. Rotteveel-de Groot

    2016-08-01

    Full Text Available Objectives: Thyroglobulin (Tg measurements are used to monitor for residual thyroid tissue in patients with differentiated thyroid cancer (DTC after thyroidectomy and radioiodine ablative therapy. In recent years highly sensitive Tg assays have been developed. In this study the analytical performance of the new Roche Elecsys Tg II assay was evaluated and compared with the well documented Access2 Tg assay (Beckman–Coulter. Design and methods: Analytical performance was examined using various Clinical and Laboratory Standards Institute (CLSI evaluation protocols. Tg negative patient sera were used to establish an upper reference limit (URL for the Elecsys Tg II assay. Results: Non-linearity, drift and carry-over according to CLSI EP10 and EP6 in a measuring range of 0.04–500 ng/mL were non-significant. Total precision according to CLSI EP5 was 10% at a Tg concentration of 0.08 ng/mL. A patient serum comparison performed according to a modified CLSI EP9 protocol showed a significant difference of a factor of approximately 1.4, despite using an identical CRM calibrator. The Elecsys Tg II assay measured Tg with a two-fold higher sensitivity than the Access2 assay. Finally, using human sera without Tg, an URL of 0.05 ng/mL was determined. Conclusions: In our hands the highly sensitive Elecsys Tg II assay shows a good analytical performance and a higher sensitivity compared to the Access2 Tg assay. An URL of 0.05 ng/mL for the Elecsys Tg II assay was determined which may improve the clinical utility of the assay for the detection of residual DTC or disease recurrence. Keywords: Thyroglobulin, Roche Elecsys Tg II assay, validation, reporting limit

  16. Framework and indicator testing protocol for developing and piloting quality indicators for the UK quality and outcomes framework

    Directory of Open Access Journals (Sweden)

    Burke Martyn

    2011-08-01

    Full Text Available Abstract Background Quality measures should be subjected to a testing protocol before being used in practice using key attributes such as acceptability, feasibility and reliability, as well as identifying issues derived from actual implementation and unintended consequences. We describe the methodologies and results of an indicator testing protocol (ITP using data from proposed quality indicators for the United Kingdom Quality and Outcomes Framework (QOF. Methods The indicator testing protocol involved a multi-step and methodological process: 1 The RAND/UCLA Appropriateness Method, to test clarity and necessity, 2 data extraction from patients' medical records, to test technical feasibility and reliability, 3 diaries, to test workload, 4 cost-effectiveness modelling, and 5 semi-structured interviews, to test acceptability, implementation issues and unintended consequences. Testing was conducted in a sample of representative family practices in England. These methods were combined into an overall recommendation for each tested indicator. Results Using an indicator testing protocol as part of piloting was seen as a valuable way of testing potential indicators in 'real world' settings. Pilot 1 (October 2009-March 2010 involved thirteen indicators across six clinical domains and twelve indicators passed the indicator testing protocol. However, the indicator testing protocol identified a number of implementation issues and unintended consequences that can be rectified or removed prior to national roll out. A palliative care indicator is used as an exemplar of the value of piloting using a multiple attribute indicator testing protocol - while technically feasible and reliable, it was unacceptable to practice staff and raised concerns about potentially causing actual patient harm. Conclusions This indicator testing protocol is one example of a protocol that may be useful in assessing potential quality indicators when adapted to specific country health

  17. A new gaseous imaging detector for the assay of lymphocyte cultures

    International Nuclear Information System (INIS)

    Bateman, J.E.; Joyce, A.; Knight, S.C.; Bedford, P.

    1991-01-01

    Tritium-labelled cell cultures used in studies of lymphocyte proliferation at the Clinical Research Centre are blotted in arrays of 10x6 spots spaced at 6 mm. An imaging detector based on the differential induction signals produced at a central amplifying electrode has been developed for the imaging and assay of these blots. A spatial resolution ≅ 2.5 mm FWHM attained over the aperture of 60 mmx36mm enables the individual spots to be reliably counted. Data is captured in a PC/AT at rates which permit an assay to be completed in typically 30-60 min. The simplicity of both the detector and the readout electronics leads to a low cost system. Images and assay results are presented. (orig.)

  18. A Direct, Competitive Enzyme-Linked Immunosorbent Assay (ELISA) as a Quantitative Technique for Small Molecules

    Science.gov (United States)

    Powers, Jennifer L.; Rippe, Karen Duda; Imarhia, Kelly; Swift, Aileen; Scholten, Melanie; Islam, Naina

    2012-01-01

    ELISA (enzyme-linked immunosorbent assay) is a widely used technique with applications in disease diagnosis, detection of contaminated foods, and screening for drugs of abuse or environmental contaminants. However, published protocols with a focus on quantitative detection of small molecules designed for teaching laboratories are limited. A…

  19. A Passive Testing Approach for Protocols in Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    Xiaoping Che

    2015-11-01

    Full Text Available Smart systems are today increasingly developed with the number of wireless sensor devices drastically increasing. They are implemented within several contexts throughout our environment. Thus, sensed data transported in ubiquitous systems are important, and the way to carry them must be efficient and reliable. For that purpose, several routing protocols have been proposed for wireless sensor networks (WSN. However, one stage that is often neglected before their deployment is the conformance testing process, a crucial and challenging step. Compared to active testing techniques commonly used in wired networks, passive approaches are more suitable to the WSN environment. While some works propose to specify the protocol with state models or to analyze them with simulators and emulators, we here propose a logic-based approach for formally specifying some functional requirements of a novel WSN routing protocol. We provide an algorithm to evaluate these properties on collected protocol execution traces. Further, we demonstrate the efficiency and suitability of our approach by its application into common WSN functional properties, as well as specific ones designed from our own routing protocol. We provide relevant testing verdicts through a real indoor testbed and the implementation of our protocol. Furthermore, the flexibility, genericity and practicability of our approach have been proven by the experimental results.

  20. Inter-assessor reliability of practice based biomechanical assessment of the foot and ankle

    Directory of Open Access Journals (Sweden)

    Jarvis Hannah L

    2012-06-01

    Full Text Available Abstract Background There is no consensus on which protocols should be used to assess foot and lower limb biomechanics in clinical practice. The reliability of many assessments has been questioned by previous research. The aim of this investigation was to (i identify (through consensus what biomechanical examinations are used in clinical practice and (ii evaluate the inter-assessor reliability of some of these examinations. Methods Part1: Using a modified Delphi technique 12 podiatrists derived consensus on the biomechanical examinations used in clinical practice. Part 2: Eleven podiatrists assessed 6 participants using a subset of the assessment protocol derived in Part 1. Examinations were compared between assessors. Results Clinicians choose to estimate rather than quantitatively measure foot position and motion. Poor inter-assessor reliability was recorded for all examinations. Intra-class correlation coefficient values (ICC for relaxed calcaneal stance position were less than 0.23 and were less than 0.14 for neutral calcaneal stance position. For the examination of ankle joint dorsiflexion, ICC values suggest moderate reliability (less than 0.61. The results of a random effects ANOVA highlight that participant (up to 5.7°, assessor (up to 5.8° and random (up to 5.7° error all contribute to the total error (up to 9.5° for relaxed calcaneal stance position, up to 10.7° for the examination of ankle joint dorsiflexion. Kappa Fleiss values for categorisation of first ray position and mobility were less than 0.05 and for limb length assessment less than 0.02, indicating slight agreement. Conclusion Static biomechanical assessment of the foot, leg and lower limb is an important protocol in clinical practice, but the key examinations used to make inferences about dynamic foot function and to determine orthotic prescription are unreliable.

  1. Methodology for benzodiazepine receptor binding assays at physiological temperature. Rapid change in equilibrium with falling temperature

    International Nuclear Information System (INIS)

    Dawson, R.M.

    1986-01-01

    Benzodiazepine receptors of rat cerebellum were assayed with [ 3 H]-labeled flunitrazepam at 37 0 C, and assays were terminated by filtration in a cold room according to one of three protocols: keeping each sample at 37 degrees C until ready for filtration, taking the batch of samples (30) into the cold room and filtering sequentially in the order 1-30, and taking the batch of 30 samples into the cold room and filtering sequentially in the order 30-1. the results for each protocol were substantially different from each other, indicating that rapid disruption of equilibrium occurred as the samples cooled in the cold room while waiting to be filtered. Positive or negative cooperativity of binding was apparent, and misleading effects of gamma-aminobutyric acid on the affinity of diazepam were observed, unless each sample was kept at 37 0 C until just prior to filtration

  2. Flow cytometry protocol to evaluate ionizing radiation effects on P-glycoprotein activity

    International Nuclear Information System (INIS)

    Santos, Neyliane Goncalves dos; Amaral, Ademir; Cavalcanti, Mariana Brayner . E-mail; Neves, Maria Amelia Batista; Machado, Cintia Gonsalves de Faria

    2008-01-01

    The aim of this work was to establish a protocol to evaluate ionizing radiation effects on P-glycoprotein (P-gp) activity. For this, human peripheral blood samples were irradiated in vitro with different doses and P-gp activity was analyzed for CD4 and CD8 T lymphocytes through rhodamine123-efflux assay by flow cytometry. By simultaneous employment of percentage and mean fluorescence index parameters, subject-by-subject analysis pointed out changes in P-gp activity for some individuals and irradiated samples. Based on this work, the proposed protocol was considered adequate for evaluating P-gp activity on cells after radioactive stress. Besides, this research suggests that P-gp activity could be an important factor to define patient-specific protocols in combined chemo- and radiotherapy, particularly when radiation exposure precedes chemical treatment. (author)

  3. Flow cytometry protocol to evaluate ionizing radiation effects on P-glycoprotein activity

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Neyliane Goncalves dos; Amaral, Ademir; Cavalcanti, Mariana Brayner [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Dept. de Energia Nuclear]. E-mail; neylisantos@yahoo.com.br; Neves, Maria Amelia Batista; Machado, Cintia Gonsalves de Faria [Fundacao de Hematologia e Hemoterapia de Pernambuco, Recife, PE (Brazil). Unidade de Laboratorios Especializados. Lab. de Imunofenotipagem

    2008-12-15

    The aim of this work was to establish a protocol to evaluate ionizing radiation effects on P-glycoprotein (P-gp) activity. For this, human peripheral blood samples were irradiated in vitro with different doses and P-gp activity was analyzed for CD4 and CD8 T lymphocytes through rhodamine123-efflux assay by flow cytometry. By simultaneous employment of percentage and mean fluorescence index parameters, subject-by-subject analysis pointed out changes in P-gp activity for some individuals and irradiated samples. Based on this work, the proposed protocol was considered adequate for evaluating P-gp activity on cells after radioactive stress. Besides, this research suggests that P-gp activity could be an important factor to define patient-specific protocols in combined chemo- and radiotherapy, particularly when radiation exposure precedes chemical treatment. (author)

  4. Treatment of attention deficit hyperactivity disorder with monoamine amino acid precursors and organic cation transporter assay interpretation

    Directory of Open Access Journals (Sweden)

    Marty Hinz

    2011-01-01

    Full Text Available Marty Hinz1, Alvin Stein2, Robert Neff3, Robert Weinberg4, Thomas Uncini51NeuroResearch Clinics Inc, Cape Coral, FL; 2Stein Orthopedic Associates, Plantation, FL; 3Mental Training Inc, Dallas, TX; 4Department of Kinesiology and Health, Miami University, Oxford, OH; 5Laboratory, Fairview Regional Medical Center-Mesabi, Hibbing, MN, USABackground: This paper documents a retrospective pilot study of a novel approach for treating attention deficit hyperactivity disorder (ADHD with amino acid precursors of serotonin and dopamine in conjunction with urinary monoamine assays subjected to organic cation transporter (OCT functional status determination. The goal of this research was to document the findings and related considerations of a retrospective chart review study designed to identify issues and areas of concern that will define parameters for a prospective controlled study.Methods: This study included 85 patients, aged 4–18 years, who were treated with a novel amino acid precursor protocol. Their clinical course during the first 8–10 weeks of treatment was analyzed retrospectively. The study team consisted of PhD clinical psychologists, individuals compiling clinical data from records, and a statistician. The patients had been treated with a predefined protocol for administering amino acid precursors of serotonin and dopamine, along with OCT assay interpretation as indicated.Results: In total, 67% of participants achieved significant improvement with only amino acid precursors of serotonin and dopamine. In patients who achieved no significant relief of symptoms with only amino acid precursors, OCT assay interpretation was utilized. In this subgroup, 30.3% achieved significant relief following two or three urine assays and dosage changes as recommended by the assay results. The total percentage of patients showing significant improvement was 77%.Conclusion: The efficacy of this novel protocol appears superior to some ADHD prescription drugs

  5. ASAP: A MAC Protocol for Dense and Time-Constrained RFID Systems

    Directory of Open Access Journals (Sweden)

    Kyounghwan Lee

    2007-08-01

    Full Text Available We introduce a novel medium access control (MAC protocol for radio frequency identification (RFID systems which exploits the statistical information collected at the reader. The protocol, termed adaptive slotted ALOHA protocol (ASAP, is motivated by the need to significantly improve the total read time performance of the currently suggested MAC protocols for RFID systems. In order to accomplish this task, ASAP estimates the dynamic tag population and adapts the frame size in the subsequent round via a simple policy that maximizes an appropriately defined efficiency function. We demonstrate that ASAP provides significant improvement in total read time performance over the current RFID MAC protocols. We next extend the design to accomplish reliable performance of ASAP in realistic scenarios such as the existence of constraints on frame size, and mobile RFID systems where tags move at constant velocity in the reader's field. We also consider the case where tags may fail to respond because of a physical breakdown or a temporary malfunction, and show the robustness in those scenarios as well.

  6. FOG: Fighting the Achilles' Heel of Gossip Protocols with Fountain Codes

    Science.gov (United States)

    Champel, Mary-Luc; Kermarrec, Anne-Marie; Le Scouarnec, Nicolas

    Gossip protocols are well known to provide reliable and robust dissemination protocols in highly dynamic systems. Yet, they suffer from high redundancy in the last phase of the dissemination. In this paper, we combine fountain codes (rateless erasure-correcting codes) together with gossip protocols for a robust and fast content dissemination in large-scale dynamic systems. The use of fountain enables to eliminate the unnecessary redundancy of gossip protocols. We propose the design of FOG, which fully exploits the first exponential growth phase (where the data is disseminated exponentially fast) of gossip protocols while avoiding the need for the shrinking phase by using fountain codes. FOG voluntarily increases the number of disseminations but limits those disseminations to the exponential growth phase. In addition, FOG creates a split-graph overlay that splits the peers between encoders and forwarders. Forwarder peers become encoders as soon as they have received the whole content. In order to benefit even further and quicker from encoders, FOG biases the dissemination towards the most advanced peers to make them complete earlier.

  7. Evaluation of the Branched-Chain DNA Assay for Measurement of RNA in Formalin-Fixed Tissues

    Science.gov (United States)

    Knudsen, Beatrice S.; Allen, April N.; McLerran, Dale F.; Vessella, Robert L.; Karademos, Jonathan; Davies, Joan E.; Maqsodi, Botoul; McMaster, Gary K.; Kristal, Alan R.

    2008-01-01

    We evaluated the branched-chain DNA (bDNA) assay QuantiGene Reagent System to measure RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. The QuantiGene Reagent System does not require RNA isolation, avoids enzymatic preamplification, and has a simple workflow. Five selected genes were measured by bDNA assay; quantitative polymerase chain reaction (qPCR) was used as a reference method. Mixed-effect statistical models were used to partition the overall variance into components attributable to xenograft, sample, and assay. For FFPE tissues, the coefficients of reliability were significantly higher for the bDNA assay (93–100%) than for qPCR (82.4–95%). Correlations between qPCRFROZEN, the gold standard, and bDNAFFPE ranged from 0.60 to 0.94, similar to those from qPCRFROZEN and qPCRFFPE. Additionally, the sensitivity of the bDNA assay in tissue homogenates was 10-fold higher than in purified RNA. In 9- to 13-year-old blocks with poor RNA quality, the bDNA assay allowed the correct identification of the overexpression of known cancer genes. In conclusion, the QuantiGene Reagent System is considerably more reliable, reproducible, and sensitive than qPCR, providing an alternative method for the measurement of gene expression in FFPE tissues. It also appears to be well suited for the clinical analysis of FFPE tissues with diagnostic or prognostic gene expression biomarker panels for use in patient treatment and management. PMID:18276773

  8. Implementation and Use of State-of-the-Art, Cell-Based In Vitro Assays.

    Science.gov (United States)

    Langer, Gernot

    2016-01-01

    The impressive advances in the generation and interpretation of functional omics data have greatly contributed to a better understanding of the (patho-)physiology of many biological systems and led to a massive increase in the number of specific targets and phenotypes to investigate in both basic and applied research. The obvious complexity revealed by these studies represents a major challenge to the research community and asks for improved target characterisation strategies with the help of reliable, high-quality assays. Thus, the use of living cells has become an integral part of many research activities because the cellular context more closely represents target-specific interrelations and activity patterns. Although still predominant, the use of traditional two-dimensional (2D) monolayer cell culture models has been gradually complemented by studies based on three-dimensional (3D) spheroid (Sutherland 1988) and other 3D tissue culture systems (Santos et al. 2012; Matsusaki et al. 2014) in an attempt to employ model systems more closely representing the microenvironment of cells in the body. Hence, quite a variety of state-of-the-art cell culture models are available for the generation of novel chemical probes or the identification of starting points for drug development in translational research and pharma drug discovery. In order to cope with these information-rich formats and their increasing technical complexity, cell-based assay development has become a scientific research topic in its own right and is used to ensure the provision of significant, reliable and high-quality data outlasting any discussions related to the current "irreproducibility epidemic" (Dolgin 2014; Prinz et al. 2011; Schatz 2014). At the same time the use of cells in microplate assay formats has become state of the art and greatly facilitates rigorous cell-based assay development by providing the researcher with the opportunity to address the multitude of factors affecting the actual

  9. Treatability and scale-up protocols for polynuclear aromatic hydrocarbon bioremediation of manufactured-gas-plant soils. Final report, September 1987-July 1991

    International Nuclear Information System (INIS)

    Blackburn, J.W.; DiGrazia, P.M.; Sanseverino, J.

    1991-07-01

    The report describes activities to develop a framework to reliably scale-up and apply challenging bioremediation processes to polynuclear aromatic hydrocarbons in Manufactured Gas Plant (MGP) soils. It includes: a discussion of the accuracy needed for competitive application of bioremediation; a framework and examples for treatability and scale-up protocols for selection, design and application of these processes; both batch and continuous testing protocols for developing predictive rate data; and special predictive relationships that may be used in process selection/scale-up. The work, coupled with subsequent work (as recommended) to develop an MGP soil desorption/diffusion protocol and new scale-up methods, and with subsequent scale-up testing should lead to the capability for improved selection of MGP sites for bioremediation and improved performance, success, and reliability of field applications. With this greater predictive reliability, bioremediation will be used more often in the field on the most favorable applications and its cost advantages over other remediation options will be realized

  10. Frequent sgRNA-barcode recombination in single-cell perturbation assays.

    Directory of Open Access Journals (Sweden)

    Shiqi Xie

    Full Text Available Simultaneously detecting CRISPR-based perturbations and induced transcriptional changes in the same cell is a powerful approach to unraveling genome function. Several lentiviral approaches have been developed, some of which rely on the detection of distally located genetic barcodes as an indirect proxy of sgRNA identity. Since barcodes are often several kilobases from their corresponding sgRNAs, viral recombination-mediated swapping of barcodes and sgRNAs is feasible. Using a self-circularization-based sgRNA-barcode library preparation protocol, we estimate the recombination rate to be ~50% and we trace this phenomenon to the pooled viral packaging step. Recombination is random, and decreases the signal-to-noise ratio of the assay. Our results suggest that alternative approaches can increase the throughput and sensitivity of single-cell perturbation assays.

  11. Problems and challenges in the development and validation of human cell-based assays to determine nanoparticle-induced immunomodulatory effects

    Directory of Open Access Journals (Sweden)

    Rossi François

    2011-02-01

    Full Text Available Abstract Background With the increasing use of nanomaterials, the need for methods and assays to examine their immunosafety is becoming urgent, in particular for nanomaterials that are deliberately administered to human subjects (as in the case of nanomedicines. To obtain reliable results, standardised in vitro immunotoxicological tests should be used to determine the effects of engineered nanoparticles on human immune responses. However, before assays can be standardised, it is important that suitable methods are established and validated. Results In a collaborative work between European laboratories, existing immunological and toxicological in vitro assays were tested and compared for their suitability to test effects of nanoparticles on immune responses. The prototypical nanoparticles used were metal (oxide particles, either custom-generated by wet synthesis or commercially available as powders. Several problems and challenges were encountered during assay validation, ranging from particle agglomeration in biological media and optical interference with assay systems, to chemical immunotoxicity of solvents and contamination with endotoxin. Conclusion The problems that were encountered in the immunological assay systems used in this study, such as chemical or endotoxin contamination and optical interference caused by the dense material, significantly affected the data obtained. These problems have to be solved to enable the development of reliable assays for the assessment of nano-immunosafety.

  12. An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay

    DEFF Research Database (Denmark)

    Johansson, Clara; Møller, Peter; Forchhammer, Lykke

    2010-01-01

    The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due...... to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet...... assay end points to number of lesions/10(6) bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA...

  13. ADAPTIVE GOSSIP BASED PROTOCOL FOR ENERGY EFFICIENT MOBILE ADHOC NETWORK

    Directory of Open Access Journals (Sweden)

    S. Rajeswari

    2012-03-01

    Full Text Available In Gossip Sleep Protocol, network performance is enhanced based on energy resource. But energy conservation is achieved with the reduced throughput. In this paper, it has been proposed a new Protocol for Mobile Ad hoc Network to achieve reliability with energy conservation. Based on the probability (p values, the value of sleep nodes is fixed initially. The probability value can be adaptively adjusted by Remote Activated Switch during the transmission process. The adaptiveness of gossiping probability is determined by the Packet Delivery Ratio. For performance comparison, we have taken Routing overhead, Packet Delivery Ratio, Number of dropped packets and Energy consumption with the increasing number of forwarding nodes. We used UDP based traffic models to analyze the performance of this protocol. We analyzed TCP based traffic models for average end to end delay. We have used the NS-2 simulator.

  14. Novel assays to assess the functional capacity of the classical, the alternative and the lectin pathways of the complement system

    DEFF Research Database (Denmark)

    Palarasah, Y; Nielsen, C; Sprogøe, U

    2011-01-01

    is therefore of great importance. In this study, we present novel improved enzyme-linked immunosorbent assays for the functional assessment of the three individual pathways of the complement system. The method is applicable at high serum concentrations and we demonstrate that it minimizes both false negative...... as well as false positive results. In particular, for the functional mannose-binding lectin activity it represents an improvement on the existing assays. In this respect, the present assays represent novel improved diagnostic protocols for patients with suspected immunodeficiencies related...

  15. Evaluation of condyle defects using different reconstruction protocols of cone-beam computed tomography

    International Nuclear Information System (INIS)

    Bastos, Luana Costa; Campos, Paulo Sergio Flores; Ramos-Perez, Flavia Maria de Moraes; Pontual, Andrea dos Anjos; Almeida, Solange Maria

    2013-01-01

    This study was conducted to investigate how well cone-beam computed tomography (CBCT) can detect simulated cavitary defects in condyles, and to test the influence of the reconstruction protocols. Defects were created with spherical diamond burs (numbers 1013, 1016, 3017) in superior and / or posterior surfaces of twenty condyles. The condyles were scanned, and cross-sectional reconstructions were performed with nine different protocols, based on slice thickness (0.2, 0.6, 1.0 mm) and on the filters (original image, Sharpen Mild, S9) used. Two observers evaluated the defects, determining their presence and location. Statistical analysis was carried out using simple Kappa coefficient and McNemar’s test to check inter- and intra-rater reliability. The chi-square test was used to compare the rater accuracy. Analysis of variance (Tukey's test) assessed the effect of the protocols used. Kappa values for inter- and intra-rater reliability demonstrate almost perfect agreement. The proportion of correct answers was significantly higher than that of errors for cavitary defects on both condyle surfaces (p < 0.01). Only in identifying the defects located on the posterior surface was it possible to observe the influence of the 1.0 mm protocol thickness and no filter, which showed a significantly lower value. Based on the results of the current study, the technique used was valid for identifying the existence of cavities in the condyle surface. However, the protocol of a 1.0 mm-thick slice and no filter proved to be the worst method for identifying the defects on the posterior surface. (author)

  16. Evaluation of condyle defects using different reconstruction protocols of cone-beam computed tomography

    Energy Technology Data Exchange (ETDEWEB)

    Bastos, Luana Costa; Campos, Paulo Sergio Flores, E-mail: bastosluana@ymail.com [Universidade Federal da Bahia (UFBA), Salvador, BA (Brazil). Fac. de Odontologia. Dept. de Radiologia Oral e Maxilofacial; Ramos-Perez, Flavia Maria de Moraes [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Fac. de Odontologia. Dept. de Clinica e Odontologia Preventiva; Pontual, Andrea dos Anjos [Universidade Federal de Pernambuco (UFPE), Camaragibe, PE (Brazil). Fac. de Odontologia. Dept. de Radiologia Oral; Almeida, Solange Maria [Universidade Estadual de Campinas (UNICAMP), Piracicaba, SP (Brazil). Fac. de Odontologia. Dept. de Radiologia Oral

    2013-11-15

    This study was conducted to investigate how well cone-beam computed tomography (CBCT) can detect simulated cavitary defects in condyles, and to test the influence of the reconstruction protocols. Defects were created with spherical diamond burs (numbers 1013, 1016, 3017) in superior and / or posterior surfaces of twenty condyles. The condyles were scanned, and cross-sectional reconstructions were performed with nine different protocols, based on slice thickness (0.2, 0.6, 1.0 mm) and on the filters (original image, Sharpen Mild, S9) used. Two observers evaluated the defects, determining their presence and location. Statistical analysis was carried out using simple Kappa coefficient and McNemar’s test to check inter- and intra-rater reliability. The chi-square test was used to compare the rater accuracy. Analysis of variance (Tukey's test) assessed the effect of the protocols used. Kappa values for inter- and intra-rater reliability demonstrate almost perfect agreement. The proportion of correct answers was significantly higher than that of errors for cavitary defects on both condyle surfaces (p < 0.01). Only in identifying the defects located on the posterior surface was it possible to observe the influence of the 1.0 mm protocol thickness and no filter, which showed a significantly lower value. Based on the results of the current study, the technique used was valid for identifying the existence of cavities in the condyle surface. However, the protocol of a 1.0 mm-thick slice and no filter proved to be the worst method for identifying the defects on the posterior surface. (author)

  17. Learned helplessness: validity and reliability of depressive-like states in mice.

    Science.gov (United States)

    Chourbaji, S; Zacher, C; Sanchis-Segura, C; Dormann, C; Vollmayr, B; Gass, P

    2005-12-01

    The learned helplessness paradigm is a depression model in which animals are exposed to unpredictable and uncontrollable stress, e.g. electroshocks, and subsequently develop coping deficits for aversive but escapable situations (J.B. Overmier, M.E. Seligman, Effects of inescapable shock upon subsequent escape and avoidance responding, J. Comp. Physiol. Psychol. 63 (1967) 28-33 ). It represents a model with good similarity to the symptoms of depression, construct, and predictive validity in rats. Despite an increased need to investigate emotional, in particular depression-like behaviors in transgenic mice, so far only a few studies have been published using the learned helplessness paradigm. One reason may be the fact that-in contrast to rats (B. Vollmayr, F.A. Henn, Learned helplessness in the rat: improvements in validity and reliability, Brain Res. Brain Res. Protoc. 8 (2001) 1-7)--there is no generally accepted learned helplessness protocol available for mice. This prompted us to develop a reliable helplessness procedure in C57BL/6N mice, to exclude possible artifacts, and to establish a protocol, which yields a consistent fraction of helpless mice following the shock exposure. Furthermore, we validated this protocol pharmacologically using the tricyclic antidepressant imipramine. Here, we present a mouse model with good face and predictive validity that can be used for transgenic, behavioral, and pharmacological studies.

  18. Qualification of a select one-stage activated partial thromboplastin time-based clotting assay and two chromogenic assays for the post-administration monitoring of nonacog beta pegol.

    Science.gov (United States)

    Tiefenbacher, S; Bohra, R; Amiral, J; Bowyer, A; Kitchen, S; Lochu, A; Rosén, S; Ezban, M

    2017-10-01

    Essentials Nonacog beta pegol (N9-GP) is an extended half-life, recombinant human factor IX (FIX). One-stage clotting (OSC) and chromogenic FIX activity assays were assessed for N9-GP recovery. OSC STA ® -Cephascreen ® , ROX FIX and BIOPHEN FIX chromogenic assays were qualified for N9-GP. Other extended half-life factor products should be assessed in a similar way prior to approval. Background Nonacog beta pegol (N9-GP) is an extended half-life, glycoPEGylated recombinant human factor IX that is under development for the prophylaxis and treatment of bleeding episodes in hemophilia B patients. Considerable reagent-dependent variability has been observed when one-stage clotting assays are used to measure the recovery of recombinant FIX products, including N9-GP. Objective To qualify select one-stage clotting and chromogenic FIX activity assays for measuring N9-GP recovery. Methods The accuracy and precision of the one-stage clotting assay (with the STA-Cephascreen activated partial thromboplastin [APTT] reagent) and the ROX Factor IX and BIOPHEN Factor IX chromogenic assays for measuring N9-GP recovery were assessed in N9-GP-spiked hemophilia B plasma samples in a systematic manner at three independent sites, with manufacturer-recommended protocols and/or site-specific assay setups, including different instruments. Results For each of the three FIX activity assays qualified on five different reagent-instrument systems, acceptable intra-assay and interassay accuracy and precision, dilution integrity, reagent robustness and freeze-thaw and short-term sample stabilities were demonstrated. The STA-Cephascreen assay showed a limited reportable range at one of the three qualification sites, and the BIOPHEN Factor IX assay showed suspect low-end sensitivity at one of the three qualification sites. An individual laboratory would account for these limitations by adjusting the assay's reportable range; thus, these findings are not considered to impact the respective

  19. A rapid qualitative assay for detection of Clostridium perfringens in canned food products.

    Science.gov (United States)

    Dave, Gayatri Ashwinkumar

    2017-01-01

    Clostridium perfringens (MTCC 1349) is a Gram-positive, anaerobic, endospore forming, and rod-shaped bacterium. This bacterium produces a variety of toxins under strict anaerobic environment. C. perfringens can grow at temperatures ranging between 20°C and 50°C. It is the major causetive agent for gas gangrene, cellulitis, septicemia, necrotic enteritis and food poisoning, which are common toxin induced conditions noted in human and animals. C. perfringens can produce produce four major types of toxins that are used for the classification of strains, classified under type A-E. Across the globe many countries, including the United States, are affected by C. perfringens food poisonings where it is ranked as one of the most common causes of food borne infections. To date, no direct one step assay for the detection of C. perfringens has been developed and only few methods are known for accurate detection of C. perfringens. Long detection and incubation time is the major consideration of these reporter assays. The prensent study proposes a rapid and reliable colorimetric assay for the detection of C. perfringens. In principale, this assay detects the para nitrophenyl (yellow colour end product) liberated due to the hydrolysis of paranitrophenyl phosphetidyl choline (PNPC) through phospholipase C (lecithinase). Constitutive secretion of phospholipase C is a charactristic feature of C. perfringens. This assay detects the presence of the extracellular lecithinse through the PNPC impragnated impregnated probe. The probe is impregnated with peranitrophenyl phosphotidyl choline ester, which is colourless substrate used by lecithinase. The designed assay is specific towards PNPC and detectes very small quantites of lecithinase under conditions used. The reaction is substrate specific, no cross reaction was observed upon incubation with other substrates. In addition, this assay gave negative results with other clostridium strains, no cross reactions were observed with other

  20. Lightweight UDP Pervasive Protocol in Smart Home Environment Based on Labview

    Science.gov (United States)

    Kurniawan, Wijaya; Hannats Hanafi Ichsan, Mochammad; Rizqika Akbar, Sabriansyah; Arwani, Issa

    2017-04-01

    TCP (Transmission Control Protocol) technology in a reliable environment was not a problem, but not in an environment where the entire Smart Home network connected locally. Currently employing pervasive protocols using TCP technology, when data transmission is sent, it would be slower because they have to perform handshaking process in advance and could not broadcast the data. On smart home environment, it does not need large size and complex data transmission between monitoring site and monitoring center required in Smart home strain monitoring system. UDP (User Datagram Protocol) technology is quick and simple on data transmission process. UDP can broadcast messages because the UDP did not require handshaking and with more efficient memory usage. LabVIEW is a programming language software for processing and visualization of data in the field of data acquisition. This paper proposes to examine Pervasive UDP protocol implementations in smart home environment based on LabVIEW. UDP coded in LabVIEW and experiments were performed on a PC and can work properly.

