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

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

2018-04-03

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

Detection of SYT-SSX mutant transcripts in formalin-fixed paraffin-embedded sarcoma tissues using one-step reverse transcriptase real-time PCR.

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Norlelawati, A T; Mohd Danial, G; Nora, H; Nadia, O; Zatur Rawihah, K; Nor Zamzila, A; Naznin, M

2016-04-01

Synovial sarcoma (SS) is a rare cancer and accounts for 5-10% of adult soft tissue sarcomas. Making an accurate diagnosis is difficult due to the overlapping histological features of SS with other types of sarcomas and the non-specific immunohistochemistry profile findings. Molecular testing is thus considered necessary to confirm the diagnosis since more than 90% of SS cases carry the transcript of t(X;18)(p11.2;q11.2). The purpose of this study is to diagnose SS at molecular level by testing for t(X;18) fusion-transcript expression through One-step reverse transcriptase real-time Polymerase Chain Reaction (PCR). Formalin-fixed paraffin-embedded tissue blocks of 23 cases of soft tissue sarcomas, which included 5 and 8 cases reported as SS as the primary diagnosis and differential diagnosis respectively, were retrieved from the Department of Pathology, Tengku Ampuan Afzan Hospital, Kuantan, Pahang. RNA was purified from the tissue block sections and then subjected to One-step reverse transcriptase real-time PCR using sequence specific hydrolysis probes for simultaneous detection of either SYT-SSX1 or SYT-SSX2 fusion transcript. Of the 23 cases, 4 cases were found to be positive for SYT-SSX fusion transcript in which 2 were diagnosed as SS whereas in the 2 other cases, SS was the differential diagnosis. Three cases were excluded due to failure of both amplification assays SYT-SSX and control β-2-microglobulin. The remaining 16 cases were negative for the fusion transcript. This study has shown that the application of One-Step reverse transcriptase real time PCR for the detection SYT-SSX transcript is feasible as an aid in confirming the diagnosis of synovial sarcoma.

Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription.

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Johnston, Stephen; Gallaher, Zachary; Czaja, Krzysztof

2012-05-15

Quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2(-∆∆Ct) normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxin selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference β-III tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.

Rapid and sensitive detection of novel avian-origin influenza A (H7N9 virus by reverse transcription loop-mediated isothermal amplification combined with a lateral-flow device.

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Yiyue Ge

Full Text Available A severe disease in humans caused by a novel avian-origin influenza A (H7N9 virus emerged in China recently, which has caused at least 128 cases and 26 deaths. Rapid detection of the novel H7N9 virus is urgently needed to differentiate the disease from other infections, and to facilitate infection control as well as epidemiologic investigations. In this study, a reverse transcription loop-mediated isothermal amplification combined with a lateral flow device (RT-LAMP-LFD assay to rapidly detect H7N9 virus was developed and evaluated. The RT-LAMP primers were designed to target the haemagglutinin (HA and neuraminidase (NA genes of H7N9 virus. Results of 10-fold dilution series assays showed that analysis of RT-LAMP products by the LFD method was as sensitive as real-time turbidity detection, and that the analytic sensitivities of the HA and NA RT-LAMP assays were both 10 copies of synthetic RNA. Furthermore, both the assays showed 100% clinical specificity for identification of H7N9 virus. The performance characteristics of the RT-LAMP-LFD assay were evaluated with 80 clinical specimens collected from suspected H7N9 patients. The NA RT-LAMP-LFD assay was more sensitive than real time RT-PCR assay. Compared with a combination of virus culture and real-time RT-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the RT-LAMP-LFD assay were all 100%. Overall, The RT-LAMP-LFD assay established in this study can be used as a reliable method for early diagnosis of the avian-origin influenza A (H7N9 virus infection.

Simultaneous detection and differentiation of dengue virus serotypes 1-4, Japanese encephalitis virus, and West Nile virus by a combined reverse-transcription loop-mediated isothermal amplification assay

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Yin Jianhua

2011-07-01

Full Text Available Abstract Background Rapid identification and differentiation of mosquito-transmitted flaviviruses in acute-phase sera of patients and field-caught vector mosquitoes are important for the prediction and prevention of large-scale epidemics. Results We developed a flexible reverse-transcription loop-mediated isothermal amplification (RT-LAMP unit for the detection and differentiation of dengue virus serotypes 1-4 (DENV1-4, Japanese encephalitis virus (JEV, and West Nile virus (WNV. The unit efficiently amplified the viral genomes specifically at wide ranges of viral template concentrations, and exhibited similar amplification curves as monitored by a real-time PCR engine. The detection limits of the RT-LAMP unit were 100-fold higher than that of RT-PCR in 5 of the six flaviviruses. The results on specificity indicated that the six viruses in the assay had no cross-reactions with each other. By examining 66 viral strains of DENV1-4 and JEV, the unit identified the viruses with 100% accuracy and did not cross-react with influenza viruses and hantaviruses. By screening a panel of specimens containing sera of 168 patients and 279 pools of field-caught blood sucked mosquitoes, results showed that this unit is high feasible in clinical settings and epidemiologic field, and it obtained results 100% correlated with real-time RT-PCR. Conclusions The RT-LAMP unit developed in this study is able to quickly detect and accurately differentiate the six kinds of flaviviruses, which makes it extremely feasible for screening these viruses in acute-phase sera of the patients and in vector mosquitoes without the need of high-precision instruments.

Enteroviruses in blood of patients with type 1 diabetes detected by integrated cell culture and reverse transcription quantitative real-time PCR.

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Alidjinou, Enagnon Kazali; Sane, Famara; Lefevre, Christine; Baras, Agathe; Moumna, Ilham; Engelmann, Ilka; Vantyghem, Marie-Christine; Hober, Didier

2017-11-01

Enteroviruses (EV) have been associated with type 1 diabetes (T1D), but EV RNA detection has been reported in only a small proportion of T1D patients. We studied whether integrated cell culture and reverse transcription real-time PCR could improve EV detection in blood samples from patients with T1D. Blood was collected from 13 patients with T1D. The presence of EV RNA in blood was investigated by using real-time RT-PCR. In addition, plasma and white blood cells (WBC) were inoculated to BGM and Vero cell line cultures. Culture supernatants and cells collected on day 7 and day 14 were tested for EV RNA by real-time RT-PCR. Enterovirus identification was performed through sequencing of the VP4/VP2 region. Enterovirus RNA was detected in blood by using real-time RT-PCR in only one out of 13 patients. The detection of EV RNA in cultures inoculated with clinical samples (plasma and/or WBC) gave positive results in five other patients. The viral loads were low, ranging from 45 to 4420 copies/ng of total RNA. One isolate was successfully identified as coxsackievirus B1. Integrated cell culture and reverse transcription real-time PCR can improve the detection rate of EV in blood samples of patients with T1D and can be useful to investigate further the relationship between EV and the disease.

Outbreak of hepatitis E virus infection in Darfur, Sudan: effectiveness of real-time reverse transcription-PCR analysis of dried blood spots.

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Mérens, Audrey; Guérin, Philippe Jean; Guthmann, Jean-Paul; Nicand, Elisabeth

2009-06-01

Biological samples collected in refugee camps during an outbreak of hepatitis E were used to compare the accuracy of hepatitis E virus RNA amplification by real-time reverse transcription-PCR (RT-PCR) for sera and dried blood spots (concordance of 90.6%). Biological profiles (RT-PCR and serology) of asymptomatic individuals were also analyzed.

Visual detection of West Nile virus using reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip

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Zengguo eCao

2016-04-01

Full Text Available West Nile virus (WNV causes a severe zoonosis, which can lead to a large number of casualties and considerable economic losses. A rapid and accurate identification methodfor WNV for use in field laboratories is urgently needed. Here, a method utilizing reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip (RT-LAMP-VF was developed to detect the envelope (E gene of WNV. The RT-LAMP-VF assay could detect 102 copies/μl ofan WNV RNA standard using a 40 min amplification reaction followed by a 2 min incubationof the amplification product on the visualization strip, and no cross-reaction with other closely related members of theFlavivirus genus was observed. The assay was further evaluated using cells and mouse brain tissues infected with a recombinant rabies virus expressing the E protein of WNV.The assay produced sensitivities of 101.5TCID50/ml and 101.33 TCID50/ml for detection of the recombinant virus in the cells and brain tissues, respectively. Overall, the RT-LAMP-VF assay developed in this study is rapid, simple and effective, and it is therefore suitable for clinical application in the field.

Rapid typing of foot-and-mouth disease serotype Asia 1 by reverse transcription loop-mediated isothermal amplification

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Chen Hao-tai

2011-10-01

Full Text Available Abstract A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP assay was rapidly used to detect serotype Asia 1 of foot-and-mouth disease virus (FMDV within 45 min at 61°C. All FMDV serotype Asia 1 reference strains were positive by RT-LAMP, while other viruses such as FMDV serotypes O, C, A and classical swine fever virus, swine vesicular disease virus, porcine reproductive and respiratory syndrome virus and Japanese encephalitis virus remained negative. Furthermore, FMDV sreotype Asia 1 positive samples were able to detect by RT-LAMP assay. This RT-LAMP assay may be suitable particularly for diagnosis of FMDV serotype Asia 1 infection in field stations.

Smartphone-Imaged HIV-1 Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP on a Chip from Whole Blood

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Gregory L. Damhorst

2015-09-01

Full Text Available Viral load measurements are an essential tool for the long-term clinical care of human immunodeficiency virus (HIV-positive individuals. The gold standards in viral load instrumentation, however, are still too limited by their size, cost, and sophisticated operation for these measurements to be ubiquitous in remote settings with poor healthcare infrastructure, including parts of the world that are disproportionately affected by HIV infection. The challenge of developing a point-of-care platform capable of making viral load more accessible has been frequently approached but no solution has yet emerged that meets the practical requirements of low cost, portability, and ease-of-use. In this paper, we perform reverse-transcription loop-mediated isothermal amplification (RT-LAMP on minimally processed HIV-spiked whole blood samples with a microfluidic and silicon microchip platform, and perform fluorescence measurements with a consumer smartphone. Our integrated assay shows amplification from as few as three viruses in a ~ 60 nL RT-LAMP droplet, corresponding to a whole blood concentration of 670 viruses per μL of whole blood. The technology contains greater power in a digital RT-LAMP approach that could be scaled up for the determination of viral load from a finger prick of blood in the clinical care of HIV-positive individuals. We demonstrate that all aspects of this viral load approach, from a drop of blood to imaging the RT-LAMP reaction, are compatible with lab-on-a-chip components and mobile instrumentation.

Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification.

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Wang, Jianchang; Wang, Jinfeng; Li, Ruiwen; Liu, Libing; Yuan, Wanzhe

2017-08-15

Canine distemper, caused by Canine distemper virus (CDV), is a highly contagious and fatal systemic disease in free-living and captive carnivores worldwide. Recombinase polymerase amplification (RPA), as an isothermal gene amplification technique, has been explored for the molecular detection of diverse pathogens. A real-time reverse transcription RPA (RT-RPA) assay for the detection of canine distemper virus (CDV) using primers and exo probe targeting the CDV nucleocapsid protein gene was developed. A series of other viruses were tested by the RT-RPA.Thirty-two field samples were further tested by RT-RPA, and the resuts were compared with those obtained by the real-time RT-PCR. The RT-RPA assay was performed successfully at 40 °C, and the results were obtained within 3 min-12 min. The assay could detect CDV, but did not show cross-detection of canine parvovirus-2 (CPV-2), canine coronavirus (CCoV), canine parainfluenza virus (CPIV), pseudorabies virus (PRV) or Newcastle disease virus (NDV), demonstrating high specificity. The analytical sensitivity of RT-RPA was 31.8 copies in vitro transcribed CDV RNA, which is 10 times lower than the real-time RT-PCR. The assay performance was validated by testing 32 field samples and compared to real-time RT-PCR. The results indicated an excellent correlation between RT-RPA and a reference real-time RT-PCR method. Both assays provided the same results, and R 2 value of the positive results was 0.947. The results demonstrated that the RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of CDV both in the laboratory and point-of-care facility, especially in the resource-limited settings.

Properties of the reverse transcription reaction in mRNA quantification

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Ståhlberg, Anders; Håkansson, Joakim; Xian, Xiaojie

2004-01-01

BACKGROUND: In most measurements of gene expression, mRNA is first reverse-transcribed into cDNA. We studied the reverse transcription reaction and its consequences for quantitative measurements of gene expression. METHODS: We used SYBR green I-based quantitative real-time PCR (QPCR) to measure...... the properties of reverse transcription reaction for the beta-tubulin, glyceraldehyde-3-phosphate dehydrogenase, Glut2, CaV1D, and insulin II genes, using random hexamers, oligo(dT), and gene-specific reverse transcription primers. RESULTS: Experimental variation in reverse transcription-QPCR (RT......-QPCR) was mainly attributable to the reverse transcription step. Reverse transcription efficiency depended on priming strategy, and the dependence was different for the five genes studied. Reverse transcription yields also depended on total RNA concentration. CONCLUSIONS: RT-QPCR gene expression measurements...

Porcine reproductive and respiratory syndrome virus: Interlaboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction detection methods

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Wernike, Kerstin; Bonilauri, Paolo; Dauber, Malte

2012-01-01

To compare the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays used for the diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV), a Europe-wide interlaboratory ring trial was conducted. A variety of PRRSV strains including North American...... (NA) and European (EU) genotype isolates were analyzed by the participants. Great differences regarding qualitative diagnostics as well as analytical sensitivity were observed between the individual RT-qPCR systems, especially when investigating strains from the EU genotype. None of the assays...

Detection of Tumor Cell-Specific mRNA in the Peripheral Blood of Patients with Breast Cancer — Evaluation of Several Markers with Real-Time Reverse Transcription-PCR

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Ulrich Andergassen

2013-01-01

Full Text Available It is widely known that cells from epithelial tumors, e.g., breast cancer, detach from their primary tissue and enter blood circulation. We show that the presence of circulating tumor cells (CTCs in samples of patients with primary and metastatic breast cancer can be detected with an array of selected tumor-marker-genes by reverse transcription real-time PCR. The focus of the presented work is on detecting differences in gene expression between healthy individuals and adjuvant and metastatic breast cancer patients, not an accurate quantification of these differences. Therefore, total RNA was isolated from blood samples of healthy donors and patients with primary or metastatic breast cancer after enrichment of mononuclear cells by density gradient centrifugation. After reverse transcription real-time PCR was carried out with a set of marker genes (BCSP, CK8, Her2, MGL, CK18, CK19. B2M and GAPDH were used as reference genes. Blood samples from patients with metastatic disease revealed increased cytokine gene levels in comparison to normal blood samples. Detection of a single gene was not sufficient to detect CTCs by reverse transcription real-time PCR. Markers used here were selected based on a recent study detecting cancer cells on different protein levels. The combination of such a marker array leads to higher and more specific discovery rates, predominantly in metastatic patients. Identification of CTCs by PCR methods may lead to better diagnosis and prognosis and could help to choose an adequate therapy.

Improved detection limit in rapid detection of human enterovirus 71 and coxsackievirus A16 by a novel reverse transcription-isothermal multiple-self-matching-initiated amplification assay.

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Ding, Xiong; Nie, Kai; Shi, Lei; Zhang, Yong; Guan, Li; Zhang, Dan; Qi, Shunxiang; Ma, Xuejun

2014-06-01

Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-foot-and-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription-isothermal multiple-self-matching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R(2) values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R(2) values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71

Development of a reverse transcription loop-mediated isothermal amplification method for the rapid detection of avian influenza virus subtype H7.

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Bao, Hongmei; Wang, Xiurong; Zhao, Yuhui; Sun, Xiaodong; Li, Yanbing; Xiong, Yongzhong; Chen, Hualan

2012-01-01

A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7 avian influenza virus (H7 AIV) isotype was developed. The minimum detection limit of the RT-LAMP assay was 0.1-0.01 PFU per reaction for H7 AIV RNA, making this assay 100-fold more sensitive than the conventional RT-PCR method. This RT-LAMP assay also has the capacity to detect both high- and low-pathogenic H7 AIV strains. Using a pool of RNAs extracted from influenza viruses corresponding to all 15 HA subtypes (in addition to other avian pathogenic viruses), the RT-LAMP system was confirmed to amplify only H7 AIV RNA. Furthermore, specific pathogen free (SPF) chickens were infected artificially with H7 AIV, throat and cloacal swabs were collected, and viral shedding was examined using viral isolation, RT-PCR and RT-LAMP. Shedding was detected following viral isolation and RT-LAMP one day after infection, whereas viral detection using RT-PCR was effective only on day 3 post-infection. These results indicate that the RT-LAMP method could facilitate epidemiological surveillance and the rapid diagnosis of the avian influenza subtype H7. Copyright © 2011 Elsevier B.V. All rights reserved.

Optimization of the elution buffer and concentration method for detecting hepatitis E virus in swine liver using a nested reverse transcription-polymerase chain reaction and real-time reverse transcription-polymerase chain reaction.

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Son, Na Ry; Seo, Dong Joo; Lee, Min Hwa; Seo, Sheungwoo; Wang, Xiaoyu; Lee, Bog-Hieu; Lee, Jeong-Su; Joo, In-Sun; Hwang, In-Gyun; Choi, Changsun

2014-09-01

The aim of this study was to develop an optimal technique for detecting hepatitis E virus (HEV) in swine livers. Here, three elution buffers and two concentration methods were compared with respect to enhancing recovery of HEV from swine liver samples. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and nested RT-PCR were performed to detect HEV RNA. When phosphate-buffered saline (PBS, pH 7.4) was used to concentrate HEV in swine liver samples using ultrafiltration, real-time RT-PCR detected HEV in 6 of the 26 samples. When threonine buffer was used to concentrate HEV using polyethylene glycol (PEG) precipitation and ultrafiltration, real-time RT-PCR detected HEV in 1 and 3 of the 26 samples, respectively. When glycine buffer was used to concentrate HEV using ultrafiltration and PEG precipitation, real-time RT-PCR detected HEV in 1 and 3 samples of the 26 samples, respectively. When nested RT-PCR was used to detect HEV, all samples tested negative regardless of the type of elution buffer or concentration method used. Therefore, the combination of real-time RT-PCR and ultrafiltration with PBS buffer was the most sensitive and reliable method for detecting HEV in swine livers. Copyright © 2014 Elsevier B.V. All rights reserved.

An Optogenetic Platform for Real-Time, Single-Cell Interrogation of Stochastic Transcriptional Regulation.

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Rullan, Marc; Benzinger, Dirk; Schmidt, Gregor W; Milias-Argeitis, Andreas; Khammash, Mustafa

2018-05-17

Transcription is a highly regulated and inherently stochastic process. The complexity of signal transduction and gene regulation makes it challenging to analyze how the dynamic activity of transcriptional regulators affects stochastic transcription. By combining a fast-acting, photo-regulatable transcription factor with nascent RNA quantification in live cells and an experimental setup for precise spatiotemporal delivery of light inputs, we constructed a platform for the real-time, single-cell interrogation of transcription in Saccharomyces cerevisiae. We show that transcriptional activation and deactivation are fast and memoryless. By analyzing the temporal activity of individual cells, we found that transcription occurs in bursts, whose duration and timing are modulated by transcription factor activity. Using our platform, we regulated transcription via light-driven feedback loops at the single-cell level. Feedback markedly reduced cell-to-cell variability and led to qualitative differences in cellular transcriptional dynamics. Our platform establishes a flexible method for studying transcriptional dynamics in single cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Evaluation of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a screening method for the detection of influenza viruses in the fecal materials of water birds.

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Yoshida, Hiromi; Sakoda, Yoshihiro; Endo, Mayumi; Motoshima, Masayuki; Yoshino, Fumi; Yamamoto, Naoki; Okamatsu, Masatoshi; Soejima, Takahiro; Senba, Syouhei; Kanda, Hidetoshi; Kida, Hiroshi

2011-06-01

Migratory water birds are a natural reservoir for influenza A viruses. Viruses replicate in the intestines of ducks and are shed with the fecal materials. Virus isolation from collected fecal materials, therefore, is an integral part of the surveillance of avian influenza in water birds. In the present study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) was assessed for its usefulness in detecting the RNA of influenza A viruses in fecal materials. It was found that, RT-LAMP specifically and sensitively detects the matrix gene of influenza A viruses. Influenza A viruses were isolated from the fecal materials in which viral RNA were detected by RT-LAMP in 35 min. The present findings indicate that RT-LAMP is useful as a high throughput screening method for field samples prior to virus isolation, allowing the processing of hundreds of samples per day.

A sensitive, reproducible, and economic real-time reverse transcription PCR detecting avian metapneumovirus subtypes A and B.

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Franzo, G; Drigo, M; Lupini, C; Catelli, E; Laconi, A; Listorti, V; Bonci, M; Naylor, C J; Martini, M; Cecchinato, M

2014-06-01

Use of real-time PCR is increasing in the diagnosis of infectious disease due to its sensitivity, specificity, and speed of detection. These characteristics make it particularly suited for the diagnosis of viral infections, like avian metapneumovirus (AMPV), for which effective control benefits from continuously updated knowledge of the epidemiological situation. Other real-time reverse transcription (RT)-PCRs have been published based on highly specific fluorescent dye-labeled probes, but they have high initial cost, complex validation, and a marked susceptibility to the genetic variability of their target sequence. With this in mind, we developed and validated a SYBR Green I-based quantitative RT-PCR for the detection of the two most prevalent AMPV subtypes (i.e., subtypes A and B). The assay demonstrated an analytical sensitivity comparable with that of a previously published real-time RT-PCR and the ability to detect RNA equivalent to approximately 0.5 infectious doses for both A and B subtypes. The high efficiency and linearity between viral titer and crossing point displayed for both subtypes make it suited for viral quantification. Optimization of reaction conditions and the implementation of melting curve analysis guaranteed the high specificity of the assay. The stable melting temperature difference between the two subtypes indicated the possibility of subtyping through melting temperature analysis. These characteristics make our assay a sensitive, specific, and rapid tool, enabling contemporaneous detection, quantification, and discrimination of AMPV subtype A and B.

On-site detection of Xylella fastidiosa in host plants and in “spy insects” using the real-time loop-mediated isothermal amplification method

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Thaer YASEEN

2015-12-01

Full Text Available A recent severe outbreak of Xylella fastidiosa associated with ‘olive quick decline syndrome’ (OQDS was reported in Apulia (Southern Italy. In this study an on-site real-time loop-mediated isothermal amplification (real-time LAMP was developed for detecting X. fastidiosa in host plants and insects. A marked simplification of the DNA extraction procedure was obtained by heating the samples in a portable Smart-Dart device and using an optimized enhancer reaction buffer. The connection to a tablet or Smartphone allowed to visualize the results of the reaction in real time. Compared to PCR and ELISA, with which it showed comparable results in terms of sensitivity and reliability in the X. fastidiosa detection, this simplified real-time LAMP procedure proved to be “user friendly”, displaying the advantages to be an on-site detection method of easy handling, rapid execution and low cost.

Establishment of reverse transcription loop-mediated isothermal amplification for rapid detection and differentiation of canine distemper virus infected and vaccinated animals.

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Liu, Da-Fei; Liu, Chun-Guo; Tian, Jin; Jiang, Yi-Tong; Zhang, Xiao-Zhan; Chai, Hong-Liang; Yang, Tian-Kuo; Yin, Xiu-Chen; Zhang, Hong-Ying; Liu, Ming; Hua, Yu-Ping; Qu, Lian-Dong

2015-06-01

Although widespread vaccination against canine distemper virus (CDV) has been conducted for many decades, several canine distemper outbreaks in vaccinated animals have been reported frequently. In order to detect and differentiate the wild-type and vaccine strains of the CDV from the vaccinated animals, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed. A set of four primers-two internal and two external-were designed to target the H gene for the specific detection of wild-type CDV variants. The CDV-H RT-LAMP assay rapidly amplified the target gene, within 60 min, using a water bath held at a constant temperature of 65°C. The assay was 100-fold more sensitive than conventional RT-PCR, with a detection limit of 10(-1)TCID50ml(-1). The system showed a preference for wild-type CDV, and exhibited less sensitivity to canine parvovirus, canine adenovirus type 1 and type 2, canine coronavirus, and canine parainfluenza virus. The assay was validated using 102 clinical samples obtained from vaccinated dog farms, and the results were comparable to a multiplex nested RT-PCR assay. The specific CDV-H RT-LAMP assay provides a simple, rapid, and sensitive tool for the detection of canines infected with wild-type CDV from canines vaccinated with attenuated vaccine. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of one-step Loop-Mediated Isothermal Amplification (LAMP) for the detection of norovirus in oysters

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The aim of this study was to develop a simple and rapid technique for detecting human norovirus (NoV). The loop-mediated isothermal amplification (LAMP) technique was evaluated and found to be sensitive, highly specific, and useful for routine oyster testing. Reverse transcription-LAMP (RT-LAMP) pri...

Detection of Cucurbit chlorotic yellows virus from Bemisia tabaci captured on sticky traps using reverse transcription loop-mediated isothermal amplification (RT-LAMP) and simple template preparation.

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Okuda, Mitsuru; Okuda, Shiori; Iwai, Hisashi

2015-09-01

Cucurbit chlorotic yellows virus (CCYV) of the genus Crinivirus within the family Closteroviridae is an emerging infectious agent of cucurbits leading to severe disease and significant economic losses. Effective detection and identification methods for this virus are urgently required. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect CCYV from its vector Bemisia tabaci. LAMP primer sets to detect CCYV were evaluated for their sensitivity and specificity, and a primer set designed from the HSP70h gene with corresponding loop primers were selected. The RT-LAMP assay was applied to detect CCYV from viruliferous B. tabaci trapped on sticky traps. A simple extraction procedure using RNAsecure™ was developed for template preparation. CCYV was detected in all of the B. tabaci 0, 1, 7 and 14 days after they were trapped. Although the rise of turbidity was delayed in reactions using RNA from B. tabaci trapped for 7 and 14 days compared with those from 0 and 1 day, the DNA amplification was sufficient to detect CCYV in all of the samples. These findings therefore present a simple template preparation method and an effective RT-LAMP assay, which can be easily and rapidly performed to monitor CCYV-viruliferous B. tabaci in the field. Copyright © 2015 Elsevier B.V. All rights reserved.

Variation in Bluetongue virus real-time reverse transcription polymerase chain reaction assay results in blood samples of sheep, cattle, and alpaca.

Science.gov (United States)

Brito, Barbara P; Gardner, Ian A; Hietala, Sharon K; Crossley, Beate M

2011-07-01

Bluetongue is a vector-borne viral disease that affects domestic and wild ruminants. The epidemiology of this disease has recently changed, with occurrence in new geographic areas. Various real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) assays are used to detect Bluetongue virus (BTV); however, the impact of biologic differences between New World camelids and domestic ruminant samples on PCR efficiency, for which the BTV real-time qRT-PCR was initially validated are unknown. New world camelids are known to have important biologic differences in whole blood composition, including hemoglobin concentration, which can alter PCR performance. In the present study, sheep, cattle, and alpaca blood were spiked with BTV serotypes 10, 11, 13, and 17 and analyzed in 10-fold dilutions by real-time qRT-PCR to determine if species affected nucleic acid recovery and assay performance. A separate experiment was performed using spiked alpaca blood subsequently diluted in 10-fold series in sheep blood to assess the influence of alpaca blood on performance efficiency of the BTV real-time qRT-PCR assay. Results showed that BTV-specific nucleic acid detection from alpaca blood was consistently 1-2 logs lower than from sheep and cattle blood, and results were similar for each of the 4 BTV serotypes analyzed.

Biochip for Real-Time Monitoring of Hepatitis B Virus (HBV) by Combined Loop-Mediated Isothermal Amplification and Solution-Phase Electrochemical Detection

Science.gov (United States)

Tien, Bui Quang; Ngoc, Nguyen Thy; Loc, Nguyen Thai; Thu, Vu Thi; Lam, Tran Dai

2017-06-01

Accurate in situ diagnostic tests play a key role in patient management and control of most infectious diseases. To achieve this, use of handheld biochips that implement sample handling, sample analysis, and result readout together is an ideal approach. We present herein a fluid-handling biochip for real-time electrochemical monitoring of nucleic acid amplification based on loop-mediated isothermal amplification and real-time electrochemical detection on a microfluidic platform. Intercalation between amplifying DNA and free redox probe in solution phase was used to monitor the number of DNA copies. The whole diagnostic process is completed within 70 min. Our platform offers a fast and easy tool for quantification of viral pathogens in shorter time and with limited risk of all potential forms of cross-contamination. Such diagnostic tools have potential to make a huge difference to the lives of millions of people worldwide.

Loop-mediated Isothermal Amplification Assay to Rapidly Detect Wheat Streak Mosaic Virus in Quarantined Plants

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Siwon Lee

2015-12-01

Full Text Available We developed a loop-mediated isothermal amplification (LAMP method to rapidly diagnose Wheat streak mosaic virus (WSMV during quarantine inspections of imported wheat, corn, oats, and millet. The LAMP method was developed as a plant quarantine inspection method for the first time, and its simplicity, quickness, specificity and sensitivity were verified compared to current reverse transcription-polymerase chain reaction (RT-PCR and nested PCR quarantine methods. We were able to quickly screen for WSMV at quarantine sites with many test samples; thus, this method is expected to contribute to plant quarantine inspections.

Preliminary validation of direct detection of foot-and-mouth disease virus within clinical samples using reverse transcription loop-mediated isothermal amplification coupled with a simple lateral flow device for detection.

Directory of Open Access Journals (Sweden)

Ryan A Waters

Full Text Available Rapid, field-based diagnostic assays are desirable tools for the control of foot-and-mouth disease (FMD. Current approaches involve either; 1 Detection of FMD virus (FMDV with immuochromatographic antigen lateral flow devices (LFD, which have relatively low analytical sensitivity, or 2 portable RT-qPCR that has high analytical sensitivity but is expensive. Loop-mediated isothermal amplification (LAMP may provide a platform upon which to develop field based assays without these drawbacks. The objective of this study was to modify an FMDV-specific reverse transcription-LAMP (RT-LAMP assay to enable detection of dual-labelled LAMP products with an LFD, and to evaluate simple sample processing protocols without nucleic acid extraction. The limit of detection of this assay was demonstrated to be equivalent to that of a laboratory based real-time RT-qPCR assay and to have a 10,000 fold higher analytical sensitivity than the FMDV-specific antigen LFD currently used in the field. Importantly, this study demonstrated that FMDV RNA could be detected from epithelial suspensions without the need for prior RNA extraction, utilising a rudimentary heat source for amplification. Once optimised, this RT-LAMP-LFD protocol was able to detect multiple serotypes from field epithelial samples, in addition to detecting FMDV in the air surrounding infected cattle, pigs and sheep, including pre-clinical detection. This study describes the development and evaluation of an assay format, which may be used as a future basis for rapid and low cost detection of FMDV. In addition it provides providing "proof of concept" for the future use of LAMP assays to tackle other challenging diagnostic scenarios encompassing veterinary and human health.

Real-Time ECG Simulation for Hybrid Mock Circulatory Loops.

Science.gov (United States)

Korn, Leonie; Rüschen, Daniel; Zander, Niklas; Leonhardt, Steffen; Walter, Marian

2018-02-01

Classically, mock circulatory loops only simulate mechanical properties of the circulation. To connect the hydraulic world with electrophysiology, we present a real-time electrical activity model of the heart and show how to integrate this model into a real-time mock loop simulation. The model incorporates a predefined conduction pathway and a simplified volume conductor to solve the bidomain equations and the forward problem of electrocardiography, resulting in a physiological simulation of the electrocardiogram (ECG) at arbitrary electrode positions. A complete physiological simulation of the heart's excitation would be too CPU intensive. Thus, in our model, complexity was reduced to allow real-time simulation of ECG-triggered medical systems in vitro; this decreases time and cost in the development process. Conversely, the presented model can still be adapted to various pathologies by locally changing the properties of the heart's conduction pathway. To simulate the ECG, the heart is divided into suitable areas, which are innervated by the hierarchically structured conduction system. To distinguish different cardiac regions, a segmentation of the heart was performed. In these regions, Prim's algorithm was applied to identify the directed minimal spanning trees for conduction orientation. Each node of the tree was assigned to a cardiac action potential generated by its hybrid automaton to represent the heart's conduction system by the spatial distribution of action potentials. To generate the ECG output, the bidomain equations were implemented and a simple model of the volume conductor of the body was used to solve the forward problem of electrocardiography. As a result, the model simulates potentials at arbitrary electrode positions in real-time. To verify the developed real-time ECG model, measurements were made within a hybrid mock circulatory loop, including a simple ECG-triggered ventricular assist device control. The model's potential value is to simulate

Development of a Reverse Transcription Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Subtype H7N9 Avian Influenza Virus

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Hongmei Bao

2014-01-01

Full Text Available A novel influenza A (H7N9 virus has emerged in China. To rapidly detect this virus from clinical samples, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP method for the detection of the H7N9 virus. The minimum detection limit of the RT-LAMP assay was 0.01 PFU H7N9 virus, making this method 100-fold more sensitive to the detection of the H7N9 virus than conventional RT-PCR. The H7N9 virus RT-LAMP assays can efficiently detect different sources of H7N9 influenza virus RNA (from chickens, pigeons, the environment, and humans. No cross-reactive amplification with the RNA of other subtype influenza viruses or of other avian respiratory viruses was observed. The assays can effectively detect H7N9 influenza virus RNA in drinking water, soil, cloacal swab, and tracheal swab samples that were collected from live poultry markets, as well as human H7N9 virus, in less than 30 min. These results suggest that the H7N9 virus RT-LAMP assays were efficient, practical, and rapid diagnostic methods for the epidemiological surveillance and diagnosis of influenza A (H7N9 virus from different resource samples.