  1. Simple algorithm for improved security in the FDDI protocol

    Science.gov (United States)

    Lundy, G. M.; Jones, Benjamin

    1993-02-01

    We propose a modification to the Fiber Distributed Data Interface (FDDI) protocol based on a simple algorithm which will improve confidential communication capability. This proposed modification provides a simple and reliable system which exploits some of the inherent security properties in a fiber optic ring network. This method differs from conventional methods in that end to end encryption can be facilitated at the media access control sublayer of the data link layer in the OSI network model. Our method is based on a variation of the bit stream cipher method. The transmitting station takes the intended confidential message and uses a simple modulo two addition operation against an initialization vector. The encrypted message is virtually unbreakable without the initialization vector. None of the stations on the ring will have access to both the encrypted message and the initialization vector except the transmitting and receiving stations. The generation of the initialization vector is unique for each confidential transmission and thus provides a unique approach to the key distribution problem. The FDDI protocol is of particular interest to the military in terms of LAN/MAN implementations. Both the Army and the Navy are considering the standard as the basis for future network systems. A simple and reliable security mechanism with the potential to support realtime communications is a necessary consideration in the implementation of these systems. The proposed method offers several advantages over traditional methods in terms of speed, reliability, and standardization.

  2. The Single-Molecule Centroid Localization Algorithm Improves the Accuracy of Fluorescence Binding Assays.

    Science.gov (United States)

    Hua, Boyang; Wang, Yanbo; Park, Seongjin; Han, Kyu Young; Singh, Digvijay; Kim, Jin H; Cheng, Wei; Ha, Taekjip

    2018-03-13

    Here, we demonstrate that the use of the single-molecule centroid localization algorithm can improve the accuracy of fluorescence binding assays. Two major artifacts in this type of assay, i.e., nonspecific binding events and optically overlapping receptors, can be detected and corrected during analysis. The effectiveness of our method was confirmed by measuring two weak biomolecular interactions, the interaction between the B1 domain of streptococcal protein G and immunoglobulin G and the interaction between double-stranded DNA and the Cas9-RNA complex with limited sequence matches. This analysis routine requires little modification to common experimental protocols, making it readily applicable to existing data and future experiments.

  3. Rapid multiple immunoenzyme assay of mycotoxins.

    Science.gov (United States)

    Urusov, Alexandr E; Zherdev, Anatoly V; Petrakova, Alina V; Sadykhov, Elchin G; Koroleva, Olga V; Dzantiev, Boris B

    2015-01-27

    Mycotoxins are low molecular weight fungal metabolites that pose a threat as toxic contaminants of food products, thereby necessitating their effective monitoring and control. Microplate ELISA can be used for this purpose, but this method is characteristically time consuming, with a duration extending to several hours. This report proposes a variant of the ELISA method for the detection and quantification of three mycotoxins, ochratoxin A, aflatoxin B1 and zearalenone, in the kinetic regime. The main requirement for the proposed kinetic protocol was to provide a rapid method that combined sensitivity and accuracy. The use of biotin with an extended spacer together with a streptavidin-polyperoxidase conjugate provided high signal levels, despite these interactions occurring under non-equilibrium conditions. Duration of the individual mycotoxin assays was 20 min, whereas the analysis of all three mycotoxins in parallel reached a maximum duration of 25 min. Recovery of at least 95% mycotoxins in water-organic extracts was shown. The developed assays were successfully validated using poultry processing products and corn samples spiked with known quantities of mycotoxins. The detection limits for aflatoxin B1, ochratoxin A and zearalenone in these substances were 0.24, 1.2 and 3 ng/g, respectively.

  4. Further Development and Validation of the Frog Embryo Teratogenesis Assay - Xenopus (FETAX). Phase III

    National Research Council Canada - National Science Library

    Bantle, John

    1996-01-01

    This interlaboratory study of the Frog Embryo Teratogenesis Assay (FETAX) was undertaken in order to assess the repeatability and reliability of data collected under the guide published by the American Society for Testing and Materials...

  5. Reliability and number of trials of Y Balance Test in adolescent athletes.

    Science.gov (United States)

    Linek, Pawel; Sikora, Damian; Wolny, Tomasz; Saulicz, Edward

    2017-10-01

    The Star Excursion Balance Test (SEBT) is commonly used to evaluate dynamic equilibrium. The Y Balance Test (Y-BT) is a shortened version of the SEBT where a Y- Balance Kit is commonly used. To date, research concerning the protocol and reliability of the SEBT and Y-BT has been conducted only for adults. The aim of the study was to assess the protocol (the necessary number of trials to stabilize the results) and reliability of the Y-BT in adolescent athletes. One-way repeated-measures analysis of variance (ANOVA) and reliability study. The sample of 38 athletes (mean age: 15.6 years) was selected from a football club. A Y-Balance test kit was applied for the evaluation of dynamic balance. The analysis used the values normalized to the relative length of the lower limbs. After six attempts, three consecutive ones achieved stability for all directions and both extremities (p > 0.05). The intraclass correlation coefficient (ICC 3,1 ), standard error of measurement and minimal detectable change values for the three attempts ranged from 0.57 to 0.82, from 3 to less than 6% and from 7.68 to 13.7%, respectively. In the study of adolescent dynamic equilibrium using the Y-BT, it is recommended to perform nine attempts (including six trial attempts and three measurements). In order to increase reliability it is recommended that the average of the three measured attempts is analysed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Comet assay as a procedure for detecting possible genotoxicity induced by non-ionizing radiation

    Directory of Open Access Journals (Sweden)

    Zsuzsanna Nemeth

    2015-05-01

    In our laboratory we use comet assay for testing genotoxicity of non-ionizing radiation for more than ten years. In the experiments we use whole blood samples (human or dog, cell lines (e.g. H295R cell line or 3 dimensional in vitro skin tissue (epidermis models. In our protocol a slightly modified alkaline Comet assay method of Singh et al. (1988 is used. On our poster there will be presented a brief summary of our experiments with exposure to different types of radiation (ELF, RF, and intermediate frequency. In our protocols the non-ionizing radiation was often combined with ionizing radiation to see whether the non-ionizing radiation can influence the repair of the DNA damage induced by ionizing radiation. For the evaluation of the slides mainly Komet 4.0 image analysis system software (Kinetic Imaging, Liverpool, UK was used, but as we got familiarized with other methods for slide evaluation like grading the comets by visual scoring into 5 categories or the CaspLab software, the comparison of these three methods will be also presented.

  7. How blockchain-timestamped protocols could improve the trustworthiness of medical science.

    Science.gov (United States)

    Irving, Greg; Holden, John

    2016-01-01

    Trust in scientific research is diminished by evidence that data are being manipulated. Outcome switching, data dredging and selective publication are some of the problems that undermine the integrity of published research. Methods for using blockchain to provide proof of pre-specified endpoints in clinical trial protocols were first reported by Carlisle. We wished to empirically test such an approach using a clinical trial protocol where outcome switching has previously been reported. Here we confirm the use of blockchain as a low cost, independently verifiable method to audit and confirm the reliability of scientific studies.

  8. Optimized efflux assay for the NorA multidrug efflux pump in Staphylococcus aureus.

    Science.gov (United States)

    Zimmermann, Saskia; Tuchscherr, Lorena; Rödel, Jürgen; Löffler, Bettina; Bohnert, Jürgen A

    2017-11-01

    Real-time fluorescent efflux assays are commonly used for measuring the efflux of bacterial pumps. Here we describe an optimized protocol for the NorA efflux pump in S. aureus using DiOC 3 instead of ethidium bromide. Glucose and sodium formate were tested as energy carriers. This novel method is fast and reproducible. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Toward development of a comprehensive external quality assurance program for polyfunctional intracellular cytokine staining assays.

    Science.gov (United States)

    Staats, Janet S; Enzor, Jennifer H; Sanchez, Ana M; Rountree, Wes; Chan, Cliburn; Jaimes, Maria; Chan, Ray Chun-Fai; Gaur, Amitabh; Denny, Thomas N; Weinhold, Kent J

    2014-07-01

    The External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program assesses the proficiency of NIH/NIAID/DAIDS-supported and potentially other interested research laboratories in performing Intracellular Cytokine Staining (ICS) assays. The goal of the EQAPOL Flow Cytometry External Quality Assurance Program (EQAP) is to provide proficiency testing and remediation for participating sites. The program is not punitive; rather, EQAPOL aims to help sites identify areas for improvement. EQAPOL utilizes a highly standardized ICS assay to minimize variability and readily identify those sites experiencing technical difficulties with their assays. Here, we report the results of External Proficiency 3 (EP3) where participating sites performed a 7-color ICS assay. On average, sites perform well in the Flow Cytometry EQAP (median score is "Good"). The most common technical issues identified by the program involve protocol adherence and data analysis; these areas have been the focus of site remediation. The EQAPOL Flow Cytometry team is now in the process of expanding the program to 8-color ICS assays. Evaluating polyfunctional ICS responses would align the program with assays currently being performed in support of HIV immune monitoring assays. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. The Reliability and Validity of Fatigue Measures During Multiple-Sprint Work: An Issue Revisited

    OpenAIRE

    Glaister, Mark; Howatson, Glyn; Pattison, John R.; McInnes, Gill

    2008-01-01

    The ability to repeatedly produce a high-power output or sprint speed is a key fitness component of most field and court sports. The aim of this study was to evaluate the validity and reliability of eight different approaches to quantify this parameter in tests of multiple-sprint performance. Ten physically active men completed two trials of each of two multiple-sprint running protocols with contrasting recovery periods. Protocol 1 consisted of 12 × 30-m sprints repeated every 35 seconds; pro...

  11. A Method for the Preparation of Chicken Liver P?t? that Reliably Destroys Campylobacters

    OpenAIRE

    Hutchison, Mike; Harrison, Dawn; Richardson, Ian; Tch?rzewska, Monika

    2015-01-01

    This study devised a protocol for the manufacture of commercial quantities of chicken liver pâté that reliably destroyed campylobacters. A literature search identified 40 pâté manufacture recipes. Recipes stages with a potential to be antimicrobial were assembled to form a new protocol that included washing with organic acid, freeze-thaw and flambé in alcohol. Naturally-contaminated, high-risk livers were obtained from clearance flocks at slaughter and the effect of each stage of the protoco...

  12. 2014 Building America House Simulation Protocols

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, E. [National Renewable Energy Lab. (NREL), Golden, CO (United States); Engebrecht-Metzger, C. [National Renewable Energy Lab. (NREL), Golden, CO (United States); Horowitz, S. [National Renewable Energy Lab. (NREL), Golden, CO (United States); Hendron, R. [National Renewable Energy Lab. (NREL), Golden, CO (United States)

    2014-03-01

    As BA has grown to include a large and diverse cross-section of the home building and retrofit industries, it has become more important to develop accurate, consistent analysis techniques to measure progress towards the program's goals. The House Simulation Protocol (HSP) document provides guidance to program partners and managers so they can compare energy savings for new construction and retrofit projects. The HSP provides the program with analysis methods that are proven to be effective and reliable in investigating the energy use of advanced energy systems and of entire houses.

  13. 2014 Building America House Simulation Protocols

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, E. [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Engebrecht, C. Metzger [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Horowitz, S. [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Hendron, R. [National Renewable Energy Laboratory (NREL), Golden, CO (United States)

    2014-03-01

    As Building America has grown to include a large and diverse cross-section of the home building and retrofit industries, it has become more important to develop accurate, consistent analysis techniques to measure progress towards the program's goals. The House Simulation Protocol (HSP) document provides guidance to program partners and managers so they can compare energy savings for new construction and retrofit projects. The HSP provides the program with analysis methods that are proven to be effective and reliable in investigating the energy use of advanced energy systems and of entire houses.

  14. Evaluation of a Noncontact, Alternative Mosquito Repellent Assay System.

    Science.gov (United States)

    Tisgratog, Rungarun; Kongmee, Monthathip; Sanguanpong, Unchalee; Prabaripai, Atchariya; Bangs, Michael J; Chareonviriyaphap, Theeraphap

    2016-09-01

    A novel noncontact repellency assay system (NCRAS) was designed and evaluated as a possible alternative method for testing compounds that repel or inhibit mosquitoes from blood feeding. Deet and Aedes aegypti were used in a controlled laboratory setting. Using 2 study designs, a highly significant difference were seen between deet-treated and untreated skin placed behind the protective screens, indicating that deet was detected and was acting as a deterrence to mosquito landing and probing behavior. However, a 2nd study showed significant differences between protected (behind a metal screen barrier) and unprotected (exposed) deet-treated forearms, indicating the screen mesh might restrict the detection of deet and thus influences landing/biting response. These findings indicate the prototype NCRAS shows good promise but requires further evaluation and possible modification in design and testing protocol to achieve more desirable operational attributes in comparison with direct skin-contact repellency mosquito assays.

  15. A Priority-aware Frequency Domain Polling MAC Protocol for OFDMA-based Networks in Cyber-physical Systems

    Institute of Scientific and Technical Information of China (English)

    Meng Zheng; Junru Lin; Wei Liang; Haibin Yu

    2015-01-01

    Wireless networking in cyber-physical systems(CPSs) is characteristically different from traditional wireless systems due to the harsh radio frequency environment and applications that impose high real-time and reliability constraints.One of the fundamental considerations for enabling CPS networks is the medium access control protocol. To this end, this paper proposes a novel priority-aware frequency domain polling medium access control(MAC) protocol, which takes advantage of an orthogonal frequency-division multiple access(OFDMA)physical layer to achieve instantaneous priority-aware polling.Based on the polling result, the proposed work then optimizes the resource allocation of the OFDMA network to further improve the data reliability. Due to the non-polynomial-complete nature of the OFDMA resource allocation, we propose two heuristic rules,based on which an efficient solution algorithm to the OFDMA resource allocation problem is designed. Simulation results show that the reliability performance of CPS networks is significantly improved because of this work.

  16. Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams

    Directory of Open Access Journals (Sweden)

    Adeline Bidault

    2015-12-01

    Full Text Available The Gram-negative bacterium Vibrio tapetis is known as the causative agent of Brown Ring Disease (BRD in the Manila clam Venerupis (=Ruditapes philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification of V. tapetis strains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600T V. tapetis strain. Quantification curves of V. tapetis strain seeded in filtered seawater (FSW or extrapallial fluids (EF samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detect V. tapetis strains down to 1.125 101 bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitor V. tapetis load both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections by V. tapetis and for designing appropriate control measures for aquaculture purposes.

  17. Analysis of Pervasive Mobile Ad Hoc Routing Protocols

    Science.gov (United States)

    Qadri, Nadia N.; Liotta, Antonio

    Mobile ad hoc networks (MANETs) are a fundamental element of pervasive networks and therefore, of pervasive systems that truly support pervasive computing, where user can communicate anywhere, anytime and on-the-fly. In fact, future advances in pervasive computing rely on advancements in mobile communication, which includes both infrastructure-based wireless networks and non-infrastructure-based MANETs. MANETs introduce a new communication paradigm, which does not require a fixed infrastructure - they rely on wireless terminals for routing and transport services. Due to highly dynamic topology, absence of established infrastructure for centralized administration, bandwidth constrained wireless links, and limited resources in MANETs, it is challenging to design an efficient and reliable routing protocol. This chapter reviews the key studies carried out so far on the performance of mobile ad hoc routing protocols. We discuss performance issues and metrics required for the evaluation of ad hoc routing protocols. This leads to a survey of existing work, which captures the performance of ad hoc routing algorithms and their behaviour from different perspectives and highlights avenues for future research.

  18. Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay.

    Science.gov (United States)

    Linares, María; Viera, Sara; Crespo, Benigno; Franco, Virginia; Gómez-Lorenzo, María G; Jiménez-Díaz, María Belén; Angulo-Barturen, Íñigo; Sanz, Laura María; Gamo, Francisco-Javier

    2015-11-05

    The emergence of Plasmodium falciparum resistance to artemisinins threatens to undermine the effectiveness of artemisinin-based combination anti-malarial therapy. Developing suitable drugs to replace artemisinins requires the identification of new compounds that display rapid parasite killing kinetics. However, no current methods fully meet the requirements to screen large compound libraries for candidates with such properties. This study describes the development and validation of an in vitro parasite viability fast assay for identifying rapidly parasiticidal anti-malarial drugs. Parasite killing kinetics were determined by first culturing unlabelled erythrocytes with P. falciparum in the presence of anti-malarial drugs for 24 or 48 h. After removing the drug, samples were added to erythrocytes pre-labelled with intracellular dye to allow their subsequent identification. The ability of viable parasites to re-establish infection in labelled erythrocytes could then be detected by two-colour flow cytometry after tagging of parasite DNA. Thus, double-stained erythrocytes (with the pre-labelled intracellular dye and the parasite DNA dye) result only after establishment of new infections by surviving parasites. The capacity of the test anti-malarial drugs to eliminate viable parasites within 24 or 48 h could, therefore, be determined. The parasite viability fast assay could be completed within 48 h following drug treatment and distinguished between rapidly parasiticidal anti-malarial drugs versus those acting more slowly. The assay was validated against ten standard anti-malarial agents with known properties and results correlated well with established methods. An abbreviated assay, suitable for adaption to medium-high throughput screening, was validated and applied against a set of 20 compounds retrieved from the publically available Medicines for Malaria Venture 'Malaria Box'. The quantification of new infections to determine parasite viability offers important

  19. Validity And Reliability Of The Stages Cycling Power Meter.

    Science.gov (United States)

    Granier, Cyril; Hausswirth, Christophe; Dorel, Sylvain; Yann, Le Meur

    2017-09-06

    This study aimed to determine the validity and the reliability of the Stages power meter crank system (Boulder, United States) during several laboratory cycling tasks. Eleven trained participants completed laboratory cycling trials on an indoor cycle fitted with SRM Professional and Stages systems. The trials consisted of an incremental test at 100W, 200W, 300W, 400W and four 7s sprints. The level of pedaling asymmetry was determined for each cycling intensity during a similar protocol completed on a Lode Excalibur Sport ergometer. The reliability of Stages and SRM power meters was compared by repeating the incremental test during a test-retest protocol on a Cyclus 2 ergometer. Over power ranges of 100-1250W the Stages system produced trivial to small differences compared to the SRM (standardized typical error values of 0.06, 0.24 and 0.08 for the incremental, sprint and combined trials, respectively). A large correlation was reported between the difference in power output (PO) between the two systems and the level of pedaling asymmetry (r=0.58, p system according to the level of pedaling asymmetry provided only marginal improvements in PO measures. The reliability of the Stages power meter at the sub-maximal intensities was similar to the SRM Professional model (coefficient of variation: 2.1 and 1.3% for Stages and SRM, respectively). The Stages system is a suitable device for PO measurements, except when a typical error of measurement power ranges of 100-1250W is expected.

  20. A global protocol for monitoring of coral bleaching

    OpenAIRE

    Oliver, J.; Setiasih, N.; Marshall, P.; Hansen, L.

    2004-01-01

    Coral bleaching and subsequent mortality represent a major threat to the future health and productivity of coral reefs. However a lack of reliable data on occurrence, severity and other characteristics of bleaching events hampers research on the causes and consequences of this important phenomenon. This article describes a global protocol for monitoring coral bleaching events, which addresses this problem and can be used by people with different levels of expertise and resources.

  1. Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection

    Science.gov (United States)

    Paudel, Damodar; Jarman, Richard; Limkittikul, Kriengsak; Klungthong, Chonticha; Chamnanchanunt, Supat; Nisalak, Ananda; Gibbons, Robert; Chokejindachai, Watcharee

    2011-01-01

    Background: Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman® assay and conventional nested PCR assay. Aims: To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conventional nested PCR (modified Lanciotti). Materials and Methods: Eight cultured virus strains were diluted in tenth dilution down to undetectable level by the PCR to optimize the primer, temperature (annealing, and extension and to detect the limit of detection of the assay. Hundred and ninety three ELISA and PCR proved dengue clinical samples were tested with real time SYBR® Green assay, real time Taqman® assay to compare the sensitivity and specificity. Results: Sensitivity and specificity of real time SYBR® green dengue assay (84% and 66%, respectively) was almost comparable to those (81% and 74%) of Taqman real time PCR dengue assay. Real time SYBR® green RT-PCR was equally sensitive in primary and secondary infection while real time Taqman was less sensitive in the secondary infection. Sensitivity of real time Taqman on DENV3 (87%) was equal to SYBR green real time PCR dengue assay. Conclusion: We developed low cost rapid diagnostic SYBR green dengue assay. Further study is needed to make duplex primer assay for the serotyping of dengue virus. PMID:22363089

  2. Evaluation of the HISCL Anti-Treponema pallidum Assay as a Screening Test for Syphilis

    OpenAIRE

    An, Jingna; Chen, Qixia; Liu, Qianqian; Rao, Chenli; Li, Dongdong; Wang, Tingting; Tao, Chuanmin; Wang, Lanlan

    2015-01-01

    The resurgence of syphilis in recent years has become a serious threat to public health worldwide, and the serological detection of specific antibodies against Treponema pallidum remains the most reliable method for laboratory diagnosis of syphilis. This study examined the performance of the recently launched HISCL anti-Treponema pallidum (anti-TP) assay as a screening test for syphilis in a high-volume laboratory. The HISCL anti-TP assay was tested in 300 preselected syphilis-positive sample...

  3. Micronucleus assay for human peripheral blood lymphocytes as a biomarker of individual sensitivity to assessing radiation health risk in different population

    International Nuclear Information System (INIS)

    Kang, C.-M.; Jeon, H.-J.; Lee, Y.-S.; Lee, S.-J.; Jin, Y.-H.; Kim, Y.-H.; Kim, T.-W.; Cho, C.-K.

    2003-01-01

    Full text: Our studies were to evaluate micronucleus (MN) assay for human peripheral blood lymphocytes (HPBL) as a biomarker of individual sensitivity to assessing radiation health risk in different population in Korea. Further studied are carried out to provide evidence for the existence of individual variations in age-dependent responses. For the MN assay, HPBLs were irradiated with doses of 0, 1, 2, 4, 8Gy 60 Co γ-rays. Spontaneous frequencies not only vary greatly between individuals, but also working or living areas because of the groups with different lifestyle living in different ecological situation and the reaction to radiation exposure. It was shown that the increased level of spontaneous cell with MN was observed with increased age. The relationship between radiosensitivity and the increased spontaneous level of MN may be in inverse proportion. Age and gender are the most important demographic variables impact on MN index with MN frequencies in female being greater than those in male by a factor of depending on the age group. For both sexes, MN frequency was significantly and positively correlated with age. The main lifestyle factors influencing the MN index in subjects are significantly and positively correlated with smoking in measuring the spontaneous frequencies of micronuclei. The described results show that the genetic damaged rate like MN index in human populations is correlated significantly with age, sex and lifestyle factors. So far, it is evident that with regard to the application of MN assay all future studies to evaluate radiation health risks in different population have to take into account the influence of age, gender, and lifestyle. The results suggested that the MN assay have a high potential to ensure appropriate quality control and standard documentation protocol which can be used to monitor a large population exposed to radiation epidemiologically. We conclude that the determination of individual radiosensitivity with MN assay is

  4. Diagnostic evaluation of assays for detection of antibodies against porcine epidemic diarrhea virus (PEDV) in pigs exposed to different PEDV strains

    DEFF Research Database (Denmark)

    Gerber, Priscilla F.; Lelli, Davide; Zhang, Jianqiang

    2016-01-01

    Porcine epidemic diarrhea virus (PEDV) has caused economic losses in the Americas, Asia and Europe in recent years. Reliable serological assays are essential for epidemiological studies and vaccine evaluation. The objective of this study was to compare the ability of five enzyme-linked immunosorb......Porcine epidemic diarrhea virus (PEDV) has caused economic losses in the Americas, Asia and Europe in recent years. Reliable serological assays are essential for epidemiological studies and vaccine evaluation. The objective of this study was to compare the ability of five enzyme...

  5. Development of an opioid self-administration assay to study drug seeking in zebrafish.

    Science.gov (United States)

    Bossé, Gabriel D; Peterson, Randall T

    2017-09-29

    The zebrafish (Danio rerio) has become an excellent tool to study mental health disorders, due to its physiological and genetic similarity to humans, ease of genetic manipulation, and feasibility of small molecule screening. Zebrafish have been shown to exhibit characteristics of addiction to drugs of abuse in non-contingent assays, including conditioned place preference, but contingent assays have been limited to a single assay for alcohol consumption. Using inexpensive electronic, mechanical, and optical components, we developed an automated opioid self-administration assay for zebrafish, enabling us to measure drug seeking and gain insight into the underlying biological pathways. Zebrafish trained in the assay for five days exhibited robust self-administration, which was dependent on the function of the μ-opioid receptor. In addition, a progressive ratio protocol was used to test conditioned animals for motivation. Furthermore, conditioned fish continued to seek the drug despite an adverse consequence and showed signs of stress and anxiety upon withdrawal of the drug. Finally, we validated our assay by confirming that self-administration in zebrafish is dependent on several of the same molecular pathways as in other animal models. Given the ease and throughput of this assay, it will enable identification of important biological pathways regulating drug seeking and could lead to the development of new therapeutic molecules to treat addiction. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Psychometric Properties of a Standardized Observation Protocol to Quantify Pediatric Physical Therapy Actions

    NARCIS (Netherlands)

    Sonderer, Patrizia; Ziegler, Schirin Akhbari; Oertle, Barbara Gressbach; Meichtry, Andre; Hadders-Algra, Mijna

    Purpose: Pediatric physical therapy (PPT) is characterized by heterogeneity. This blurs the evaluation of effective components of PPT. The Groningen Observation Protocol (GOP) was developed to quantify contents of PPT. This study assesses the reliability and completeness of the GOP. Methods: Sixty

  7. Comparison of Different Double Immunostaining Protocols for Paraffin Embedded Liver Tissue

    Directory of Open Access Journals (Sweden)

    Alexander Schütz

    1999-01-01

    Full Text Available Most of the double immunostaining protocols that have been introduced so far have been developed for application on fresh frozen material or based on different species antibodies. In liver tissue, general problems of double immunostaining techniques are further complicated by tissue‐specific difficulties, such as necrosis or high intracellular protein content. To assess a reliable double immunostaining protocol for archived, paraffin embedded liver tissue, different protocols based on the use of same species primary antibodies were evaluated in terms of sensitivity, specificity and non‐specific background staining in pathological liver specimens. We compared peroxidase–anti‐peroxidase, alkaline phosphatase–anti‐alkaline phosphatase (PAP/APAP, labelled‐avidin–biotin (LAB/LAB and digoxigenin–anti‐digoxigenin (dig–a‐dig/PAP techniques using different cytokeratin antibodies and an antibody against PCNA. Comparison of the double immunostaining techniques revealed a high sensitivity and specificity in all procedures. Sections, which were stained employing PAP/APAP‐technique, displayed a higher background staining compared to sections which were treated with the LAB/LAB or dig–a‐dig/PAP protocol. In contrast to the dig–a‐dig/PAP protocol, the LAB/LAB technique provides a better time/cost relationship. Therefore, we would like to recommend a modified LAB/LAB protocol for simultaneous detection of different antigens in archived liver tissue.

  8. Comparison of two commercial carbapenemase gene confirmatory assays in multiresistant Enterobacteriaceae and Acinetobacter baumannii-complex.

    Science.gov (United States)

    Rösner, Stephan; Gehlweiler, Kevin; Küsters, Uta; Kolbert, Mathias; Hübner, Kirsten; Pfennigwerth, Niels; Mack, Dietrich

    2018-01-01

    Multidrug-resistant Gram-negative bacilli (MDR-GNB) producing carbapenemases are increasing at an alarming speed. Rapid confirmation of carbapenemase type will be an important diagnostic step in clinical microbiology laboratories not only to reduce the risk of transmissions but also for optimising antibiotic therapy in the future. We compared diagnostic reliability of two commercially available molecular assays (Check-Direct CPE vs. AID line probe assay) for detection and typing of carbapenemase genes in 80 well-characterized isolates of MDR-GNB. Respective strains were isolated in various clinical specimens at our clinical microbiology laboratory. The reference standard included confirmation of carbapenemase-production at the molecular level at the German National Reference Laboratory for Multidrug-resistant Gram-negative bacteria (Ruhr-University Bochum, Germany). 53 Enterobacteriaceae and 27 members of the A. baumannii-complex were used in this study. The tested assays appeared highly reliable to confirm carbapenemase-producing Enterobacteriaceae (CPE) with respective sensitivities of 97.7%, but are currently unsuitable for analysis of members of the A. baumannii-complex. Both assays are easy to perform and rapid tools for confirmation and typing of the most common carbapenemase genes in Enterobacteriaceae. Implementation should be possible for any clinical microbiology laboratory with Check-Direct CPE being easier to handle and having less technological requirements.

  9. Comparison of two commercial carbapenemase gene confirmatory assays in multiresistant Enterobacteriaceae and Acinetobacter baumannii-complex.

    Directory of Open Access Journals (Sweden)

    Stephan Rösner

    Full Text Available Multidrug-resistant Gram-negative bacilli (MDR-GNB producing carbapenemases are increasing at an alarming speed. Rapid confirmation of carbapenemase type will be an important diagnostic step in clinical microbiology laboratories not only to reduce the risk of transmissions but also for optimising antibiotic therapy in the future. We compared diagnostic reliability of two commercially available molecular assays (Check-Direct CPE vs. AID line probe assay for detection and typing of carbapenemase genes in 80 well-characterized isolates of MDR-GNB. Respective strains were isolated in various clinical specimens at our clinical microbiology laboratory. The reference standard included confirmation of carbapenemase-production at the molecular level at the German National Reference Laboratory for Multidrug-resistant Gram-negative bacteria (Ruhr-University Bochum, Germany. 53 Enterobacteriaceae and 27 members of the A. baumannii-complex were used in this study. The tested assays appeared highly reliable to confirm carbapenemase-producing Enterobacteriaceae (CPE with respective sensitivities of 97.7%, but are currently unsuitable for analysis of members of the A. baumannii-complex. Both assays are easy to perform and rapid tools for confirmation and typing of the most common carbapenemase genes in Enterobacteriaceae. Implementation should be possible for any clinical microbiology laboratory with Check-Direct CPE being easier to handle and having less technological requirements.