Laboratory evaluation of a quantitative real-time reverse transcription PCR assay for the detection and identification of the four subgroups of avian metapneumovirus.

Science.gov (United States)

Guionie, O; Toquin, D; Sellal, E; Bouley, S; Zwingelstein, F; Allée, C; Bougeard, S; Lemière, S; Eterradossi, N

2007-02-01

Avian metapneumovirus (AMPV) is an important pathogen causing respiratory diseases and egg drops in several avian species. Four AMPV subgroups have been identified. The laboratory diagnosis of AMPV infections relies on serological methods, on labour-intensive virus isolation procedures, and on recently developed subgroup specific reverse transcription PCR (RT-PCR) protocols. In the present study, both the specificity and sensitivity of a commercial real-time reverse transcription PCR (RRT-PCR) for the detection and identification of the four AMPV subgroups were evaluated. Fifteen non-AMPV avian viruses belonging to 7 genera and 32 AMPV belonging to the 4 subgroups were tested. No non-AMPV virus was detected, whereas all AMPV viruses were identified in agreement with their previous molecular and antigenic subgroup assignment. The sensitivity and quantitating ability of the RRT-PCR assay were determined using serial dilutions of RNA derived either from AMPV virus stocks or from runoff transcripts. In all cases, linear dose/responses were observed. The detection limits of the different subgroups ranged from 500 to 5000 RNA copies and from 0.03 to 3.16TCID50/ml. The results were reproducible under laboratory conditions, thus showing that quantitative RRT-PCR is a new and powerful tool for the rapid and sensitive detection, identification and quantitation of AMPVs.

Loop mediated isothermal amplification assay using hydroxy naphthol blue, conventional polymerase chain reaction and real-time PCR in the diagnosis of intraocular tuberculosis

Directory of Open Access Journals (Sweden)

P K Balne

2015-01-01

Full Text Available This study is a comparative evaluation (Chi-square test of a closed tube loop mediated isothermal amplification assay using hydroxy naphthol blue dye (HNB-LAMP, real-time polymerase chain reaction (PCR and conventional PCR in the diagnosis of intraocular tuberculosis. Considering clinical presentation as the gold standard in 33 patients, the sensitivity of HNB-LAMP assay (75.8% was higher (not significant, P value 0.2 than conventional PCR (57.6% and lower than real-time PCR (90.9%. Specificity was 100% by all three methods. No amplification was observed in negative controls (n = 20 by all three methods. The cost of the HNB-LAMP assay was Rs. 500.00 and it does not require thermocycler, therefore, it can be used as an alternative to conventional PCR in resource-poor settings.

Loop-R : Real-Time Video Interface

OpenAIRE

Pereira, Rui

2007-01-01

Loop-R is a real-time video performance tool, based in the exploration of low-tech, used technology and human engineering research. With this tool its , , author is giving a shout to industry, using existing and mistreated technology in innovative ways, combining concepts and interfaces: blending segregated interfaces (GUI and Physical) into one. After graspable interfaces and the "end" of WIMP interfaces, hardware and software blend themselves in a new genre providing free control of video-l...

LDB1-mediated enhancer looping can be established independent of mediator and cohesin.

Science.gov (United States)

Krivega, Ivan; Dean, Ann

2017-08-21

Mechanistic studies in erythroid cells indicate that LDB1, as part of a GATA1/TAL1/LMO2 complex, brings erythroid-expressed genes into proximity with enhancers for transcription activation. The role of co-activators in establishing this long-range interaction is poorly understood. Here we tested the contributions of the RNA Pol II pre-initiation complex (PIC), mediator and cohesin to establishment of locus control region (LCR)/β-globin proximity. CRISPR/Cas9 editing of the β-globin promoter to eliminate the RNA Pol II PIC by deleting the TATA-box resulted in loss of transcription, but enhancer-promoter interaction was unaffected. Additional deletion of the promoter GATA1 site eliminated LDB1 complex and mediator occupancy and resulted in loss of LCR/β-globin proximity. To separate the roles of LDB1 and mediator in LCR looping, we expressed a looping-competent but transcription-activation deficient form of LDB1 in LDB1 knock down cells: LCR/β-globin proximity was restored without mediator core occupancy. Further, Cas9-directed tethering of mutant LDB1 to the β-globin promoter forced LCR loop formation in the absence of mediator or cohesin occupancy. Moreover, ENCODE data and our chromatin immunoprecipitation results indicate that cohesin is almost completely absent from validated and predicted LDB1-regulated erythroid enhancer-gene pairs. Thus, lineage specific factors largely mediate enhancer-promoter looping in erythroid cells independent of mediator and cohesin. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

Reference genes for gene expression analysis by real-time reverse transcription polymerase chain reaction of renal cell carcinoma.

Science.gov (United States)

Bjerregaard, Henriette; Pedersen, Shona; Kristensen, Søren Risom; Marcussen, Niels

2011-12-01

Differentiation between malignant renal cell carcinoma and benign oncocytoma is of great importance to choose the optimal treatment. Accurate preoperative diagnosis of renal tumor is therefore crucial; however, existing imaging techniques and histologic examinations are incapable of providing an optimal differentiation profile. Analysis of gene expression of molecular markers is a new possibility but relies on appropriate standardization to compare different samples. The aim of this study was to identify stably expressed reference genes suitable for the normalization of results extracted from gene expression analysis of renal tumors. Expression levels of 8 potential reference genes (ATP5J, HMBS, HPRT1, PPIA, TBP, 18S, GAPDH, and POLR2A) were examined by real-time reverse transcription polymerase chain reaction in tumor and normal tissue from removed kidneys from 13 patients with renal cell carcinoma and 5 patients with oncocytoma. The expression levels of genes were compared by gene stability value M, average gene stability M, pairwise variation V, and coefficient of variation CV. More candidates were not suitable for the purpose, but a combination of HMBS, PPIA, ATP5J, and TBP was found to be the best combination with an average gene stability value M of 0.9 and a CV of 0.4 in the 18 tumors and normal tissues. A combination of 4 genes, HMBS, PPIA, ATP5J, and TBP, is a possible reference in renal tumor gene expression analysis by reverse transcription polymerase chain reaction. A combination of four genes, HMBS, PPIA, ATP5J and TBP, being stably expressed in tissues from RCC is possible reference genes for gene expression analysis.

Development of field-based real-time reverse transcription-polymerase chain reaction assays for detection of Chikungunya and O'nyong-nyong viruses in mosquitoes.

Science.gov (United States)

Smith, Darci R; Lee, John S; Jahrling, Jordan; Kulesh, David A; Turell, Michael J; Groebner, Jennifer L; O'Guinn, Monica L

2009-10-01

Chikungunya (CHIK) and O'nyong-nyong (ONN) are important emerging arthropod-borne diseases. Molecular diagnosis of these two viruses in mosquitoes has not been evaluated, and the effects of extraneous mosquito tissue on assay performance have not been tested. Additionally, no real-time reverse transcription-polymerase chain reaction (RT-PCR) assay exists for detecting ONN virus (ONNV) RNA. We describe the development of sensitive and specific real-time RT-PCR assays for detecting CHIK and ONN viral RNA in mosquitoes, which have application for field use. In addition, we compared three methods for primer/probe design for assay development by evaluating their sensitivity and specificity. This comparison resulted in development of virus-specific assays that could detect less than one plaque-forming unit equivalent of each of the viruses in mosquitoes. The use of these assays will aid in arthropod-borne disease surveillance and in the control of the associated diseases.

Classical swine fever virus detection: results of a real-time reverse transcription polymerase chain reaction ring trial conducted in the framework of the European network of excellence for epizootic disease diagnosis and control

DEFF Research Database (Denmark)

Hoffmann, Bernd; Blome, Sandra; Bonilauri, Paolo

2011-01-01

and specificity values. Nevertheless, some in-house systems had unspecific reactions or suboptimal sensitivity with only a single CSFV genotype. Follow-up actions involved either improvement of suboptimal assays or replacement of specific laboratory assays with the FLI protocol, with or without modifications......The current study reports on a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) ring trial for the detection of Classical swine fever virus (CSFV) genomic RNA undertaken by 10 European laboratories. All laboratories were asked to use their routine in-house real-time RT...

Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results.

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Andreas Garcia-Bardon

Full Text Available Real-time reverse transcription polymerase chain reaction (PCR is the gold standard for expression analysis. Designed to improve reproducibility and sensitivity, commercial kits are commonly used for the critical step of cDNA synthesis. The present study was designed to determine the impact of these kits. mRNA from mouse brains were pooled to create serial dilutions ranging from 0.0625 μg to 2 μg, which were transcribed into cDNA using four different commercial reverse-transcription kits. Next, we transcribed mRNA from brain tissue after acute brain injury and naïve mice into cDNA for qPCR. Depending on tested genes, some kits failed to show linear results in dilution series and revealed strong variations in cDNA yield. Absolute expression data in naïve and trauma settings varied substantially between these kits. Normalization with a housekeeping gene failed to reduce kit-dependent variations, whereas normalization eliminated differences when naïve samples from the same region were used. The study shows strong evidence that choice of commercial cDNA synthesis kit has a major impact on PCR results and, consequently, on comparability between studies. Additionally, it provides a solution to overcome this limitation by normalization with data from naïve samples. This simple step helps to compare mRNA expression data between different studies and groups.

Reverse transcriptase real-time PCR for detection and quantification of viable Campylobacter jejuni directly from poultry faecal samples

DEFF Research Database (Denmark)

Bui, Thanh Xuan; Wolff, Anders; Madsen, Mogens

2012-01-01

Campylobacter spp. is the most common cause of bacterial diarrhoea in humans worldwide. Therefore, rapid and reliable methods fordetection and quantification of this pathogen are required. In this study, we have developed a reverse transcription quantitative real-time PCR(RT-qPCR) for detection a...

Real time hardware-in-loop simulation of ESMO satellite attitude control system

Directory of Open Access Journals (Sweden)

Rune Finnset

2006-04-01

Full Text Available This paper studies attitude control of the ESMO satellite using six reaction thrusters. Bang-bang control with dead-zone and Pulse-Width Modulation (PWM for the modulation of the on-time of the thrusters are treated. Closed loop hardware-in-loop simulations, using themicrocontroller unit (MCU Microchip PIC18F452 for implementation of attitude control and MatLab in a standard PC for simulating satellite dynamics, are carried out. Results for real time simulation are compared with autonomous simulations. The controller gives a satisfactory performance in the real time environment.

Evaluation of a duplex reverse-transcription real-time PCR assay for the detection of encephalomyocarditis virus.

Science.gov (United States)

Qin, Shaomin; Underwood, Darren; Driver, Luke; Kistler, Carol; Diallo, Ibrahim; Kirkland, Peter D

2018-06-01

We evaluated a fluorogenic probe-based assay for the detection of encephalomyocarditis virus (EMCV) by comparing a set of published primers and probe to a new set of primers and probe. The published reagents failed to amplify a range of Australian isolates and an Italian reference strain of EMCV. In contrast, an assay based on 2 new sets of primers and probes that were run in a duplex reverse-transcription real-time PCR (RT-rtPCR) worked well, with high amplification efficiency. The analytical sensitivity was ~100-fold higher than virus isolation in cell culture. The intra-assay variation was 0.21-4.90%. No cross-reactivity was observed with a range of other porcine viruses. One hundred and twenty-two clinical specimens were tested simultaneously by RT-rtPCR and virus isolation in cell culture; 72 specimens gave positive results by RT-rtPCR, and 63 of these were also positive by virus isolation. Of 245 archived cell culture isolates of EMCV that were tested in the RT-rtPCR, 242 samples were positive. The new duplex RT-rtPCR assay is a reliable tool for the detection of EMCV in clinical specimens and for use in epidemiologic investigations.

Visual detection of human enterovirus 71 subgenotype C4 and Coxsackievirus A16 by reverse transcription loop-mediated isothermal amplification with the hydroxynaphthol blue dye.

Science.gov (United States)

Nie, Kai; Zhang, Yong; Luo, Le; Yang, Meng-Jie; Hu, Xiu-Mei; Wang, Miao; Zhu, Shuang-Li; Han, Feng; Xu, Wen-Bo; Ma, Xue-Jun

2011-08-01

A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of human enterovirus 71 subgenotype C4 (EV71-C4) and Coxsackievirus A16 (CVA16) infection, respectively. The reaction was performed in one step in a single tube at 65°C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limits of the RT-LAMP assay were 0.33 and 1.58 of a 50% tissue culture infective dose (TCID(50)) per reaction based on 10-fold dilutions of a titrated EV71 or CVA16 strain, respectively. No cross-reaction was observed with Coxsackievirus A (CVA) viruses (CVA2, 4, 5, 7, 9, 10, 14, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3, 6, 11, and 19). The assay was further evaluated with 47 clinical stool specimens diagnosed previously with EV71, CVA16 or other human enterovirus infections. Virus isolates from stool samples were confirmed by virus neutralization testing and sequencing. RT-LAMP with HNB dye was demonstrated to be a sensitive and cost-effective assay for rapid visual detection of human EV71-C4 and CVA16. Copyright © 2011 Elsevier B.V. All rights reserved.

Power in the loop real time simulation platform for renewable energy generation

Science.gov (United States)

Li, Yang; Shi, Wenhui; Zhang, Xing; He, Guoqing

2018-02-01

Nowadays, a large scale of renewable energy sources has been connecting to power system and the real time simulation platform is widely used to carry out research on integration control algorithm, power system stability etc. Compared to traditional pure digital simulation and hardware in the loop simulation, power in the loop simulation has higher accuracy and degree of reliability. In this paper, a power in the loop analog digital hybrid simulation platform has been built and it can be used not only for the single generation unit connecting to grid, but also for multiple new energy generation units connecting to grid. A wind generator inertia control experiment was carried out on the platform. The structure of the inertia control platform was researched and the results verify that the platform is up to need for renewable power in the loop real time simulation.

Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in Guinea

Science.gov (United States)

Kurosaki, Yohei; Magassouba, N’Faly; Oloniniyi, Olamide K.; Cherif, Mahamoud S.; Sakabe, Saori; Takada, Ayato; Hirayama, Kenji; Yasuda, Jiro

2016-01-01

Given the current absence of specific drugs or vaccines for Ebola virus disease (EVD), rapid, sensitive, and reliable diagnostic methods are required to stem the transmission chain of the disease. We have developed a rapid detection assay for Zaire ebolavirus based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and coupled with a novel portable isothermal amplification and detection platform. The RT-LAMP assay is based on primer sets that target the untranscribed trailer region or nucleoprotein coding region of the viral RNA. The test could specifically detect viral RNAs of Central and West African Ebola virus strains within 15 minutes with no cross-reactivity to other hemorrhagic fever viruses and arboviruses, which cause febrile disease. The assay was evaluated using a total of 100 clinical specimens (serum, n = 44; oral swab, n = 56) collected from suspected EVD cases in Guinea. The specificity of this diagnostic test was 100% for both primer sets, while the sensitivity was 100% and 97.9% for the trailer and nucleoprotein primer sets, respectively, compared with a reference standard RT-PCR test. These observations suggest that our diagnostic assay is useful for identifying EVD cases, especially in the field or in settings with insufficient infrastructure. PMID:26900929

Matching-range-constrained real-time loop closure detection with CNNs features.

Science.gov (United States)

Bai, Dongdong; Wang, Chaoqun; Zhang, Bo; Yi, Xiaodong; Tang, Yuhua

2016-01-01

The loop closure detection (LCD) is an essential part of visual simultaneous localization and mapping systems (SLAM). LCD is capable of identifying and compensating the accumulation drift of localization algorithms to produce an consistent map if the loops are checked correctly. Deep convolutional neural networks (CNNs) have outperformed state-of-the-art solutions that use traditional hand-crafted features in many computer vision and pattern recognition applications. After the great success of CNNs, there has been much interest in applying CNNs features to robotic fields such as visual LCD. Some researchers focus on using a pre-trained CNNs model as a method of generating an image representation appropriate for visual loop closure detection in SLAM. However, there are many fundamental differences and challenges involved in character between simple computer vision applications and robotic applications. Firstly, the adjacent images in the dataset of loop closure detection might have more resemblance than the images that form the loop closure. Secondly, real-time performance is one of the most critical demands for robots. In this paper, we focus on making use of the feature generated by CNNs layers to implement LCD in real environment. In order to address the above challenges, we explicitly provide a value to limit the matching range of images to solve the first problem; meanwhile we get better results than state-of-the-art methods and improve the real-time performance using an efficient feature compression method.

Detection of feline Coronavirus in effusions of cats with and without feline infectious peritonitis using loop-mediated isothermal amplification.

Science.gov (United States)

Günther, Sonja; Felten, Sandra; Wess, Gerhard; Hartmann, Katrin; Weber, Karin

2018-06-01

Feline infectious peritonitis (FIP) is a fatal disease in cats worldwide. The aim of this study was to test two commercially available reaction mixtures in a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect feline Coronavirus (FCoV) in body cavity effusions of cats with and without FIP, in order to minimize the time from sampling to obtaining results. RNA was extracted from body cavity effusion samples of 71 cats, including 34 samples from cats with a definitive diagnosis of FIP, and 37 samples of control cats with similar clinical signs but other confirmed diseases. Two reaction mixtures (Isothermal Mastermix, OptiGene Ltd.and PCRun™ Molecular Detection Mix, Biogal) were tested using the same primers, which were designed to bind to a conserved region of the FCoV membrane protein gene. Both assays were conducted under isothermal conditions (61 °C-62 °C). Using the Isothermal Mastermix of OptiGene Ltd., amplification times ranged from 4 and 39 min with a sensitivity of 35.3% and a specificity of 94.6% for the reported sample group. Using the PCRun™ Molecular Detection Mix of Biogal, amplification times ranged from 18 to 77 min with a sensitivity of 58.8% and a specificity of 97.3%. Although the RT-LAMP assay is less sensitive than real time reverse transcription PCR (RT-PCR), it can be performed without the need of expensive equipment and with less hands-on time. Further modifications of primers might lead to a suitable in-house test and accelerate the diagnosis of FIP. Copyright © 2018 Elsevier B.V. All rights reserved.

Real-time Control Mediation in Agile Distributed Software Development

DEFF Research Database (Denmark)

Persson, John Stouby; Aaen, Ivan; Mathiassen, Lars

2008-01-01

Agile distributed environments pose particular challenges related to control of quality and collaboration in software development. Moreover, while face-to-face interaction is fundamental in agile development, distributed environments must rely extensively on mediated interactions. On this backdrop...... control was mediated over distance by technology through real-time exchanges. Contrary to previous research, the analysis suggests that both formal and informal elements of real-time mediated control were used; that evolving goals and adjustment of expectations were two of the main issues in real......-time mediated control exchanges; and, that the actors, despite distances in space and culture, developed a clan-like pattern mediated by technology to help control quality and collaboration in software development....

Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples

NARCIS (Netherlands)

Adams, Emily R.; Schoone, Gerard J.; Ageed, Al Farazdag; El Safi, Sayda; Schallig, Henk D. F. H.

2010-01-01

Here we describe a generic, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) assay, for the identification of Leishmania species from clinical samples. LAMP is an isothermal reaction recently developed as a point-of-care diagnostic tool. Primers were designed in the conserved

Concentration and length dependence of DNA looping in transcriptional regulation.

Directory of Open Access Journals (Sweden)

Lin Han

2009-05-01

Full Text Available In many cases, transcriptional regulation involves the binding of transcription factors at sites on the DNA that are not immediately adjacent to the promoter of interest. This action at a distance is often mediated by the formation of DNA loops: Binding at two or more sites on the DNA results in the formation of a loop, which can bring the transcription factor into the immediate neighborhood of the relevant promoter. These processes are important in settings ranging from the historic bacterial examples (bacterial metabolism and the lytic-lysogeny decision in bacteriophage, to the modern concept of gene regulation to regulatory processes central to pattern formation during development of multicellular organisms. Though there have been a variety of insights into the combinatorial aspects of transcriptional control, the mechanism of DNA looping as an agent of combinatorial control in both prokaryotes and eukaryotes remains unclear. We use single-molecule techniques to dissect DNA looping in the lac operon. In particular, we measure the propensity for DNA looping by the Lac repressor as a function of the concentration of repressor protein and as a function of the distance between repressor binding sites. As with earlier single-molecule studies, we find (at least two distinct looped states and demonstrate that the presence of these two states depends both upon the concentration of repressor protein and the distance between the two repressor binding sites. We find that loops form even at interoperator spacings considerably shorter than the DNA persistence length, without the intervention of any other proteins to prebend the DNA. The concentration measurements also permit us to use a simple statistical mechanical model of DNA loop formation to determine the free energy of DNA looping, or equivalently, the for looping.

Rapid detection of Piper yellow mottle virus and Cucumber mosaic virus infecting black pepper (Piper nigrum) by loop-mediated isothermal amplification (LAMP).

Science.gov (United States)

Bhat, A I; Siljo, A; Deeshma, K P

2013-10-01

The loop-mediated isothermal amplification (LAMP) assay for Piper yellow mottle virus and the reverse transcription (RT) LAMP assay for Cucumber mosaic virus each consisted of a set of five primers designed against the conserved sequences in the viral genome. Both RNA and DNA isolated from black pepper were used as a template for the assay. The results were assessed visually by checking turbidity, green fluorescence and pellet formation in the reaction tube and also by gel electrophoresis. The assay successfully detected both viruses in infected plants whereas no cross-reactions were recorded with healthy plants. Optimum conditions for successful amplification were determined in terms of the concentrations of magnesium sulphate and betaine, temperature, and duration. The detection limit for both LAMP and RT-LAMP was up to 100 times that for conventional PCR and up to one-hundredth of that for real-time PCR. The optimal conditions arrived at were validated by testing field samples of infected vines of three species from different regions. Copyright © 2013 Elsevier B.V. All rights reserved.

Comparison of DNA Microarray, Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of Fusarium spp. Obtained from Patients with Hematologic Malignancies.

Science.gov (United States)

de Souza, Marcela; Matsuzawa, Tetsuhiro; Sakai, Kanae; Muraosa, Yasunori; Lyra, Luzia; Busso-Lopes, Ariane Fidelis; Levin, Anna Sara Shafferman; Schreiber, Angélica Zaninelli; Mikami, Yuzuru; Gonoi, Tohoru; Kamei, Katsuhiko; Moretti, Maria Luiza; Trabasso, Plínio

2017-08-01

The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.

Real-time display of flow-pressure-volume loops.

Science.gov (United States)

Morozoff, P E; Evans, R W

1992-01-01

Graphic display of respiratory waveforms can be valuable for monitoring the progress of ventilated patients. A system has been developed that can display flow-pressure-volume loops as derived from a patient's respiratory circuit in real time. It can also display, store, print, and retrieve ventilatory waveforms. Five loops can be displayed at once: current, previous, reference, "ideal," and previously saved. Two components, the data-display device (DDD) and the data-collection device (DCD), comprise the system. An IBM 286/386 computer with a graphics card (VGA) and bidirectional parallel port is used for the DDD; an eight-bit microprocessor card and an A/D convertor card make up the DCD. A real-time multitasking operating system was written to control the DDD, while the DCD operates from in-line assembly code. The DCD samples the pressure and flow sensors at 100 Hz and looks for a complete flow waveform pattern based on flow slope. These waveforms are then passed to the DDD via the mutual parallel port. Within the DDD a process integrates the flow to create a volume signal and performs a multilinear regression on the pressure, flow, and volume data to calculate the elastance, resistance, pressure offset, and coefficient of determination. Elastance, resistance, and offset are used to calculate Pr and Pc where: Pr[k] = P[k]-offset-(elastance.V[k]) and Pc[k] = P[k]-offset-(resistance.F[k]). Volume vs. Pc and flow vs. Pr can be displayed in real time. Patient data from previous clinical tests were loaded into the device to verify the software calculations. An analog waveform generator was used to simulate flow and pressure waveforms that validated the system.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil

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Marilia Farignoli Romeiro

2016-06-01

Full Text Available Abstract: INTRODUCTION: The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR method for the diagnosis of the Flavivirus genus. METHODS: We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. RESULTS: The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410 of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×107to 3.4×103copies per ml. CONCLUSIONS: The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies.

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR "Double Check" Strategy

DEFF Research Database (Denmark)

Hoffmann, B.; Freuling, C. M.; Wakeley, P. R.

2010-01-01

To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus...... sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany......), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome...

Development of loop-mediated isothermal amplification method for ...

African Journals Online (AJOL)

A novel assay method to detect the highly virulent Porcine reproductive and respiratory syndrome virus (PRRSV) termed reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), was reported by using hydroxynaphthol blue (HNB) as the LAMP product colorimetric judgment. By the set of special primers, ...

Real-time reverse transcription-polymerase chain reaction assays for identification of wild poliovirus 1 & 3.

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Sharma, Deepa K; Nalavade, Uma P; Deshpande, Jagadish M

2015-10-01

The poliovirus serotype identification and intratypic differentiation by real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay is suitable for serotype mixtures but not for intratypic mixtures of wild and vaccine poliovirus strains. This study was undertaken to develop wild poliovirus 1 and 3 (WPV1 and WPV3) specific rRT-PCR assays for use. Specific primers and probes for rRT-PCR were designed based on VP1 sequences of WPV1 and WPV3 isolated in India since 2000. The specificity of the rRT-PCR assays was evaluated using WPV1 and WPV3 of different genetic lineages, non-polio enteroviruses (NPEVs) and mixtures of wild/wild and wild/Sabin vaccine strains. The sensitivity of the assays was determined by testing serial 10-fold dilutions of wild poliovirus 1 and 3 stock suspensions of known titre. No cross-reactivity with Sabin strains, intertypic wild poliovirus isolates or 27 types of NPEVs across all the four Enterovirus species was found for both the wild poliovirus 1 and 3 rRT-PCR assays. All WPV1 and WPV3 strains isolated since 2000 were successfully amplified. The rRT-PCR assays detected 10 4.40 CCID 50 /ml of WPV1 and 10 4.00 CCID 50 /ml of WPV3, respectively either as single isolate or mixture with Sabin vaccine strains or intertypic wild poliovirus. rRT-PCR assays for WPV1 and WPV3 have been validated to detect all the genetic variations of the WPV1 and WPV3 isolated in India for the last decade. When used in combination with the current rRT-PCR assay testing was complete for confirmation of the presence of wild poliovirus in intratypic mixtures.

Mediator can regulate mitotic entry and direct periodic transcription in fission yeast.

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Banyai, Gabor; Lopez, Marcela Davila; Szilagyi, Zsolt; Gustafsson, Claes M

2014-11-01

Cdk8 is required for correct timing of mitotic progression in fission yeast. How the activity of Cdk8 is regulated is unclear, since the kinase is not activated by T-loop phosphorylation and its partner, CycC, does not oscillate. Cdk8 is, however, a component of the multiprotein Mediator complex, a conserved coregulator of eukaryotic transcription that is connected to a number of intracellular signaling pathways. We demonstrate here that other Mediator components regulate the activity of Cdk8 in vivo and thereby direct the timing of mitotic entry. Deletion of Mediator components Med12 and Med13 leads to higher cellular Cdk8 protein levels, premature phosphorylation of the Cdk8 target Fkh2, and earlier entry into mitosis. We also demonstrate that Mediator is recruited to clusters of mitotic genes in a periodic fashion and that the complex is required for the transcription of these genes. We suggest that Mediator functions as a hub for coordinated regulation of mitotic progression and cell cycle-dependent transcription. The many signaling pathways and activator proteins shown to function via Mediator may influence the timing of these cell cycle events. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Comparison of reverse transcriptase PCR, reverse transcriptase loop-mediated isothermal amplification, and culture-based assays for Salmonella detection from pork processing environments.

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Techathuvanan, Chayapa; Draughon, Frances Ann; D'Souza, Doris Helen

2011-02-01

Novel rapid Salmonella detection assays without the need for sophisticated equipment or labor remain in high demand. Real-time reverse transcriptase PCR (RT-PCR) assays, though rapid and sensitive, require expensive thermocyclers, while a novel RT loop-mediated isothermal amplification (RT-LAMP) method requires only a simple water bath. Our objective was to compare the detection sensitivity of Salmonella Typhimurium from the pork processing environment by RT-LAMP, RT-PCR, and culture-based assays. Carcass and surface swabs and carcass rinses were obtained from a local processing plant. Autoclaved carcass rinses (500 ml) were spiked with Salmonella Typhimurium and filtered. Filters were placed in stomacher bags containing tetrathionate broth (TTB) and analyzed with or without 10-h enrichment at 37 °C. Natural swabs were stomached with buffered peptone water, and natural carcass rinses were filtered, preenriched, and further enriched in TTB. Serially-diluted enriched samples were enumerated by spread plating on xylose lysine Tergitol 4 agar. RNA was extracted from 5 ml of enriched TTB with TRIzol. RT-LAMP assay using previously described invA primers was conducted at 62 °C for 90 min in a water bath with visual detection and by gel electrophoresis. SYBR Green I-based-real-time RT-PCR was carried out with invA primers followed by melt temperature analysis. The results of RT-LAMP detection for spiked carcass rinses were comparable to those of RT-PCR and cultural plating, with detection limits of 1 log CFU/ml, although they were obtained significantly faster, within 24 h including preenrichment and enrichment. RT-LAMP showed 4 of 12 rinse samples positive, while RT-PCR showed 1 of 12 rinse samples positive. For swabs, 6 of 27 samples positive by RT-LAMP and 5 of 27 by RT-PCR were obtained. This 1-day RT-LAMP assay shows promise for routine Salmonella screening by the pork industry. Copyright ©, International Association for Food Protection

Noncoding transcription by alternative rna polymerases dynamically regulates an auxin-driven chromatin loop

KAUST Repository

Ariel, Federico D.; Jé gu, Teddy; Latrasse, David; Romero-Barrios, Natali; Christ, Auré lie; Benhamed, Moussa; Crespi, Martí n D.

2014-01-01

The eukaryotic epigenome is shaped by the genome topology in three-dimensional space. Dynamic reversible variations in this epigenome structure directly influence the transcriptional responses to developmental cues. Here, we show that the Arabidopsis long intergenic noncoding RNA (lincRNA) APOLO is transcribed by RNA polymerases II and V in response to auxin, a phytohormone controlling numerous facets of plant development. This dual APOLO transcription regulates the formation of a chromatin loop encompassing the promoter of its neighboring gene PID, a key regulator of polar auxin transport. Altering APOLO expression affects chromatin loop formation, whereas RNA-dependent DNA methylation, active DNA demethylation, and Polycomb complexes control loop dynamics. This dynamic chromatin topology determines PID expression patterns. Hence, the dual transcription of a lincRNA influences local chromatin topology and directs dynamic auxin-controlled developmental outputs on neighboring genes. This mechanism likely underscores the adaptive success of plants in diverse environments and may be widespread in eukaryotes. © 2014 Elsevier Inc.

Noncoding transcription by alternative rna polymerases dynamically regulates an auxin-driven chromatin loop

KAUST Repository

Ariel, Federico D.

2014-08-01

The eukaryotic epigenome is shaped by the genome topology in three-dimensional space. Dynamic reversible variations in this epigenome structure directly influence the transcriptional responses to developmental cues. Here, we show that the Arabidopsis long intergenic noncoding RNA (lincRNA) APOLO is transcribed by RNA polymerases II and V in response to auxin, a phytohormone controlling numerous facets of plant development. This dual APOLO transcription regulates the formation of a chromatin loop encompassing the promoter of its neighboring gene PID, a key regulator of polar auxin transport. Altering APOLO expression affects chromatin loop formation, whereas RNA-dependent DNA methylation, active DNA demethylation, and Polycomb complexes control loop dynamics. This dynamic chromatin topology determines PID expression patterns. Hence, the dual transcription of a lincRNA influences local chromatin topology and directs dynamic auxin-controlled developmental outputs on neighboring genes. This mechanism likely underscores the adaptive success of plants in diverse environments and may be widespread in eukaryotes. © 2014 Elsevier Inc.

Real-time reverse transcription polymerase chain reaction method for detection of Canine distemper virus modified live vaccine shedding for differentiation from infection with wild-type strains.

Science.gov (United States)

Wilkes, Rebecca P; Sanchez, Elena; Riley, Matthew C; Kennedy, Melissa A

2014-01-01

Canine distemper virus (CDV) remains a common cause of infectious disease in dogs, particularly in high-density housing situations such as shelters. Vaccination of all dogs against CDV is recommended at the time of admission to animal shelters and many use a modified live virus (MLV) vaccine. From a diagnostic standpoint for dogs with suspected CDV infection, this is problematic because highly sensitive diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) tests are able to detect MLV virus in clinical samples. Real-time PCR can be used to quantitate amount of virus shedding and can differentiate vaccine strains from wild-type strains when shedding is high. However, differentiation by quantitation is not possible in vaccinated animals during acute infection, when shedding is low and could be mistaken for low level vaccine virus shedding. While there are gel-based RT-PCR assays for differentiation of vaccine strains from field strains based on sequence differences, the sensitivity of these assays is unable to match that of the real-time RT-PCR assay currently used in the authors' laboratory. Therefore, a real-time RT-PCR assay was developed that detects CDV MLV vaccine strains and distinguishes them from wild-type strains based on nucleotide sequence differences, rather than the amount of viral RNA in the sample. The test is highly sensitive, with detection of as few as 5 virus genomic copies (corresponding to 10(-1) TCID(50)). Sequencing of the DNA real-time products also allows phylogenetic differentiation of the wild-type strains. This test will aid diagnosis during outbreaks of CDV in recently vaccinated animals.