  10. Interrater and Intrarater Reliability of the Tuck Jump Assessment by Health Professionals of Varied Educational Backgrounds

    Directory of Open Access Journals (Sweden)

    Lisa A. Dudley

    2013-01-01

    Full Text Available Objective. The Tuck Jump Assessment (TJA, a clinical plyometric assessment, identifies 10 jumping and landing technique flaws. The study objective was to investigate TJA interrater and intrarater reliability with raters of different educational and clinical backgrounds. Methods. 40 participants were video recorded performing the TJA using published protocol and instructions. Five raters of varied educational and clinical backgrounds scored the TJA. Each score of the 10 technique flaws was summed for the total TJA score. Approximately one month later, 3 raters scored the videos again. Intraclass correlation coefficients determined interrater (5 and 3 raters for first and second session, resp. and intrarater (3 raters reliability. Results. Interrater reliability with 5 raters was poor (ICC = 0.47; 95% confidence intervals (CI 0.33–0.62. Interrater reliability between 3 raters who completed 2 scoring sessions improved from 0.52 (95% CI 0.35–0.68 for session one to 0.69 (95% CI 0.55–0.81 for session two. Intrarater reliability was poor to moderate, ranging from 0.44 (95% CI 0.22–0.68 to 0.72 (95% CI 0.55–0.84. Conclusion. Published protocol and training of raters were insufficient to allow consistent TJA scoring. There may be a learned effect with the TJA since interrater reliability improved with repetition. TJA instructions and training should be modified and enhanced before clinical implementation.

  11. An ultra low-power and traffic-adaptive medium access control protocol for wireless body area network.

    Science.gov (United States)

    Ullah, Sana; Kwak, Kyung Sup

    2012-06-01

    Wireless Body Area Network (WBAN) consists of low-power, miniaturized, and autonomous wireless sensor nodes that enable physicians to remotely monitor vital signs of patients and provide real-time feedback with medical diagnosis and consultations. It is the most reliable and cheaper way to take care of patients suffering from chronic diseases such as asthma, diabetes and cardiovascular diseases. Some of the most important attributes of WBAN is low-power consumption and delay. This can be achieved by introducing flexible duty cycling techniques on the energy constraint sensor nodes. Stated otherwise, low duty cycle nodes should not receive frequent synchronization and control packets if they have no data to send/receive. In this paper, we introduce a Traffic-adaptive MAC protocol (TaMAC) by taking into account the traffic information of the sensor nodes. The protocol dynamically adjusts the duty cycle of the sensor nodes according to their traffic-patterns, thus solving the idle listening and overhearing problems. The traffic-patterns of all sensor nodes are organized and maintained by the coordinator. The TaMAC protocol is supported by a wakeup radio that is used to accommodate emergency and on-demand events in a reliable manner. The wakeup radio uses a separate control channel along with the data channel and therefore it has considerably low power consumption requirements. Analytical expressions are derived to analyze and compare the performance of the TaMAC protocol with the well-known beacon-enabled IEEE 802.15.4 MAC, WiseMAC, and SMAC protocols. The analytical derivations are further validated by simulation results. It is shown that the TaMAC protocol outperforms all other protocols in terms of power consumption and delay.

  12. Development of kits for radioimmunometric assays for tumour markers. Final report of a co-ordinated research project 1997-2001

    International Nuclear Information System (INIS)

    2002-08-01

    Many tumour marker assays have been reported over the years and their role is well recognized and acknowledged in the follow-up of known cancer cases. However, their true potential for use in primary diagnosis or screening of high risk groups is still to be fully realized due to the need to achieve better specificity. Among the various tumour markers, the one for prostate cancer - prostate specific antigen (PSA) - appears to have better specificity, coming close to a tumour specific antigen. Prostate cancer is a commonly encountered cancer in men, and can be effectively treated if detected early. PSA levels in serum appear to provide good correlation with tumour burden. Estimation of free PSA in serum is reported to further improve the diagnosis. In several developed countries routine screening of men above 50 years of age for prostate cancer using serum PSA as marker is recommended. Radioimmunometric assay techniques offer themselves as attractive candidates for measurement of tumour markers. They are robust, economical and didactic, thus eminently suitable for technology transfer, training and teaching. Preparation of primary reagents is relatively easy. The methodology is flexible. As a result of co-operation projects of the IAEA, many developing Member States have built up indigenous capabilities to perform radioimmunometric assays, which can be extended to development of kits for tumour marker assays. Considering the need for indigenous development of capabilities to produce reliable kits for radioimmunometric assays for PSA, in 1997 the IAEA initiated a Co-ordinated Research Project (CRP) on Development of Kits for Radioimmunometric Assays for Tumour Markers. Even though the focus of the project was PSA, it was expected that the expertise to be gained by the participants would also help them undertake development of kits for other tumour markers, essentially using the same methodology. Ten laboratories from Europe, Asia, Africa and the Americas participated

  13. A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

    Science.gov (United States)

    Pooley, Hannah B.; de Silva, Kumudika; Purdie, Auriol C.; Begg, Douglas J.; Whittington, Richard J.

    2016-01-01

    ABSTRACT Determining the viability of bacteria is a key outcome of in vitro cellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viable M. avium subsp. paratuberculosis cells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and dead M. avium subsp. paratuberculosis organisms and their accuracy at low bacterial concentrations. Using the culture-based method, M. avium subsp. paratuberculosis growth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viable M. avium subsp. paratuberculosis cells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples from in vitro cellular infection assays. IMPORTANCE Rapid quantification of the viability of Mycobacterium avium subsp. paratuberculosis in samples from in vitro cellular infection assays is important, as it allows these assays to be carried out on a large scale. In vitro cellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regarding M. avium subsp. paratuberculosis viability after an in vitro infection assay; however, with the quantitative PCR and liquid culture method

  14. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

    Science.gov (United States)

    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

    2014-07-01

    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. HIV-1 viral load measurement in venous blood and fingerprick blood using Abbott RealTime HIV-1 DBS assay.

    Science.gov (United States)

    Tang, Ning; Pahalawatta, Vihanga; Frank, Andrea; Bagley, Zowie; Viana, Raquel; Lampinen, John; Leckie, Gregor; Huang, Shihai; Abravaya, Klara; Wallis, Carole L

    2017-07-01

    HIV RNA suppression is a key indicator for monitoring success of antiretroviral therapy. From a logistical perspective, viral load (VL) testing using Dried Blood Spots (DBS) is a promising alternative to plasma based VL testing in resource-limited settings. To evaluate the analytical and clinical performance of the Abbott RealTime HIV-1 assay using a fully automated one-spot DBS sample protocol. Limit of detection (LOD), linearity, lower limit of quantitation (LLQ), upper limit of quantitation (ULQ), and precision were determined using serial dilutions of HIV-1 Virology Quality Assurance stock (VQA Rush University), or HIV-1-containing armored RNA, made in venous blood. To evaluate correlation, bias, and agreement, 497 HIV-1 positive adult clinical samples were collected from Ivory Coast, Uganda and South Africa. For each HIV-1 participant, DBS-fingerprick, DBS-venous and plasma sample results were compared. Correlation and bias values were obtained. The sensitivity and specificity were analyzed at a threshold of 1000 HIV-1 copies/mL generated using the standard plasma protocol. The Abbott HIV-1 DBS protocol had an LOD of 839 copies/mL, a linear range from 500 to 1×10 7 copies/mL, an LLQ of 839 copies/mL, a ULQ of 1×10 7 copies/mL, and an inter-assay SD of ≤0.30 log copies/mL for all tested levels within this range. With clinical samples, the correlation coefficient (r value) was 0.896 between DBS-fingerprick and plasma and 0.901 between DBS-venous and plasma, and the bias was -0.07 log copies/mL between DBS-fingerprick and plasma and -0.02 log copies/mL between DBS-venous and plasma. The sensitivity of DBS-fingerprick and DBS-venous was 93%, while the specificity of both DBS methods was 95%. The results demonstrated that the Abbott RealTime HIV-1 assay with DBS sample protocol is highly sensitive, specific and precise across a wide dynamic range and correlates well with plasma values. The Abbott RealTime HIV-1 assay with DBS sample protocol provides an

  16. Impact of ubiquitous inhibitors on the GUS gene reporter system: evidence from the model plants Arabidopsis, tobacco and rice and correction methods for quantitative assays of transgenic and endogenous GUS

    Directory of Open Access Journals (Sweden)

    Gerola Paolo D

    2009-12-01

    Full Text Available Abstract Background The β-glucuronidase (GUS gene reporter system is one of the most effective and employed techniques in the study of gene regulation in plant molecular biology. Improving protocols for GUS assays have rendered the original method described by Jefferson amenable to various requirements and conditions, but the serious limitation caused by inhibitors of the enzyme activity in plant tissues has thus far been underestimated. Results We report that inhibitors of GUS activity are ubiquitous in organ tissues of Arabidopsis, tobacco and rice, and significantly bias quantitative assessment of GUS activity in plant transformation experiments. Combined with previous literature reports on non-model species, our findings suggest that inhibitors may be common components of plant cells, with variable affinity towards the E. coli enzyme. The reduced inhibitory capacity towards the plant endogenous GUS discredits the hypothesis of a regulatory role of these compounds in plant cells, and their effect on the bacterial enzyme is better interpreted as a side effect due to their interaction with GUS during the assay. This is likely to have a bearing also on histochemical analyses, leading to inaccurate evaluations of GUS expression. Conclusions In order to achieve reliable results, inhibitor activity should be routinely tested during quantitative GUS assays. Two separate methods to correct the measured activity of the transgenic and endogenous GUS are presented.

  17. Reliability and validity of the Mywellness Key physical activity monitor

    Directory of Open Access Journals (Sweden)

    Sieverdes JC

    2013-01-01

    Full Text Available John C Sieverdes,1 Eric E Wickel,2 Gregory A Hand,3 Marco Bergamin,4 Robert R Moran,5 Steven N Blair3,51Medical University of South Carolina, College of Nursing and Medicine, Charleson, SC, 2University of Tulsa, Exercise and Sport Science, Tulsa, OK, 3University of South Carolina, Department of Exercise Science, Division of Health Aspects of Physical Activity, Arnold School of Public Health, Columbia, SC, USA; 4University of Padova, Department of Medicine, Sports Medicine Division, Padova, Italy; 5University of South Carolina, Department of Epidemiology and Biostatistics, Arnold School of Public Health, Columbia, SC, USABackground: This study evaluated the reliability and criterion validity of the Mywellness Key accelerometer (MWK using treadmill protocols and indirect calorimetry.Methods: Twenty-five participants completed two four-stage 20-minute treadmill protocols while wearing two MWK accelerometers. Reliability was assessed using raw counts. Validity was assessed by comparing the estimated VO2 calculated from the MWK with values from respiratory gas exchange.Results: Good overall and point estimates of reliability were found for the MWK (all intraclass correlations > 0.93. Generalizability theory coefficients showed lower values for running speed (0.70 versus walking speed (all > 0.84, with the majority of the overall percentage of variability derived from the participant (68%–88% of the total 100%. Acceptable validity was found overall (Pearson’s r = 0.895–0.902, P < 0.0001, with an overall mean absolute error of 16.22% and a coefficient of variance of 16.92%. Bland-Altman plots showed an overestimation of energy expenditure during the running speed, but total kilocalories were underestimated during the protocol by approximately 10%.Conclusion: Good validity was found during light and moderate walking, while running was slightly overestimated. The MWK may be useful for clinicians and researchers interested in promotion or assessment

  18. 75 FR 4323 - Additional Quantitative Fit-testing Protocols for the Respiratory Protection Standard

    Science.gov (United States)

    2010-01-27

    ...-mask and full face piece respirators are normally considered two different types of air purifying... article published in a peer-reviewed industrial-hygiene journal describing the protocol and explaining how... article from an industrial- hygiene journal describing the accuracy and reliability of these proposed...

  19. An Enhanced Reservation-Based MAC Protocol for IEEE 802.15.4 Networks

    Science.gov (United States)

    Afonso, José A.; Silva, Helder D.; Macedo, Pedro; Rocha, Luis A.

    2011-01-01

    The IEEE 802.15.4 Medium Access Control (MAC) protocol is an enabling standard for wireless sensor networks. In order to support applications requiring dedicated bandwidth or bounded delay, it provides a reservation-based scheme named Guaranteed Time Slot (GTS). However, the GTS scheme presents some drawbacks, such as inefficient bandwidth utilization and support to a maximum of only seven devices. This paper presents eLPRT (enhanced Low Power Real Time), a new reservation-based MAC protocol that introduces several performance enhancing features in comparison to the GTS scheme. This MAC protocol builds on top of LPRT (Low Power Real Time) and includes various mechanisms designed to increase data transmission reliability against channel errors, improve bandwidth utilization and increase the number of supported devices. A motion capture system based on inertial and magnetic sensors has been used to validate the protocol. The effectiveness of the performance enhancements introduced by each of the new features is demonstrated through the provision of both simulation and experimental results. PMID:22163826

  20. Performance Comparison of Wireless Sensor Network Standard Protocols in an Aerospace Environment: ISA100.11a and ZigBee Pro

    Science.gov (United States)

    Wagner, Raymond S.; Barton, Richard J.

    2011-01-01

    Standards-based wireless sensor network (WSN) protocols are promising candidates for spacecraft avionics systems, offering unprecedented instrumentation flexibility and expandability. Ensuring reliable data transport is key, however, when migrating from wired to wireless data gathering systems. In this paper, we conduct a rigorous laboratory analysis of the relative performances of the ZigBee Pro and ISA100.11a protocols in a representative crewed aerospace environment. Since both operate in the 2.4 GHz radio frequency (RF) band shared by systems such as Wi-Fi, they are subject at times to potentially debilitating RF interference. We compare goodput (application-level throughput) achievable by both under varying levels of 802.11g Wi-Fi traffic. We conclude that while the simpler, more inexpensive ZigBee Pro protocol performs well under moderate levels of interference, the more complex and costly ISA100.11a protocol is needed to ensure reliable data delivery under heavier interference. This paper represents the first published, rigorous analysis of WSN protocols in an aerospace environment that we are aware of and the first published head-to-head comparison of ZigBee Pro and ISA100.11a.

  1. Reliable Freestanding Position-Based Routing in Highway Scenarios

    Science.gov (United States)

    Galaviz-Mosqueda, Gabriel A.; Aquino-Santos, Raúl; Villarreal-Reyes, Salvador; Rivera-Rodríguez, Raúl; Villaseñor-González, Luis; Edwards, Arthur

    2012-01-01

    Vehicular Ad Hoc Networks (VANETs) are considered by car manufacturers and the research community as the enabling technology to radically improve the safety, efficiency and comfort of everyday driving. However, before VANET technology can fulfill all its expected potential, several difficulties must be addressed. One key issue arising when working with VANETs is the complexity of the networking protocols compared to those used by traditional infrastructure networks. Therefore, proper design of the routing strategy becomes a main issue for the effective deployment of VANETs. In this paper, a reliable freestanding position-based routing algorithm (FPBR) for highway scenarios is proposed. For this scenario, several important issues such as the high mobility of vehicles and the propagation conditions may affect the performance of the routing strategy. These constraints have only been partially addressed in previous proposals. In contrast, the design approach used for developing FPBR considered the constraints imposed by a highway scenario and implements mechanisms to overcome them. FPBR performance is compared to one of the leading protocols for highway scenarios. Performance metrics show that FPBR yields similar results when considering freespace propagation conditions, and outperforms the leading protocol when considering a realistic highway path loss model. PMID:23202159

  2. Intrarater and interrater reliability for measurements in videofluoroscopy of swallowing

    International Nuclear Information System (INIS)

    Baijens, Laura; Barikroo, Ali; Pilz, Walmari

    2013-01-01

    Objective: Intrarater and interrater reliability is crucial to the quality of diagnostic and therapy-effect studies. This paper reports on a systematic review of studies on intrarater and interrater reliability for measurements in videofluoroscopy of swallowing. The aim of this review was to summarize and qualitatively analyze published studies on that topic. Materials and methods: Those published up to March 2013 were found through a comprehensive electronic database search using PubMed, Embase, and The Cochrane Library. Two reviewers independently assessed the studies using strict inclusion criteria. Results: Nineteen studies were included and then qualitatively analyzed. In several of these, methodological problems were found. Moreover, intrarater and interrater reliability varied with the measure applied. A meta-analysis was not carried out as studies were not of sufficient quality to warrant doing so. Conclusion: In order to achieve reliable measurements in videofluoroscopy of swallowing, it is recommended that raters use well-defined guidelines for the levels of ordinal visuoperceptual variables. Furthermore, in order to make the measurements reliable (intrarater and interrater) it is recommended that, following protocolled pre-experimental training, the raters should have maximum consensus about the definition of the measured variables

  3. Intrarater and interrater reliability for measurements in videofluoroscopy of swallowing

    Energy Technology Data Exchange (ETDEWEB)

    Baijens, Laura, E-mail: laura.baijens@mumc.nl [Department of Otorhinolaryngology, Head and Neck Surgery, Maastricht University Medical Center, Maastricht (Netherlands); Barikroo, Ali, E-mail: a.Barikroo@ufl.edu [Swallowing Research Laboratory, Department of Speech, Language and Hearing Sciences, College of Public Health and Health Professions, University of Florida, Gainesville, FL (United States); Pilz, Walmari, E-mail: walmari.pilz@mumc.nl [Department of Otorhinolaryngology, Head and Neck Surgery, Maastricht University Medical Center, Maastricht (Netherlands)

    2013-10-01

    Objective: Intrarater and interrater reliability is crucial to the quality of diagnostic and therapy-effect studies. This paper reports on a systematic review of studies on intrarater and interrater reliability for measurements in videofluoroscopy of swallowing. The aim of this review was to summarize and qualitatively analyze published studies on that topic. Materials and methods: Those published up to March 2013 were found through a comprehensive electronic database search using PubMed, Embase, and The Cochrane Library. Two reviewers independently assessed the studies using strict inclusion criteria. Results: Nineteen studies were included and then qualitatively analyzed. In several of these, methodological problems were found. Moreover, intrarater and interrater reliability varied with the measure applied. A meta-analysis was not carried out as studies were not of sufficient quality to warrant doing so. Conclusion: In order to achieve reliable measurements in videofluoroscopy of swallowing, it is recommended that raters use well-defined guidelines for the levels of ordinal visuoperceptual variables. Furthermore, in order to make the measurements reliable (intrarater and interrater) it is recommended that, following protocolled pre-experimental training, the raters should have maximum consensus about the definition of the measured variables.

  4. Computer network time synchronization the network time protocol on earth and in space

    CERN Document Server

    Mills, David L

    2010-01-01

    Carefully coordinated, reliable, and accurate time synchronization is vital to a wide spectrum of fields-from air and ground traffic control, to buying and selling goods and services, to TV network programming. Ill-gotten time could even lead to the unimaginable and cause DNS caches to expire, leaving the entire Internet to implode on the root servers.Written by the original developer of the Network Time Protocol (NTP), Computer Network Time Synchronization: The Network Time Protocol on Earth and in Space, Second Edition addresses the technological infrastructure of time dissemination, distrib

  5. Recommendations for safety testing with the in vivo comet assay.

    Science.gov (United States)

    Vasquez, Marie Z

    2012-08-30

    While the in vivo comet assay increases its role in regulatory safety testing, deliberations about the interpretation of comet data continue. Concerns can arise regarding comet assay publications with limited data from non-blind testing of positive control compounds and using protocols (e.g. dose concentrations, sample times, and tissues) known to give an expected effect. There may be a tendency towards bias when the validation or interpretation of comet assay data is based on results generated by widely accepted but non-validated assays. The greatest advantages of the comet assay are its sensitivity and its ability to detect genotoxicity in tissues and at sample times that could not previously be evaluated. Guidelines for its use and interpretation in safety testing should take these factors into account. Guidelines should be derived from objective review of data generated by blind testing of unknown compounds dosed at non-toxic concentrations and evaluated in a true safety-testing environment, where the experimental design and conclusions must be defensible. However, positive in vivo comet findings with such compounds are rarely submitted to regulatory agencies and this data is typically unavailable for publication due to its proprietary nature. To enhance the development of guidelines for safety testing with the comet assay, and with the permission of several sponsors, this paper presents and discusses relevant data from multiple GLP comet studies conducted blind, with unknown pharmaceuticals and consumer products. Based on these data and the lessons we have learned through the course of conducting these studies, I suggest significant adjustments to the current conventions, and I provide recommendations for interpreting in vivo comet assay results in situations where risk must be evaluated in the absence of carcinogenicity or clinical data. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Performance Analysis of the IEEE 802.11p Multichannel MAC Protocol in Vehicular Ad Hoc Networks.

    Science.gov (United States)

    Song, Caixia

    2017-12-12

    Vehicular Ad Hoc Networks (VANETs) employ multichannel to provide a variety of safety and non-safety applications, based on the IEEE 802.11p and IEEE 1609.4 protocols. The safety applications require timely and reliable transmissions, while the non-safety applications require efficient and high throughput. In the IEEE 1609.4 protocol, operating interval is divided into alternating Control Channel (CCH) interval and Service Channel (SCH) interval with an identical length. During the CCH interval, nodes transmit safety-related messages and control messages, and Enhanced Distributed Channel Access (EDCA) mechanism is employed to allow four Access Categories (ACs) within a station with different priorities according to their criticality for the vehicle's safety. During the SCH interval, the non-safety massages are transmitted. An analytical model is proposed in this paper to evaluate performance, reliability and efficiency of the IEEE 802.11p and IEEE 1609.4 protocols. The proposed model improves the existing work by taking serval aspects and the character of multichannel switching into design consideration. Extensive performance evaluations based on analysis and simulation help to validate the accuracy of the proposed model and analyze the capabilities and limitations of the IEEE 802.11p and IEEE 1609.4 protocols, and enhancement suggestions are given.

  7. Industrial wireless sensor networks applications, protocols, and standards

    CERN Document Server

    Güngör, V Çagri

    2013-01-01

    The collaborative nature of industrial wireless sensor networks (IWSNs) brings several advantages over traditional wired industrial monitoring and control systems, including self-organization, rapid deployment, flexibility, and inherent intelligent processing. In this regard, IWSNs play a vital role in creating more reliable, efficient, and productive industrial systems, thus improving companies' competitiveness in the marketplace. Industrial Wireless Sensor Networks: Applications, Protocols, and Standards examines the current state of the art in industrial wireless sensor networks and outline

  8. A review on transport layer protocol performance for delivering video on an adhoc network

    Science.gov (United States)

    Suherman; Suwendri; Al-Akaidi, Marwan

    2017-09-01

    The transport layer protocol is responsible for the end to end data transmission. Transmission control protocol (TCP) provides a reliable connection and user datagram protocol (UDP) offers fast but unguaranteed data transfer. Meanwhile, the 802.11 (wireless fidelity/WiFi) networks have been widely used as internet hotspots. This paper evaluates TCP, TCP variants and UDP performances for video transmission on an adhoc network. The transport protocol - medium access cross-layer is proposed by prioritizing TCP acknowledgement to reduce delay. The NS-2 evaluations show that the average delays increase linearly for all the evaluated protocols and the average packet losses grow logarithmically. UDP produces the lowest transmission delay; 5.4% and 5.8% lower than TCP and TCP variant, but experiences the highest packet loss. Both TCP and TCP Vegas maintain packet loss as low as possible. The proposed cross-layer successfully decreases TCP and TCP Vegas delay about 0.12 % and 0.15%, although losses remain similar.

  9. Evaluation of two real-time multiplex PCR screening assays detecting fetal RHD in plasma from RhD negative women to ascertain the requirement for antenatal RhD prophylaxis

    DEFF Research Database (Denmark)

    Clausen, Frederik Banch; Krog, Grethe Risum; Rieneck, Klaus

    2011-01-01

    and 5. We used the same fluorescent dye for the exon 7 and 10 probes to increase sensitivity; exon 5 was VIC labeled. We evaluated possible inhibition of DNA amplification with dilution experiments. We then tested the two multiplex assays with DNA extracted from 97 plasma samples from 38 RhD negative......OBJECTIVE: To evaluate two different multiplex real-time PCR assays detecting fetal RHD for screening of RhD negative women in relation to antenatal RhD prophylaxis. METHODS: We designed a duplex assay for the detection of RHD exon 7 and 10 and a triplex assay for the detection of RHD exon 7, 10...... assay (exon 7/10), accuracy was 94.2%. Detection of exon 5 was less reliable. CONCLUSION: The duplex assay using exon 7/10 was the most reliable for prenatal prediction of fetal RhD type as a candidate assay for screening of RhD negative women in relation to antenatal RhD prophylaxis. The triplex assay...

  10. Protocol: validation of the INCODE barometer to measure the innovation compe-tence through the Rasch Measurement Theory

    Directory of Open Access Journals (Sweden)

    Lidia Sanchez

    2017-06-01

    Full Text Available This communication presents a protocol in order to show the different phases that must be followed in order to validate the INCODE barometer, which is used to measure the innovation competence, with Rasch Measurement Theory. Five phases are stated: dimensionality analysis, individual reliability and validity analysis of ítems and persons, global reliability and validity analysis, and cathegory analysis.

  11. Candida albicans Germ-Tube Antibody: Evaluation of a New Automatic Assay for Diagnosing Invasive Candidiasis in ICU Patients.

    Science.gov (United States)

    Parra-Sánchez, Manuel; Zakariya-Yousef Breval, Ismail; Castro Méndez, Carmen; García-Rey, Silvia; Loza Vazquez, Ana; Úbeda Iglesias, Alejandro; Macías Guerrero, Desiree; Romero Mejías, Ana; León Gil, Cristobal; Martín-Mazuelos, Estrella

    2017-08-01

    Testing for Candida albicans germ-tube antibody IFA IgG assay (CAGTA) is used to detect invasive candidiasis infection. However, most suitable assays lack automation and rapid single-sample testing. The CAGTA assay was adapted in an automatic monotest system (invasive candidiasis [CAGTA] VirClia ® IgG monotest (VirClia ® ), a chemiluminescence assay with ready-to-use reagents that provides a rapid objective result. CAGTA assay was compared with the monotest automatic VirClia ® assay in order to establish the diagnostic reliability, accuracy, and usefulness of this method. A prospective study with 361 samples from 179 non-neutropenic critically ill adults patients was conducted, including 21 patients with candidemia, 18 with intra-abdominal candidiasis, 84 with Candida spp. colonization, and 56 with culture-negative samples, as well as samples from ten healthy subjects. Overall agreement between the two assays (CAGTA and VirCLIA) was 85.3%. These assays were compared with the gold-standard method to determine the sensitivity, specificity as well as positive and negative predictive values. In patients with candidemia, values for CAGTA and VirCLIA assays were 76.2 versus 85.7%, 80.3 versus 75.8%, 55.2 versus 52.9%, and 91.4 versus 94.3%, respectively. The corresponding values in patients with intra-abdominal candidiasis were 61.1 versus 66.7%, 80.3 versus 75.8%, 45.8 versus 42.9%, and 88.3 versus 89.3%, respectively. No differences were found according to the species of Candida isolated in culture, except for Candida albicans and C. parapsilosis, for which VirClia ® was better than CAGTA. According to these results, the automated VirClia ® assay was a reliable, rapid, and very easy to perform technique as tool for the diagnosis invasive candidiasis.

  12. Combining Cytotoxicity Assessment and Xenopus laevis Phenotypic Abnormality Assay as a Predictor of Nanomaterial Safety.

    Science.gov (United States)

    Al-Yousuf, Karamallah; Webster, Carl A; Wheeler, Grant N; Bombelli, Francesca Baldelli; Sherwood, Victoria

    2017-08-04

    The African clawed frog, Xenopus laevis, has been used as an efficient pre-clinical screening tool to predict drug safety during the early stages of the drug discovery process. X. laevis is a relatively inexpensive model that can be used in whole organism high-throughput assays whilst maintaining a high degree of homology to the higher vertebrate models often used in scientific research. Despite an ever-increasing volume of biomedical nanoparticles (NPs) in development, their unique physico-chemical properties challenge the use of standard toxicology assays. Here, we present a protocol that directly compares the sensitivity of X. laevis development as a tool to assess potential NP toxicity by observation of embryo phenotypic abnormalities/lethality after NP exposure, to in vitro cytotoxicity obtained using mammalian cell lines. In combination with conventional cytotoxicity assays, the X. laevis phenotypic assay provides accurate data to efficiently assess the safety of novel biomedical NPs. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 by John Wiley & Sons, Inc.

  13. Application of statistical process control to qualitative molecular diagnostic assays.

    Science.gov (United States)

    O'Brien, Cathal P; Finn, Stephen P

    2014-01-01

    Modern pathology laboratories and in particular high throughput laboratories such as clinical chemistry have developed a reliable system for statistical process control (SPC). Such a system is absent from the majority of molecular laboratories and where present is confined to quantitative assays. As the inability to apply SPC to an assay is an obvious disadvantage this study aimed to solve this problem by using a frequency estimate coupled with a confidence interval calculation to detect deviations from an expected mutation frequency. The results of this study demonstrate the strengths and weaknesses of this approach and highlight minimum sample number requirements. Notably, assays with low mutation frequencies and detection of small deviations from an expected value require greater sample numbers to mitigate a protracted time to detection. Modeled laboratory data was also used to highlight how this approach might be applied in a routine molecular laboratory. This article is the first to describe the application of SPC to qualitative laboratory data.

  14. Application of statistical process control to qualitative molecular diagnostic assays.

    Directory of Open Access Journals (Sweden)

    Cathal P O'brien

    2014-11-01

    Full Text Available Modern pathology laboratories and in particular high throughput laboratories such as clinical chemistry have developed a reliable system for statistical process control. Such a system is absent from the majority of molecular laboratories and where present is confined to quantitative assays. As the inability to apply statistical process control to assay is an obvious disadvantage this study aimed to solve this problem by using a frequency estimate coupled with a confidence interval calculation to detect deviations from an expected mutation frequency. The results of this study demonstrate the strengths and weaknesses of this approach and highlight minimum sample number requirements. Notably, assays with low mutation frequencies and detection of small deviations from an expected value require greater samples with a resultant protracted time to detection. Modelled laboratory data was also used to highlight how this approach might be applied in a routine molecular laboratory. This article is the first to describe the application of statistical process control to qualitative laboratory data.

  15. Application of statistical process control to qualitative molecular diagnostic assays

    LENUS (Irish Health Repository)

    O'Brien, Cathal P.

    2014-11-01

    Modern pathology laboratories and in particular high throughput laboratories such as clinical chemistry have developed a reliable system for statistical process control (SPC). Such a system is absent from the majority of molecular laboratories and where present is confined to quantitative assays. As the inability to apply SPC to an assay is an obvious disadvantage this study aimed to solve this problem by using a frequency estimate coupled with a confidence interval calculation to detect deviations from an expected mutation frequency. The results of this study demonstrate the strengths and weaknesses of this approach and highlight minimum sample number requirements. Notably, assays with low mutation frequencies and detection of small deviations from an expected value require greater sample numbers to mitigate a protracted time to detection. Modeled laboratory data was also used to highlight how this approach might be applied in a routine molecular laboratory. This article is the first to describe the application of SPC to qualitative laboratory data.

  16. A Framework for Reliable Reception of Wireless Metering Data using Protocol Side Information

    DEFF Research Database (Denmark)

    Melchior Jacobsen, Rasmus; Popovski, Petar

    2013-01-01

    the deterministic protocol structure to obtain side information and group the packets from the same meter. We derive the probability of falsely pairing packets from different senders in the simple case of no channel errors, and show through simulation and data from an experimental deployment the probability...... of false pairing with channel errors. The pairing is an essential step towards recovery of metering data from as many as possible meters under harsh channel conditions. From the experiment we find that more than 15% of all conducted pairings are between two erroneous packets, which sets an upper bound...