Combining loop unrolling strategies and code predication to reduce the worst-case execution time of real-time software

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Andreu Carminati

2017-07-01

Full Text Available Worst-case execution time (WCET is a parameter necessary to guarantee timing constraints on real-time systems. The higher the worst-case execution time of tasks, the higher will be the resource demand for the associated system. The goal of this paper is to propose a different way to perform loop unrolling on data-dependent loops using code predication targeting WCET reduction, because existing techniques only consider loops with fixed execution counts. We also combine our technique with existing unrolling approaches. Results showed that this combination can produce aggressive WCET reductions when compared with the original code.

GMO detection using a bioluminescent real time reporter (BART of loop mediated isothermal amplification (LAMP suitable for field use

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Kiddle Guy

2012-04-01

Full Text Available Abstract Background There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART occurs at a constant temperature and only requires a simple light detection and integration device. Results Loop mediated isothermal amplification (LAMP shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART for determination of genetically modified (GM maize target DNA at low levels of contamination (0.1-5.0% GM using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation. Conclusions LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading

Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection.

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Laurent Dacheux

2016-07-01

Full Text Available The definitive diagnosis of lyssavirus infection (including rabies in animals and humans is based on laboratory confirmation. The reference techniques for post-mortem rabies diagnosis are still based on direct immunofluorescence and virus isolation, but molecular techniques, such as polymerase chain reaction (PCR based methods, are increasingly being used and now constitute the principal tools for diagnosing rabies in humans and for epidemiological analyses. However, it remains a key challenge to obtain relevant specificity and sensitivity with these techniques while ensuring that the genetic diversity of lyssaviruses does not compromise detection. We developed a dual combined real-time reverse transcription polymerase chain reaction (combo RT-qPCR method for pan-lyssavirus detection. This method is based on two complementary technologies: a probe-based (TaqMan RT-qPCR for detecting the RABV species (pan-RABV RT-qPCR and a second reaction using an intercalating dye (SYBR Green to detect other lyssavirus species (pan-lyssa RT-qPCR. The performance parameters of this combined assay were evaluated with a large panel of primary animal samples covering almost all the genetic variability encountered at the viral species level, and they extended to almost all lyssavirus species characterized to date. This method was also evaluated for the diagnosis of human rabies on 211 biological samples (positive n = 76 and negative n = 135 including saliva, skin and brain biopsies. It detected all 41 human cases of rabies tested and confirmed the sensitivity and the interest of skin biopsy (91.5% and saliva (54% samples for intra-vitam diagnosis of human rabies. Finally, this method was successfully implemented in two rabies reference laboratories in enzootic countries (Cambodia and Morocco. This combined RT-qPCR method constitutes a relevant, useful, validated tool for the diagnosis of rabies in both humans and animals, and represents a promising tool for

Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection.

Science.gov (United States)

Dacheux, Laurent; Larrous, Florence; Lavenir, Rachel; Lepelletier, Anthony; Faouzi, Abdellah; Troupin, Cécile; Nourlil, Jalal; Buchy, Philippe; Bourhy, Herve

2016-07-01

The definitive diagnosis of lyssavirus infection (including rabies) in animals and humans is based on laboratory confirmation. The reference techniques for post-mortem rabies diagnosis are still based on direct immunofluorescence and virus isolation, but molecular techniques, such as polymerase chain reaction (PCR) based methods, are increasingly being used and now constitute the principal tools for diagnosing rabies in humans and for epidemiological analyses. However, it remains a key challenge to obtain relevant specificity and sensitivity with these techniques while ensuring that the genetic diversity of lyssaviruses does not compromise detection. We developed a dual combined real-time reverse transcription polymerase chain reaction (combo RT-qPCR) method for pan-lyssavirus detection. This method is based on two complementary technologies: a probe-based (TaqMan) RT-qPCR for detecting the RABV species (pan-RABV RT-qPCR) and a second reaction using an intercalating dye (SYBR Green) to detect other lyssavirus species (pan-lyssa RT-qPCR). The performance parameters of this combined assay were evaluated with a large panel of primary animal samples covering almost all the genetic variability encountered at the viral species level, and they extended to almost all lyssavirus species characterized to date. This method was also evaluated for the diagnosis of human rabies on 211 biological samples (positive n = 76 and negative n = 135) including saliva, skin and brain biopsies. It detected all 41 human cases of rabies tested and confirmed the sensitivity and the interest of skin biopsy (91.5%) and saliva (54%) samples for intra-vitam diagnosis of human rabies. Finally, this method was successfully implemented in two rabies reference laboratories in enzootic countries (Cambodia and Morocco). This combined RT-qPCR method constitutes a relevant, useful, validated tool for the diagnosis of rabies in both humans and animals, and represents a promising tool for lyssavirus

A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system.

Science.gov (United States)

D'Souza, Alicia D; Belotserkovskii, Boris P; Hanawalt, Philip C

2018-02-01

The selective inhibition of transcription of a chosen gene by an artificial agent has numerous applications. Usually, these agents are designed to bind a specific nucleotide sequence in the promoter or within the transcribed region of the chosen gene. However, since optimal binding sites might not exist within the gene, it is of interest to explore the possibility of transcription inhibition when the agent is designed to bind at other locations. One of these possibilities arises when an additional transcription initiation site (e.g. secondary promoter) is present upstream from the primary promoter of the target gene. In this case, transcription inhibition might be achieved by inducing the formation of an RNA-DNA hybrid (R-loop) upon transcription from the secondary promoter. The R-loop could extend into the region of the primary promoter, to interfere with promoter recognition by RNA polymerase and thereby inhibit transcription. As a sequence-specific R-loop-inducing agent, a peptide nucleic acid (PNA) could be designed to facilitate R-loop formation by sequestering the non-template DNA strand. To investigate this mode for transcription inhibition, we have employed a model system in which a PNA binding site is localized between the T3 and T7 phage RNA polymerase promoters, which respectively assume the roles of primary and secondary promoters. In accord with our model, we have demonstrated that with PNA-bound DNA substrates, transcription from the T7 promoter reduces transcription from the T3 promoter by 30-fold, while in the absence of PNA binding there is no significant effect of T7 transcription upon T3 transcription. Copyright © 2018 Elsevier B.V. All rights reserved.

Detection of influenza viruses by coupling multiplex reverse-transcription loop-mediated isothermal amplification with cascade invasive reaction using nanoparticles as a sensor

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Ge Y

2017-04-01

Full Text Available Yiyue Ge,1 Qiang Zhou,2 Kangchen Zhao,1 Ying Chi,1 Bin Liu,3 Xiaoyan Min,4 Zhiyang Shi,1 Bingjie Zou,2 Lunbiao Cui1 1Institute of Pathogenic Microbiology, Key Laboratories of Enteric Pathogenic Microbiology (Ministry of Health, Jiangsu Provincial Center for Disease Control and Prevention, 2Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, 3Department of Biomedical Engineering, Nanjing Medical University, 4Department of Geriatrics, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of China Abstract: Influenza virus infections represent a worldwide public health and economic problem due to the significant morbidity and mortality caused by seasonal epidemics and pandemics. Sensitive and convenient methodologies for detection of influenza viruses are essential for further disease control. Loop-mediated isothermal amplification (LAMP is the most commonly used method of nucleic acid isothermal amplification. However, with regard to multiplex LAMP, differentiating the ladder-like LAMP products derived from multiple targets is still challenging today. The requirement of specialized instruments has further hindered the on-site application of multiplex LAMP. We have developed an integrated assay coupling multiplex reverse transcription LAMP with cascade invasive reaction using nanoparticles (mRT-LAMP-CIRN as a sensor for the detection of three subtypes of influenza viruses: A/H1N1pdm09, A/H3 and influenza B. The analytic sensitivities of the mRT-LAMP-CIRN assay were 101 copies of RNA for both A/H1N1pdm09 and A/H3, and 102 copies of RNA for influenza B. This assay demonstrated highly specific detection of target viruses and could differentiate them from other genetically or clinically related viruses. Clinical specimen analysis showed the mRT-LAMP-CIRN assay had an overall sensitivity and specificity of 98.3% and 100%, respectively. In summary, the mRT-LAMP-CIRN assay is

Loop versus end colostomy reversal: has anything changed?

Science.gov (United States)

Bruns, B R; DuBose, J; Pasley, J; Kheirbek, T; Chouliaras, K; Riggle, A; Frank, M K; Phelan, H A; Holena, D; Inaba, K; Diaz, J; Scalea, T M

2015-10-01

Though primary repair of colon injuries is preferred, certain injury patterns require colostomy creation. Colostomy reversal is associated with significant morbidity and healthcare cost. Complication rates may be influenced by technique of diversion (loop vs. end colostomy), though this remains ill-defined. We hypothesized that reversal of loop colostomies is associated with fewer complications than end colostomies. This is a retrospective, multi-institutional study (four, level-1 trauma centers) of patients undergoing colostomy takedown for trauma during the time period 1/2006-12/2012. Data were collected from index trauma admission and subsequent admission for reversal and included demographics and complications of reversal. Student's t test was used to compare continuous variables against loop versus end colostomy. Discrete variables were compared against both groups using Chi-squared tests. Over the 6-year study period, 218 patients underwent colostomy takedown after trauma with a mean age of 30; 190 (87%) were male, 162 (74%) had penetrating injury as their indication for colostomy, and 98 (45%) experienced at least one complication. Patients in the end colostomy group (n = 160) were more likely to require midline laparotomy (145 vs. 18, p colostomy (n = 58). Local takedown of a loop colostomy is safe and leads to shorter hospital stays, less intra-operative blood loss, and fewer complications when compared to end colostomy.

Investigations on the frequency of norovirus contamination of ready-to-eat food items in Istanbul, Turkey, by using real-time reverse transcription PCR.

Science.gov (United States)

Yilmaz, Aysun; Bostan, Kamil; Altan, Eda; Muratoglu, Karlo; Turan, Nuri; Tan, Derya; Helps, Christopher; Yilmaz, Huseyin

2011-05-01

Investigation of norovirus (NoV) contamination of food items is important because many outbreaks occur after consumption of contaminated shellfish, vegetables, fruits, and water. The frequency of NoV contamination in food items has not previously been investigated in Turkey. The aim of this study was to investigate the frequency of human NoV genogroups (G) I and II in ready-to-eat tomatoes, parsley, green onion, lettuce, mixed salads, and cracked wheat balls. RNA was extracted with the RNeasy Mini Kit, and a real-time reverse transcription (RT) PCR assay was performed using primers specific for NoV GI and GII. Among the 525 samples analyzed, NoV GII was detected in 1 green onion sample and 1 tomato sample by both SYBR Green and TaqMan real-time RT-PCR assays; no GI virus was detected. The Enterobactericaeae and Escherichia coli levels in the NoV-positive green onion were 6.56 and 1.28 log CFU/g, and those in the tomato were 5.55 and 1.30 log CFU/g, respectively. No significant difference in the bacterial levels was found between the NoV-positive and NoV-negative samples. This study is the first in which NoV GII was found in ready-to-eat food collected from Istanbul, Turkey; thus, these foods may be considered a risk to human health. Epidemiological studies and measures to prevent NoV infection should be considered.

A real-time and closed-loop control algorithm for cascaded multilevel inverter based on artificial neural network.

Science.gov (United States)

Wang, Libing; Mao, Chengxiong; Wang, Dan; Lu, Jiming; Zhang, Junfeng; Chen, Xun

2014-01-01

In order to control the cascaded H-bridges (CHB) converter with staircase modulation strategy in a real-time manner, a real-time and closed-loop control algorithm based on artificial neural network (ANN) for three-phase CHB converter is proposed in this paper. It costs little computation time and memory. It has two steps. In the first step, hierarchical particle swarm optimizer with time-varying acceleration coefficient (HPSO-TVAC) algorithm is employed to minimize the total harmonic distortion (THD) and generate the optimal switching angles offline. In the second step, part of optimal switching angles are used to train an ANN and the well-designed ANN can generate optimal switching angles in a real-time manner. Compared with previous real-time algorithm, the proposed algorithm is suitable for a wider range of modulation index and results in a smaller THD and a lower calculation time. Furthermore, the well-designed ANN is embedded into a closed-loop control algorithm for CHB converter with variable direct voltage (DC) sources. Simulation results demonstrate that the proposed closed-loop control algorithm is able to quickly stabilize load voltage and minimize the line current's THD (<5%) when subjecting the DC sources disturbance or load disturbance. In real design stage, a switching angle pulse generation scheme is proposed and experiment results verify its correctness.

A Real-Time and Closed-Loop Control Algorithm for Cascaded Multilevel Inverter Based on Artificial Neural Network

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Libing Wang

2014-01-01

Full Text Available In order to control the cascaded H-bridges (CHB converter with staircase modulation strategy in a real-time manner, a real-time and closed-loop control algorithm based on artificial neural network (ANN for three-phase CHB converter is proposed in this paper. It costs little computation time and memory. It has two steps. In the first step, hierarchical particle swarm optimizer with time-varying acceleration coefficient (HPSO-TVAC algorithm is employed to minimize the total harmonic distortion (THD and generate the optimal switching angles offline. In the second step, part of optimal switching angles are used to train an ANN and the well-designed ANN can generate optimal switching angles in a real-time manner. Compared with previous real-time algorithm, the proposed algorithm is suitable for a wider range of modulation index and results in a smaller THD and a lower calculation time. Furthermore, the well-designed ANN is embedded into a closed-loop control algorithm for CHB converter with variable direct voltage (DC sources. Simulation results demonstrate that the proposed closed-loop control algorithm is able to quickly stabilize load voltage and minimize the line current’s THD (<5% when subjecting the DC sources disturbance or load disturbance. In real design stage, a switching angle pulse generation scheme is proposed and experiment results verify its correctness.

A Precise and Real-Time Loop-closure Detection for SLAM Using the RSOM Tree

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Siyang Song

2015-06-01

Full Text Available In robotic applications of visual simultaneous localization and mapping (SLAM techniques, loop-closure detection detects whether or not a current location has previously been visited. We present an online and incremental approach to detect loops when images come from an already visited scene and learn new information from the environment. Instead of utilizing a bag-of-words model, the attributed graph model is applied to represent images and measure the similarity between pairs of images in our method. In order to position a camera in visual environments in real-time, the method demands retrieval of images from the database through a clustering tree that we call RSOM (recursive self-organizing feature map. As long as the match is found between the current graph and several graphs in the database, a threshold will be chosen to judge whether loop-closure is accepted or rejected. The results demonstrate the method's accuracy and real-time performance by testing several videos collected from a digital camera fixed on vehicles in indoor and outdoor environments.

Rapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification (RT-LAMP.

Directory of Open Access Journals (Sweden)

Amanda E Calvert

Full Text Available Zika virus (ZIKV has emerged as a major global public health concern in the last two years due to its link as a causative agent of human birth defects. Its rapid expansion into the Western Hemisphere as well as the ability to be transmitted from mother to fetus, through sexual transmission and possibly through blood transfusions has increased the need for a rapid and expansive public health response to this unprecedented epidemic. A non-invasive and rapid ZIKV diagnostic screening assay that can be performed in a clinical setting throughout pregnancy is vital for prenatal care of women living in areas of the world where exposure to the virus is possible. To meet this need we have developed a sensitive and specific reverse transcriptase loop-mediated isothermal amplification (RT-LAMP assay to detect ZIKV RNA in urine and serum with a simple visual detection. RT-LAMP results were shown to have a limit of detection 10-fold higher than qRT-PCR. As little as 1.2 RNA copies/μl was detected by RT-LAMP from a panel of 178 diagnostic specimens. The assay was shown to be highly specific for ZIKV RNA when tested with diagnostic specimens positive for dengue virus (DENV and chikungunya virus (CHIKV. The assay described here illustrates the potential for a fast, reliable, sensitive and specific assay for the detection of ZIKV from urine or serum that can be performed in a clinical or field setting with minimal equipment and technological expertise.

Screening suitable reference genes for normalization in reverse transcription quantitative real-time PCR analysis in melon.