  17. Rapid molecular assays for the detection of yellow fever virus in low-resource settings.

    Science.gov (United States)

    Escadafal, Camille; Faye, Oumar; Sall, Amadou Alpha; Faye, Ousmane; Weidmann, Manfred; Strohmeier, Oliver; von Stetten, Felix; Drexler, Josef; Eberhard, Michael; Niedrig, Matthias; Patel, Pranav

    2014-03-01

    Yellow fever (YF) is an acute viral hemorrhagic disease transmitted by Aedes mosquitoes. The causative agent, the yellow fever virus (YFV), is found in tropical and subtropical areas of South America and Africa. Although a vaccine is available since the 1930s, YF still causes thousands of deaths and several outbreaks have recently occurred in Africa. Therefore, rapid and reliable diagnostic methods easy to perform in low-resources settings could have a major impact on early detection of outbreaks and implementation of appropriate response strategies such as vaccination and/or vector control. The aim of this study was to develop a YFV nucleic acid detection method applicable in outbreak investigations and surveillance studies in low-resource and field settings. The method should be simple, robust, rapid and reliable. Therefore, we adopted an isothermal approach and developed a recombinase polymerase amplification (RPA) assay which can be performed with a small portable instrument and easy-to-use lyophilized reagents. The assay was developed in three different formats (real-time with or without microfluidic semi-automated system and lateral-flow assay) to evaluate their application for different purposes. Analytical specificity and sensitivity were evaluated with a wide panel of viruses and serial dilutions of YFV RNA. Mosquito pools and spiked human plasma samples were also tested for assay validation. Finally, real-time RPA in portable format was tested under field conditions in Senegal. The assay was able to detect 20 different YFV strains and demonstrated no cross-reactions with closely related viruses. The RPA assay proved to be a robust, portable method with a low detection limit (<21 genome equivalent copies per reaction) and rapid processing time (<20 min). Results from real-time RPA field testing were comparable to results obtained in the laboratory, thus confirming our method is suitable for YFV detection in low-resource settings.

  18. Rapid molecular assays for the detection of yellow fever virus in low-resource settings.

    Directory of Open Access Journals (Sweden)

    Camille Escadafal

    2014-03-01

    Full Text Available BACKGROUND: Yellow fever (YF is an acute viral hemorrhagic disease transmitted by Aedes mosquitoes. The causative agent, the yellow fever virus (YFV, is found in tropical and subtropical areas of South America and Africa. Although a vaccine is available since the 1930s, YF still causes thousands of deaths and several outbreaks have recently occurred in Africa. Therefore, rapid and reliable diagnostic methods easy to perform in low-resources settings could have a major impact on early detection of outbreaks and implementation of appropriate response strategies such as vaccination and/or vector control. METHODOLOGY: The aim of this study was to develop a YFV nucleic acid detection method applicable in outbreak investigations and surveillance studies in low-resource and field settings. The method should be simple, robust, rapid and reliable. Therefore, we adopted an isothermal approach and developed a recombinase polymerase amplification (RPA assay which can be performed with a small portable instrument and easy-to-use lyophilized reagents. The assay was developed in three different formats (real-time with or without microfluidic semi-automated system and lateral-flow assay to evaluate their application for different purposes. Analytical specificity and sensitivity were evaluated with a wide panel of viruses and serial dilutions of YFV RNA. Mosquito pools and spiked human plasma samples were also tested for assay validation. Finally, real-time RPA in portable format was tested under field conditions in Senegal. CONCLUSION/SIGNIFICANCE: The assay was able to detect 20 different YFV strains and demonstrated no cross-reactions with closely related viruses. The RPA assay proved to be a robust, portable method with a low detection limit (<21 genome equivalent copies per reaction and rapid processing time (<20 min. Results from real-time RPA field testing were comparable to results obtained in the laboratory, thus confirming our method is suitable for

  19. Fluorescence-based assay as a new screening tool for toxic chemicals

    Science.gov (United States)

    Moczko, Ewa; Mirkes, Evgeny M.; Cáceres, César; Gorban, Alexander N.; Piletsky, Sergey

    2016-09-01

    Our study involves development of fluorescent cell-based diagnostic assay as a new approach in high-throughput screening method. This highly sensitive optical assay operates similarly to e-noses and e-tongues which combine semi-specific sensors and multivariate data analysis for monitoring biochemical processes. The optical assay consists of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns distinctive for particular physico-chemical and physiological conditions. Using chemometric techniques the optical signal is processed providing qualitative information about analytical characteristics of the samples. This integrated approach has been successfully applied (with sensitivity of 93% and specificity of 97%) in assessing whether particular chemical agents are irritating or not for human skin. It has several advantages compared with traditional biochemical or biological assays and can impact the new way of high-throughput screening and understanding cell activity. It also can provide reliable and reproducible method for assessing a risk of exposing people to different harmful substances, identification active compounds in toxicity screening and safety assessment of drugs, cosmetic or their specific ingredients.

  20. Stability-Indicating HPLC Assay for Determination of Idebenone in Pharmaceutical Forms

    Directory of Open Access Journals (Sweden)

    Sonoube Kombath

    2015-01-01

    Full Text Available A stability-indicating method was validated for the determination in pharmaceutical forms of idebenone a coenzyme Q10-like compound. The assay was achieved by liquid chromatography analysis using a reversed-phase C18 column and a detector set at 480 nm. The optimized mobile phase consisted of isocratic flow rate at 1.0 mL/min for 3 min with methanol. The linearity of the assay was demonstrated in the range of 3.0 to 8.0 mg/mL with a correlation coefficient r2>0.998. The limits of detection and quantification were 0.03 and 0.05 mg/mL, respectively. The intraday and interday precisions were less than 1.0%. Accuracy of the method ranged from 98.6 to 101.5% with RSD < 0.6%. Specificity of the assay showed no interference from tablets components and breakdown products formed by alkaline, acidic, oxidative, sunlight, and high temperature conditions. This method allows accurate and reliable determination of idebenone for drug stability assay in pharmaceutical studies.

  1. Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies

    DEFF Research Database (Denmark)

    Hum, S.; Quinn, K.; Brunner, J.

    1997-01-01

    methods were attributed to methodological differences used in various laboratories. Conclusion Our results indicate that misidentification of C fetus in routine diagnostic laboratories may be relatively common. The PCR assay evaluated gave rapid and reproducible results and is thus a valuable adjunctive......Objective To evaluate a polymerase chain reaction assay for identification of Campylobacter fetus and differentiation of the defined subspecies. Design Characterisation of bacterial strains by traditional phenotyping, polymerase chain reaction, a probabilistic identification scheme...... by traditional phenotypic methods and the PCR assay was found to be 80.8%. The polymerase chain reaction proved to be a reliable technique for the species and subspecies identification of C fetus; equivocal results were obtained in only two instances. Initial misidentifications by conventional phenotyping...

  2. International network for comparison of HIV neutralization assays: the NeutNet report.

    Science.gov (United States)

    Fenyö, Eva Maria; Heath, Alan; Dispinseri, Stefania; Holmes, Harvey; Lusso, Paolo; Zolla-Pazner, Susan; Donners, Helen; Heyndrickx, Leo; Alcami, Jose; Bongertz, Vera; Jassoy, Christian; Malnati, Mauro; Montefiori, David; Moog, Christiane; Morris, Lynn; Osmanov, Saladin; Polonis, Victoria; Sattentau, Quentin; Schuitemaker, Hanneke; Sutthent, Ruengpung; Wrin, Terri; Scarlatti, Gabriella

    2009-01-01

    Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet) involving 18 independent participants was organized to compare different assays. Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4) at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (virus infectivity assays, VI assays), or their Env-pseudotyped (gp160) derivatives produced in 293T cells (PSV assays) from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression. PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent. The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of neutralizing

  3. Reliability of ultrasound for measurement of selected foot structures.

    Science.gov (United States)

    Crofts, G; Angin, S; Mickle, K J; Hill, S; Nester, C J

    2014-01-01

    Understanding the relationship between the lower leg muscles, foot structures and function is essential to explain how disease or injury may relate to changes in foot function and clinical pathology. The aim of this study was to investigate the inter-operator reliability of an ultrasound protocol to quantify features of: rear, mid and forefoot sections of the plantar fascia (PF); flexor hallucis brevis (FHB); flexor digitorum brevis (FDB); abductor hallucis (AbH); flexor digitorum longus (FDL); flexor hallucis longus (FHL); tibialis anterior (TA); and peroneus longus and brevis (PER). A sample of 6 females and 4 males (mean age 29.1 ± 7.2 years, mean BMI 25.5 ± 4.8) was recruited from a university student and staff population. Scans were obtained using a portable Venue 40 musculoskeletal ultrasound system (GE Healthcare UK) with a 5-13 MHz wideband linear array probe with a 12.7 mm × 47.1mm footprint by two operators in the same scanning session. Intraclass Correlation Coefficients (ICC) values for muscle thickness (ICC range 0.90-0.97), plantar fascia thickness (ICC range 0.94-0.98) and cross sectional muscle measurements (ICC range 0.91-0.98) revealed excellent inter-operator reliability. The limits of agreement, relative to structure size, ranged from 9.0% to 17.5% for muscle thickness, 11.0-18.0% for plantar fascia, and 11.0-26.0% for cross sectional area measurements. The ultrasound protocol implemented in this work has been shown to be reliable. It therefore offers the opportunity to quantify the structures concerned and better understand their contributions to foot function. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  4. Rapid Multiple Immunoenzyme Assay of Mycotoxins

    Directory of Open Access Journals (Sweden)

    Alexandr E. Urusov

    2015-01-01

    Full Text Available Mycotoxins are low molecular weight fungal metabolites that pose a threat as toxic contaminants of food products, thereby necessitating their effective monitoring and control. Microplate ELISA can be used for this purpose, but this method is characteristically time consuming, with a duration extending to several hours. This report proposes a variant of the ELISA method for the detection and quantification of three mycotoxins, ochratoxin A, aflatoxin B1 and zearalenone, in the kinetic regime. The main requirement for the proposed kinetic protocol was to provide a rapid method that combined sensitivity and accuracy. The use of biotin with an extended spacer together with a streptavidin–polyperoxidase conjugate provided high signal levels, despite these interactions occurring under non-equilibrium conditions. Duration of the individual mycotoxin assays was 20 min, whereas the analysis of all three mycotoxins in parallel reached a maximum duration of 25 min. Recovery of at least 95% mycotoxins in water-organic extracts was shown. The developed assays were successfully validated using poultry processing products and corn samples spiked with known quantities of mycotoxins. The detection limits for aflatoxin B1, ochratoxin A and zearalenone in these substances were 0.24, 1.2 and 3 ng/g, respectively.

  5. Evaluation of the Thermo Scientific™ SureTect™ Listeria species Assay.

    Science.gov (United States)

    Cloke, Jonathan; Evans, Katharine; Crabtree, David; Hughes, Annette; Simpson, Helen; Holopainen, Jani; Wickstrand, Nina; Kauppinen, Mikko

    2014-03-01

    The Thermo Scientific™ SureTect™ Listeria species Assay is a new real-time PCR assay for the detection of all species of Listeria in food and environmental samples. This validation study was conducted using the AOAC Research Institute (RI) Performance Tested MethodsSM program to validate the SureTect Listeria species Assay in comparison to the reference method detailed in International Organization for Standardization 11290-1:1996 including amendment 1:2004 in a variety of foods plus plastic and stainless steel. The food matrixes validated were smoked salmon, processed cheese, fresh bagged spinach, cantaloupe, cooked prawns, cooked sliced turkey meat, cooked sliced ham, salami, pork frankfurters, and raw ground beef. All matrixes were tested by Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. In addition, three matrixes (pork frankfurters, fresh bagged spinach, and stainless steel surface samples) were analyzed independently as part of the AOAC-RI-controlled independent laboratory study by the University of Guelph, Canada. Using probability of detection statistical analysis, a significant difference in favour of the SureTect assay was demonstrated between the SureTect and reference method for high level spiked samples of pork frankfurters, smoked salmon, cooked prawns, stainless steel, and low-spiked samples of salami. For all other matrixes, no significant difference was seen between the two methods during the study. Inclusivity testing was conducted with 68 different isolates of Listeria species, all of which were detected by the SureTect Listeria species Assay. None of the 33 exclusivity isolates were detected by the SureTect Listeria species Assay. Ruggedness testing was conducted to evaluate the performance of the assay with specific method deviations outside of the recommended parameters open to variation, which demonstrated that the assay gave reliable performance. Accelerated stability testing was additionally conducted, validating the

  6. Evaluation of the Thermo Scientific SureTect Listeria monocytogenes Assay.

    Science.gov (United States)

    Cloke, Jonathan; Leon-Velarde, Carlos; Larson, Nathan; Dave, Keron; Evans, Katharine; Crabtree, David; Hughes, Annette; Hopper, Craig; Simpson, Helen; Withey, Sophie; Oleksiuk, Milena; Holopainen, Jani; Wickstrand, Nina; Kauppinen, Mikko

    2014-01-01

    The Thermo Scientific SureTect Listeria monocytogenes Assay is a new real-time PCR assay for the detection of Listeria monocytogenes in food and environmental samples. This assay was validated using the AOAC Research Institute (AOAC-RI) Performance Tested Methods program in comparison to the reference method detailed in International Organization for Standardization 11290-1:1996, including Amendment 1:2004 with the following foods and food contact surfaces: smoked salmon, processed cheese, fresh bagged spinach, fresh cantaloupe, cooked prawns (chilled product), cooked sliced turkey meat (chilled product), ice cream, pork frankfurters, salami, ground raw beef meat (12% fat), plastic, and stainless steel. All matrixes were tested by Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. In addition, three matrixes (pork frankfurters, bagged lettuce, and stainless steel) were analyzed independently as part of the AOAC-RI controlled laboratory study by the University of Guelph, Canada. Using probability of detection (POD) statistical analysis, a significant difference was demonstrated between the candidate and reference methods for salami, cooked sliced turkey and ice cream in favor of the SureTect assay. For all other matrixes, no significant difference by POD was seen between the two methods during the study. Inclusivity and exclusivity testing was also conducted with 53 and 30 isolates, respectively, which demonstrated that the SureTect assay was able to detect all serotypes of L. monocytogenes. None of the exclusivity isolates analyzed were detected by the SureTect assay. Ruggedness testing was conducted to evaluate the performance of the assay with specific method deviations outside the recommended parameters open to variation, i.e., enrichment time and temperature and lysis temperature, which demonstrated that the assay gave reliable performance. Accelerated stability testing was also conducted, validating the assay shelf life.

  7. MsLDR-creator: a web service to design msLDR assays.

    Science.gov (United States)

    Bormann, Felix; Dahl, Andreas; Sers, Christine

    2012-03-01

    MsLDR-creator is a free web service to design assays for the new DNA methylation detection method msLDR. The service provides the user with all necessary information about the oligonucleotides required for the measurement of a given CpG within a sequence of interest. The parameters are calculated by the nearest neighbour approach to achieve optimal behaviour during the experimental procedure. In addition, to guarantee a good start using msLDR, further information, like protocols and hints and tricks, are provided.

  8. A sensitive radioimmunosorbent assay for the detection of plant viruses

    International Nuclear Information System (INIS)

    Ghabrial, S.A.; Shepherd, R.J.

    1980-01-01

    A simple and highly sensitive radioimmunosorbent assay (RISA) for the detection of plant viruses is described. The RISA procedure is a microplate method based on the principle of 'double-antibody sandwich' and follows essentially the protocol of the enzyme-linked immunosorbent assay (ELISA) (Clark and Adams, 1977), with the exception that 125 I-labelled γ-globulin is substituted for the γ-globulin enzyme conjugate; the bound 125 I-γ-globulin is dissociated by acidification from the double-antibody sandwich. The radioactivity is proportional to virus concentration, and cauliflower mosaic virus (CaMV) and lettuce mosaic virus (LMV) could be detected at concentrations as low as 5 and 2 ng/ml, respectively. Direct evidence of the adverse effects of conjugation with enzyme on the binding abilities of antibodies is presented. The RISA procedure should prove valuable with viruses for which the ELISA values are too low to be dependable. (author)

  9. Evaluation of environmental genotoxicity by comet assay in Columba livia.

    Science.gov (United States)

    González-Acevedo, Anahi; García-Salas, Juan A; Gosálvez, Jaime; Fernández, José Luis; Dávila-Rodríguez, Martha I; Cerda-Flores, Ricardo M; Méndez-López, Luis F; Cortés-Gutiérrez, Elva I

    2016-01-01

    The concentrations of recognized or suspected genotoxic and carcinogenic agents found in the air of large cities and, in particular, developing countries, have raised concerns about the potential for chronic health effects in the populations exposed to them. The biomonitoring of environmental genotoxicity requires the selection of representative organisms as "sentinels," as well as the development of suitable and sensitive assays, such as those aimed at assessing DNA damage. The aim of this study was to evaluate DNA damage levels in erythrocytes from Columba livia living in the metropolitan area of Monterrey, Mexico, compared with control animals via comet assay, and to confirm the results via Micronuclei test (MN) and DNA breakage detection-fluorescence in situ hybridization (DBD-FISH). Our results showed a significant increase in DNA migration in animals from the area assayed compared with that observed in control animals sampled in non-contaminated areas. These results were confirmed by MN test and DBD-FISH. In conclusion, these observations confirm that the examination of erythrocytes from Columba livia via alkaline comet assay provides a sensitive and reliable end point for the detection of environmental genotoxicants.

  10. A continuous spectrophotometric assay for monitoring adenosine 5'-monophosphate production.

    Science.gov (United States)

    First, Eric A

    2015-08-15

    A number of biologically important enzymes release adenosine 5'-monophosphate (AMP) as a product, including aminoacyl-tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5'-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5'-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD(+)) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. The comet assay: Reflections on its development, evolution and applications.

    Science.gov (United States)

    Singh, Narendra P

    2016-01-01

    The study of DNA damage and its repair is critical to our understanding of human aging and cancer. This review reflects on the development of a simple technique, now known as the comet assay, to study the accumulation of DNA damage and its repair. It describes my journey into aging research and the need for a method that sensitively quantifies DNA damage on a cell-by-cell basis and on a day-by-day basis. My inspirations, obstacles and successes on the path to developing this assay and improving its reliability and sensitivity are discussed. Recent modifications, applications, and the process of standardizing the technique are also described. What was once untried and unknown has become a technique used around the world for understanding and monitoring DNA damage. The comet assay's use has grown exponentially in the new millennium, as emphasis on studying biological phenomena at the single-cell level has increased. I and others have applied the technique across cell types (including germ cells) and species (including bacteria). As it enters new realms and gains clinical relevance, the comet assay may very well illuminate human aging and its prevention. Copyright © 2016. Published by Elsevier B.V.

  12. Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay.

    Science.gov (United States)

    Pessina, A; Albella, B; Bueren, J; Brantom, P; Casati, S; Gribaldo, L; Croera, C; Gagliardi, G; Foti, P; Parchment, R; Parent-Massin, D; Sibiril, Y; Van Den Heuvel, R

    2001-12-01

    This report describes an international prevalidation study conducted to optimise the Standard Operating Procedure (SOP) for detecting myelosuppressive agents by CFU-GM assay and to study a model for predicting (by means of this in vitro hematopoietic assay) the acute xenobiotic exposure levels that cause maximum tolerated decreases in absolute neutrophil counts (ANC). In the first phase of the study (Protocol Refinement), two SOPs were assessed, by using two cell culture media (Test A, containing GM-CSF; and Test B, containing G-CSF, GM-CSF, IL-3, IL-6 and SCF), and the two tests were applied to cells from both human (bone marrow and umbilical cord blood) and mouse (bone marrow) CFU-GM. In the second phase (Protocol Transfer), the SOPs were transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility. After a further phase (Protocol Performance), dedicated to a training set of six anticancer drugs (adriamycin, flavopindol, morpholino-doxorubicin, pyrazoloacridine, taxol and topotecan), a model for predicting neutropenia was verified. Results showed that the assay is linear under SOP conditions, and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories. Valid tests represented 95% of all tests attempted. The 90% inhibitory concentration values (IC(90)) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs, respectively, that were selected as prototype xenobiotics. As expected, both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant. It is concluded that Test A offers significant cost advantages compared to Test B, without any loss of performance or predictive accuracy. On the basis of these results, we proposed a formal Phase II validation study using the Test A SOP for 16-18 additional

  13. Tumor markers kits development for use in radioimmunometric assays

    International Nuclear Information System (INIS)

    Ahmed, B.

    1997-01-01

    The immunoassays such as RIA and IRMA are now widely used through the world for the quantitation of a variety of substances in the biological fluid for their high sensibility and specificity which required simple equipments. These techniques are also very used in Algeria for an effective amelioration of public heath The assays kits of RIA/IRMA of thyroid hormones are the most used, followed by peptidic hormones, steroids hormones and IRMA Tumor Markers (T.M) kits. In spite of the important demand, of tumor markers kits for the diagnosis and follow up of cancers their use are always insufficient due to the high cost. The research contract programme proposed by IAEA on the theme 'The Developments of IRMA Tumor Markers Kits' of prostate specific Antigen (PSA) and Tissue Polypeptide Specific Antigen (TPS) will allowed us to produce locally with best quality-price, the main reagents for PSA and TPS IRMA assays kits for diagnosis and follow up the prostate and breast cancers which are very spready in the country. This report include the following points: Generalities on the use of tumor markers in Algeria, programme for the Development of the PSA IRMA assay (schedule of protocols applied for each reagents; annual planning for assessing the programme activities) and conclusion

  14. Relative sensitivity of conventional and real-time PCR assays for detection of SFG Rickettsia in blood and tissue samples from laboratory animals.

    Science.gov (United States)

    Zemtsova, Galina E; Montgomery, Merrill; Levin, Michael L

    2015-01-01

    Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87). The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays.

  15. Reliable rapid blood test for the exclusion of venous thromboembolism in symptomatic outpatients

    NARCIS (Netherlands)

    Turkstra, F.; van Beek, E. J.; ten Cate, J. W.; Büller, H. R.

    1996-01-01

    In this study we assessed the reliability of a rapid bed-side whole blood D-dimer assay prospectively in patients with clinically suspected venous thromboembolism, referred to the Academic Medical Centre, Amsterdam. In consecutive outpatients with clinically suspected deep vein thrombosis or

  16. An Efficient Data-Gathering Routing Protocol for Underwater Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    Nadeem Javaid

    2015-11-01

    Full Text Available Most applications of underwater wireless sensor networks (UWSNs demand reliable data delivery over a longer period in an efficient and timely manner. However, the harsh and unpredictable underwater environment makes routing more challenging as compared to terrestrial WSNs. Most of the existing schemes deploy mobile sensors or a mobile sink (MS to maximize data gathering. However, the relatively high deployment cost prevents their usage in most applications. Thus, this paper presents an autonomous underwater vehicle (AUV-aided efficient data-gathering (AEDG routing protocol for reliable data delivery in UWSNs. To prolong the network lifetime, AEDG employs an AUV for data collection from gateways and uses a shortest path tree (SPT algorithm while associating sensor nodes with the gateways. The AEDG protocol also limits the number of associated nodes with the gateway nodes to minimize the network energy consumption and to prevent the gateways from overloading. Moreover, gateways are rotated with the passage of time to balance the energy consumption of the network. To prevent data loss, AEDG allows dynamic data collection at the AUV depending on the limited number of member nodes that are associated with each gateway. We also develop a sub-optimal elliptical trajectory of AUV by using a connected dominating set (CDS to further facilitate network throughput maximization. The performance of the AEDG is validated via simulations, which demonstrate the effectiveness of AEDG in comparison to two existing UWSN routing protocols in terms of the selected performance metrics.

  17. An Efficient Data-Gathering Routing Protocol for Underwater Wireless Sensor Networks.

    Science.gov (United States)

    Javaid, Nadeem; Ilyas, Naveed; Ahmad, Ashfaq; Alrajeh, Nabil; Qasim, Umar; Khan, Zahoor Ali; Liaqat, Tayyaba; Khan, Majid Iqbal

    2015-11-17

    Most applications of underwater wireless sensor networks (UWSNs) demand reliable data delivery over a longer period in an efficient and timely manner. However, the harsh and unpredictable underwater environment makes routing more challenging as compared to terrestrial WSNs. Most of the existing schemes deploy mobile sensors or a mobile sink (MS) to maximize data gathering. However, the relatively high deployment cost prevents their usage in most applications. Thus, this paper presents an autonomous underwater vehicle (AUV)-aided efficient data-gathering (AEDG) routing protocol for reliable data delivery in UWSNs. To prolong the network lifetime, AEDG employs an AUV for data collection from gateways and uses a shortest path tree (SPT) algorithm while associating sensor nodes with the gateways. The AEDG protocol also limits the number of associated nodes with the gateway nodes to minimize the network energy consumption and to prevent the gateways from overloading. Moreover, gateways are rotated with the passage of time to balance the energy consumption of the network. To prevent data loss, AEDG allows dynamic data collection at the AUV depending on the limited number of member nodes that are associated with each gateway. We also develop a sub-optimal elliptical trajectory of AUV by using a connected dominating set (CDS) to further facilitate network throughput maximization. The performance of the AEDG is validated via simulations, which demonstrate the effectiveness of AEDG in comparison to two existing UWSN routing protocols in terms of the selected performance metrics.

  18. Feasibility evaluation of 3 automated cellular drug screening assays on a robotic workstation.

    Science.gov (United States)

    Soikkeli, Anne; Sempio, Cristina; Kaukonen, Ann Marie; Urtti, Arto; Hirvonen, Jouni; Yliperttula, Marjo

    2010-01-01

    This study presents the implementation and optimization of 3 cell-based assays on a TECAN Genesis workstation-the Caspase-Glo 3/7 and sulforhodamine B (SRB) screening assays and the mechanistic Caco-2 permeability protocol-and evaluates their feasibility for automation. During implementation, the dispensing speed to add drug solutions and fixative trichloroacetic acid and the aspiration speed to remove the supernatant immediately after fixation were optimized. Decontamination steps for cleaning the tips and pipetting tubing were also added. The automated Caspase-Glo 3/7 screen was successfully optimized with Caco-2 cells (Z' 0.7, signal-to-base ratio [S/B] 1.7) but not with DU-145 cells. In contrast, the automated SRB screen was successfully optimized with the DU-145 cells (Z' 0.8, S/B 2.4) but not with the Caco-2 cells (Z' -0.8, S/B 1.4). The automated bidirectional Caco-2 permeability experiments separated successfully low- and high-permeability compounds (Z' 0.8, S/B 84.2) and passive drug permeation from efflux-mediated transport (Z' 0.5, S/B 8.6). Of the assays, the homogeneous Caspase-Glo 3/7 assay benefits the most from automation, but also the heterogeneous SRB assay and Caco-2 permeability experiments gain advantages from automation.

  19. Use of limited MR protocol (coronal STIR) in the evaluation of patients with hip pain

    International Nuclear Information System (INIS)

    Khoury, N.J.; Birjawi, G.A.; Hourani, M.H.; Chaaya, M.

    2003-01-01

    To assess the role of a limited MR protocol (coronal STIR) as the initial part of the MR examination in patients with hip pain. Eighty-five patients presenting with hip pain, and normal radiographs of the pelvis, and who underwent our full MR protocol for hips were included retrospectively in the study. The full protocol consists of coronal T1-weighted and short tau inversion-recovery (STIR), and axial T2-weighted sequences. Ninety-three MR examinations were performed. Two radiologists interpreted the STIR (limited) examinations and the full studies separately, masked to each other's findings and to the final diagnosis. Comparison between the two protocols was then undertaken. For both readers, all normal MR examinations on the coronal STIR limited protocol were normal on the full protocol, with an interobserver reliability of 0.96. The STIR protocol was able to detect the presence or absence of an abnormality in 100% of cases (sensitivity). The STIR-only protocol provided a specific diagnosis in only 65% of cases (specificity). A normal coronal STIR study of the hips in patients with hip pain and normal radiographs precludes the need for further pelvic MR sequences. Any abnormality detected on this limited protocol should be further assessed by additional MR sequences. (orig.)

  20. Validation of 2 commercially available enzyme-linked immunosorbent assays for adiponectin determination in canine serum samples.

    Science.gov (United States)

    Tvarijonaviciute, Asta; Martínez-Subiela, Silvia; Ceron, José J

    2010-10-01

    The aim of this study was to validate 2 commercially available enzyme-linked immunosorbent assays (ELISAs) for adiponectin in dogs, 1 canine-specific and 1 originally designed for measurements in humans. Intra-assay and interassay precision was evaluated by multiple measurements in canine serum samples, and assay accuracy was indirectly determined by linearity under dilution. Interference caused by hemolysis and lipemia was also studied. Both assays were subsequently used for measuring adiponectin concentrations in clinically healthy dogs and those with different grades of obesity. The intra-assay and inter-assay precision was less than 7.5% and 13.5% in serum samples with low and high adiponectin concentrations, respectively. Lipemia and hemolysis did not affect the results of any of the assays. Both assays were able to differentiate lean dogs from those that were overweight or obese on the basis of the measured adiponectin concentrations. From these results it can be concluded that canine adiponectin concentrations can be measured reliably by means of the 2 ELISAs evaluated in this study.

  1. Assessing estuarine quality: A cost-effective in situ assay with amphipods.

    Science.gov (United States)

    Martinez-Haro, Monica; Acevedo, Pelayo; Pais-Costa, Antónia Juliana; Taggart, Mark A; Martins, Irene; Ribeiro, Rui; Marques, João Carlos

    2016-05-01

    In situ assays based on feeding depression can be powerful ecotoxicological tools that can link physiological organism-level responses to population and/or community-level effects. Amphipods are traditional target species for toxicity tests due to their high sensitivity to contaminants, availability in the field and ease of handling. However, cost-effective in situ assays based on feeding depression are not yet available for amphipods that inhabit estuarine ecosystems. The aim of this work was to assess a short-term in situ assay based on postexposure feeding rates on easily quantifiable food items with an estuarine amphipod. Experiments were carried out under laboratory conditions using juvenile Echinogammarus marinus as the target individual. When 60 Artemia franciscana nauplii (as prey) were provided per individual for a period of 30 min in dark conditions, feeding rates could be easily quantified. As an endpoint, postexposure feeding inhibition in E. marinus was more sensitive to cadmium contamination than mortality. Assay calibration under field conditions demonstrated the relevance of sediment particle size in explaining individual feeding rates in uncontaminated water bodies. An evaluation of the 48-h in situ bioassay based on postexposure feeding rates indicated that it is able to discriminate between unpolluted and polluted estuarine sites. Using the harmonized protocol described here, the in situ postexposure feeding assay with E. marinus was found to be a potentially useful, cost-effective tool for assessing estuarine sediment and water quality. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Reliability and Validity of the Clinical Dementia Rating for Community-Living Elderly Subjects without an Informant

    Directory of Open Access Journals (Sweden)

    Ma Shwe Zin Nyunt

    2013-10-01

    Full Text Available Background: The Clinical Dementia Rating (CDR scale is widely used to assess cognitive impairment in Alzheimer's disease. It requires collateral information from a reliable informant who is not available in many instances. We adapted the original CDR scale for use with elderly subjects without an informant (CDR-NI and evaluated its reliability and validity for assessing mild cognitive impairment (MCI and dementia among community-dwelling elderly subjects. Method: At two consecutive visits 1 week apart, nurses trained in CDR assessment interviewed, observed and rated cognitive and functional performance according to a protocol in 90 elderly subjects with suboptimal cognitive performance [Mini-Mental State Examination (MMSE Results: The CDR-NI scores (0, 0.5, 1 showed good internal consistency (Crohnbach's a 0.83-0.84, inter-rater reliability (κ 0.77-1.00 for six domains and 0.95 for global rating and test-retest reliability (κ 0.75-1.00 for six domains and 0.80 for global rating, good agreement (κ 0.79 with the clinical assessment status of MCI (n = 37 and dementia (n = 4 and significant differences in the mean scores for MMSE, MOCA and Instrumental Activities of Daily Living (ANOVA global p Conclusion: Owing to the protocol of the interviews, assessments and structured observations gathered during the two visits, CDR-NI provides valid and reliable assessment of MCI and dementia in community-living elderly subjects without an informant.

  3. Evaluation of the radioimmunoassay, indirect enzyme linked immunosorbent assay, and dot blot assay for the identification of Xanthomonas campestris pv. phaseoli

    Energy Technology Data Exchange (ETDEWEB)

    Malin, E; Belden, E L; Roth, D

    1985-09-01

    A radioimmunoassay (RIA), an indirect competitive enzyme-linked immunosorbent assay (ELISA), and a dot-blot modification of the ELISA were evaluated for detection and identification of Xanthomonas campestris pv. phaseoli (X. c. pv. phaseoli). RIA and the dot blot tests were specific for X. c. pv. phaseoli; however, significant cross reactions occurred in the indirect competitive ELISA when using anti-X. c. pv. phaseoli antiserum against other closely related bacteria. The sensitivity level of all procedures for X. c. pv. phaseoli was approximately l0/sup 5/ colony forming unitsmL. All procedures were unsatisfactory in reliably detecting low levels of X. c. pv. phaseoli directly from extracts of bean seed. However when used in conjunction with ilution plating the dot blot assay and the RIA would be useful in specifically identifying X. c. pv. phaseoli. The relative merits of these tests for identification of X. c. pv. phaseoli are discussed.

  4. How to best freeze liver samples to perform the in vivo mammalian alkaline comet assay

    Directory of Open Access Journals (Sweden)

    José Manuel Enciso Gadea

    2015-06-01

    None of the different methods used was capable of giving good results, except immersing the liver samples in liquid nitrogen, followed by Jackson’s et al. (2013 thawing protocol, suggesting that the thawing process may be as critical as the freezing process. To sum up, these results highlight the importance of deepening the possibility to perform the comet assay with frozen tissue.

  5. Modification of Rat Lung Decellularization Protocol Based on Dynamic Conductometry of Working Solution.

    Science.gov (United States)

    Kuevda, E V; Gubareva, E A; Gumenyuk, I S; Sotnichenko, A S; Gilevich, I V; Nakokhov, R Z; Rusinova, T V; Yudina, T G; Red'ko, A N; Alekseenko, S N

    2017-03-01

    We modified the protocol of obtaining of biological scaffolds of rat lungs based on dynamic recording of specific resistivity of working detergent solution (conductometry) during perfusion decellularization. Termination of sodium deoxycholate exposure after attaining ionic equilibrium plateau did not impair the quality of decellularization and preserved structural matrix components, which was confirmed by morphological analysis and quantitative assay of residual DNA.

  6. Standardized protocol for artery-only fingertip replantation.

    Science.gov (United States)

    Buntic, Rudolf F; Brooks, Darrell

    2010-09-01

    Artery-only fingertip replantation can be reliable if low-resistance flow through the replant is maintained until venous outflow is restored naturally. Injuring the tip of the replant to promote ongoing bleeding augmented with anticoagulation usually accomplishes this; however, such management results in prolonged hospitalization. In this study, we analyzed the outcomes of artery-only fingertip replantation using a standardized postoperative protocol consisting of dextran-40, heparin, and leech therapy. Between 2001 and 2008, we performed 19 artery-only fingertip replants for 17 patients. All patients had the replanted nail plate removed and received intravenous dextran-40, heparin, and aspirin to promote fingertip bleeding and vascular outflow. Anticoagulation was titrated to promote a controlled bleed until physiologic venous outflow was restored by neovascularization. We used medicinal leeches and mechanical heparin scrubbing for acute decongestion. By postoperative day 6, bleeding was no longer promoted. We initiated fluorescent dye perfusion studies to assess circulatory competence and direct further anticoagulant intervention if necessary. The absence of bleeding associated with an initial rise followed by an appropriate fall in fluorescent dye concentration would trigger a weaning of anticoagulation. All of the 19 replants survived. The average length of hospital stay was 9 days (range, 7-17 d). Eleven patients received blood transfusions. The average transfusion was 1.8 units (range, 0-9 units). All patients were happy with the decision to replant, and the cosmetic result. A protocol that promotes temporary, controlled bleeding from the fingertip is protective of artery-only replants distal to the distal interphalangeal joint until physiologic venous outflow is restored. The protocol described is both safe and reliable. The patient should be informed that such replant attempts may result in the need for transfusions and extended hospital stays, factors that

  7. Intrajudge and Interjudge Reliability of the Stuttering Severity Instrument-Fourth Edition.

    Science.gov (United States)

    Davidow, Jason H; Scott, Kathleen A

    2017-11-08

    The Stuttering Severity Instrument (SSI) is a tool used to measure the severity of stuttering. Previous versions of the instrument have known limitations (e.g., Lewis, 1995). The present study examined the intra- and interjudge reliability of the newest version, the Stuttering Severity Instrument-Fourth Edition (SSI-4) (Riley, 2009). Twelve judges who were trained on the SSI-4 protocol participated. Judges collected SSI-4 data while viewing 4 videos of adults who stutter at Time 1 and 4 weeks later at Time 2. Data were analyzed for intra- and interjudge reliability of the SSI-4 subscores (for Frequency, Duration, and Physical Concomitants), total score, and final severity rating. Intra- and interjudge reliability across the subscores and total score concurred with the manual's reported reliability when reliability was calculated using the methods described in the manual. New calculations of judge agreement produced different values from those in the manual-for the 3 subscores, total score, and final severity rating-and provided data absent from the manual. Clinicians and researchers who use the SSI-4 should carefully consider the limitations of the instrument. Investigation into the multitasking demands of the instrument may provide information on whether separating the collection of data for specific variables will improve intra- and interjudge reliability of those variables.

  8. Gateway-Assisted Retransmission for Lightweight and Reliable IoT Communications.

    Science.gov (United States)

    Chang, Hui-Ling; Wang, Cheng-Gang; Wu, Mong-Ting; Tsai, Meng-Hsun; Lin, Chia-Ying

    2016-09-22

    Message Queuing Telemetry Transport for Sensor Networks (MQTT-SN) and Constrained Application Protocol (CoAP) are two protocols supporting publish/subscribe models for IoT devices to publish messages to interested subscribers. Retransmission mechanisms are introduced to compensate for the lack of data reliability. If the device does not receive the acknowledgement (ACK) before retransmission timeout (RTO) expires, the device will retransmit data. Setting an appropriate RTO is important because the delay may be large or retransmission may be too frequent when the RTO is inappropriate. We propose a Gateway-assisted CoAP (GaCoAP) to dynamically compute RTO for devices. Simulation models are proposed to investigate the performance of GaCoAP compared with four other methods. The experiment results show that GaCoAP is more suitable for IoT devices.

  9. Patched Skin Bilirubin Assay to Monitor Neonates Born Extremely Preterm Undergoing Phototherapy.

    Science.gov (United States)

    De Luca, Daniele; Dell'Orto, Valentina

    2017-09-01

    To verify the reliability and safety of transcutaneous bilirubin (TcB) measurements in patched skin areas in neonates born extremely preterm under phototherapy. Sixty neonates (bilirubin (TSB), lactate, pH, hemoglobin, and skin temperature were measured within 10 minutes of the TcB assay. Clinicians were blinded to TcB values, and clinical decisions about phototherapy were made with the TSB measurement only. TcB and TSB significantly were correlated (r = 0.84; P bilirubin passage. TcB overestimated TSB, and this may expose infants born preterm to unnecessary phototherapy, although it could spare approximately 65% of TSB assays. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Psychometric Properties of a Standardized Observation Protocol to Quantify Pediatric Physical Therapy Actions.

    Science.gov (United States)

    Sonderer, Patrizia; Akhbari Ziegler, Schirin; Gressbach Oertle, Barbara; Meichtry, André; Hadders-Algra, Mijna

    2017-07-01

    Pediatric physical therapy (PPT) is characterized by heterogeneity. This blurs the evaluation of effective components of PPT. The Groningen Observation Protocol (GOP) was developed to quantify contents of PPT. This study assesses the reliability and completeness of the GOP. Sixty infant PPT sessions were video-taped. Two random samples of 10 videos were used to determine interrater and intrarater reliability using interclass correlation coefficients (ICCs) with 95% confidence intervals. Completeness of GOP 2.0 was based on 60 videos. Interrater reliability of quantifying PPT actions was excellent (ICC, 0.75-1.0) in 71% and sufficient to good (ICC, 0.4-0.74) in 24% of PPT actions. Intrarater reliability was excellent in 94% and sufficient to good in 6% of PPT actions. Completeness was good for greater than 90% of PPT actions. GOP 2.0 has good reliability and completeness. After appropriate training, it is a useful tool to quantify PPT for children with developmental disorders.

  11. A histidine-rich protein 2-based malaria drug sensitivity assay for field use.

    Science.gov (United States)

    Noedl, Harald; Attlmayr, Bernhard; Wernsdorfer, Walther H; Kollaritsch, Herwig; Miller, Robert S

    2004-12-01

    With the spread of antimalarial drug resistance, simple and reliable tools for the assessment of antimalarial drug resistance, particularly in endemic regions and under field conditions, have become more important than ever before. We therefore developed a histidine-rich protein 2 (HRP2)-based drug sensitivity assay for testing of fresh isolates of Plasmodium falciparum in the field. In contrast to the HRP2 laboratory assay, the field assay uses a procedure that further simplifies the handling and culturing of malaria parasites by omitting centrifugation, washing, the use of serum, and dilution with uninfected red blood cells. A total of 40 fresh Plasmodium falciparum isolates were successfully tested for their susceptibility to dihydroartemisinin, mefloquine, quinine, and chloroquine (50% inhibitory concentration [IC50] = 3.43, 61.89, 326.75, and 185.31 nM, respectively). Results very closely matched those obtained with a modified World Health Organization schizont maturation assay (R2 = 0.96, P < 0.001; mean log difference at IC50 = 0.054).

  12. A MIQE-compliant real-time PCR assay for Aspergillus detection.

    Directory of Open Access Journals (Sweden)

    Gemma L Johnson

    Full Text Available The polymerase chain reaction (PCR is widely used as a diagnostic tool in clinical laboratories and is particularly effective for detecting and identifying infectious agents for which routine culture and microscopy methods are inadequate. Invasive fungal disease (IFD is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA, variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published "Minimum Information for the publication of real-time Quantitative PCR Experiments" (MIQE guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95-107%, a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness

  13. Application of Peptide Nucleic Acid-based Assays Toward Detection of Somatic Mosaicism

    Directory of Open Access Journals (Sweden)

    Christopher S Hong

    2016-01-01

    Full Text Available Peptide nucleic acids (PNAs are synthetic oligonucleotides with many applications. Compared with DNA, PNAs bind their complementary DNA strand with higher specificity and strength, an attribute that can make it an effective polymerase chain reaction clamp. A growing body of work has demonstrated the utility of PNAs in detecting low levels of mutant DNA, particularly in the detection of circulating mutated tumor cells in the peripheral blood. The PNA-based assay has greater sensitivity than direct sequencing and is significantly more affordable and rapid than next-generation deep sequencing. We have previously demonstrated that PNAs can successfully detect somatic mosaicism in patients with suspected disease phenotypes. In this report, we detail our methodology behind PNA design and application. We describe our protocol for optimizing the PNA for sequencing use and for determining the sensitivity of the PNA-based assay. Lastly, we discuss the potential applications of our assay for future laboratory and clinical purposes and highlight the role of PNAs in the detection of somatic mosaicism.

  14. Cyber Security Vulnerability Impact on I and C Reliability

    International Nuclear Information System (INIS)

    Hadley, Mark D.; McBride, Justin B.

    2006-01-01

    We present a discussion of the cyber security vulnerability impact on instrument and control reliability. In the discussion we demonstrate the likely vector of attack and vulnerabilities associated with commodity hardware, protocols and communication media. The current fleet of nuclear power plants in the United States utilizes aging analog instrument and control systems which are more frequently suffering from obsolescence and failure. The commodity equipment available now and in the near future incorporates features from information technology systems which compound cyber vulnerabilities

  15. A Robust PCR Protocol for HIV Drug Resistance Testing on Low-Level Viremia Samples

    Directory of Open Access Journals (Sweden)

    Shivani Gupta

    2017-01-01

    Full Text Available The prevalence of drug resistance (DR mutations in people with HIV-1 infection, particularly those with low-level viremia (LLV, supports the need to improve the sensitivity of amplification methods for HIV DR genotyping in order to optimize antiretroviral regimen and facilitate HIV-1 DR surveillance and relevant research. Here we report on a fully validated PCR-based protocol that achieves consistent amplification of the protease (PR and reverse transcriptase (RT regions of HIV-1 pol gene across many HIV-1 subtypes from LLV plasma samples. HIV-spiked plasma samples from the External Quality Assurance Program Oversight Laboratory (EQAPOL, covering various HIV-1 subtypes, as well as clinical specimens were used to optimize and validate the protocol. Our results demonstrate that this protocol has a broad HIV-1 subtype coverage and viral load span with high sensitivity and reproducibility. Moreover, the protocol is robust even when plasma sample volumes are limited, the HIV viral load is unknown, and/or the HIV subtype is undetermined. Thus, the protocol is applicable for the initial amplification of the HIV-1 PR and RT genes required for subsequent genotypic DR assays.

  16. Reliability and Validity of SERVQUAL Scores Used To Evaluate Perceptions of Library Service Quality.

    Science.gov (United States)

    Thompson, Bruce; Cook, Colleen

    Research libraries are increasingly supplementing collection counts with perceptions of service quality as indices of status and productivity. The present study was undertaken to explore the reliability and validity of scores from the SERVQUAL measurement protocol (A. Parasuraman and others, 1991), which has previously been used in this type of…

  17. Performance characteristics of bioassay, radioenzymatic assay, homogeneous enzyme immunoassay, and high-performance liquid chromatographic determination of serum gentamicin

    International Nuclear Information System (INIS)

    Delaney, C.J.; Opheim, K.E.; Smith, A.L.; Plorde, J.J.

    1982-01-01

    We compared the accuracy, precision, and between-method error of the microbiological assay, the radioenzymatic assay, the homogeneous enzyme immunoassay, and the high-performance liquid chromatographic assay for the quantitation of gentamicin in serum. Precision and accuracy were evaluated by reference samples prepared to contain 0.0 to 32.7 micrograms of gentamicin per ml. Correlations between the methods utilized patient sera with gentamicin concentrations ranging from 0.6 to 13.3 micrograms/ml. All methods were reliable within acceptable limits for routine clinical use; intermethod correlation coefficients exceeded 0.96. Relative to the microbiological assay, the alternative methods offer the advantage of rapid analysis. The elapsed times for acquiring data on a set of 10 specimens under routine operating conditions were 0.5 h by the enzyme immunoassay, 4 h by the radioenzymatic assay, 5 h by the high-performance liquid chromatographic assay, and 10 h by the microbiological assay

  18. The Comet assay in insects--Status, prospects and benefits for science.

    Science.gov (United States)

    Augustyniak, Maria; Gladysz, Marcin; Dziewięcka, Marta

    2016-01-01

    The Comet assay has been recently adapted to investigate DNA damage in insects. The first reports of its use in Drosophila melanogaster appeared in 2002. Since then, the interest in the application of the Comet assay to studies of insects has been rapidly increasing. Many authors see substantial potential in the use of the Comet assay in D. melanogaster for medical toxicology studies. This application could allow the testing of drugs and result in an understanding of the mechanisms of action of toxins, which could significantly influence the limited research that has been performed on vertebrates. The possible perspectives and benefits for science are considered in this review. In the last decade, the use of the Comet assay has been described in insects other than D. melanogaster. Specifically, methods to prepare a cell suspension from insect tissues, which is a difficult task, were analyzed and compared in detail. Furthermore, attention was paid to any differences and modifications in the research protocols, such as the buffer composition and electrophoresis conditions. Various scientific fields in addition to toxicological and ecotoxicological research were considered. We expect the Comet assay to be used in environmental risk assessments and to improve our understanding of many important phenomena of insect life, such as metamorphosis, molting, diapause and quiescence. The use of this method to study species that are of key importance to humans, such as pests and beneficial insects, appears to be highly probable and very promising. The use of the Comet assay for DNA stability testing in insects will most likely rapidly increase in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Multicenter Evaluation of Cystatin C Measurement after Assay Standardization.

    Science.gov (United States)

    Bargnoux, Anne-Sophie; Piéroni, Laurence; Cristol, Jean-Paul; Kuster, Nils; Delanaye, Pierre; Carlier, Marie-Christine; Fellahi, Soraya; Boutten, Anne; Lombard, Christine; González-Antuña, Ana; Delatour, Vincent; Cavalier, Etienne

    2017-04-01

    Since 2010, a certified reference material ERM-DA471/IFCC has been available for cystatin C (CysC). This study aimed to assess the sources of uncertainty in results for clinical samples measured using standardized assays. This evaluation was performed in 2015 and involved 7 clinical laboratories located in France and Belgium. CysC was measured in a panel of 4 serum pools using 8 automated assays and a candidate isotope dilution mass spectrometry reference measurement procedure. Sources of uncertainty (imprecision and bias) were evaluated to calculate the relative expanded combined uncertainty for each CysC assay. Uncertainty was judged against the performance specifications derived from the biological variation model. Only Siemens reagents on the Siemens systems and, to a lesser extent, DiaSys reagents on the Cobas system, provided results that met the minimum performance criterion calculated according to the intraindividual and interindividual biological variations. Although the imprecision was acceptable for almost all assays, an increase in the bias with concentration was observed for Gentian reagents, and unacceptably high biases were observed for Abbott and Roche reagents on their own systems. This comprehensive picture of the market situation since the release of ERM-DA471/IFCC shows that bias remains the major component of the combined uncertainty because of possible problems associated with the implementation of traceability. Although some manufacturers have clearly improved their calibration protocols relative to ERM-DA471, most of them failed to meet the criteria for acceptable CysC measurements. © 2016 American Association for Clinical Chemistry.

  20. New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter.

    Directory of Open Access Journals (Sweden)

    Joanna Jońca

    Full Text Available Butyrylcholinesterase (BChE activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman's method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.

  1. Inter- and intra-observer reliability of masking in plantar pressure measurement analysis.

    Science.gov (United States)

    Deschamps, K; Birch, I; Mc Innes, J; Desloovere, K; Matricali, G A

    2009-10-01

    Plantar pressure measurement is an important tool in gait analysis. Manual placement of small masks (masking) is increasingly used to calculate plantar pressure characteristics. Little is known concerning the reliability of manual masking. The aim of this study was to determine the reliability of masking on 2D plantar pressure footprints, in a population with forefoot deformity (i.e. hallux valgus). Using a random repeated-measure design, four observers identified the third metatarsal head on a peak-pressure barefoot footprint, using a small mask. Subsequently, the location of all five metatarsal heads was identified, using the same size of masks and the same protocol. The 2D positional variation of the masks and the peak pressure (PP) and pressure time integral (PTI) values of each mask were calculated. For single-masking the lowest inter-observer reliability was found for the distal-proximal direction, causing a clear, adverse impact on the reliability of the pressure characteristics (PP and PTI). In the medial-lateral direction the inter-observer reliability could be scored as high. Intra-observer reliability was better and could be scored as high or good for both directions, with a correlated improved reliability of the pressure characteristics. Reliability of multi-masking showed a similar pattern, but overall values tended to be lower. Therefore, small sized masking in order to define pressure characteristics in the forefoot should be done with care.

  2. International network for comparison of HIV neutralization assays: the NeutNet report.

    Directory of Open Access Journals (Sweden)

    Eva Maria Fenyö

    Full Text Available Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1 vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet involving 18 independent participants was organized to compare different assays.Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4 at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs (virus infectivity assays, VI assays, or their Env-pseudotyped (gp160 derivatives produced in 293T cells (PSV assays from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression.PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent.The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of

  3. A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

    Science.gov (United States)

    Kim, G G; Donnenberg, V S; Donnenberg, A D; Gooding, W; Whiteside, T L

    2007-08-31

    Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

  4. Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells.

    Science.gov (United States)

    Stapelfeldt, Karsten; Ehrke, Eric; Steinmeier, Johann; Rastedt, Wiebke; Dringen, Ralf

    2017-12-01

    Cellular reduction of tetrazolium salts to their respective formazans is frequently used to determine the metabolic activity of cultured cells as an indicator of cell viability. For membrane-impermeable tetrazolium salts such as WST1 the application of a membrane-permeable electron cycler is usually required to mediate the transfer of intracellular electrons for extracellular WST1 reduction. Here we demonstrate that in addition to the commonly used electron cycler M-PMS, menadione can also serve as an efficient electron cycler for extracellular WST1 reduction in cultured neural cells. The increase in formazan absorbance in glial cell cultures for the WST1 reduction by menadione involves enzymatic menadione reduction and was twice that recorded for the cytosolic enzyme-independent WST1 reduction in the presence of M-PMS. The optimized WST1 reduction assay allowed within 30 min of incubation a highly reliable detection of compromised cell metabolism caused by 3-bromopyruvate and impaired membrane integrity caused by Triton X-100, with a sensitivity as good as that of spectrophotometric assays which determine cellular MTT reduction or lactate dehydrogenase release. The short incubation period of 30 min and the observed good sensitivity make this optimized menadione-mediated WST1 reduction assay a quick and reliable alternative to other viability and toxicity assays. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Who Shot Ya? How Emergency Departments Can Collect Reliable Police Shooting Data.

    Science.gov (United States)

    Richardson, Joseph B; St Vil, Christopher; Cooper, Carnell

    2016-04-01

    This paper examines an alternative solution for collecting reliable police shooting data. One alternative is the collection of police shooting data from hospital trauma units, specifically hospital-based violence intervention programs. These programs are situated in Level I trauma units in many major cities in USA. While the intent of these programs is to reduce the risk factors associated with trauma recidivism among victims of violent injury, they also collect reliable data on the number of individuals treated for gunshot wounds. While most trauma units do a great job collecting data on mode of injury, many do not collect data on the circumstances surrounding the injury, particularly police-involved shootings. Research protocol on firearm-related injury conducted in emergency departments typically does not allow researchers to interview victims of violent injury who are under arrest. Most victims of nonfatal police-involved shootings are under arrest at the time they are treated by the ED for their injury. Research protocol on victims of violent injury often excludes individuals under arrest; they fall under the exclusion criteria when recruiting potential participants for research on violence. Researchers working in hospital emergency departments are prohibited from recruited individuals under arrests. The trauma staff, particularly ED physicians and nurses, are in a strategic position to collect this kind of data. Thus, this paper examines how trauma units can serve as an alternative in the reliable collection of police shooting data.

  6. Brief communication: effect of coca-leaf chewing on salivary progesterone assays.

    Science.gov (United States)

    Vitzthum, V J; von Dornum, M; Ellison, P T

    1993-12-01

    Although there is evidence for reduced fertility in Andean and Himalayan populations at higher altitudes, factors other than hypoxia may be primarily responsible. A valuable approach in the investigation of these fertility determinants is the use of salivary steroid assays. However, coca-leaf chewing--a ubiquitous practice among high altitude Andean populations--has negative consequences for the accurate measurement of ovarian steroids. This report evaluates the effects of coca-leaf chewing on assays of salivary progesterone. Study participants include naive and habitual users of coca leaf from La Paz and El Alto, Bolivia. Approximately 300 saliva samples were collected immediately before, during, and after coca-leaf chewing. The series includes samples with and without the alkaloid enhancer typically used by coca-leaf chewers. Coca chewing produces false salivary progesterone values that mimic luteal phase values. On the basis of this study, an appropriate protocol is developed for the collection of salivary samples in coca-leaf chewing populations. These results verify the feasibility of salivary assays, even for very difficult field conditions, and highlight the necessity of establishing suitable collection procedures before full field implementation of saliva sampling.

  7. Reliable multicast for the Grid: a case study in experimental computer science.

    Science.gov (United States)

    Nekovee, Maziar; Barcellos, Marinho P; Daw, Michael

    2005-08-15

    In its simplest form, multicast communication is the process of sending data packets from a source to multiple destinations in the same logical multicast group. IP multicast allows the efficient transport of data through wide-area networks, and its potentially great value for the Grid has been highlighted recently by a number of research groups. In this paper, we focus on the use of IP multicast in Grid applications, which require high-throughput reliable multicast. These include Grid-enabled computational steering and collaborative visualization applications, and wide-area distributed computing. We describe the results of our extensive evaluation studies of state-of-the-art reliable-multicast protocols, which were performed on the UK's high-speed academic networks. Based on these studies, we examine the ability of current reliable multicast technology to meet the Grid's requirements and discuss future directions.

  8. Validation of the Filovirus Plaque Assay for Use in Preclinical Studies

    Directory of Open Access Journals (Sweden)

    Amy C. Shurtleff

    2016-04-01

    Full Text Available A plaque assay for quantitating filoviruses in virus stocks, prepared viral challenge inocula and samples from research animals has recently been fully characterized and standardized for use across multiple institutions performing Biosafety Level 4 (BSL-4 studies. After standardization studies were completed, Good Laboratory Practices (GLP-compliant plaque assay method validation studies to demonstrate suitability for reliable and reproducible measurement of the Marburg Virus Angola (MARV variant and Ebola Virus Kikwit (EBOV variant commenced at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID. The validation parameters tested included accuracy, precision, linearity, robustness, stability of the virus stocks and system suitability. The MARV and EBOV assays were confirmed to be accurate to ±0.5 log10 PFU/mL. Repeatability precision, intermediate precision and reproducibility precision were sufficient to return viral titers with a coefficient of variation (%CV of ≤30%, deemed acceptable variation for a cell-based bioassay. Intraclass correlation statistical techniques for the evaluation of the assay’s precision when the same plaques were quantitated by two analysts returned values passing the acceptance criteria, indicating high agreement between analysts. The assay was shown to be accurate and specific when run on Nonhuman Primates (NHP serum and plasma samples diluted in plaque assay medium, with negligible matrix effects. Virus stocks demonstrated stability for freeze-thaw cycles typical of normal usage during assay retests. The results demonstrated that the EBOV and MARV plaque assays are accurate, precise and robust for filovirus titration in samples associated with the performance of GLP animal model studies.

  9. Wellness protocol for smart homes an integrated framework for ambient assisted living

    CERN Document Server

    Ghayvat, Hemant

    2017-01-01

    This book focuses on the development of wellness protocols for smart home monitoring, aiming to forecast the wellness of individuals living in ambient assisted living (AAL) environments. It describes in detail the design and implementation of heterogeneous wireless sensors and networks as applied to data mining and machine learning, which the protocols are based on. Further, it shows how these sensor and actuator nodes are deployed in the home environment, generating real-time data on object usage and other movements inside the home, and therefore demonstrates that the protocols have proven to offer a reliable, efficient, flexible, and economical solution for smart home systems. Documenting the approach from sensor to decision making and information generation, the book addresses various issues concerning interference mitigation, errors, security and large data handling. As such, it offers a valuable resource for researchers, students and practitioners interested in interdisciplinary studies at the intersecti...

  10. Reliability of Phase Velocity Measurements of Flexural Acoustic Waves in the Human Tibia In-Vivo.

    Science.gov (United States)

    Vogl, Florian; Schnüriger, Karin; Gerber, Hans; Taylor, William R

    2016-01-01

    Axial-transmission acoustics have shown to be a promising technique to measure individual bone properties and detect bone pathologies. With the ultimate goal being the in-vivo application of such systems, quantification of the key aspects governing the reliability is crucial to bring this method towards clinical use. This work presents a systematic reliability study quantifying the sources of variability and their magnitudes of in-vivo measurements using axial-transmission acoustics. 42 healthy subjects were measured by an experienced operator twice per week, over a four-month period, resulting in over 150000 wave measurements. In a complementary study to assess the influence of different operators performing the measurements, 10 novice operators were trained, and each measured 5 subjects on a single occasion, using the same measurement protocol as in the first part of the study. The estimated standard error for the measurement protocol used to collect the study data was ∼ 17 m/s (∼ 4% of the grand mean) and the index of dependability, as a measure of reliability, was Φ = 0.81. It was shown that the method is suitable for multi-operator use and that the reliability can be improved efficiently by additional measurements with device repositioning, while additional measurements without repositioning cannot improve the reliability substantially. Phase velocity values were found to be significantly higher in males than in females (p < 10-5) and an intra-class correlation coefficient of r = 0.70 was found between the legs of each subject. The high reliability of this non-invasive approach and its intrinsic sensitivity to mechanical properties opens perspectives for the rapid and inexpensive clinical assessment of bone pathologies, as well as for monitoring programmes without any radiation exposure for the patient.

  11. Relative sensitivity of conventional and real-time PCR assays for detection of SFG Rickettsia in blood and tissue samples from laboratory animals.

    Directory of Open Access Journals (Sweden)

    Galina E Zemtsova

    Full Text Available Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87. The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays.

  12. A critical assessment for the value of markers to gate-out undesired events in HLA-peptide multimer staining protocols

    Directory of Open Access Journals (Sweden)

    Odunsi Kunle

    2011-07-01

    Full Text Available Abstract Background The introduction of antibody markers to identify undesired cell populations in flow-cytometry based assays, so called DUMP channel markers, has become a practice in an increasing number of labs performing HLA-peptide multimer assays. However, the impact of the introduction of a DUMP channel in multimer assays has so far not been systematically investigated across a broad variety of protocols. Methods The Cancer Research Institute's Cancer Immunotherapy Consortium (CRI-CIC conducted a multimer proficiency panel with a specific focus on the impact of DUMP channel use. The panel design allowed individual laboratories to use their own protocol for thawing, staining, gating, and data analysis. Each experiment was performed twice and in parallel, with and without the application of a dump channel strategy. Results The introduction of a DUMP channel is an effective measure to reduce the amount of non-specific MULTIMER binding to T cells. Beneficial effects for the use of a DUMP channel were observed across a wide range of individual laboratories and for all tested donor-antigen combinations. In 48% of experiments we observed a reduction of the background MULTIMER-binding. In this subgroup of experiments the median background reduction observed after introduction of a DUMP channel was 0.053%. Conclusions We conclude that appropriate use of a DUMP channel can significantly reduce background staining across a large fraction of protocols and improve the ability to accurately detect and quantify the frequency of antigen-specific T cells by multimer reagents. Thus, use of a DUMP channel may become crucial for detecting low frequency antigen-specific immune responses. Further recommendations on assay performance and data presentation guidelines for publication of MULTIMER experimental data are provided.

  13. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

    2002-01-01

    A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD......) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect ... 6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro...

  14. Automated two-site immunofluorescent assay for the measurement of serum chromogranin A.

    Science.gov (United States)

    Popovici, Théodora; Moreira, Baptiste; Schlageter, Marie-Hélène; Bories, Phuong-Nhi

    2014-01-01

    Chromogranin A (CgA) is the best-characterized biological marker common to neuroendocrine tumours and is therefore recommended for their diagnosis. The measurement of serum CgA is of great importance for reaching an early diagnosis and thus reducing the delay before treatment is instigated. The Kryptor CgA assay is the first fully automated assay available. The aim of this study was to evaluate its analytical performance. The imprecision and linearity of the Kryptor CgA assay were evaluated. This assay was compared with the Cis Bio CgA RIA assay in 78 serum samples. Its clinical utility was assessed in serum from 229 patients. The study performed on imprecision of Kryptor measurements showed intra- and inter-run CVs ≤ 5%. The study of linearity showed a satisfactory recovery rate for CgA concentrations up to 1200 μg/L. The Kryptor and RIA assays agreed well on the basis of the cut-off values provided by the two manufacturers. The Bland and Altman plot of the values obtained (range: 20-5560 μg/L) provided a mean difference of -10.1 μg/L (SD: 116). The clinical sensitivities of Kryptor CgA for diagnosis of pheochromocytoma and paraganglioma (n 20) and gastroenteropancreatic NETs (n 17) were respectively 100 and 94%. The Kryptor assay for CgA shows reliable analytical and clinical characteristics and allows a fast delivery of results. © 2013.