Directory of Open Access Journals (Sweden)

Qiusheng Kong

Full Text Available Melon (Cucumis melo. L is not only an economically important cucurbitaceous crop but also an attractive model for studying many biological characteristics. Screening appropriate reference genes is essential to reverse transcription quantitative real-time PCR (RT-qPCR, which is key to many studies involving gene expression analysis. In this study, 14 candidate reference genes were selected, and the variations in their expression in roots and leaves of plants subjected to biotic stress, abiotic stress, and plant growth regulator treatment were assessed by RT-qPCR. The stability of the expression of the selected genes was determined and ranked using geNorm and NormFinder. geNorm identified the two most stable genes for each set of conditions: CmADP and CmUBIep across all samples, CmUBIep and CmRPL in roots, CmRAN and CmACT in leaves, CmADP and CmRPL under abiotic stress conditions, CmTUA and CmACT under biotic stress conditions, and CmRAN and CmACT under plant growth regulator treatments. NormFinder determined CmRPL to be the best reference gene in roots and under biotic stress conditions and CmADP under the other experimental conditions. CmUBC2 and CmPP2A were not found to be suitable under many experimental conditions. The catalase family genes CmCAT1, CmCAT2, and CmCAT3 were identified in melon genome and used as target genes to validate the reliability of identified reference genes. The catalase family genes showed the most upregulation 3 days after inoculation with Fusarium wilt in roots, after which they were downregulated. Their levels of expression were significantly overestimated when the unsuitable reference gene was used for normalization. These results not only provide guidelines for the selection of reference genes for gene expression analyses in melons but may also provide valuable information for studying the functions of catalase family genes in stress responses.

Comparison of the Diagnostic Value Between Real-Time Reverse Transcription-Polymerase Chain Reaction Assay and Histopathologic Examination in Sentinel Lymph Nodes for Patients With Gastric Carcinoma.

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Kwak, Yoonjin; Nam, Soo Kyung; Shin, Eun; Ahn, Sang-Hoon; Lee, Hee Eun; Park, Do Joong; Kim, Woo Ho; Kim, Hyung-Ho; Lee, Hye Seung

2016-05-01

Sentinel lymph node (SLN)-based diagnosis in gastric cancers has shown varied sensitivities and false-negative rates in several studies. Application of the reverse transcription-polymerase chain reaction (RT-PCR) in SLN diagnosis has recently been proposed. A total of 155 SLNs from 65 patients with cT1-2, N0 gastric cancer were examined. The histopathologic results were compared with results obtained by real-time RT-PCR for detecting molecular RNA (mRNA) of cytokeratin (CK)19, carcinoembryonic antigen (CEA), and CK20. The sensitivity and specificity of the multiple marker RT-PCR assay standardized against the results of the postoperative histological examination were 0.778 (95% confidence interval [CI], 0.577-0.914) and 0.781 (95% CI, 0.700-0.850), respectively. In comparison, the sensitivity and specificity of intraoperative diagnosis were 0.819 (95% CI, 0.619-0.937) and 1.000 (95% CI, 0.972-1.000), respectively. The positive predictive value of the multiple-marker RT-PCR assay was 0.355 (95% CI, 0.192-0.546) for predicting non-SLN metastasis, which was lower than that of intraoperative diagnosis (0.813, 95% CI, 0.544-0.960). The real-time RT-PCR assay could detect SLN metastasis in gastric cancer. However, the predictive value of the real-time RT-PCR assay was lower than that of precise histopathologic examination and did not outweigh that of our intraoperative SLN diagnosis. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Tip-enhanced fluorescence with radially polarized illumination for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA

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Wei, Shih-Chung; Chuang, Tsung-Liang; Wang, Da-Shin; Lu, Hui-Hsin; Gu, Frank X.; Sung, Kung-Bin; Lin, Chii-Wann

2015-02-01

A tip nanobiosensor for monitoring DNA replication was presented. The effects of excitation power and polarization on tip-enhanced fluorescence (TEF) were assessed with the tip immersed in fluorescein isothiocyanate solution first. The photon count rose on average fivefold with radially polarized illumination at 50 mW. We then used polymerase-functionalized tips for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA. The amplicon-SYBR Green I complex was detected and compared to real-time loop-mediated isothermal amplification. The signals of the reaction using 4 and 0.004 ng/μl templates were detected 10 and 30 min earlier, respectively. The results showed the potential of TEF in developing a nanobiosensor for real-time DNA amplification.

A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods.

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Nixon, Gavin J; Svenstrup, Helle F; Donald, Carol E; Carder, Caroline; Stephenson, Judith M; Morris-Jones, Stephen; Huggett, Jim F; Foy, Carole A

2014-12-01

Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These 'isothermal' methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

Real-time PCR analysis of carbon catabolite repression of cellobiose gene transcription in Trametes versicolor

Energy Technology Data Exchange (ETDEWEB)

Stapleton, P. C.; O' Mahoney, J.; Dobson, A. D. W. [National University of Ireland, Microbiology Department, Cork (Ireland)

2004-02-01

Previous reports indicate that in white rot fungi such as Trametes versicolor, the production of cellobiose dehydrogenase (CDH), an extracellular haemo-flavo-enzyme, is subject to carbon catabolite repression by both glucose and maltose, and that the repression is mediated at the transcriptional level. This paper describes the results of an investigation of CDH gene transcription in cellulolytic cultures of T. versicolor, in the presence of other additional carbon sources such as glucose, arabinose, and xylose. Using real time polymerase chain reaction (RT-PCR) assay methods in the presence of these other additional carbon sources, the levels of repression observed are quantitatively determined in an effort to obtain more accurate measurements of carbon catabolite repression of CDH production in this ligninolytic fungus. Ninety-six hours after addition, results of the analysis showed reduction in CDH transcript levels of 19-fold for galactose, 92-fold for arabinose and 114-fold for xylose. The greatest repressive effect was exhibited by glucose. In this case the reduction in CDH transcript levels was 3400-fold. CDH plays an important role in lignin degradation, and there is also substantial interest in the biotechnological applications of CDH, most particularly in the pulp and paper industry. 24 refs., 4 figs.

Development of a real-time closed-loop micro-/nano-positioning system embedded with a capacitive sensor

International Nuclear Information System (INIS)

Shiou, Fang-Jung; Chiang, Chia-Jui; Liou, Ke-Jhen; Liao, Shu-Chung; Chen, Chao-Jung; Liou, Huay-Chung

2010-01-01

The hysteresis and nonlinearity of the PZT is an actual problem in the piezo-driven micro-/nano-positioning stage, especially for the open-loop positioning stage. The study presents the development of an NI cRIO9074-based real-time closed-loop micro-/nano-positioning system, to overcome the problem of the hysteresis and nonlinearity of a PZT and to increase the positioning speed of the positioning stage. The developed system mainly consists of a piezoelectric actuator, a bridge-type hinge mechanism for displacement magnification, a micro-/nano-positioning stage body, a capacitive sensor system, an NI cRIO9074 real-time control unit with FPGA chip and a PC. After executing the optimization analysis of the displacement, stress and the frequency, using the ANSYS software, the dimensions of the stage body have been designed and determined. A set of software written with the LabView programming language was developed to construct the real-time PID closed-loop control of the developed positioning system. Based on the test results, the designed closed-loop micro-/nano-positioning system was capable of precision positioning within the travel of 119.08 µm with maximum stage tilting angle at 25 µrad. The steady-state positioning deviation of the stage is about ±2 nm in the step-positioning test. In the transient slope-tracing test at a tracing speed of 5 µm s −1 , an error of about ±100 nm is observed

R-loops in bacterial transcription: their causes and consequences.

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Gowrishankar, J; Leela, J Krishna; Anupama, K

2013-01-01

Nascent untranslated transcripts in bacteria are prone to generating RNA-DNA hybrids (R-loops); Rho-dependent transcription termination acts to reduce their prevalence. Here we discuss the mechanisms of R-loop formation and growth inhibition in bacteria.

Activating RNAs associate with Mediator to enhance chromatin architecture and transcription.

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Lai, Fan; Orom, Ulf A; Cesaroni, Matteo; Beringer, Malte; Taatjes, Dylan J; Blobel, Gerd A; Shiekhattar, Ramin

2013-02-28

Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms. Although the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X-chromosome inactivation and imprinting, different classes of lncRNAs may have varied biological functions. We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their neighbouring genes using a cis-mediated mechanism. To define the precise mode by which such enhancer-like RNAs function, we depleted factors with known roles in transcriptional activation and assessed their role in RNA-dependent activation. Here we report that depletion of the components of the co-activator complex, Mediator, specifically and potently diminished the ncRNA-induced activation of transcription in a heterologous reporter assay using human HEK293 cells. In vivo, Mediator is recruited to ncRNA-a target genes and regulates their expression. We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10. The Mediator complex harbouring disease- displays diminished ability to associate with activating ncRNAs. Chromosome conformation capture confirmed the presence of DNA looping between the ncRNA-a loci and its targets. Importantly, depletion of Mediator subunits or ncRNA-a reduced the chromatin looping between the two loci. Our results identify the human Mediator complex as the transducer of activating ncRNAs and highlight the importance of Mediator and activating ncRNA association in human disease.

Development of Reverse Transcription Thermostable Helicase-Dependent DNA Amplification for the Detection of Tomato Spotted Wilt Virus.

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Wu, Xinghai; Chen, Chanfa; Xiao, Xizhi; Deng, Ming Jun

2016-11-01

A protocol for the reverse transcription-helicase-dependent amplification (RT-HDA) of isothermal DNA was developed for the detection of tomato spotted wilt virus (TSWV). Specific primers, which were based on the highly conserved region of the N gene sequence in TSWV, were used for the amplification of virus's RNA. The LOD of RT-HDA, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP), and reverse transcriptase-polymerase chain reaction (RT-PCR) assays were conducted using 10-fold serial dilution of RNA eluates. TSWV sensitivity in RT-HDA and RT-LAMP was 4 pg RNA compared with 40 pg RNA in RT-PCR. The specificity of RT-HDA for TSWV was high, showing no cross-reactivity with other tomato and Tospovirus viruses including cucumber mosaic virus (CMV), tomato black ring virus (TBRV), tomato mosaic virus (ToMV), or impatiens necrotic spot virus (INSV). The RT-HDA method is effective for the detection of TSWV in plant samples and is a potential tool for early and rapid detection of TSWV.

A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods

Directory of Open Access Journals (Sweden)

Gavin J. Nixon

2014-12-01

Full Text Available Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR. There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These ‘isothermal’ methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT, akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

Real-time observation of the initiation of RNA polymerase II transcription.

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Fazal, Furqan M; Meng, Cong A; Murakami, Kenji; Kornberg, Roger D; Block, Steven M

2015-09-10

Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a 'closed' preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an 'open' complex; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers. Contrary to expectation, scanning driven by the transcription factor IIH involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.

Rapid Newcastle Disease Virus Detection Based on Loop-Mediated Isothermal Amplification and Optomagnetic Readout

DEFF Research Database (Denmark)

Tian, Bo; Ma, Jing; Zardán Gómez de la Torre, Teresa

2016-01-01

Rapid and sensitive diagnostic methods based on isothermal amplification are ideal substitutes for PCR in out-of-lab settings. However, there are bottlenecks in terms of establishing low-cost and user-friendly readout methods for isothermal amplification schemes. Combining the high amplification...... efficiency of loop-mediated isothermal amplification (LAMP) with an optomagnetic nanoparticle-based readout system, we demonstrate ultrasensitive and rapid detection of Newcastle disease virus RNA. Biotinylated amplicons of LAMP and reverse transcription LAMP (RT-LAMP) bind to streptavidin-coated magnetic...... nanoparticles (MNPs) resulting in a dramatical increase in the hydrodynamic size of the MNPs. This increase was measured by an optomagnetic readout system and provided quantitative information on the amount of LAMP target sequence. Our assay resulted in a limit of detection of 10 aM of target sequence...

Sensitivity and specificity of real-time reverse transcription polymerase chain reaction, histopathology, and immunohistochemical labeling for the detection of Rift Valley fever virus in naturally infected cattle and sheep.

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Odendaal, Lieza; Fosgate, Geoffrey T; Romito, Marco; Coetzer, Jacobus A W; Clift, Sarah J

2014-01-01

Real-time reverse transcription polymerase chain reaction (real-time RT-PCR), histopathology, and immunohistochemical labeling (IHC) were performed on liver specimens from 380 naturally infected cattle and sheep necropsied during the 2010 Rift Valley fever (RVF) epidemic in South Africa. Sensitivity (Se) and specificity (Sp) of real-time RT-PCR, histopathology, and IHC were estimated in a latent-class model using a Bayesian framework. The Se and Sp of real-time RT-PCR were estimated as 97.4% (95% confidence interval [CI] = 95.2-98.8%) and 71.7% (95% CI = 65-77.9%) respectively. The Se and Sp of histopathology were estimated as 94.6% (95% CI = 91-97.2%) and 92.3% (95% CI = 87.6-95.8%), respectively. The Se and Sp of IHC were estimated as 97.6% (95% CI = 93.9-99.8%) and 99.4% (95% CI = 96.9-100%), respectively. Decreased Sp of real-time RT-PCR was ascribed to cross-contamination of samples. Stratified analysis of the data suggested variations in test accuracy with fetuses and severely autolyzed specimens. The Sp of histopathology in fetuses (83%) was 9.3% lower than the sample population (92.3%). The Se of IHC decreased from 97.6% to 81.5% in the presence of severe autolysis. The diagnostic Se and Sp of histopathology was higher than expected, confirming the value of routine postmortem examinations and histopathology of liver specimens. Aborted fetuses, however, should be screened using a variety of tests in areas endemic for RVF, and results from severely autolyzed specimens should be interpreted with caution. The most feasible testing option for countries lacking suitably equipped laboratories seems to be routine histology in combination with IHC.

The Mediator complex: a central integrator of transcription

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Allen, Benjamin L.; Taatjes, Dylan J.

2016-01-01

The RNA polymerase II (pol II) enzyme transcribes all protein-coding and most non-coding RNA genes and is globally regulated by Mediator, a large, conformationally flexible protein complex with variable subunit composition (for example, a four-subunit CDK8 module can reversibly associate). These biochemical characteristics are fundamentally important for Mediator's ability to control various processes important for transcription, including organization of chromatin architecture and regulation of pol II pre-initiation, initiation, re-initiation, pausing, and elongation. Although Mediator exists in all eukaryotes, a variety of Mediator functions appear to be specific to metazoans, indicative of more diverse regulatory requirements. PMID:25693131

Development of loop-mediated isothermal amplification and SYBR green real-time PCR methods for the detection of Citrus yellow mosaic badnavirus in citrus species.

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Anthony Johnson, A M; Dasgupta, I; Sai Gopal, D V R

2014-07-01

Citrus yellow mosaic badnavirus (CMBV) is an important pathogen in southern India spread by infected citrus propagules. One of the measures to arrest the spread of CMBV is to develop methods to screen and certify citrus propagules as CMBV-free. The methods loop-mediated isothermal amplification (LAMP) and SYBR green real-time PCR (SGRTPCR) have been developed for the efficient detection of CMBV in citrus propagules. This paper compares the sensitivities of LAMP and SGRTPCR with polymerase chain reaction (PCR) for the detection of CMBV. Whereas PCR and LAMP were able to detect CMBV from a minimum of 10 ng of total DNA of infected leaf samples, SGRTPCR could detect the same from 1 ng of total DNA. Using SGRTPCR, the viral titres were estimated to be the highest in rough lemon and lowest in Nagpur Mandarin of the five naturally infected citrus species tested. The results will help in designing suitable strategies for the sensitive detection of CMBV from citrus propagules. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of Capillary Loop Convective Polymerase Chain Reaction Platform with Real-Time Fluorescence Detection

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Wen-Pin Chou

2017-02-01

Full Text Available Polymerase chain reaction (PCR has been one of the principal techniques of molecular biology and diagnosis for decades. Conventional PCR platforms, which work by rapidly heating and cooling the whole vessel, need complicated hardware designs, and cause energy waste and high cost. On the other hand, partial heating on the various locations of vessels to induce convective solution flows by buoyancy have been used for DNA amplification in recent years. In this research, we develop a new convective PCR platform, capillary loop convective polymerase chain reaction (clcPCR, which can generate one direction flow and make the PCR reaction more stable. The U-shaped loop capillaries with 1.6 mm inner diameter are designed as PCR reagent containers. The clcPCR platform utilizes one isothermal heater for heating the bottom of the loop capillary and a CCD device for detecting real-time amplifying fluorescence signals. The stable flow was generated in the U-shaped container and the amplification process could be finished in 25 min. Our experiments with different initial concentrations of DNA templates demonstrate that clcPCR can be applied for precise quantification. Multiple sample testing and real-time quantification will be achieved in future studies.