  15. Strategy for Developing Expert-System-Based Internet Protocols (TCP/IP)

    Science.gov (United States)

    Ivancic, William D.

    1997-01-01

    The Satellite Networks and Architectures Branch of NASA's Lewis Research is addressing the issue of seamless interoperability of satellite networks with terrestrial networks. One of the major issues is improving reliable transmission protocols such as TCP over long latency and error-prone links. Many tuning parameters are available to enhance the performance of TCP including segment size, timers and window sizes. There are also numerous congestion avoidance algorithms such as slow start, selective retransmission and selective acknowledgment that are utilized to improve performance. This paper provides a strategy to characterize the performance of TCP relative to various parameter settings in a variety of network environments (i.e. LAN, WAN, wireless, satellite, and IP over ATM). This information can then be utilized to develop expert-system-based Internet protocols.

  16. Microbubble Enzyme-Linked Immunosorbent Assay for the Detection of Targeted Microbubbles in in Vitro Static Binding Assays.

    Science.gov (United States)

    Wischhusen, Jennifer; Padilla, Frederic

    2017-07-01

    Targeted microbubbles (MBs) are ultrasound contrast agents that are functionalized with a ligand for ultrasound molecular imaging of endothelial markers. Novel targeted MBs are characterized in vitro by incubation in protein-coated wells, followed by binding quantification by microscopy or ultrasound imaging. Both methods provide operator-dependent results: Between 3 and 20 fields of view from a heterogeneous sample are typically selected for analysis by microscopy, and in ultrasound imaging, different acoustic settings affect signal intensities. This study proposes a new method to reproducibly quantify MB binding based on enzyme-linked immunosorbent assay (ELISA), in which bound MBs are revealed with an enzyme-linked antibody. MB-ELISA was adapted to in vitro static binding assays, incubating the MBs in inverted position or by agitation, and compared with microscopy. The specificity and sensitivity of MB-ELISA enable the reliable quantification of MB binding in a rapid, high-throughput and whole-well analysis, facilitating the characterization of new targeted contrast agents. Copyright © 2017 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  17. Development and evaluation of an interferon gamma assay for the diagnosis of tuberculosis in red deer experimentally infected with Mycobacterium bovis.

    Science.gov (United States)

    Risalde, María Ángeles; Thomas, Jobin; Sevilla, Iker; Serrano, Miriam; Ortíz, Jose Antonio; Garrido, Joseba; Domínguez, Mercedes; Domínguez, Lucas; Gortázar, Christian; Ruíz-Fons, Jose Francisco

    2017-11-16

    Red deer (Cervus elaphus) is regarded as an epidemiologically relevant host for Mycobacterium bovis (M. bovis) and closely related members of the Mycobacterium tuberculosis complex that cause animal tuberculosis (TB). The standard antemortem screening test for the detection of TB in deer is the intradermal tuberculin skin test, but the detection of interferon-gamma (IFNγ) produced by white blood cells exposed to M. bovis antigens can be used as an alternative or supplemental assay in most TB eradication/control programs. This study aims to develop an in-house sandwich ELISA for deer IFNγ, based on the cross-reactivity of the antibodies to both cervid and bovine IFNγ, and to evaluate the potential of this assay to detect M. bovis-infected red deer in response to the in vitro stimulation of whole-blood cells with bovine purified protein derivative (bPPD), p22 protein complex derived from bPPD or using the specific tuberculous mycobacterial proteins ESAT-6/CFP-10, Rv3615c and Rv3020c. The positive control stimulant used in this study was pokeweed mitogen, which resulted in a consistent induction of IFNγ in samples from red deer, thus allowing the interpretation of the assay. The percentage of animals correctly classified by this technique as M. bovis non-infected was 100%. The detection of infected animals as positive was high and ranged widely depending upon the antigen and the cut-off value applied, as well as the time after infection. Our findings indicate that this protocol may serve as a reliable assay for the antemortem diagnosis of TB from the initial stage of M. bovis-infection, and may also be adequately sensitive. The suggested optimal antigens and cut-off are bPPD, p22 and the combination of ESAT-6/CFP-10 and Rv3020c with a 0.05 Δ optical density, which yielded a up to 100% correct classification of TB positive and negatve red deer under our experimental conditions. This technique will aid in TB testing of farmed and translocated deer. Future studies

  18. Development and optimization of a direct plaque assay for human and avian metapneumoviruses

    Science.gov (United States)

    Zhang, Yu; Wei, Yongwei; Li, Junan; Li, Jianrong

    2012-01-01

    The genus Metapneumovirus within the subfamily Pneumovirinae and family Paramyxoviridae includes only two viruses, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), which cause respiratory disease in humans and birds, respectively. These two viruses grow poorly in cell culture and other quantitation methods, such as indirect immuno-staining and immuno-fluorescent assays, are expensive, time consuming, and do not allow for plaque purification of the virus. In order to enhance research efforts for studying these two viruses, a direct plaque assay for both hMPV and aMPV has been developed. By optimizing the chemical components of the agarose overlay, it was found that both hMPV with a trypsin-independent F cleavage site and aMPV formed clear and countable plaques in a number of mammalian cell lines (such as Vero-E6 and LLC-MK2 cells) after 5 days of incubation. The plaque forming assay has similar sensitivity and reliability as the currently used immunological methods for viral quantitation. The plaque assay is also a more simple, rapid, and economical method compared to immunological assays, and in addition allows for plaque purification of the viruses. The direct plaque assay will be a valuable method for the quantitation and evaluation of the biological properties of some metapneumoviruses. PMID:22684013

  19. Monoclonal antibody-based dipstick assay: a reliable field applicable technique for diagnosis of Schistosoma mansoni infection using human serum and urine samples.

    Science.gov (United States)

    Demerdash, Zeinab; Mohamed, Salwa; Hendawy, Mohamed; Rabia, Ibrahim; Attia, Mohy; Shaker, Zeinab; Diab, Tarek M

    2013-02-01

    A field applicable diagnostic technique, the dipstick assay, was evaluated for its sensitivity and specificity in diagnosing human Schistosoma mansoni infection. A monoclonal antibody (mAb) against S. mansoni adult worm tegumental antigen (AWTA) was employed in dipstick and sandwich ELISA for detection of circulating schistosome antigen (CSA) in both serum and urine samples. Based on clinical and parasitological examinations, 60 S. mansoni-infected patients, 30 patients infected with parasites other than schistosomiasis, and 30 uninfected healthy individuals were selected. The sensitivity and specificity of dipstick assay in urine samples were 86.7% and 90.0%, respectively, compared to 90.0% sensitivity and 91.7% specificity of sandwich ELISA. In serum samples, the sensitivity and specificity were 88.3% and 91.7% for dipstick assay vs. 91.7% and 95.0% for sandwich ELISA, respectively. The diagnostic efficacy of dipstick assay in urine and serum samples was 88.3% and 90.0%, while it was 90.8% and 93.3% for sandwich ELISA, respectively. The diagnostic indices of dipstick assay and ELISA either in serum or in urine were statistically comparable (P>0.05). In conclusion, the dipstick assay offers an alternative simple, rapid, non-invasive technique in detecting CSA or complement to stool examinations especially in field studies.

  20. Gateway-Assisted Retransmission for Lightweight and Reliable IoT Communications

    Directory of Open Access Journals (Sweden)

    Hui-Ling Chang

    2016-09-01

    Full Text Available Message Queuing Telemetry Transport for Sensor Networks (MQTT-SN and Constrained Application Protocol (CoAP are two protocols supporting publish/subscribe models for IoT devices to publish messages to interested subscribers. Retransmission mechanisms are introduced to compensate for the lack of data reliability. If the device does not receive the acknowledgement (ACK before retransmission timeout (RTO expires, the device will retransmit data. Setting an appropriate RTO is important because the delay may be large or retransmission may be too frequent when the RTO is inappropriate. We propose a Gateway-assisted CoAP (GaCoAP to dynamically compute RTO for devices. Simulation models are proposed to investigate the performance of GaCoAP compared with four other methods. The experiment results show that GaCoAP is more suitable for IoT devices.

  1. LNA probe-based assay for the detection of Tomato black ring virus isolates.

    Science.gov (United States)

    Hasiów-Jaroszewska, Beata; Rymelska, Natalia; Borodynko, Natasza

    2015-02-01

    Tomato black ring virus (TBRV) infects a wide range of economically important plant species worldwide. In the present study we developed a locked nucleic acid (LNA) real-time RT-PCR assay for accurate detection of genetically diverse TBRV isolates collected from different hosts. The assay based on the LNA probe has a wide detection range, high sensitivity, stability and amplification efficiency. The assay amplified all tested TBRV isolates, but no signal was observed for the RNA from other nepoviruses and healthy plant species. Under optimum reaction conditions, the detection limit was estimated around 17 copies of the TBRV target region in total RNA. Real-time RT-PCR with the LNA probe described in this paper will serve as a valuable tool for robust, sensitive and reliable detection of TBRV isolates. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. The use of comet assay in plant toxicology: recent advances

    Directory of Open Access Journals (Sweden)

    Conceição LV Santos

    2015-06-01

    Full Text Available The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g. Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage.

  3. The use of comet assay in plant toxicology: recent advances

    Science.gov (United States)

    Santos, Conceição L. V.; Pourrut, Bertrand; Ferreira de Oliveira, José M. P.

    2015-01-01

    The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g., Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage. PMID:26175750

  4. Protocols to test the activity of antimicrobial peptides against the honey bee pathogen Paenibacillus larvae.

    Science.gov (United States)

    Khilnani, Jasmin C; Wing, Helen J

    2015-10-01

    Paenibacillus larvae is the causal agent of the honey bee disease American Foulbrood. Two enhanced protocols that allow the activity of antimicrobial peptides to be tested against P. larvae are presented. Proof of principle experiments demonstrate that the honey bee antimicrobial peptide defensin 1 is active in both assays. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Absolute counting of neutrophils in whole blood using flow cytometry.

    Science.gov (United States)

    Brunck, Marion E G; Andersen, Stacey B; Timmins, Nicholas E; Osborne, Geoffrey W; Nielsen, Lars K

    2014-12-01

    Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked-in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucount™ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual-platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra-assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time-course study of chemotherapy induced neutropenia using four drug regimens. © 2014 International Society for Advancement of Cytometry.

  6. Quality Improvement to Demonstrate the Lack of Reliability of the Human Papillomavirus mRNA Assay to Identify Women With Latent Human Papillomavirus Infections.

    Science.gov (United States)

    Cotton, Sarah; Brown, Robert E; Nugent, Elizabeth K; Robazetti, Sonia C; Berens, Pamela D; Smith, Judith A

    2018-04-01

    To assess the consistency between human papillomavirus (HPV) mRNA testing in women with a history of previous HPV infections diagnosed by HPV DNA assay and the potential effects on follow-up HPV screening. This was a quality improvement study that used data from a pathology laboratory software database reviewed from November 2014 to June 2016 to identify female patients aged 30 years or older with greater than one HPV-positive result, including one or more HPV mRNA assay results and one or more documented HPV DNA assay results for comparison. Previous correlative cytology and colposcopic histopathology were also documented. American College of Obstetricians and Gynecologists' cervical cancer screening guidelines were used to compare potential differences in follow-up recommendations. Four hundred twenty-five charts for female patients 30 years of age or older were identified with one or more prior high-risk HPV infections by DNA assay. There was a 69.3% difference in HPV mRNA results compared with previous HPV DNA-positive results. There was a potential change in follow-up for 71.7% of patients with one prior high-risk-HPV-positive result and 60.0% of patients with two or more prior high-risk HPV-positive results. There were 231 colposcopy reports evaluated in this study. Of these, 62 (26.8%) were abnormal colposcopy reports, including 45 low-grade squamous intraepithelial lesions, 15 high-grade squamous intraepithelial lesions, and two cancers. Twenty-five (40.3%) abnormal colposcopy findings were in patients with a history of at least than two prior HPV DNA-positive results and a report of currently being HPV-negative with the mRNA assay. The HPV mRNA assays are less sensitive for detection of latent HPV infections compared with HPV DNA assays. Based on these data and the potential change in follow-up care, the HPV mRNA assay should not be used for a primary screening tool for cervical cancer. Many pathology laboratories have shifted to using the HPV mRNA assay

  7. Real-Time QoS Routing Protocols in Wireless Multimedia Sensor Networks: Study and Analysis.

    Science.gov (United States)

    Alanazi, Adwan; Elleithy, Khaled

    2015-09-02

    Many routing protocols have been proposed for wireless sensor networks. These routing protocols are almost always based on energy efficiency. However, recent advances in complementary metal-oxide semiconductor (CMOS) cameras and small microphones have led to the development of Wireless Multimedia Sensor Networks (WMSN) as a class of wireless sensor networks which pose additional challenges. The transmission of imaging and video data needs routing protocols with both energy efficiency and Quality of Service (QoS) characteristics in order to guarantee the efficient use of the sensor nodes and effective access to the collected data. Also, with integration of real time applications in Wireless Senor Networks (WSNs), the use of QoS routing protocols is not only becoming a significant topic, but is also gaining the attention of researchers. In designing an efficient QoS routing protocol, the reliability and guarantee of end-to-end delay are critical events while conserving energy. Thus, considerable research has been focused on designing energy efficient and robust QoS routing protocols. In this paper, we present a state of the art research work based on real-time QoS routing protocols for WMSNs that have already been proposed. This paper categorizes the real-time QoS routing protocols into probabilistic and deterministic protocols. In addition, both categories are classified into soft and hard real time protocols by highlighting the QoS issues including the limitations and features of each protocol. Furthermore, we have compared the performance of mobility-aware query based real-time QoS routing protocols from each category using Network Simulator-2 (NS2). This paper also focuses on the design challenges and future research directions as well as highlights the characteristics of each QoS routing protocol.

  8. Integrated Evaluation of Reliability and Power Consumption of Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    Antônio Dâmaso

    2017-11-01

    Full Text Available Power consumption is a primary interest in Wireless Sensor Networks (WSNs, and a large number of strategies have been proposed to evaluate it. However, those approaches usually neither consider reliability issues nor the power consumption of applications executing in the network. A central concern is the lack of consolidated solutions that enable us to evaluate the power consumption of applications and the network stack also considering their reliabilities. To solve this problem, we introduce a fully automatic solution to design power consumption aware WSN applications and communication protocols. The solution presented in this paper comprises a methodology to evaluate the power consumption based on the integration of formal models, a set of power consumption and reliability models, a sensitivity analysis strategy to select WSN configurations and a toolbox named EDEN to fully support the proposed methodology. This solution allows accurately estimating the power consumption of WSN applications and the network stack in an automated way.

  9. Integrated Evaluation of Reliability and Power Consumption of Wireless Sensor Networks

    Science.gov (United States)

    Dâmaso, Antônio; Maciel, Paulo

    2017-01-01

    Power consumption is a primary interest in Wireless Sensor Networks (WSNs), and a large number of strategies have been proposed to evaluate it. However, those approaches usually neither consider reliability issues nor the power consumption of applications executing in the network. A central concern is the lack of consolidated solutions that enable us to evaluate the power consumption of applications and the network stack also considering their reliabilities. To solve this problem, we introduce a fully automatic solution to design power consumption aware WSN applications and communication protocols. The solution presented in this paper comprises a methodology to evaluate the power consumption based on the integration of formal models, a set of power consumption and reliability models, a sensitivity analysis strategy to select WSN configurations and a toolbox named EDEN to fully support the proposed methodology. This solution allows accurately estimating the power consumption of WSN applications and the network stack in an automated way. PMID:29113078

  10. Shortened protocol in practical [11C]SA4503-PET studies for sigma1 receptor quantification

    International Nuclear Information System (INIS)

    Sakata, Muneyuki; Kimura, Yuichi; Ishikawa, Masatomo; Oda, Keiichi; Ishii, Kenji; Ishiwata, Kiichi; Naganawa, Mika; Hashimoto, Kenji; Chihara, Kunihiro

    2008-01-01

    In practical positron emission tomography (PET) diagnosis, a shortened protocol is preferred for patients with brain disorders. In this study, the applicability of a shortened protocol as an alternative to the 90-min PET scan with [ 11 C]SA4503 for quantitative sigma 1 receptor measurement was investigated. Tissue time-activity curves of 288 regions of interest in the brain from 32 [ 11 C]SA4503-PET scans of 16 healthy subjects prior to and following administration of a selective serotonin reuptake inhibitor (fluvoxamine or paroxetine) were applied to two algorithms of quantitative analysis; binding potential (BP) was derived from compartmental analysis based on nonlinear estimation, and total distribution volume (tDV) was derived from Logan plot analysis. As a result, although both BP and tDV tended to be underestimated by the shortened method, the estimates from the shortened protocol had good linear relationships with those of the full-length protocol. In conclusion, if approximately 10% differences in the estimated results are acceptable for a specific purpose, then a 60-min measurement protocol is capable of providing reliable results. (author)

  11. Moderate reagent mixing on an orbital shaker reduces the incubation time of enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Kumar, Saroj; Ahirwar, Rajesh; Rehman, Ishita; Nahar, Pradip

    2017-07-01

    Rapid diagnostic tests can be developed using ELISA for detection of diseases in emergency conditions. Conventional ELISA takes 1-2 days, making it unsuitable for rapid diagnostics. Here, we report the effect of reagents mixing via shaking or vortexing on the assay timing of ELISA. A 48-min protocol of ELISA involving 12-min incubations with reagent mixing at 750 rpm for every step was optimized. Contrary to this, time-optimized control ELISA performed without mixing produced similar results in 8 h, leaving a time gain of 7 h using the developed protocol. Collectively, the findings suggest the development of ELISA-based rapid diagnostics. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. A Data Analysis Pipeline Accounting for Artifacts in Tox21 Quantitative High-Throughput Screening Assays.

    Science.gov (United States)

    Hsieh, Jui-Hua; Sedykh, Alexander; Huang, Ruili; Xia, Menghang; Tice, Raymond R

    2015-08-01

    A main goal of the U.S. Tox21 program is to profile a 10K-compound library for activity against a panel of stress-related and nuclear receptor signaling pathway assays using a quantitative high-throughput screening (qHTS) approach. However, assay artifacts, including nonreproducible signals and assay interference (e.g., autofluorescence), complicate compound activity interpretation. To address these issues, we have developed a data analysis pipeline that includes an updated signal noise-filtering/curation protocol and an assay interference flagging system. To better characterize various types of signals, we adopted a weighted version of the area under the curve (wAUC) to quantify the amount of activity across the tested concentration range in combination with the assay-dependent point-of-departure (POD) concentration. Based on the 32 Tox21 qHTS assays analyzed, we demonstrate that signal profiling using wAUC affords the best reproducibility (Pearson's r = 0.91) in comparison with the POD (0.82) only or the AC(50) (i.e., half-maximal activity concentration, 0.81). Among the activity artifacts characterized, cytotoxicity is the major confounding factor; on average, about 8% of Tox21 compounds are affected, whereas autofluorescence affects less than 0.5%. To facilitate data evaluation, we implemented two graphical user interface applications, allowing users to rapidly evaluate the in vitro activity of Tox21 compounds. © 2015 Society for Laboratory Automation and Screening.

  13. A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors

    Directory of Open Access Journals (Sweden)

    Hidenobu Yaku

    2013-09-01

    Full Text Available An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR on magnetic beads (MBs and subsequent application of cycling probe technology (CPT is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences (5'-(TTAGGGn-3' of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5' end and a quencher at the 3' end is hydrolyzed by RNase H in the presence of the target DNA. The catalyst-mediated cleavage of the probe RNA enhances fluorescence from the 5' end of the probe. The assay allowed us to successfully detect HeLa cells selectively over normal human dermal fibroblast (NHDF cells. Importantly, this selectivity produced identical results with regard to detection of HeLa cells in the absence and presence of excess NHDF cells; therefore, this assay can be used for practical clinical applications. The lower limit of detection for HeLa cells was 50 cells, which is lower than that achieved with a conventional telomeric repeat amplification protocol assay. Our assay also eliminated false-negative results caused by PCR inhibitors. Furthermore, we show that this assay is appropriate for screening among G-quadruplex ligands to find those that inhibit telomerase activity.

  14. A Reliable Method for the Evaluation of the Anaphylactoid Reaction Caused by Injectable Drugs

    Directory of Open Access Journals (Sweden)

    Fang Wang

    2016-10-01

    Full Text Available Adverse reactions of injectable drugs usually occur at first administration and are closely associated with the dosage and speed of injection. This phenomenon is correlated with the anaphylactoid reaction. However, up to now, study methods based on antigen detection have still not gained wide acceptance and single physiological indicators cannot be utilized to differentiate anaphylactoid reactions from allergic reactions and inflammatory reactions. In this study, a reliable method for the evaluation of anaphylactoid reactions caused by injectable drugs was established by using multiple physiological indicators. We used compound 48/80, ovalbumin and endotoxin as the sensitization agents to induce anaphylactoid, allergic and inflammatory reactions. Different experimental animals (guinea pig and nude rat and different modes of administration (intramuscular, intravenous and intraperitoneal injection and different times (15 min, 30 min and 60 min were evaluated to optimize the study protocol. The results showed that the optimal way to achieve sensitization involved treating guinea pigs with the different agents by intravenous injection for 30 min. Further, seven related humoral factors including 5-HT, SC5b-9, Bb, C4d, IL-6, C3a and histamine were detected by HPLC analysis and ELISA assay to determine their expression level. The results showed that five of them, including 5-HT, SC5b-9, Bb, C4d and IL-6, displayed significant differences between anaphylactoid, allergic and inflammatory reactions, which indicated that their combination could be used to distinguish these three reactions. Then different injectable drugs were used to verify this method and the results showed that the chosen indicators exhibited good correlation with the anaphylactoid reaction which indicated that the established method was both practical and reliable. Our research provides a feasible method for the diagnosis of the serious adverse reactions caused by injectable drugs which

  15. Reliability of Various Measurement Stations for Determining Plantar Fascia Thickness and Echogenicity

    Directory of Open Access Journals (Sweden)

    Adebisi Bisi-Balogun

    2016-04-01

    Full Text Available This study aimed to determine the relative and absolute reliability of ultrasound (US measurements of the thickness and echogenicity of the plantar fascia (PF at different measurement stations along its length using a standardized protocol. Twelve healthy subjects (24 feet were enrolled. The PF was imaged in the longitudinal plane. Subjects were assessed twice to evaluate the intra-rater reliability. A quantitative evaluation of the thickness and echogenicity of the plantar fascia was performed using Image J, a digital image analysis and viewer software. A sonography evaluation of the thickness and echogenicity of the PF showed a high relative reliability with an Intra class correlation coefficient of ≥0.88 at all measurement stations. However, the measurement stations for both the PF thickness and echogenicity which showed the highest intraclass correlation coefficient (ICCs did not have the highest absolute reliability. Compared to other measurement stations, measuring the PF thickness at 3 cm distal and the echogenicity at a region of interest 1 cm to 2 cm distal from its insertion at the medial calcaneal tubercle showed the highest absolute reliability with the least systematic bias and random error. Also, the reliability was higher using a mean of three measurements compared to one measurement. To reduce discrepancies in the interpretation of the thickness and echogenicity measurements of the PF, the absolute reliability of the different measurement stations should be considered in clinical practice and research rather than the relative reliability with the ICC.

  16. Reliability of Various Measurement Stations for Determining Plantar Fascia Thickness and Echogenicity.

    Science.gov (United States)

    Bisi-Balogun, Adebisi; Cassel, Michael; Mayer, Frank

    2016-04-13

    This study aimed to determine the relative and absolute reliability of ultrasound (US) measurements of the thickness and echogenicity of the plantar fascia (PF) at different measurement stations along its length using a standardized protocol. Twelve healthy subjects (24 feet) were enrolled. The PF was imaged in the longitudinal plane. Subjects were assessed twice to evaluate the intra-rater reliability. A quantitative evaluation of the thickness and echogenicity of the plantar fascia was performed using Image J, a digital image analysis and viewer software. A sonography evaluation of the thickness and echogenicity of the PF showed a high relative reliability with an Intra class correlation coefficient of ≥0.88 at all measurement stations. However, the measurement stations for both the PF thickness and echogenicity which showed the highest intraclass correlation coefficient (ICCs) did not have the highest absolute reliability. Compared to other measurement stations, measuring the PF thickness at 3 cm distal and the echogenicity at a region of interest 1 cm to 2 cm distal from its insertion at the medial calcaneal tubercle showed the highest absolute reliability with the least systematic bias and random error. Also, the reliability was higher using a mean of three measurements compared to one measurement. To reduce discrepancies in the interpretation of the thickness and echogenicity measurements of the PF, the absolute reliability of the different measurement stations should be considered in clinical practice and research rather than the relative reliability with the ICC.

  17. Reliability Testing the Die-Attach of CPV Cell Assemblies

    Energy Technology Data Exchange (ETDEWEB)

    Bosco, N.; Sweet, C.; Kurtz, S.

    2011-02-01

    Results and progress are reported for a course of work to establish an efficient reliability test for the die-attach of CPV cell assemblies. Test vehicle design consists of a ~1 cm2 multijunction cell attached to a substrate via several processes. A thermal cycling sequence is developed in a test-to-failure protocol. Methods of detecting a failed or failing joint are prerequisite for this work; therefore both in-situ and non-destructive methods, including infrared imaging techniques, are being explored as a method to quickly detect non-ideal or failing bonds.

  18. Novel Route for Agmatine Catabolism in Aspergillus niger: 4-Guanidinobutyrase Assay.

    Science.gov (United States)

    Saragadam, Tejaswani; Punekar, Narayan S

    2018-01-01

    The enzyme 4-guanidinobutyrase (GBase) catalyzes the hydrolysis of 4-guanidinobutyric acid (GB) to 4-aminobutyric acid (GABA) and urea. Here we describe methods to estimate urea and GABA that were suitably adapted from the published literature. The urea is determined by colorimetric assay using modified Archibald's method. However, the low sensitivity of this method often renders it impractical to perform fine kinetic analysis. To overcome this limitation, a high sensitive method for detecting GABA is exploited that can even detect 1 μM of GABA in the assay mixture. The samples are deproteinized by perchloric acid (PCA) and potassium hydroxide treatment prior to HPLC analysis of GABA. The method involves a pre-column derivatization with o-phthalaldehyde (OPA) in combination with the thiol 3-mercaptopropionic acid (MPA). The fluorescent GABA derivative is then detected after reversed phase high performance liquid chromatography (RP-HPLC) using isocratic elution. The protocols described here are broadly applicable to other biological samples involving urea and GABA as metabolites.

  19. A feedback-retransmission based asynchronous frequency hopping MAC protocol for military aeronautical ad hoc networks

    Directory of Open Access Journals (Sweden)

    Jinhui TANG

    2018-05-01

    Full Text Available Attacking time-sensitive targets has rigid demands for the timeliness and reliability of information transmission, while typical Media Access Control (MAC designed for this application works well only in very light-load scenarios; as a consequence, the performances of system throughput and channel utilization are degraded. For this problem, a feedback-retransmission based asynchronous FRequency hopping Media Access (FRMA control protocol is proposed. Burst communication, asynchronous Frequency Hopping (FH, channel coding, and feedback retransmission are utilized in FRMA. With the mechanism of asynchronous FH, immediate packet transmission and multi-packet reception can be realized, and thus the timeliness is improved. Furthermore, reliability can be achieved via channel coding and feedback retransmission. With theories of queuing theory, Markov model, packets collision model, and discrete Laplace transformation, the formulas of packet success probability, system throughput, average packet end-to-end delay, and delay distribution are obtained. The approximation accuracy of theoretical derivation is verified by experimental results. Within a light-load network, the proposed FRMA has the ability of millisecond delay and 99% reliability as well as outperforms the non-feedback-retransmission based asynchronous frequency hopping media access control protocol. Keywords: Ad hoc networks, Aeronautical communications, Frequency hopping, Media Access Control (MAC, Time-sensitive

  20. Department of Defense need for a micro-electromechanical systems (MEMS) reliability assessment program

    Science.gov (United States)

    Zunino, James L., III; Skelton, Donald

    2005-01-01

    As the United States (U.S.) Army transforms into a lighter, more lethal, and more agile force, the technologies that support both legacy and emerging weapon systems must decrease in size while increasing in intelligence. Micro-electromechanical systems (MEMS) are one such technology that the Army as well as entire DOD will heavily rely on in achieving these objectives. Current and future military applications of MEMS devices include safety and arming devices, guidance systems, sensors/detectors, inertial measurement units, tracking devices, radio frequency devices, wireless radio frequency identification (RFID), etc. Even though the reliance on MEMS devices has been increasing, there have been no studies performed to determine their reliability and failure mechanisms. Furthermore, no standardized test protocols exist for assessing reliability. Accordingly, the U.S. Army Corrosion Office at Picatinny, NJ has initiated the MEMS Reliability Assessment Program to address this issue.

  1. Performance Analysis of Routing Protocols in Ad-hoc and Sensor Networking Environments

    Directory of Open Access Journals (Sweden)

    L. Gavrilovska

    2009-06-01

    Full Text Available Ad-hoc and sensor networks are becoming an increasingly popular wireless networking concepts lately. This paper analyzes and compares prominent routing schemes in these networking environments. The knowledge obtained can serve users to better understand short range wireless network solutions thus leading to options for implementation in various scenarios. In addition, it should aid researchers develop protocol improvements reliable for the technologies of interest.

  2. The Development of DNA Based Methods for the Reliable and Efficient Identification of Nicotiana tabacum in Tobacco and Its Derived Products

    Directory of Open Access Journals (Sweden)

    Sukumar Biswas

    2016-01-01

    Full Text Available Reliable methods are needed to detect the presence of tobacco components in tobacco products to effectively control smuggling and classify tariff and excise in tobacco industry to control illegal tobacco trade. In this study, two sensitive and specific DNA based methods, one quantitative real-time PCR (qPCR assay and the other loop-mediated isothermal amplification (LAMP assay, were developed for the reliable and efficient detection of the presence of tobacco (Nicotiana tabacum in various tobacco samples and commodities. Both assays targeted the same sequence of the uridine 5′-monophosphate synthase (UMPS, and their specificities and sensitivities were determined with various plant materials. Both qPCR and LAMP methods were reliable and accurate in the rapid detection of tobacco components in various practical samples, including customs samples, reconstituted tobacco samples, and locally purchased cigarettes, showing high potential for their application in tobacco identification, particularly in the special cases where the morphology or chemical compositions of tobacco have been disrupted. Therefore, combining both methods would facilitate not only the detection of tobacco smuggling control, but also the detection of tariff classification and of excise.

  3. The Impact of Packet Loss Behavior in 802.11g on the Cooperation Gain in Reliable Multicast

    DEFF Research Database (Denmark)

    Heide, Janus; Vingelmann, Peter; Pedersen, Morten Videbæk

    2012-01-01

    In group-oriented applications for wireless networks, reliable multicast strategies are important in order to efficiently distribute data, e.g. in Wireless Mesh Networks (WMNs) and Mobile Ad-hoc NETworks (MANETs). To ensure that developed protocols and systems will operate as expected when deploy...

  4. Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

    Science.gov (United States)

    Ogrean, Christy; Jackson, Ben; Covino, James

    2010-01-01

    The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to >98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts. Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process. Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method. PMID:20567213

  5. Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli

    Science.gov (United States)

    SOLANKY, DIPESH; HAYDEL, SHELLEY E.