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

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

2016-06-11

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

Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

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Zhang, Xin; Zhang, He; Pu, Jinji; Qi, Yanxiang; Yu, Qunfang; Xie, Yixian; Peng, Jun

2013-01-01

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3) spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.

Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

Directory of Open Access Journals (Sweden)

Xin Zhang

Full Text Available Fusarium oxysporum f. sp. cubense (Foc, the causal agent of Fusarium wilt (Panama disease, is one of the most devastating diseases of banana (Musa spp.. The Foc tropical race 4 (TR4 is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3 spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05. Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.

Comparison of reverse-transcription real-time PCR and immunohistochemistry for the detection of canine distemper virus infection in raccoons in Ontario, Canada.

Science.gov (United States)

Nemeth, Nicole M; Oesterle, Paul T; Campbell, G Douglas; Ojkic, Davor; Jardine, Claire M

2018-03-01

Canine distemper virus (CDV) is a widespread morbillivirus that causes subclinical to fatal infections in domestic and wild carnivores. Raccoons ( Procyon lotor) are CDV reservoirs and suffer from associated disease. Aspects of pathogenesis may lead to difficulty in the interpretation of commonly used testing modalities, such as reverse-transcription real-time (RT-rt)PCR and immunohistochemistry (IHC). The reliance upon such tests is greater for wildlife, which are often submitted as carcasses with no clinical history. We compared CDV RT-rtPCR results to immunohistochemistry (the gold standard) in tissues from 74 raccoons. These tests had high kappa agreement (lymph node: 0.9335; lung: 0.8671) and a negative correlation between IHC score and threshold cycle (Ct) value for lymph node and lung (Spearman rank correlation coefficient [ r s ] = -0.8555 and -0.8179, respectively; p < 0.00001). An RT-rtPCR Ct value of 30 in lung and lymph node with sensitivity and specificity of 92.3 and 92.6% and 86.8 and 96.4%, respectively, was suitable for determining CDV involvement. Conjunctival swabs provide an alternative for distemper diagnosis, as there was a strong correlation between Ct values of conjunctival swabs and tissues ( r s = -0.8498, p < 0.00001, n = 46). This information will aid in more efficient and accurate diagnoses in individuals, small-scale outbreaks, and epidemiologic investigations in wildlife.

Design of a Bioelectronics Hybrid System in Real Time and in Closed Loop

Directory of Open Access Journals (Sweden)

Sylvie Renaud

2012-12-01

Full Text Available Hynets, for Hybrid (living-artificial Networks, are an efficient and adaptable experimental support to explore the dynamics and the adaptation process of biological systems. We present in this paper an innovative platform performing a real-time closed-loop between a cultured network (e.g. neurons and an artificial processing (e.g. software processing or a robotic interface. The system gathers bioware, hardware, and software components and ensures the closed-loop data processing in less than 50 µs. We describe also a methodology that may help to standardize the description of some experiments. This method is associated to a full custom Graphical User Interface. We detail here the system choices, components, and performances.

Direct duplex real-time loop mediated isothermal amplification assay for the simultaneous detection of cow and goat species origin of milk and yogurt products for field use.

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Kim, Mi-Ju; Kim, Hae-Yeong

2018-04-25

A multiple loop-mediated isothermal amplification (LAMP) method was developed to detect cow and goat milk in the field using a portable fluorescence device. For rapid on-site detection, this duplex LAMP assay was used in combination with direct amplification, without DNA extraction. The cow- and goat-specific LAMP primer sets were designed based on the mitochondrial cytochrome b gene, and showed specificity against 13 other animal species in the reactions. The sensitivity of the duplex LAMP assay for cow and goat was 0.1 and 1 pg, respectively. The detection limit for both target species in milk mixtures was 2%. This assay successfully amplified and identified the two target species in 24 samples of commercial milk and yogurt products, with 30 min sampling-to-result analysis time. Therefore, this direct duplex real-time LAMP assay is useful for on-site simultaneous detection of cow and goat milk in commercial products, a capability needed to confirm accurate labeling. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transcription-induced DNA supercoiling: New roles of intranucleosomal DNA loops in DNA repair and transcription.

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Gerasimova, N S; Pestov, N A; Kulaeva, O I; Clark, D J; Studitsky, V M

2016-05-26

RNA polymerase II (Pol II) transcription through chromatin is accompanied by formation of small intranucleosomal DNA loops. Pol II captured within a small loop drives accumulation of DNA supercoiling, facilitating further transcription. DNA breaks relieve supercoiling and induce Pol II arrest, allowing detection of DNA damage hidden in chromatin structure.

Reverse logistics and closed-loop supply chain

DEFF Research Database (Denmark)

Govindan, Kannan; Soleimani, Hamed; Kannan, Devika

2015-01-01

Based on environmental, legal, social, and economic factors, reverse logistics and closed-loop supply chain issues have attracted attention among both academia and practitioners. This attention is evident by the vast number of publications in scientific journals which have been published in recent...... years. Hence, a comprehensive literature review of recent and state-of-the-art papers is vital to draw a framework of the past, and to shed light on future directions. The aim of this paper is to review recently published papers in reverse logistic and closed-loop supply chain in scientific journals...

Detection of negative and positive RNA strand of poliovirus Sabin 1 and echovirus E19 by a stem-loop reverse transcription PCR.

Science.gov (United States)

Fikatas, A; Dimitriou, T G; Kyriakopoulou, Z; Moschonas, G D; Amoutzias, G D; Mossialos, D; Gartzonika, C; Levidiotou-Stefanou, S; Markoulatos, P

2017-09-01

In this report a strand specific RT-PCR was established for the detection of the replicative negative RNA strand of poliovirus sabin 1 (Sabin1) and Echovirus 19 (E19) strains. The key for the successful conduction of the assay was the use of a specific reverse transcription primer targeting the 5'-UTR of enteroviruses that consisted of a stem-loop structure at the 5'-end and an enteroviral-specific sequence at the 3'-end. The stem loop RT-PCR was found to be an accurate and sensitive method, detecting even 10 -2 CCID 50 of poliovirus sabin 1 (Sabin1) and E19 strains 6 h postinfection (p.i.), while CPE appeared 3 days later. This assay was also validated in SiHa and Caski cell lines that are not used for the detection of enteroviruses. The negative RNA strand was detected 6 h and 12 h p.i. in SiHa and Caski cells, when these cell lines were inoculated with 10 5 and 1 CCID 50 respectively, whereas CPE was observed 5 days p.i for SiHa cells and 8 days p.i for Caski cells and that only at 10 5 CCID 50 . The results show that this approach may be used for replacing the time-consuming cell cultures in order to detect the active replication of enteroviruses. Enteroviruses are positive stranded RNA viruses that may cause severe diseases. The conventional method for detection of active viral replication involves virus isolation in sensitive cell cultures followed by titration and seroneutralization. In this report, we describe the use of a stem-loop secondary structured oligonucleotide in RT-PCR assay for the detection of the replicative negative strand of the positive-stranded RNA of poliovirus sabin 1 and E19 strains. This approach proved to be a useful tool that may be used for replacing the time-consuming cell culture assays in order to detect the active replication of enteroviruses. © 2017 The Society for Applied Microbiology.

Real-Time PCR (qPCR) Primer Design Using Free Online Software

Science.gov (United States)

Thornton, Brenda; Basu, Chhandak

2011-01-01

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

Estimating medication stopping fraction and real-time prevalence of drug use in pharmaco-epidemiologic databases. An application of the reverse waiting time distribution

DEFF Research Database (Denmark)

Støvring, Henrik; Pottegård, Anton; Hallas, Jesper

2017-01-01

Purpose: To introduce the reverse waiting time distribution (WTD) and show how it can be used to estimate stopping fractions and real-time prevalence of treatment in pharmacoepidemiological studies. Methods: The reverse WTD is the distribution of time from the last dispensed prescription of each......-hoc decision rules for automated implementations, and it yields estimates of real-time prevalence....... patient within a time window to the end of it. It is a mirrored version of the ordinary WTD, which considers the first dispensed prescription of patients within a time window. Based on renewal process theory, the reverse WTD can be analyzed as an ordinary WTD with maximum likelihood estimation. Based...

A real-time loop-mediated isothermal amplification assay for rapid detection of Shigella species.

Science.gov (United States)

Liew, P S; Teh, C S J; Lau, Y L; Thong, K L

2014-12-01

Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 10(5) CFU/ml, while PCR displayed a limit of 5.9 x 10(7) CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 10(4) CFU/g, whereas PCR was 3.6 x 10(5) CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.

Preliminary results on ghrelin mRNA quantification in buffalo calves during fasting and refeeding by real-time reverse transcription PCR assay

Directory of Open Access Journals (Sweden)

G. Neglia

2010-02-01

Full Text Available The aim of this trial was to evaluate ghrelin response to milk administration in 20 days old buffalo calves. The trial was carried out on 5 female buffalo calves with a mean age of 21.2±2.8 days. Five blood samples were collected from each animal into EDTA tubes, starting at 07.00 until 15.00, at 2-h intervals. At 09.00, after the second blood sample, replaced milk was administered to the calves. Blood samples were immediately placed at 4°C until processing, which was performed on the same day. We used real-time reverse transcription PCR system to detect the expression of ghrelin mRNA levels in blood of buffalo calves. Two calves showed a low ghrelin concentration at the start of the trial (Group A = low ghrelin concentration and three calves a high ghrelin concentration (Group B = high ghrelin concentration. Ghrelin expression was significantly higher either two hours (P<0.01 and just before feeding (P<0.05 in Group B vs. Group A. However, in both cases, a significant (P<0.05 difference was observed within each group between -2 and 6 hours after feeding. Therefore, ghrelin concentration tended to increase in animals that showed low levels and, similarly, it lowered in animals that showed high concentration. If these results will be confirmed, may represent the evidence that also in buffalo calves the ghrelin system may affect feed intake. Further studies are needed in order to better evaluate the ghrelin system in buffalo calves.

Real-Time Monitoring and Prediction of the Pilot Vehicle System (PVS) Closed-Loop Stability

Science.gov (United States)

Mandal, Tanmay Kumar

Understanding human control behavior is an important step for improving the safety of future aircraft. Considerable resources are invested during the design phase of an aircraft to ensure that the aircraft has desirable handling qualities. However, human pilots exhibit a wide range of control behaviors that are a function of external stimulus, aircraft dynamics, and human psychological properties (such as workload, stress factor, confidence, and sense of urgency factor). This variability is difficult to address comprehensively during the design phase and may lead to undesirable pilot-aircraft interaction, such as pilot-induced oscillations (PIO). This creates the need to keep track of human pilot performance in real-time to monitor the pilot vehicle system (PVS) stability. This work focused on studying human pilot behavior for the longitudinal axis of a remotely controlled research aircraft and using human-in-the-loop (HuIL) simulations to obtain information about the human controlled system (HCS) stability. The work in this dissertation is divided into two main parts: PIO analysis and human control model parameters estimation. To replicate different flight conditions, this study included time delay and elevator rate limiting phenomena, typical of actuator dynamics during the experiments. To study human control behavior, this study employed the McRuer model for single-input single-output manual compensatory tasks. McRuer model is a lead-lag controller with time delay which has been shown to adequately model manual compensatory tasks. This dissertation presents a novel technique to estimate McRuer model parameters in real-time and associated validation using HuIL simulations to correctly predict HCS stability. The McRuer model parameters were estimated in real-time using a Kalman filter approach. The estimated parameters were then used to analyze the stability of the closed-loop HCS and verify them against the experimental data. Therefore, the main contribution of

Real time interrogation technique for fiber Bragg grating enhanced fiber loop ringdown sensors array.

Science.gov (United States)

Zhang, Yunlong; Li, Ruoming; Shi, Yuechun; Zhang, Jintao; Chen, Xiangfei; Liu, Shengchun

2015-06-01

A novel fiber Bragg grating aided fiber loop ringdown (FLRD) sensor array and the wavelength-time multiplexing based interrogation technique for the FLRD sensors array are proposed. The interrogation frequency of the system is formulated and the interrelationships among the parameters of the system are analyzed. To validate the performance of the proposed system, a five elements array is experimentally demonstrated, and the system shows the capability of real time monitoring every FLRD element with interrogation frequency of 125.5 Hz.

Evaluation of the PrimerDesign™ genesig real-time reverse transcription-polymerase chain reaction assay and the INFINITI® Respiratory Viral Panel Plus assay for the detection of human metapneumovirus in Kuwait.

Science.gov (United States)

Al-Turab, Mariam; Chehadeh, Wassim; Al-Mulla, Fahd; Al-Nakib, Widad

2012-04-01

Human metapneumovirus (hMPV) is a respiratory pathogen that was discovered in 2001 and is considered a major cause of both upper and lower respiratory tract infections. A sensitive, fast, and high-throughput diagnostic test is needed for the detection of hMPV that may assist in the clinical management as well as in the reduction of inappropriate therapy. Therefore, a comparison assessment was performed in this study between the PrimerDesign™ genesig real-time reverse transcription-polymerase chain reaction (RT-PCR) Assay and the INFINITI(®) Respiratory Viral Panel Plus Assay (RVP-Plus) for the detection of hMPV infection in patients with respiratory tract infections. A total of 200 respiratory samples were collected from 185 hospitalized patients, during the winter season in Kuwait. Of 185 patients, 10 (5.4%) were positive for hMPV RNA by the in-house RT-PCR assay, while 7 (4%) were positive for hMPV RNA by the real-time RT-PCR assay and 9 (5%) were positive for hMPV RNA by the INFINITI(®) RVP-Plus assay. The high incidence rate (60%) of hMPV infection was in January 2011. The sensitivity of the real-time RT-PCR and INFINITI(®) RVP-Plus assays was 70% and 90%, respectively, with specificity of 100% for both assays. hMPV types A and B could be identified in this study; however, discordant genotyping results were found between the direct sequencing method and the INFINITI(®) RVP-Plus assay in 33% of hMPV-positive patients. Copyright © 2012 Elsevier Inc. All rights reserved.

Propulsion Powertrain Real-Time Simulation Using Hardware-in-the-Loop (HIL) for Aircraft Electric Propulsion System

Science.gov (United States)

Choi, Benjamin B.; Brown, Gerald V.

2017-01-01

It is essential to design a propulsion powertrain real-time simulator using the hardware-in-the-loop (HIL) system that emulates an electrified aircraft propulsion (EAP) systems power grid. This simulator would enable us to facilitate in-depth understanding of the system principles, to validate system model analysis and performance prediction, and to demonstrate the proof-of-concept of the EAP electrical system. This paper describes how subscale electrical machines with their controllers can mimic the power components in an EAP powertrain. In particular, three powertrain emulations are presented to mimic 1) a gas turbo-=shaft engine driving a generator, consisting of two permanent magnet (PM) motors with brushless motor drives, coupled by a shaft, 2) a motor driving a propulsive fan, and 3) a turbo-shaft engine driven fan (turbofan engine) operation. As a first step towards the demonstration, experimental dynamic characterization of the two motor drive systems, coupled by a mechanical shaft, were performed. The previously developed analytical motor models1 were then replaced with the experimental motor models to perform the real-time demonstration in the predefined flight path profiles. This technique can convert the plain motor system into a unique EAP power grid emulator that enables rapid analysis and real-time simulation performance using hardware-in-the-loop (HIL).

PNA binding to the non-template DNA strand interferes with transcription, suggesting a blockage mechanism mediated by R-loop formation.

Science.gov (United States)

Belotserkovskii, Boris P; Hanawalt, Philip C

2015-11-01

Peptide Nucleic Acids (PNAs) are artificial DNA mimics with superior nucleic acid binding capabilities. T7 RNA polymerase (T7 RNAP) transcription upon encountering PNA bound to the non-template DNA strand was studied in vitro. A characteristic pattern of blockage signals was observed, extending downstream from the PNA binding site, similar to that produced by G-rich homopurine-homopyrimidine (hPu-hPy) sequences and likely caused by R-loop formation. Since blocked transcription complexes in association with stable R-loops may interfere with replication and in some cases trigger apoptosis, targeted R-loop formation might be employed to inactivate selected cells, such as those in tumors, based upon their unique complement of expressed genes. © 2014 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

Transcription regulation by the Mediator complex.

Science.gov (United States)

Soutourina, Julie

2018-04-01

Alterations in the regulation of gene expression are frequently associated with developmental diseases or cancer. Transcription activation is a key phenomenon in the regulation of gene expression. In all eukaryotes, mediator of RNA polymerase II transcription (Mediator), a large complex with modular organization, is generally required for transcription by RNA polymerase II, and it regulates various steps of this process. The main function of Mediator is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery, which is assembled at promoters as the preinitiation complex (PIC) to control transcription initiation. Recent functional studies of Mediator with the use of structural biology approaches and functional genomics have revealed new insights into Mediator activity and its regulation during transcription initiation, including how Mediator is recruited to transcription regulatory regions and how it interacts and cooperates with PIC components to assist in PIC assembly. Novel roles of Mediator in the control of gene expression have also been revealed by showing its connection to the nuclear pore and linking Mediator to the regulation of gene positioning in the nuclear space. Clear links between Mediator subunits and disease have also encouraged studies to explore targeting of this complex as a potential therapeutic approach in cancer and fungal infections.