    2012-01-01

    This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated CB, by investigating the induction of DNA double-strand breaks (DSBs) in Escherichia coli. To quantify DNA damage upon exposure to soluble antimicrobial compounds, we modified a bacterial neutral comet assay, which primarily associates the general length of an electrophoresed chromosome, or comet, with the degree of DSB-associated DNA damage. To appropriately account for antimicrobial-mediated strand fragmentation, suitable control reactions consisting of exposures to water, ethanol, kanamycin, and bleomycin were developed and optimized for the assay. Bacterial exposure to the CB clay resulted in significantly longer comet lengths, compared to water and kanamycin exposures, suggesting that the induction of DNA DSBs contributes to the killing activity of this antibacterial clay mineral mixture. The comet assay protocol described herein provides a general technique for evaluating soluble antimicrobial-derived DNA damage and for comparing DNA fragmentation between experimental and control assays. PMID:22940101

  6. Reliability research based experience with systems and events at the Kozloduy NPP units 1-4

    Energy Technology Data Exchange (ETDEWEB)

    Khristova, R; Kaltchev, B; Dimitrov, B [Energoproekt, Sofia (Bulgaria); Nedyalkova, D; Sonev, A [Kombinat Atomna Energetika, Kozloduj (Bulgaria)

    1996-12-31

    An overview of equipment reliability based on operational data of selected safety systems at the Kozloduy NPP is presented. Conclusions are drawn on reliability of the service water system, feed water system, emergency power supply - category 2, emergency high pressure ejection system and spray system. For the units 1-4 all recorded accident protocols in the period 1974-1993 have been processed and the main initiators identified. A list with 39 most frequent initiators of accidents/incidents is compiled. The human-caused errors account for 27% of all events. The reliability characteristics and frequencies have been calculated for all initiating events. It is concluded that there have not been any accidents with consequences for fuel integrity or radioactive release. 14 refs.

  7. Reliability research based experience with systems and events at the Kozloduy NPP units 1-4

    International Nuclear Information System (INIS)

    Khristova, R.; Kaltchev, B.; Dimitrov, B.; Nedyalkova, D.; Sonev, A.

    1995-01-01

    An overview of equipment reliability based on operational data of selected safety systems at the Kozloduy NPP is presented. Conclusions are drawn on reliability of the service water system, feed water system, emergency power supply - category 2, emergency high pressure ejection system and spray system. For the units 1-4 all recorded accident protocols in the period 1974-1993 have been processed and the main initiators identified. A list with 39 most frequent initiators of accidents/incidents is compiled. The human-caused errors account for 27% of all events. The reliability characteristics and frequencies have been calculated for all initiating events. It is concluded that there have not been any accidents with consequences for fuel integrity or radioactive release. 14 refs

  8. Evaluation of the Thermo Scientific™ SureTect™ Salmonella species Assay.

    Science.gov (United States)

    Cloke, Jonathan; Clark, Dorn; Radcliff, Roy; Leon-Velarde, Carlos; Larson, Nathan; Dave, Keron; Evans, Katharine; Crabtree, David; Hughes, Annette; Simpson, Helen; Holopainen, Jani; Wickstrand, Nina; Kauppinen, Mikko

    2014-03-01

    (enrichment time and temperature, and lysis temperature), which demonstrated that the assay gave reliable performance. Accelerated stability testing was additionally conducted, validating the assay shelf life.

  9. Surface reactivity measurements as required for grouping and read-across: An advanced FRAS protocol

    International Nuclear Information System (INIS)

    Gandon, Arnaud; Werle, Kai; Neubauer, Nicole; Wohlleben, Wendel

    2017-01-01

    Oxidative stress is a widely accepted paradigm associated with different adverse outcomes of particulate matter, including nanomaterials. It has frequently been identified in in vitro and in vivo studies and different assays have been developed for this purpose. Here we describe a newly developed multi-dose protocol of the FRAS assay (Ferric Reduction Ability of Serum). The purpose of this SOP is the measurement of the surface reactivity of nanomaterials under physiological conditions. Antioxidative components as present in human blood serum (HBS) serve as reporter molecules. The assay separates the oxidative damage from the read-out of the reporter molecules. The results show significantly enhanced repeatability with better sensitivity towards low reactivity, enabling application of FRAS both to a rough grouping by reactive vs. passive nanomaterials and further to substantiation of read-across by enhanced resolution of the similarity between different nanoforms of the same substance. (paper)

  10. Surface reactivity measurements as required for grouping and read-across: An advanced FRAS protocol

    Science.gov (United States)

    Gandon, Arnaud; Werle, Kai; Neubauer, Nicole; Wohlleben, Wendel

    2017-06-01

    Oxidative stress is a widely accepted paradigm associated with different adverse outcomes of particulate matter, including nanomaterials. It has frequently been identified in in vitro and in vivo studies and different assays have been developed for this purpose. Here we describe a newly developed multi-dose protocol of the FRAS assay (Ferric Reduction Ability of Serum). The purpose of this SOP is the measurement of the surface reactivity of nanomaterials under physiological conditions. Antioxidative components as present in human blood serum (HBS) serve as reporter molecules. The assay separates the oxidative damage from the read-out of the reporter molecules. The results show significantly enhanced repeatability with better sensitivity towards low reactivity, enabling application of FRAS both to a rough grouping by reactive vs. passive nanomaterials and further to substantiation of read-across by enhanced resolution of the similarity between different nanoforms of the same substance.

  11. A real-time PCR assay for detection and quantification of Verticillium dahliae in spinach seed.

    Science.gov (United States)

    Duressa, Dechassa; Rauscher, Gilda; Koike, Steven T; Mou, Beiquan; Hayes, Ryan J; Maruthachalam, Karunakaran; Subbarao, Krishna V; Klosterman, Steven J

    2012-04-01

    Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.

  12. Reliability engineering

    International Nuclear Information System (INIS)

    Lee, Chi Woo; Kim, Sun Jin; Lee, Seung Woo; Jeong, Sang Yeong

    1993-08-01

    This book start what is reliability? such as origin of reliability problems, definition of reliability and reliability and use of reliability. It also deals with probability and calculation of reliability, reliability function and failure rate, probability distribution of reliability, assumption of MTBF, process of probability distribution, down time, maintainability and availability, break down maintenance and preventive maintenance design of reliability, design of reliability for prediction and statistics, reliability test, reliability data and design and management of reliability.

  13. Improved protocol and data analysis for accelerated shelf-life estimation of solid dosage forms.

    Science.gov (United States)

    Waterman, Kenneth C; Carella, Anthony J; Gumkowski, Michael J; Lukulay, Patrick; MacDonald, Bruce C; Roy, Michael C; Shamblin, Sheri L

    2007-04-01

    To propose and test a new accelerated aging protocol for solid-state, small molecule pharmaceuticals which provides faster predictions for drug substance and drug product shelf-life. The concept of an isoconversion paradigm, where times in different temperature and humidity-controlled stability chambers are set to provide a critical degradant level, is introduced for solid-state pharmaceuticals. Reliable estimates for temperature and relative humidity effects are handled using a humidity-corrected Arrhenius equation, where temperature and relative humidity are assumed to be orthogonal. Imprecision is incorporated into a Monte-Carlo simulation to propagate the variations inherent in the experiment. In early development phases, greater imprecision in predictions is tolerated to allow faster screening with reduced sampling. Early development data are then used to design appropriate test conditions for more reliable later stability estimations. Examples are reported showing that predicted shelf-life values for lower temperatures and different relative humidities are consistent with the measured shelf-life values at those conditions. The new protocols and analyses provide accurate and precise shelf-life estimations in a reduced time from current state of the art.

  14. Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

    Science.gov (United States)

    Johnson, Christopher J.; Carlson, Christina M.; Morawski, Aaron R.; Manthei, Alyson; Cashman, Neil R.

    2015-01-01

    Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitroprion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host’s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host’s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

  15. The comet assay as a tool for human biomonitoring studies: the ComNet project.

    Science.gov (United States)

    Collins, Andrew; Koppen, Gudrun; Valdiglesias, Vanessa; Dusinska, Maria; Kruszewski, Marcin; Møller, Peter; Rojas, Emilio; Dhawan, Alok; Benzie, Iris; Coskun, Erdem; Moretti, Massimo; Speit, Günter; Bonassi, Stefano

    2014-01-01

    The comet assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. Studies often involve small numbers of subjects, and design may be sub-optimal in other respects. In addition, comet assay protocols in use in different laboratories vary significantly. In spite of these difficulties, it is appropriate to carry out a pooled analysis of all available comet assay biomonitoring data, in order to establish baseline parameters of DNA damage, and to investigate associations between comet assay measurements and factors such as sex, age, smoking status, nutrition, lifestyle, etc. With this as its major objective, the ComNet project has recruited almost 100 research groups willing to share datasets. Here we provide a background to this project, discussing the history of the comet assay and practical issues that can critically affect its performance. We survey its diverse applications in biomonitoring studies, including environmental and occupational exposure to genotoxic agents, genoprotection by dietary and other factors, DNA damage associated with various diseases, and intrinsic factors that affect DNA damage levels in humans. We examine in depth the quality of data from a random selection of studies, from an epidemiological and statistical point of view. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Ana Ramón-Laca

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

  17. Neuroimaging for non-accidental head injury in childhood: A proposed protocol

    International Nuclear Information System (INIS)

    Jaspan, T.; Griffiths, P.D.; McConachie, N.S.; Punt, J.A.G.

    2003-01-01

    Non-accidental head injury (NAHI) is a major cause of neurological disability and death during infancy. Radiological imaging plays a crucial role in evaluating craniospinal injury, both for guiding medical management and the forensic aspects of abusive trauma. The damage sustained is varied, complex and may be accompanied by an evolving pattern of brain injury secondary to a cascade of metabolic and physiological derangements. Regrettably, many cases are poorly or incompletely evaluated leading to diagnostic errors and difficulties in executing subsequent child care or criminal proceedings. It is evident, from cases referred to the authors, that imaging protocols for NAHI are lacking (or only loosely adhered to, if present) in many centres throughout the U.K. Future research in this field will also be hampered if there is a lack of consistent and reliable radiological data. There is no nationally agreed protocol for imaging NAHI. We propose such a protocol, based upon a wide experience in the medical management of child abuse and extensive involvement in the medicolegal aspects of NAHI. Jaspan, T., et al. (2003). Clinical Radiology58, 44--53

  18. Neuroimaging for non-accidental head injury in childhood: A proposed protocol

    Energy Technology Data Exchange (ETDEWEB)

    Jaspan, T.; Griffiths, P.D.; McConachie, N.S.; Punt, J.A.G

    2003-01-01

    Non-accidental head injury (NAHI) is a major cause of neurological disability and death during infancy. Radiological imaging plays a crucial role in evaluating craniospinal injury, both for guiding medical management and the forensic aspects of abusive trauma. The damage sustained is varied, complex and may be accompanied by an evolving pattern of brain injury secondary to a cascade of metabolic and physiological derangements. Regrettably, many cases are poorly or incompletely evaluated leading to diagnostic errors and difficulties in executing subsequent child care or criminal proceedings. It is evident, from cases referred to the authors, that imaging protocols for NAHI are lacking (or only loosely adhered to, if present) in many centres throughout the U.K. Future research in this field will also be hampered if there is a lack of consistent and reliable radiological data. There is no nationally agreed protocol for imaging NAHI. We propose such a protocol, based upon a wide experience in the medical management of child abuse and extensive involvement in the medicolegal aspects of NAHI. Jaspan, T., et al. (2003). Clinical Radiology58, 44--53.

  19. Secure and Reliable IPTV Multimedia Transmission Using Forward Error Correction

    Directory of Open Access Journals (Sweden)

    Chi-Huang Shih

    2012-01-01

    Full Text Available With the wide deployment of Internet Protocol (IP infrastructure and rapid development of digital technologies, Internet Protocol Television (IPTV has emerged as one of the major multimedia access techniques. A general IPTV transmission system employs both encryption and forward error correction (FEC to provide the authorized subscriber with a high-quality perceptual experience. This two-layer processing, however, complicates the system design in terms of computational cost and management cost. In this paper, we propose a novel FEC scheme to ensure the secure and reliable transmission for IPTV multimedia content and services. The proposed secure FEC utilizes the characteristics of FEC including the FEC-encoded redundancies and the limitation of error correction capacity to protect the multimedia packets against the malicious attacks and data transmission errors/losses. Experimental results demonstrate that the proposed scheme obtains similar performance compared with the joint encryption and FEC scheme.

  20. Genetic point-of-care diagnosis of Mycoplasma pneumoniae infection using LAMP assay.

    Science.gov (United States)

    Kakuya, Fujio; Kinebuchi, Takahiro; Fujiyasu, Hiroaki; Tanaka, Ryosuke; Kano, Hiroki

    2014-08-01

    Mycoplasma pneumoniae (MP) is a major pathogen of lower respiratory tract infection (LRTI) in children. A rapid diagnostic method during the acute phase is required for the prescription of effective antibiotics. A prospective, single-centered study was conducted on community-acquired LRTI in children. We regarded the day of fever onset as the first day of illness. In part 1, we studied 191 patients with signs of LRTI. We compared diagnostic reliability using loop-mediated isothermal amplification (LAMP) assay and serological testing at the first visit. In part 2, we evaluated the clinical characteristics of 117 patients with positive LAMP assay. In part 1, 31 patients met the definite MP infection criteria. LAMP assay had a sensitivity of 96.8% and specificity of 100%, whereas enzyme immunoassay had a sensitivity of 38.7% and specificity of 76.9%, and particle agglutination test had a sensitivity of 19.4% and specificity of 93.1%. In part 2, of 106 patients with fever, 100 patients were diagnosed by the day 7 of illness. The diagnosis was made a mean of 3.5 ± 2.1 days after the onset of fever. LAMP assay had excellent sensitivity and specificity for the detection of acute MP infection at the first visit. This assay can diagnose MP infection during the very acute phase. LAMP assay is appropriate for genetic point-of-care diagnosis of MP infection in hospital laboratories. © 2014 Japan Pediatric Society.

  1. Protocol Implementation Generator

    DEFF Research Database (Denmark)

    Carvalho Quaresma, Jose Nuno; Probst, Christian W.

    2010-01-01

    Users expect communication systems to guarantee, amongst others, privacy and integrity of their data. These can be ensured by using well-established protocols; the best protocol, however, is useless if not all parties involved in a communication have a correct implementation of the protocol and a...... Generator framework based on the LySatool and a translator from the LySa language into C or Java....... necessary tools. In this paper, we present the Protocol Implementation Generator (PiG), a framework that can be used to add protocol generation to protocol negotiation, or to easily share and implement new protocols throughout a network. PiG enables the sharing, verification, and translation...

  2. Validation and determination of a reference interval for canine HbA1c using an immunoturbidimetric assay.

    Science.gov (United States)

    Goemans, Anne F; Spence, Susanna J; Ramsey, Ian K

    2017-06-01

    Hemoglobin A1c (HbA1c) provides a reliable measure of glycemic control over 2-3 months in human diabetes mellitus. In dogs, presence of HbA1c has been demonstrated, but there are no validated commercial assays. The purpose of the study was to validate a commercially available automated immunoturbidimetric assay for canine HbA1c and determine an RI in a hospital population. The specificity of the assay was assessed by inducing glycosylation in vitro using isolated canine hemoglobin, repeatability by measuring canine samples 5 times in succession, long term inter-assay imprecision by measuring supplied control materials, stability using samples stored at 4°C over 5 days and -20°C over 8 weeks, linearity by mixing samples of known HbA1c in differing proportions, and the effect of anticoagulants with paired samples. An RI was determined using EDTA-anticoagulated blood samples from 60 nondiabetic hospitalized animals of various ages and breeds. Hemoglobin A1c was also measured in 10 diabetic dogs. The concentration of HbA1c increased proportionally with glucose concentration in vitro. For repeat measurements, the CV was 4.08% (range 1.16-6.10%). Samples were stable for 5 days at 4°C. The assay was linear within the assessed range. Heparin- and EDTA-anticoagulated blood provided comparable results. The RI for HbA1c was 9-18.5 mmol/mol. There was no apparent effect of age or breed on HbA1c. In diabetic dogs, HbA1c ranged from 14 to 48 mmol/mol. The assay provides a reliable method for canine HbA1c measurement with good analytic performance. © 2017 American Society for Veterinary Clinical Pathology.

  3. Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing.

    Science.gov (United States)

    Aigrain, Louise; Gu, Yong; Quail, Michael A

    2016-06-13

    The emergence of next-generation sequencing (NGS) technologies in the past decade has allowed the democratization of DNA sequencing both in terms of price per sequenced bases and ease to produce DNA libraries. When it comes to preparing DNA sequencing libraries for Illumina, the current market leader, a plethora of kits are available and it can be difficult for the users to determine which kit is the most appropriate and efficient for their applications; the main concerns being not only cost but also minimal bias, yield and time efficiency. We compared 9 commercially available library preparation kits in a systematic manner using the same DNA sample by probing the amount of DNA remaining after each protocol steps using a new droplet digital PCR (ddPCR) assay. This method allows the precise quantification of fragments bearing either adaptors or P5/P7 sequences on both ends just after ligation or PCR enrichment. We also investigated the potential influence of DNA input and DNA fragment size on the final library preparation efficiency. The overall library preparations efficiencies of the libraries show important variations between the different kits with the ones combining several steps into a single one exhibiting some final yields 4 to 7 times higher than the other kits. Detailed ddPCR data also reveal that the adaptor ligation yield itself varies by more than a factor of 10 between kits, certain ligation efficiencies being so low that it could impair the original library complexity and impoverish the sequencing results. When a PCR enrichment step is necessary, lower adaptor-ligated DNA inputs leads to greater amplification yields, hiding the latent disparity between kits. We describe a ddPCR assay that allows us to probe the efficiency of the most critical step in the library preparation, ligation, and to draw conclusion on which kits is more likely to preserve the sample heterogeneity and reduce the need of amplification.

  4. Energy Efficient Medium Access Control Protocol for Clustered Wireless Sensor Networks with Adaptive Cross-Layer Scheduling.

    Science.gov (United States)

    Sefuba, Maria; Walingo, Tom; Takawira, Fambirai

    2015-09-18

    This paper presents an Energy Efficient Medium Access Control (MAC) protocol for clustered wireless sensor networks that aims to improve energy efficiency and delay performance. The proposed protocol employs an adaptive cross-layer intra-cluster scheduling and an inter-cluster relay selection diversity. The scheduling is based on available data packets and remaining energy level of the source node (SN). This helps to minimize idle listening on nodes without data to transmit as well as reducing control packet overhead. The relay selection diversity is carried out between clusters, by the cluster head (CH), and the base station (BS). The diversity helps to improve network reliability and prolong the network lifetime. Relay selection is determined based on the communication distance, the remaining energy and the channel quality indicator (CQI) for the relay cluster head (RCH). An analytical framework for energy consumption and transmission delay for the proposed MAC protocol is presented in this work. The performance of the proposed MAC protocol is evaluated based on transmission delay, energy consumption, and network lifetime. The results obtained indicate that the proposed MAC protocol provides improved performance than traditional cluster based MAC protocols.

  5. Modification of the BAX System PCR assay for detecting Salmonella in beef, produce, and soy protein isolate. Performance Tested Method 100201.

    Science.gov (United States)

    Peng, Linda X; Wallace, Morgan; Andaloro, Bridget; Fallon, Dawn; Fleck, Lois; Delduco, Dan; Tice, George

    2011-01-01

    The BAX System PCR assay for Salmonella detection in foods was previously validated as AOAC Research Institute (RI) Performance Tested Method (PTM) 100201. New studies were conducted on beef and produce using the same media and protocol currently approved for the BAX System PCR assay for E. coli O157:H7 multiplex (MP). Additionally, soy protein isolate was tested for matrix extension using the U.S. Food and Drug Administration-Bacteriological Analytical Manual (FDA-BAM) enrichment protocols. The studies compared the BAX System method to the U.S. Department of Agriculture culture method for detecting Salmonella in beef and the FDA-BAM culture method for detecting Salmonella in produce and soy protein isolate. Method comparison studies on low-level inoculates showed that the BAX System assay for Salmonella performed as well as or better than the reference method for detecting Salmonella in beef and produce in 8-24 h enrichment when the BAX System E. coli O157:H7 MP media was used, and soy protein isolate in 20 h enrichment with lactose broth followed by 3 h regrowth in brain heart infusion broth. An inclusivity panel of 104 Salmonella strains with diverse serotypes was tested by the BAX System using the proprietary BAX System media and returned all positive results. Ruggedness factors involved in the enrichment phase were also evaluated by testing outside the specified parameters, and none of the factors examined affected the performance of the assay.

  6. High-throughput respirometric assay identifies predictive toxicophore of mitochondrial injury

    Energy Technology Data Exchange (ETDEWEB)

    Wills, Lauren P. [MitoHealth Inc., Charleston, SC 29403 (United States); Beeson, Gyda C.; Trager, Richard E.; Lindsey, Christopher C. [Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Beeson, Craig C. [MitoHealth Inc., Charleston, SC 29403 (United States); Peterson, Yuri K. [Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Schnellmann, Rick G., E-mail: schnell@musc.edu [Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Ralph H. Johnson VA Medical Center, Charleston, SC 29401 (United States)

    2013-10-15

    Many environmental chemicals and drugs negatively affect human health through deleterious effects on mitochondrial function. Currently there is no chemical library of mitochondrial toxicants, and no reliable methods for predicting mitochondrial toxicity. We hypothesized that discrete toxicophores defined by distinct chemical entities can identify previously unidentified mitochondrial toxicants. We used a respirometric assay to screen 1760 compounds (5 μM) from the LOPAC and ChemBridge DIVERSet libraries. Thirty-one of the assayed compounds decreased uncoupled respiration, a stress test for mitochondrial dysfunction, prior to a decrease in cell viability and reduced the oxygen consumption rate in isolated mitochondria. The mitochondrial toxicants were grouped by chemical similarity and two clusters containing four compounds each were identified. Cheminformatic analysis of one of the clusters identified previously uncharacterized mitochondrial toxicants from the ChemBridge DIVERSet. This approach will enable the identification of mitochondrial toxicants and advance the prediction of mitochondrial toxicity for both drug discovery and risk assessment. - Highlights: • Respirometric assay conducted in RPTC to create mitochondrial toxicant database. • Chemically similar mitochondrial toxicants aligned as mitochondrial toxicophores • Mitochondrial toxicophore identifies five novel mitochondrial toxicants.

  7. High-throughput respirometric assay identifies predictive toxicophore of mitochondrial injury

    International Nuclear Information System (INIS)

    Wills, Lauren P.; Beeson, Gyda C.; Trager, Richard E.; Lindsey, Christopher C.; Beeson, Craig C.; Peterson, Yuri K.; Schnellmann, Rick G.

    2013-01-01

    Many environmental chemicals and drugs negatively affect human health through deleterious effects on mitochondrial function. Currently there is no chemical library of mitochondrial toxicants, and no reliable methods for predicting mitochondrial toxicity. We hypothesized that discrete toxicophores defined by distinct chemical entities can identify previously unidentified mitochondrial toxicants. We used a respirometric assay to screen 1760 compounds (5 μM) from the LOPAC and ChemBridge DIVERSet libraries. Thirty-one of the assayed compounds decreased uncoupled respiration, a stress test for mitochondrial dysfunction, prior to a decrease in cell viability and reduced the oxygen consumption rate in isolated mitochondria. The mitochondrial toxicants were grouped by chemical similarity and two clusters containing four compounds each were identified. Cheminformatic analysis of one of the clusters identified previously uncharacterized mitochondrial toxicants from the ChemBridge DIVERSet. This approach will enable the identification of mitochondrial toxicants and advance the prediction of mitochondrial toxicity for both drug discovery and risk assessment. - Highlights: • Respirometric assay conducted in RPTC to create mitochondrial toxicant database. • Chemically similar mitochondrial toxicants aligned as mitochondrial toxicophores • Mitochondrial toxicophore identifies five novel mitochondrial toxicants

  8. Reliability, Validity, and Sensitivity of a Novel Smartphone-Based Eccentric Hamstring Strength Test in Professional Football Players.

    Science.gov (United States)

    Lee, Justin W Y; Cai, Ming-Jing; Yung, Patrick S H; Chan, Kai-Ming

    2018-05-01

    To evaluate the test-retest reliability, sensitivity, and concurrent validity of a smartphone-based method for assessing eccentric hamstring strength among male professional football players. A total of 25 healthy male professional football players performed the Chinese University of Hong Kong (CUHK) Nordic break-point test, hamstring fatigue protocol, and isokinetic hamstring strength test. The CUHK Nordic break-point test is based on a Nordic hamstring exercise. The Nordic break-point angle was defined as the maximum point where the participant could no longer support the weight of his body against gravity. The criterion for the sensitivity test was the presprinting and postsprinting difference of the Nordic break-point angle with a hamstring fatigue protocol. The hamstring fatigue protocol consists of 12 repetitions of the 30-m sprint with 30-s recoveries between sprints. Hamstring peak torque of the isokinetic hamstring strength test was used as the criterion for validity. A high test-retest reliability (intraclass correlation coefficient = .94; 95% confidence interval, .82-.98) was found in the Nordic break-point angle measurements. The Nordic break-point angle significantly correlated with isokinetic hamstring peak torques at eccentric action of 30°/s (r = .88, r 2  = .77, P hamstring strength measures among male professional football players.

  9. Implementing the Kyoto protocol in Europe: Interactions between international and Community controls

    International Nuclear Information System (INIS)

    Tabau, Anne-Sophie

    2011-07-01

    This bibliographical note presents a book which discusses the coexistence of the Kyoto protocol and of a regional regime within the European Union for the actual application of rules requiring mechanisms of control. The international regime implements a continuous monitoring which combines conventional techniques and more intrusive procedures. The European Community introduced a non-contentious mechanism with a large and strong law basis and sanction ability. The author assesses the ability of the monitoring system as a whole to ensure the very credibility of the Protocol. She also assesses the reliability of international and community economic tools which aim at reducing greenhouse gas emissions at a minimum cost. She also discusses the desirable evolutions of the regime of struggle against climate changes

  10. Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides

    Directory of Open Access Journals (Sweden)

    Lyerly Herbert K

    2008-03-01

    Full Text Available Abstract Background Single-cell assays of immune function are increasingly used to monitor T cell responses in immunotherapy clinical trials. Standardization and validation of such assays are therefore important to interpretation of the clinical trial data. Here we assess the levels of intra-assay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFNγ-based ELISPOT and cytokine flow cytometry (CFC, as well as tetramer assays. Results Precision was measured in cryopreserved PBMC with a low, medium, or high response level to a CMV pp65 peptide or peptide mixture. Intra-assay precision was assessed using 6 replicates per assay; inter-assay precision was assessed by performing 8 assays on different days; and inter-operator precision was assessed using 3 different operators working on the same day. Percent CV values ranged from 4% to 133% depending upon the assay and response level. Linearity was measured by diluting PBMC from a high responder into PBMC from a non-responder, and yielded R2 values from 0.85 to 0.99 depending upon the assay and antigen. Conclusion These data provide target values for precision and linearity of single-cell assays for those wishing to validate these assays in their own laboratories. They also allow for comparison of the precision and linearity of ELISPOT, CFC, and tetramer across a range of response levels. There was a trend toward tetramer assays showing the highest precision, followed closely by CFC, and then ELISPOT; while all three assays had similar linearity. These findings are contingent upon the use of optimized protocols for each assay.

  11. The impacts of electricity dispatch protocols on the emission reductions due to wind power and carbon tax.

    Science.gov (United States)

    Yu, Yang; Rajagopal, Ram

    2015-02-17

    Two dispatch protocols have been adopted by electricity markets to deal with the uncertainty of wind power but the effects of the selection between the dispatch protocols have not been comprehensively analyzed. We establish a framework to compare the impacts of adopting different dispatch protocols on the efficacy of using wind power and implementing a carbon tax to reduce emissions. We suggest that a market has high potential to achieve greater emission reduction by adopting the stochastic dispatch protocol instead of the static protocol when the wind energy in the market is highly uncertain or the market has enough adjustable generators, such as gas-fired combustion generators. Furthermore, the carbon-tax policy is more cost-efficient for reducing CO2 emission when the market operates according to the stochastic protocol rather than the static protocol. An empirical study, which is calibrated according to the data from the Electric Reliability Council of Texas market, confirms that using wind energy in the Texas market results in a 12% CO2 emission reduction when the market uses the stochastic dispatch protocol instead of the 8% emission reduction associated with the static protocol. In addition, if a 6$/ton carbon tax is implemented in the Texas market operated according to the stochastic protocol, the CO2 emission is similar to the emission level from the same market with a 16$/ton carbon tax operated according to the static protocol. Correspondingly, the 16$/ton carbon tax associated with the static protocol costs 42.6% more than the 6$/ton carbon tax associated with the stochastic protocol.

  12. Interaction of nucleic acids with Coomassie Blue G-250 in the Bradford assay.

    Science.gov (United States)

    Wenrich, Broc R; Trumbo, Toni A

    2012-09-15

    The Bradford assay has been used reliably for decades to quantify protein in solution. The analyte is incubated in acidic solution of Coomassie Blue G-250 dye, during which reversible ionic and nonionic binding interactions form. Bradford assay color yields were determined for salmon, bovine, shrimp, and kiwi fruit genomic DNA; baker's yeast RNA; bovine serum albumin (BSA); and hen egg lysozyme. Pure DNA and RNA bound the dye, with color yields of 0.0017 mg⁻¹ cm⁻¹ and 0.0018 mg⁻¹ cm⁻¹, respectively. The nucleic acid-Coomassie Blue response was significant, at roughly 9% of that for BSA and 18% of that for lysozyme. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. A CO-FISH assay to assess sister chromatid segregation patterns in mitosis of mouse embryonic stem cells.

    Science.gov (United States)

    Sauer, Stephan; Burkett, Sandra S; Lewandoski, Mark; Klar, Amar J S

    2013-05-01

    Sister chromatids contain identical DNA sequence but are chiral with respect to both their helical handedness and their replication history. Emerging evidence from various model organisms suggests that certain stem cells segregate sister chromatids nonrandomly to either maintain genome integrity or to bias cellular differentiation in asymmetric cell divisions. Conventional methods for tracing of old vs. newly synthesized DNA strands generally lack resolution for individual chromosomes and employ halogenated thymidine analogs with profound cytotoxic effects on rapidly dividing cells. Here, we present a modified chromosome orientation fluorescence in situ hybridization (CO-FISH) assay, where identification of individual chromosomes and their replication history is achieved in subsequent hybridization steps with chromosome-specific DNA probes and PNA telomere probes. Importantly, we tackle the issue of BrdU cytotoxicity and show that our method is compatible with normal mouse ES cell biology, unlike a recently published related protocol. Results from our CO-FISH assay show that mitotic segregation of mouse chromosome 7 is random in ES cells, which contrasts previously published results from our laboratory and settles a controversy. Our straightforward protocol represents a useful resource for future studies on chromatid segregation patterns of in vitro-cultured cells from distinct model organisms.

  14. Measuring Bioenergetics in T Cells Using a Seahorse Extracellular Flux Analyzer.

    Science.gov (United States)

    van der Windt, Gerritje J W; Chang, Chih-Hao; Pearce, Erika L

    2016-04-01

    This unit contains several protocols to determine the energy utilization of T cells in real-time using a Seahorse Extracellular Flux Analyzer (http://www.seahorsebio.com). The advantages to using this machine over traditional metabolic assays include the simultaneous measurement of glycolysis and mitochondrial respiration, in real-time, on relatively small numbers of cells, without any radioactivity. The Basic Protocol describes a standard mitochondrial stress test on the XF(e) 96, which yields information about oxidative phosphorylation and glycolysis, two energy-generating pathways. The alternate protocols provide examples of adaptations to the Basic Protocol, including adjustments for the use of the XF(e) 24. A protocol for real-time bioenergetic responses to T cell activation allows for the analysis of immediate metabolic changes after T cell receptor stimulation. Specific substrate utilization can be determined by the use of differential assay media, or the injection of drugs that specifically affect certain metabolic processes. Accurate cell numbers, purity, and viability are critical to obtain reliable results. Copyright © 2016 John Wiley & Sons, Inc.

  15. Vertical Protocol Composition

    DEFF Research Database (Denmark)

    Groß, Thomas; Mödersheim, Sebastian Alexander

    2011-01-01

    The security of key exchange and secure channel protocols, such as TLS, has been studied intensively. However, only few works have considered what happens when the established keys are actually used—to run some protocol securely over the established “channel”. We call this a vertical protocol.......e., that the combination cannot introduce attacks that the individual protocols in isolation do not have. In this work, we prove a composability result in the symbolic model that allows for arbitrary vertical composition (including self-composition). It holds for protocols from any suite of channel and application...

  16. Addendum to the Building America House Simulation Protocols

    Energy Technology Data Exchange (ETDEWEB)

    Engebrecht-Metzger, C.; Wilson, E.; Horowitz, S.

    2012-12-01

    As Building America (BA) has grown to include a large and diverse cross-section of the home building and retrofit industries, it has become more important to develop accurate, consistent analysis techniques to measure progress towards the program's goals. The House Simulation Protocols (HSP) provides guidance to program partners and managers so that energy savings for new construction and retrofit projects can be compared alongside each other. The HSP provides the program with analysis methods that are proven to be effective and reliable in investigating the energy use of advanced energy systems and of entire houses.

  17. Addendum to the Building America House Simulation Protocols

    Energy Technology Data Exchange (ETDEWEB)

    Engebrecht, C. Metzger [National Renewable Energy Lab. (NREL), Golden, CO (United States); Wilson, E. [National Renewable Energy Lab. (NREL), Golden, CO (United States); Horowitz, S. [National Renewable Energy Lab. (NREL), Golden, CO (United States)

    2012-12-01

    As DOE's Building America program has grown to include a large and diverse cross-section of the home building and retrofit industries, it has become more important to develop accurate, consistent analysis techniques to measure progress towards the program’s goals. The House Simulation Protocols (HSP) provide guidance to program partners and managers so that energy savings for new construction and retrofit projects can be compared alongside each other. The HSP provides the program with analysis methods that are proven to be effective and reliable in investigating the energy use of advanced energy systems and of entire houses.

  18. Framework and indicator testing protocol for developing and piloting quality indicators for the UK quality and outcomes framework

    NARCIS (Netherlands)

    Campbell, S.M.; Kontopantelis, E.; Hannon, K.; Burke, M.; Barber, A.; Lester, H.E.

    2011-01-01

    BACKGROUND: Quality measures should be subjected to a testing protocol before being used in practice using key attributes such as acceptability, feasibility and reliability, as well as identifying issues derived from actual implementation and unintended consequences. We describe the methodologies

  19. Determination of the folate content in cladodes of nopal (Opuntia ficus indica) by microbiological assay utilizing Lactobacillus casei (ATCC 7469) and enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Ortiz-Escobar, Tania Breshkovskaya; Valverde-González, Maria Elena; Paredes-López, Octavio

    2010-05-26

    Prickly pear cactus has been an important food source in Mexico since ancient times due to its economical and ecological benefits and potential nutraceutical value. Nevertheless, studies on the nutritional aspects and health benefits have been scarce. The purpose of this study was to assess, apparently for the first time, the folate contents of cladodes of nopal by a microbiological assay, using Lactobacillus casei (ATCC 7469) in extracts that were enzymatically treated to release the bound vitamin, employing single, dual, and trienzymatic procedures, and using the enzyme-linked immunosorbent assay (ELISA). We used Opuntia cladodes of different length sizes. The microbiological assay showed some differences among enzyme treatments and sizes of nopal; the trienzyme treatment (alpha-amylase-protease-conjugase) was more efficient in determining the folate content in nopal, giving 5.0 ng/g in the small size cladodes at 54 h of testing time, while ELISA showed no significant differences in the folate content among sizes of cladodes (5.5-5.62 ng/g at 0 min testing time). Both techniques may be used for the assessment of folate content in cladodes, but ELISA is more rapid and reliable.

  20. Surface Enhanced Raman Spectroscopy (SERS) methods for endpoint and real-time quantification of miRNA assays

    Science.gov (United States)

    Restaino, Stephen M.; White, Ian M.

    2017-03-01

    Surface Enhanced Raman spectroscopy (SERS) provides significant improvements over conventional methods for single and multianalyte quantification. Specifically, the spectroscopic fingerprint provided by Raman scattering allows for a direct multiplexing potential far beyond that of fluorescence and colorimetry. Additionally, SERS generates a comparatively low financial and spatial footprint compared with common fluorescence based systems. Despite the advantages of SERS, it has remained largely an academic pursuit. In the field of biosensing, techniques to apply SERS to molecular diagnostics are constantly under development but, most often, assay protocols are redesigned around the use of SERS as a quantification method and ultimately complicate existing protocols. Our group has sought to rethink common SERS methodologies in order to produce translational technologies capable of allowing SERS to compete in the evolving, yet often inflexible biosensing field. This work will discuss the development of two techniques for quantification of microRNA, a promising biomarker for homeostatic and disease conditions ranging from cancer to HIV. First, an inkjet-printed paper SERS sensor has been developed to allow on-demand production of a customizable and multiplexable single-step lateral flow assay for miRNA quantification. Second, as miRNA concentrations commonly exist in relatively low concentrations, amplification methods (e.g. PCR) are therefore required to facilitate quantification. This work presents a novel miRNA assay alongside a novel technique for quantification of nuclease driven nucleic acid amplification strategies that will allow SERS to be used directly with common amplification strategies for quantification of miRNA and other nucleic acid biomarkers.

  1. The KRAS Strip Assay for detection of KRAS mutation in Egyptian patients with colorectal cancer (CRC): A pilot study

    International Nuclear Information System (INIS)

    Abd El Kader, Y.; Safwat, E.; Kassem, H.A.; Kassem, N.M.; Emera, G.

    2013-01-01

    Background: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefltinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. Purpose: Detection of KRAS mutation in Egyptian colorectal cancer (CRC) patients by the KRAS Strip Assay. Methods: Examination of 20 colorectal cancer (CRC) patients is done to detect KRAS mutations by KRAS Strip Assay. For the Strip Assay, a mutant-enriched PCR was followed by hybridization to KRAS-specific probes bound to a nitrocellulose strip. Results: Among 20 patients, KRAS mutations were identified in 80% of patients by the KRAS Strip Assay. Conclusions: Our preliminary results suggest that KRAS Strip Assay is an alternative to protocols currently in use for KRAS mutation detection

  2. Remote control of the industry processes. POWERLINK protocol application

    Science.gov (United States)

    Wóbel, A.; Paruzel, D.; Paszkiewicz, B.

    2017-08-01

    The present technological development enables the use of solutions characterized by a lower failure rate, and work with greater precision. This allows you to obtain the most efficient production, high speed production and reliability of individual components. The main scope of this article was POWERLINK protocol application for communication with the controller B & R through communication Ethernet for recording process parameters. This enables control of run production cycle using an internal network connected to the PC industry. Knowledge of the most important parameters of the production in real time allows detecting of a failure immediately after occurrence. For this purpose, the position of diagnostic use driver X20CP1301 B&R to record measurement data such as pressure, temperature valve between the parties and the torque required to change the valve setting was made. The use of POWERLINK protocol allows for the transmission of information on the status of every 200 μs.

  3. Implementation and Analysis of Real-Time Streaming Protocols.

    Science.gov (United States)

    Santos-González, Iván; Rivero-García, Alexandra; Molina-Gil, Jezabel; Caballero-Gil, Pino

    2017-04-12

    Communication media have become the primary way of interaction thanks to the discovery and innovation of many new technologies. One of the most widely used communication systems today is video streaming, which is constantly evolving. Such communications are a good alternative to face-to-face meetings, and are therefore very useful for coping with many problems caused by distance. However, they suffer from different issues such as bandwidth limitation, network congestion, energy efficiency, cost, reliability and connectivity. Hence, the quality of service and the quality of experience are considered the two most important issues for this type of communication. This work presents a complete comparative study of two of the most used protocols of video streaming, Real Time Streaming Protocol (RTSP) and the Web Real-Time Communication (WebRTC). In addition, this paper proposes two new mobile applications that implement those protocols in Android whose objective is to know how they are influenced by the aspects that most affect the streaming quality of service, which are the connection establishment time and the stream reception time. The new video streaming applications are also compared with the most popular video streaming applications for Android, and the experimental results of the analysis show that the developed WebRTC implementation improves the performance of the most popular video streaming applications with respect to the stream packet delay.

  4. A Secure Cluster-Based Multipath Routing Protocol for WMSNs

    Directory of Open Access Journals (Sweden)

    Jamal N. Al-Karaki

    2011-04-01

    Full Text Available The new characteristics of Wireless Multimedia Sensor Network (WMSN and its design issues brought by handling different traffic classes of multimedia content (video streams, audio, and still images as well as scalar data over the network, make the proposed routing protocols for typical WSNs not directly applicable for WMSNs. Handling real-time multimedia data requires both energy efficiency and QoS assurance in order to ensure efficient utility of different capabilities of sensor resources and correct delivery of collected information. In this paper, we propose a Secure Cluster-based Multipath Routing protocol for WMSNs, SCMR, to satisfy the requirements of delivering different data types and support high data rate multimedia traffic. SCMR exploits the hierarchical structure of powerful cluster heads and the optimized multiple paths to support timeliness and reliable high data rate multimedia communication with minimum energy dissipation. Also, we present a light-weight distributed security mechanism of key management in order to secure the communication between sensor nodes and protect the network against different types of attacks. Performance evaluation from simulation results demonstrates a significant performance improvement comparing with existing protocols (which do not even provide any kind of security feature in terms of average end-to-end delay, network throughput, packet delivery ratio, and energy consumption.

  5. The comet assay in Environmental Risk Assessment of marine pollutants: applications, assets and handicaps of surveying genotoxicity in non-model organisms.

    Science.gov (United States)

    Martins, Marta; Costa, Pedro M

    2015-01-01

    Determining the genotoxic effects of pollutants has long been a priority in Environmental Risk Assessment (ERA) for coastal ecosystems, especially of complex areas such as estuaries and other confined waterbodies. The acknowledged link between DNA damage, mutagenicity and carcinogenicity to the exposure to certain toxicants has been responsible to the growing interest in determining the genotoxic effects of xenobiotics to wildlife as a measure of environmental risk. The comet assay, although widely employed in in vivo and in vitro toxicology, still holds many constraints in ERA, in large part owing to difficulties in obtaining conclusive cause-effect relationships from complex environments. Nevertheless, these challenges do not hinder the attempts to apply the alkaline comet assay on sentinel organisms, wild or subjected to bioassays in or ex situ (from fish to molluscs) as well to standardise protocols and establish general guidelines to the interpretation of findings. Fish have been regarded as an appealing subject due to the ease of performing the comet assay in whole blood. However, the application of the comet assay is becoming increasingly common in invertebrates (e.g. in molluscan haemocytes and solid tissues such as gills). Virtually all sorts of results have been obtained from the application of the comet assay in ERA (null, positive and inconclusive). However, it has become clear that interpreting DNA damage data from wild organisms is particularly challenging due to their ability to adapt to continuous environmental stressors, including toxicants. Also, the comet assay in non-model organisms for the purpose of ERA implies different constraints, assumptions and interpretation of findings, compared with the in vitro procedures from which most guidelines have been derived. This paper critically reviews the application of the comet assay in ERA, focusing on target organisms and tissues; protocol developments, case studies plus data handling and

  6. Random assay in radioimmunoassay: Feasibility and application compared with batch assay

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jung Min; Lee, Hwan Hee; Park, Sohyun; Kim, Tae Sung; Kim, Seok Ki [Dept. of Nuclear MedicineNational Cancer Center, Goyang (Korea, Republic of)

    2016-12-15

    The batch assay has been conventionally used for radioimmunoassay (RIA) because of its technical robustness and practical convenience. However, it has limitations in terms of the relative lag of report time due to the necessity of multiple assays in a small number of samples compared with the random assay technique. In this study, we aimed to verify whether the random assay technique can be applied in RIA and is feasible in daily practice. The coefficients of variation (CVs) of eight standard curves within a single kit were calculated in a CA-125 immunoradiometric assay (IRMA) for the reference of the practically ideal CV of the CA-125 kit. Ten standard curves of 10 kits from 2 prospectively collected lots (pLot) and 85 standard curves of 85 kits from 3 retrospectively collected lots (Lot) were obtained. Additionally, the raw measurement data of both 170 control references and 1123 patients' sera were collected retrospectively between December 2015 and January 2016. A standard curve of the first kit of each lot was used as a master standard curve for a random assay. The CVs of inter-kits were analyzed in each lot, respectively. All raw measurements were normalized by decay and radioactivity. The CA-125 values from control samples and patients' sera were compared using the original batch assay and random assay. In standard curve analysis, the CVs of inter-kits in pLots and Lots were comparable to those within a single kit. The CVs from the random assay with normalization were similar to those from the batch assay in the control samples (CVs % of low/high concentration; Lot1 2.71/1.91, Lot2 2.35/1.83, Lot3 2.83/2.08 vs. Lot1 2.05/1.21, Lot2 1.66/1.48, Lot3 2.41/2.14). The ICCs between the batch assay and random assay using patients' sera were satisfactory (Lot1 1.00, Lot2 0.999, Lot3 1.00). The random assay technique could be successfully applied to the conventional CA-125 IRMA kits. The random assay showed strong agreement with the batch assay. The

  7. From human monocytes to genome-wide binding sites--a protocol for small amounts of blood: monocyte isolation/ChIP-protocol/library amplification/genome wide computational data analysis.

    Directory of Open Access Journals (Sweden)

    Sebastian Weiterer

    Full Text Available Chromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state of the art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplification for next generation sequencing. We developed a highly reproducible protocol to present a guideline from the first step of isolating monocytes from a blood sample to analyse the distribution of histone modifications in a genome-wide manner.The protocol describes the whole work flow from isolating monocytes from human blood samples followed by a high-sensitivity and small-scale chromatin immunoprecipitation assay with guidance for generating libraries compatible with next generation sequencing from small amounts of immunoprecipitated DNA.

  8. Development of a systematic observation protocol of physical exposure of the back: a preliminary study.

    Science.gov (United States)

    Tousignant, M; Tougas, G; Rossignol, M; Goulet, L

    2002-04-01

    At present there is no systematic observation protocol for the assessment of the multi-factorial aspects of physical exposure related to the back used within the constraints of occupational epidemiological research. In this context, a new preliminary systematic observation protocol is proposed to assess exposure to physical loading of the back using nine categories of physical risk factors: the SOPE back protocol. The objective of this study was to investigate whether the new protocol can correctly identify the level of exposure related to measured physical loading of the back. The subjects of this closed cohort study were 451 manual workers at a natural gas distribution company. The assessment of exposure was made with the protocol using groups with different job titles. The workers were followed for a 2 yr period to establish the risk of a new occurrence of complete disability related to the back (NOCD back injury) in each job grouping. Based on the median of the total scores derived from the protocol, two levels of exposure were identified (high and low). Taking into account the limitations of this study, the protocol in development may be a good tool to establish two levels of exposure to physical loading of the back in large epidemiological studies of occupational low back pain. Further research is needed to replicate these results with larger samples and to test the reliability and predictive validity of the protocol.

  9. Clinical assessment of scapular positioning in musicians: an intertester reliability study.

    Science.gov (United States)

    Struyf, Filip; Nijs, Jo; De Coninck, Kris; Giunta, Marco; Mottram, Sarah; Meeusen, Romain

    2009-01-01

    The reliability of the measurement of the distance between the posterior border of the acromion and the wall and the reliability of the modified lateral scapular slide test have not been studied. Overall, the reliability of the clinical tools used to assess scapular positioning has not been studied in musicians. To examine the intertester reliability of scapular observation and 2 clinical tests for the assessment of scapular positioning in musicians. Intertester reliability study. University research laboratory. Thirty healthy student musicians at a single university. Two assessors performed a standardized observation protocol, the measurement of the distance between the posterior border of the acromion and the wall, and the modified lateral scapular slide test. Each assessor was blinded to the other's findings. The intertester reliability coefficients (kappa) for the observation in relaxed position, during unloaded movement, and during loaded movement were 0.41, 0.63, and 0.36, respectively. The kappa values for the observation of tilting and winging at rest were 0.48 and 0.42, respectively; during unloaded movement, the kappa values were 0.52 and 0.78, respectively; and with a 1-kg load, the kappa values were 0.24 and 0.50, respectively. The intraclass correlation coefficient (ICC) of the measurement of the acromial distance was 0.72 in relaxed position and 0.75 with the participant actively retracting both shoulders. The ICCs for the modified lateral scapular slide test varied between 0.63 and 0.58. Our results demonstrated that the modified lateral scapular slide test was not a reliable tool to assess scapular positioning in these participants. Our data indicated that scapular observation in the relaxed position and during unloaded abduction in the frontal plane was a reliable assessment tool. The reliability of the measurement of the distance between the posterior border of the acromion and the wall in healthy musicians was moderate.

  10. An optimized protocol for DNA extraction from wheat seeds and Loop-Mediated Isothermal Amplification (LAMP) to detect Fusarium graminearum contamination of wheat grain.

    Science.gov (United States)

    Abd-Elsalam, Kamel; Bahkali, Ali; Moslem, Mohamed; Amin, Osama E; Niessen, Ludwig

    2011-01-01

    A simple, rapid, and efficient method for isolating genomic DNA from germinated seeds of wheat that is free from polysaccharides and polyphenols is reported. DNA was extracted, treated with RNase, measured and tested for completeness using agarose gel electrophoresis. DNA purification from wheat grains yielded abundant, amplifiable DNA with yields typically between 100 and 200 ng DNA/mg. The effectiveness and reliability of the method was tested by assessing quantity and quality of the isolated DNA using three PCR-based markers. Inter-simple sequence repeats (ISSRs) were used to assess the genetic diversity between different wheat varieties. Specific PCR primer pair Tox5-1/Tox5-2 and a loop-mediated isothermal amplification (LAMP) procedure were used to detect genomic DNA of Fusarium graminearum in contaminated wheat seeds. In this method there is no need to use liquid nitrogen for crushing germinated seedlings. The protocol takes approximately one hour to prepare high quality DNA. In combination with the LAMP assay it is a fast and cost-effective alternative to traditional diagnostic methods for the early detection of toxigenic fusaria in cereals.

  11. High reliable and Real-time Data Communication Network Technology for Nuclear Power Plant

    International Nuclear Information System (INIS)

    Jeong, K. I.; Lee, J. K.; Choi, Y. R.; Lee, J. C.; Choi, Y. S.; Cho, J. W.; Hong, S. B.; Jung, J. E.; Koo, I. S.

    2008-03-01

    As advanced digital Instrumentation and Control (I and C) system of NPP(Nuclear Power Plant) are being introduced to replace analog systems, a Data Communication Network(DCN) is becoming the important system for transmitting the data generated by I and C systems in NPP. In order to apply the DCNs to NPP I and C design, DCNs should conform to applicable acceptance criteria and meet the reliability and safety goals of the system. As response time is impacted by the selected protocol, network topology, network performance, and the network configuration of I and C system, DCNs should transmit a data within time constraints and response time required by I and C systems to satisfy response time requirements of I and C system. To meet these requirements, the DCNs of NPP I and C should be a high reliable and real-time system. With respect to high reliable and real-time system, several reports and techniques having influences upon the reliability and real-time requirements of DCNs are surveyed and analyzed

  12. Reliability of knee joint range of motion and circumference measurements after total knee arthroplasty: does tester experience matter?

    DEFF Research Database (Denmark)

    Jakobsen, Thomas Linding; Christensen, Malene; Christensen, Stine Sommer

    2010-01-01

    : The design was an intra-tester, inter-tester and intra-day reliability study. Nineteen outpatients (10 females) having received a TKA were examined by an inexperienced and an experienced physiotherapist. Following a standardized protocol, active and passive knee joint ROM and circumference measurements were...

  13. Lipase assay in soils by copper soap colorimetry.

    Science.gov (United States)

    Saisuburamaniyan, N; Krithika, L; Dileena, K P; Sivasubramanian, S; Puvanakrishnan, R

    2004-07-01

    A simple and sensitive method for the estimation of lipase activity in soils is reported. In this method, 50mg of soil is incubated with emulsified substrate, the fatty acids liberated are treated with cupric acetate-pyridine reagent, and the color developed is measured at 715 nm. Use of olive oil in this protocol leads to an estimation of true lipase activity in soils. The problem of released fatty acids getting adsorbed onto the soil colloids is obviated by the use of isooctane, and separate standards for different soils need not be developed. Among the various surfactants used for emulsification, polyvinyl alcohol is found to be the most effective. Incubation time of 20 min, soil concentration of 50 mg, pH 6.5, and incubation temperature of 37 degrees C were found to be the most suitable conditions for this assay. During the process of enrichment of the soils with oil, interference by the added oil is avoided by the maintenance of a suitable control, wherein 50 mg of soil is added after stopping the reaction. This assay is sensitive and it could be adopted to screen for lipase producers from enriched soils and oil-contaminated soils before resorting to isolation of the microbes by classical screening methods.

  14. Influenza A virus H5-specific antibodies in mute swans (Cygnus olor) in the USA.

    Science.gov (United States)

    Kistler, Whitney M; Stallknecht, David E; Lebarbenchon, Camille; Pedersen, Kerri; Marks, David R; Mickley, Randy; DeLiberto, Thomas J; Yabsley, Michael J

    2015-04-01

    The use of serologic assays for influenza A virus (IAV) surveillance in wild birds has increased because of the availability of commercial enzyme-linked immunosorbent assays (ELISAs). Recently, an H5-specific blocking ELISA (bELISA) was shown to reliably detect H5-specific antibodies to low- and high-pathogenic H5 viruses in experimentally infected waterfowl. Mute Swans (Cygnus olor) were frequently associated with highly pathogenic H5N1 outbreaks in Europe and may have a similar role if highly pathogenic H5N1 is introduced into North America. We measured the prevalence of antibodies to the nucleoprotein and H5 protein in Mute Swans using three serologic assays. We collected 340 serum samples from Mute Swans in Michigan, New Jersey, New York, and Rhode Island, US. We detected antibodies to the IAV nucleoprotein in 66.2% (225/340) of the samples. We detected H5-specific antibodies in 62.9% (214/340) and 18.8% (64/340) using a modified H5 bELISA protocol and hemagglutination inhibition (HI) assay, respectively. The modified H5 bELISA protocol detected significantly more positive samples than did the manufacturer's protocol. We also tested 46 samples using virus neutralization. Neutralization results had high agreement with the modified H5 bELISA protocol and detected a higher prevalence than did the HI assay. These results indicate that North American Mute Swans have high nucleoprotein and H5 antibody prevalences.

  15. Evaluation of a real-time PCR assay based on the single-copy SAG1 gene for the detection of Toxoplasma gondii.

    Science.gov (United States)

    Yu, Haijie; Huang, Bin; Zhuo, Xunhui; Chen, Xueqiu; Du, Aifang

    2013-11-08

    Real-time PCR-based detection of Toxoplasma gondii is very sensitive and convenient for diagnosing toxoplasmosis. However, the performance of the PCR assays could be influenced by the target gene chosen. Here we evaluate a real-time PCR assay using double-stranded DNA dyes (SYBR(®) Green I assay) with a new set of primers targeting the SAG1 gene for the fast and specific detection of T. gondii. The assay showed higher sensitivity than conventional PCR protocols using T. gondii DNA as template. The detection limit of the developed real-time PCR assay was in the order of 1 tachyzoite. The assay was also assessed by experimentally infected mice and showed positive results for blood (25%), spleen (50%) and lung (50%) as early as 1 dpi. The specificity of the assay was confirmed by using DNA from Neospora caninum, Escherichia coli, Babesia bovis, Trypanosoma brucei, Cryptosporidium parvum, and Toxocara canis. Assay applicability was successfully tested in blood samples collected from slaughtered pigs. These results indicate that, based on SYBR(®) green I, the quantitative SAG1 assay may also be useful in the study of the pathogenicity, immunoprophylaxis, and treatment of T. gondii. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Matrix effects of TRU [transuranic] assays using the SWEPP PAN assay system

    International Nuclear Information System (INIS)

    Smith, J.R.

    1990-08-01

    The Drum Assay System (DAS) at the Stored Waste Experimental Pilot Plant (SWEPP) is a second-generation active-passive neutron assay system. It has been used to assay over 5000 208-liter drums of transuranic waste from the Rocky Flats Plant (RFP). Data from these assays have been examined and compared with the assays performed at Rocky Flats, mainly utilize counting of 239 Pu gamma rays. For the most part the passive assays are in very good agreement with the Rocky Flats assays. The active assays are strongly correlated with the results of the other two methods, but require matrix-dependent correction factors beyond those provided by the system itself. A set of matrix-dependent correction factors has been developed from the study of the assay results. 3 refs., 4 figs., 3 tabs

  17. Reliability and Validity of the Inline Skating Skill Test

    Science.gov (United States)

    Radman, Ivan; Ruzic, Lana; Padovan, Viktoria; Cigrovski, Vjekoslav; Podnar, Hrvoje

    2016-01-01

    This study aimed to examine the reliability and validity of the inline skating skill test. Based on previous skating experience forty-two skaters (26 female and 16 male) were randomized into two groups (competitive level vs. recreational level). They performed the test four times, with a recovery time of 45 minutes between sessions. Prior to testing, the participants rated their skating skill using a scale from 1 to 10. The protocol included performance time measurement through a course, combining different skating techniques. Trivial changes in performance time between the repeated sessions were determined in both competitive females/males and recreational females/males (-1.7% [95% CI: -5.8–2.6%] – 2.2% [95% CI: 0.0–4.5%]). In all four subgroups, the skill test had a low mean within-individual variation (1.6% [95% CI: 1.2–2.4%] – 2.7% [95% CI: 2.1–4.0%]) and high mean inter-session correlation (ICC = 0.97 [95% CI: 0.92–0.99] – 0.99 [95% CI: 0.98–1.00]). The comparison of detected typical errors and smallest worthwhile changes (calculated as standard deviations × 0.2) revealed that the skill test was able to track changes in skaters’ performances. Competitive-level skaters needed shorter time (24.4–26.4%, all p skating skills in amateur competitive and recreational level skaters. Further studies are needed to evaluate the reproducibility of this skill test in different populations including elite inline skaters. Key points Study evaluated the reliability and construct validity of a newly developed inline skating skill test. Evaluated test is a first protocol designed to assess specific inline skating skill. Two groups of amateur skaters with different skating proficiency repeated the skill test in four separate occasions. The results suggest that evaluated test is reliable and valid to evaluate inline skating skill in amateur skaters. PMID:27803616

  18. Cloud-assisted mutual authentication and privacy preservation protocol for telecare medical information systems.

    Science.gov (United States)

    Li, Chun-Ta; Shih, Dong-Her; Wang, Chun-Cheng

    2018-04-01

     With the rapid development of wireless communication technologies and the growing prevalence of smart devices, telecare medical information system (TMIS) allows patients to receive medical treatments from the doctors via Internet technology without visiting hospitals in person. By adopting mobile device, cloud-assisted platform and wireless body area network, the patients can collect their physiological conditions and upload them to medical cloud via their mobile devices, enabling caregivers or doctors to provide patients with appropriate treatments at anytime and anywhere. In order to protect the medical privacy of the patient and guarantee reliability of the system, before accessing the TMIS, all system participants must be authenticated.  Mohit et al. recently suggested a lightweight authentication protocol for cloud-based health care system. They claimed their protocol ensures resilience of all well-known security attacks and has several important features such as mutual authentication and patient anonymity. In this paper, we demonstrate that Mohit et al.'s authentication protocol has various security flaws and we further introduce an enhanced version of their protocol for cloud-assisted TMIS, which can ensure patient anonymity and patient unlinkability and prevent the security threats of report revelation and report forgery attacks.  The security analysis proves that our enhanced protocol is secure against various known attacks as well as found in Mohit et al.'s protocol. Compared with existing related protocols, our enhanced protocol keeps the merits of all desirable security requirements and also maintains the efficiency in terms of computation costs for cloud-assisted TMIS.  We propose a more secure mutual authentication and privacy preservation protocol for cloud-assisted TMIS, which fixes the mentioned security weaknesses found in Mohit et al.'s protocol. According to our analysis, our authentication protocol satisfies most functionality features

  19. Preclinical detection of porcine circovirus type 2 infection using an ultrasensitive nanoparticle DNA probe-based PCR assay.

    Directory of Open Access Journals (Sweden)

    Yong Huang

    Full Text Available Porcine circovirus type 2 (PCV2 has emerged as one of the most important pathogens affecting swine production globally. Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR for PCV2 detection. Magnetic microparticles coated with PCV2 specific DNA probes were used to enrich PCV2 DNA from samples, then gold nanoparticles coated with PCV2 specific oligonucleotides were added to form a sandwich nucleic acid-complex. After the complex was formed, the oligonucleotides were released and characterized by PCR. This assay exhibited about 500-fold more sensitive than conventional PCR, with a detection limit of 2 copies of purified PCV2 genomic DNA and 10 viral copies of PCV2 in serum. The assay has a wide detection range for all of PCV2 genotypes with reliable reproducibility. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1, porcine parvovirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus and classical swine fever virus. The positive detection rate of PCV2 specific UNDP-PCR in 40 preclinical field samples was 27.5%, which appeared greater than that by conventional and real-time PCR and appeared application potency in evaluation of the viral loads levels of preclinical infection samples. The UNDP-PCR assay reported here can reliably rule out false negative results from antibody-based assays, provide a nucleic acid extraction free, specific, ultrasensitive, economic and rapid diagnosis method for preclinical PCV2 infection in field, which may help prevent large-scale outbreaks.

  20. Development of an assay for a biomarker of pregnancy and early fetal loss

    International Nuclear Information System (INIS)

    Canfield, R.E.; O'Connor, J.F.; Birken, S.; Krichevsky, A.; Wilcox, A.J.

    1987-01-01

    Human chorionic gonadotropin (hCG) is a glycoprotein hormone, secreted by the syncytiotrophoblast cells of the fertilized ovum, that enters the maternal circulation at the time of endometrial implantation. It is composed of two nonidentical subunits; α and β, with molecular weights of 14 kD and 23 kD, respectively. Human chorionic gonadotropin binds to the same receptor as hLH and displays the same biological response, namely, to stimulate the declining function of the corpus luteum to produce progestins and estrogen late in the menstrual cycle. The differences in the structures of hCG and hLH have been exploited to develop antibodies that can measure hCG specifically in the presence of hLH. Two-site antibody binding assays have been developed, based on a surface immunological concept of hCG epitopes, that involve four distinct regions to which antibodies against hCG can bind simultaneously. Antibody cooperative effects, in conjunction with kinetic advantages derived from the concentration factors by use of the sandwich assay technique (immunoradiometric assay, IRMA), have enabled development of extremely sensitive and specific measurement protocols for urinary hCG. The assay described herein permits the detection of pregnancy on an average 25.4 days after the first day of the preceding menses, as opposed to 29.5 days for conventional radioimmunoassay techniques. In addition, the greater sensitivity and specificity of this assay method has permitted the detection of episodes of fetal loss not detected by radioimmunoassay of urine specimens. A large scale epidemiological study is in progress using this assay technique as a way to identify pregnancies that are lost before becoming clinically apparent