Real-Time Closed Loop Modulated Turbine Cooling

Science.gov (United States)

Shyam, Vikram; Culley, Dennis E.; Eldridge, Jeffrey; Jones, Scott; Woike, Mark; Cuy, Michael

2014-01-01

It has been noted by industry that in addition to dramatic variations of temperature over a given blade surface, blade-to-blade variations also exist despite identical design. These variations result from manufacturing variations, uneven wear and deposition over the life of the part as well as limitations in the uniformity of coolant distribution in the baseline cooling design. It is proposed to combine recent advances in optical sensing, actuation, and film cooling concepts to develop a workable active, closed-loop modulated turbine cooling system to improve by 10 to 20 the turbine thermal state over the flight mission, to improve engine life and to dramatically reduce turbine cooling air usage and aircraft fuel burn. A reduction in oxides of nitrogen (NOx) can also be achieved by using the excess coolant to improve mixing in the combustor especially for rotorcraft engines. Recent patents filed by industry and universities relate to modulating endwall cooling using valves. These schemes are complex, add weight and are limited to the endwalls. The novelty of the proposed approach is twofold 1) Fluidic diverters that have no moving parts are used to modulate cooling and can operate under a wide range of conditions and environments. 2) Real-time optical sensing to map the thermal state of the turbine has never been attempted in realistic engine conditions.

Gait adaptation to visual kinematic perturbations using a real-time closed-loop brain-computer interface to a virtual reality avatar.

Science.gov (United States)

Luu, Trieu Phat; He, Yongtian; Brown, Samuel; Nakagame, Sho; Contreras-Vidal, Jose L

2016-06-01

The control of human bipedal locomotion is of great interest to the field of lower-body brain-computer interfaces (BCIs) for gait rehabilitation. While the feasibility of closed-loop BCI systems for the control of a lower body exoskeleton has been recently shown, multi-day closed-loop neural decoding of human gait in a BCI virtual reality (BCI-VR) environment has yet to be demonstrated. BCI-VR systems provide valuable alternatives for movement rehabilitation when wearable robots are not desirable due to medical conditions, cost, accessibility, usability, or patient preferences. In this study, we propose a real-time closed-loop BCI that decodes lower limb joint angles from scalp electroencephalography (EEG) during treadmill walking to control a walking avatar in a virtual environment. Fluctuations in the amplitude of slow cortical potentials of EEG in the delta band (0.1-3 Hz) were used for prediction; thus, the EEG features correspond to time-domain amplitude modulated potentials in the delta band. Virtual kinematic perturbations resulting in asymmetric walking gait patterns of the avatar were also introduced to investigate gait adaptation using the closed-loop BCI-VR system over a period of eight days. Our results demonstrate the feasibility of using a closed-loop BCI to learn to control a walking avatar under normal and altered visuomotor perturbations, which involved cortical adaptations. The average decoding accuracies (Pearson's r values) in real-time BCI across all subjects increased from (Hip: 0.18 ± 0.31; Knee: 0.23 ± 0.33; Ankle: 0.14 ± 0.22) on Day 1 to (Hip: 0.40 ± 0.24; Knee: 0.55 ± 0.20; Ankle: 0.29 ± 0.22) on Day 8. These findings have implications for the development of a real-time closed-loop EEG-based BCI-VR system for gait rehabilitation after stroke and for understanding cortical plasticity induced by a closed-loop BCI-VR system.

Gait adaptation to visual kinematic perturbations using a real-time closed-loop brain-computer interface to a virtual reality avatar

Science.gov (United States)

Phat Luu, Trieu; He, Yongtian; Brown, Samuel; Nakagome, Sho; Contreras-Vidal, Jose L.

2016-06-01

Objective. The control of human bipedal locomotion is of great interest to the field of lower-body brain-computer interfaces (BCIs) for gait rehabilitation. While the feasibility of closed-loop BCI systems for the control of a lower body exoskeleton has been recently shown, multi-day closed-loop neural decoding of human gait in a BCI virtual reality (BCI-VR) environment has yet to be demonstrated. BCI-VR systems provide valuable alternatives for movement rehabilitation when wearable robots are not desirable due to medical conditions, cost, accessibility, usability, or patient preferences. Approach. In this study, we propose a real-time closed-loop BCI that decodes lower limb joint angles from scalp electroencephalography (EEG) during treadmill walking to control a walking avatar in a virtual environment. Fluctuations in the amplitude of slow cortical potentials of EEG in the delta band (0.1-3 Hz) were used for prediction; thus, the EEG features correspond to time-domain amplitude modulated potentials in the delta band. Virtual kinematic perturbations resulting in asymmetric walking gait patterns of the avatar were also introduced to investigate gait adaptation using the closed-loop BCI-VR system over a period of eight days. Main results. Our results demonstrate the feasibility of using a closed-loop BCI to learn to control a walking avatar under normal and altered visuomotor perturbations, which involved cortical adaptations. The average decoding accuracies (Pearson’s r values) in real-time BCI across all subjects increased from (Hip: 0.18 ± 0.31 Knee: 0.23 ± 0.33 Ankle: 0.14 ± 0.22) on Day 1 to (Hip: 0.40 ± 0.24 Knee: 0.55 ± 0.20 Ankle: 0.29 ± 0.22) on Day 8. Significance. These findings have implications for the development of a real-time closed-loop EEG-based BCI-VR system for gait rehabilitation after stroke and for understanding cortical plasticity induced by a closed-loop BCI-VR system.

A review of reverse logistics and closed-loop supply chains

DEFF Research Database (Denmark)

Govindan, Kannan; Soleimani, Hamed

2016-01-01

Today, the task of developing reverse logistics and closed-loop supply chains in both developed and developing industries is accepted as a vital need in our societies. The growing interest in reverse issues can be clearly seen in the large number of publications, especially the ones that consider...... case studies in various industries. Based on its missions, Journal of Cleaner Production (JCP) is one of the most active journals in this field. Therefore, a comprehensive review is undertaken of the publications in the field of reverse logistics and closed-loop supply chains; the selection process...... December 2014 have been selected and reviewed. The selected papers are then analyzed based on their content and the appropriate developed categories. The results clarify the main trends in reverse logistics and closed-loop supply chain subjects for the Journal of Cleaner Production and the evaluations...

Precipitation of PEG/Carboxyl-Modified Gold Nanoparticles with Magnesium Pyrophosphate: A New Platform for Real-Time Monitoring of Loop-Mediated Isothermal Amplification.

Science.gov (United States)

Qin, Ailin; Fu, Lok Tin; Wong, Jacky K F; Chau, Li Yin; Yip, Shea Ping; Lee, Thomas M H

2017-03-29

Gold nanoparticles have proven to be promising for decentralized nucleic acid testing by virtue of their simple visual readout and absorbance-based quantification. A major challenge toward their practical application is to achieve ultrasensitive detection without compromising simplicity. The conventional strategy of thermocycling amplification is unfavorable (because of both instrumentation and preparation of thermostable oligonucleotide-modified gold nanoparticle probes). Herein, on the basis of a previously unreported co-precipitation phenomenon between thiolated poly(ethylene glycol)/11-mercaptoundecanoic acid co-modified gold nanoparticles and magnesium pyrophosphate crystals (an isothermal DNA amplification reaction byproduct), a new ultrasensitive and simple DNA assay platform is developed. The binding mechanism underlying the co-precipitation phenomenon is found to be caused by the complexation of carboxyl and pyrophosphate with free magnesium ions. Remarkably, poly(ethylene glycol) does not hinder the binding and effectively stabilizes gold nanoparticles against magnesium ion-induced aggregation (without pyrophosphate). In fact, a similar phenomenon is observed in other poly(ethylene glycol)- and carboxyl-containing nanomaterials. When the gold nanoparticle probe is incorporated into a loop-mediated isothermal amplification reaction, it remains as a red dispersion for a negative sample (in the absence of a target DNA sequence) but appears as a red precipitate for a positive sample (in the presence of a target). This results in a first-of-its-kind gold nanoparticle-based DNA assay platform with isothermal amplification and real-time monitoring capabilities.

Advanced Research and Education in Electrical Drives by Using Digital Real-Time Hardware-in-the-Loop Simulation

DEFF Research Database (Denmark)

Bojoi, R.; Profumo, F.; Griva, G.

2002-01-01

The authors present in this paper a digital real-time hardware-in-the-loop simulation of a three-phase induction motor drive. The main real-time simulation tool is the dSPACE DS1103 PPC Controller Board which simulates the power and signal conditioning parts. The control algorithm of the virtual...... drive has been implemented on the Evaluation Board of TMS320F240 DSP. The experimental results validate this solution as a powerful tool to be used in research and advanced education. Thus, the students can put in practic the theory without spending too much time with details concerning the hardware...

Real-time dynamic analysis for complete loop of direct steam generation solar trough collector

International Nuclear Information System (INIS)

Guo, Su; Liu, Deyou; Chu, Yinghao; Chen, Xingying; Shen, Bingbing; Xu, Chang; Zhou, Ling; Wang, Pei

2016-01-01

Highlights: • A nonlinear distribution parameter dynamic model has been developed. • Real-time local heat transfer coefficient and friction coefficient are adopted. • The dynamic behavior of the solar trough collector loop are simulated. • High-frequency chattering of outlet fluid flow are analyzed and modeled. • Irradiance disturbance at subcooled water region generates larger influence. - Abstract: Direct steam generation is a potential approach to further reduce the levelized electricity cost of solar trough. Dynamic modeling of the collector loop is essential for operation and control of direct steam generation solar trough. However, the dynamic behavior of fluid based on direct steam generation is complex because of the two-phase flow in the pipeline. In this work, a nonlinear distribution parameter model has been developed to model the dynamic behaviors of direct steam generation parabolic trough collector loops under either full or partial solar irradiance disturbance. Compared with available dynamic model, the proposed model possesses two advantages: (1) real-time local values of heat transfer coefficient and friction resistance coefficient, and (2) considering of the complete loop of collectors, including subcooled water region, two-phase flow region and superheated steam region. The proposed model has shown superior performance, particularly in case of sensitivity study of fluid parameters when the pipe is partially shaded. The proposed model has been validated using experimental data from Solar Thermal Energy Laboratory of University of New South Wales, with an outlet fluid temperature relative error of only 1.91%. The validation results show that: (1) The proposed model successfully outperforms two reference models in predicting the behavior of direct steam generation solar trough. (2) The model theoretically predicts that, during solar irradiance disturbance, the discontinuities of fluid physical property parameters and the moving back and

Real-Time Simulation and Hardware-in-the-Loop Testbed for Distribution Synchrophasor Applications

Directory of Open Access Journals (Sweden)

Matthias Stifter

2018-04-01

Full Text Available With the advent of Distribution Phasor Measurement Units (D-PMUs and Micro-Synchrophasors (Micro-PMUs, the situational awareness in power distribution systems is going to the next level using time-synchronization. However, designing, analyzing, and testing of such accurate measurement devices are still challenging. Due to the lack of available knowledge and sufficient history for synchrophasors’ applications at the power distribution level, the realistic simulation, and validation environments are essential for D-PMU development and deployment. This paper presents a vendor agnostic PMU real-time simulation and hardware-in-the-Loop (PMU-RTS-HIL testbed, which helps in multiple PMUs validation and studies. The network of real and virtual PMUs was built in a full time-synchronized environment for PMU applications’ validation. The proposed testbed also includes an emulated communication network (CNS layer to replicate bandwidth, packet loss and collisions conditions inherent to the PMUs data streams’ issues. Experimental results demonstrate the flexibility and scalability of the developed PMU-RTS-HIL testbed by producing large amounts of measurements under typical normal and abnormal distribution grid operation conditions.

Intracytoplasmic maturation of the human immunodeficiency virus type 1 reverse transcription complexes determines their capacity to integrate into chromatin

Directory of Open Access Journals (Sweden)

Kashanchi Fatah

2006-01-01

Full Text Available Abstract Background The early events of the HIV-1 life cycle include entry of the viral core into target cell, assembly of the reverse transcription complex (RTCs performing reverse transcription, its transformation into integration-competent complexes called pre-integration complexes (PICs, trafficking of complexes into the nucleus, and finally integration of the viral DNA into chromatin. Molecular details and temporal organization of these processes remain among the least investigated and most controversial problems in the biology of HIV. Results To quantitatively evaluate maturation and nuclear translocation of the HIV-1 RTCs, nucleoprotein complexes isolated from the nucleus (nRTC and cytoplasm (cRTC of HeLa cells infected with MLV Env-pseudotyped HIV-1 were analyzed by real-time PCR. While most complexes completed reverse transcription in the cytoplasm, some got into the nucleus before completing DNA synthesis. The HIV-specific RNA complexes could get into the nucleus when reverse transcription was blocked by reverse transcriptase inhibitor, although nuclear import of RNA complexes was less efficient than of DNA-containing RTCs. Analysis of the RTC nuclear import in synchronized cells infected in the G2/M phase of the cell cycle showed enrichment in the nuclei of RTCs containing incomplete HIV-1 DNA compared to non-synchronized cells, where RTCs with complete reverse transcripts prevailed. Immunoprecipitation assays identified viral proteins IN, Vpr, MA, and cellular Ini1 and PML associated with both cRTCs and nRTCs, whereas CA was detected only in cRTCs and RT was diminished in nRTCs. Cytoplasmic maturation of the complexes was associated with increased immunoreactivity with anti-Vpr and anti-IN antibodies, and decreased reactivity with antibodies to RT. Both cRTCs and nRTCs carried out endogenous reverse transcription reaction in vitro. In contrast to cRTCs, in vitro completion of reverse transcription in nRTCs did not increase their

Transcript Lifetime Is Balanced between Stabilizing Stem-Loop Structures and Degradation-Promoting Polyadenylation in Plant Mitochondria

Science.gov (United States)

Kuhn, Josef; Tengler, Ulrike; Binder, Stefan

2001-01-01

To determine the influence of posttranscriptional modifications on 3′ end processing and RNA stability in plant mitochondria, pea atp9 and Oenothera atp1 transcripts were investigated for the presence and function of 3′ nonencoded nucleotides. A 3′ rapid amplification of cDNA ends approach initiated at oligo(dT)-adapter primers finds the expected poly(A) tails predominantly attached within the second stem or downstream of the double stem-loop structures at sites of previously mapped 3′ ends. Functional studies in a pea mitochondrial in vitro processing system reveal a rapid removal of the poly(A) tails up to termini at the stem-loop structure but little if any influence on further degradation of the RNA. In contrast 3′ poly(A) tracts at RNAs without such stem-loop structures significantly promote total degradation in vitro. To determine the in vivo identity of 3′ nonencoded nucleotides more accurately, pea atp9 transcripts were analyzed by a direct anchor primer ligation-reverse transcriptase PCR approach. This analysis identified maximally 3-nucleotide-long nonencoded extensions most frequently of adenosines combined with cytidines. Processing assays with substrates containing homopolymer stretches of different lengths showed that 10 or more adenosines accelerate RNA processivity, while 3 adenosines have no impact on RNA life span. Thus polyadenylation can generally stimulate the decay of RNAs, but processivity of degradation is almost annihilated by the stabilizing effect of the stem-loop structures. These antagonistic actions thus result in the efficient formation of 3′ processed and stable transcripts. PMID:11154261

Detection of three porcine vesicular viruses using multiplex real-time primer-probe energy transfer

DEFF Research Database (Denmark)

Rasmussen, Thomas Bruun; Uttenthal, Åse; Aguero, M.

2006-01-01

Rapid identification of the etiologic agent in infected animals is important for the control of an outbreak of vesicular disease in livestock. We have in the present study developed a multiplex real-time reverse transcription-PCR, based on primer-probe energy transfer (PriProET), for simultaneous...

Electro-optic chaotic system based on the reverse-time chaos theory and a nonlinear hybrid feedback loop.

Science.gov (United States)

Jiang, Xingxing; Cheng, Mengfan; Luo, Fengguang; Deng, Lei; Fu, Songnian; Ke, Changjian; Zhang, Minming; Tang, Ming; Shum, Ping; Liu, Deming

2016-12-12

A novel electro-optic chaos source is proposed on the basis of the reverse-time chaos theory and an analog-digital hybrid feedback loop. The analog output of the system can be determined by the numeric states of shift registers, which makes the system robust and easy to control. The dynamical properties as well as the complexity dependence on the feedback parameters are investigated in detail. The correlation characteristics of the system are also studied. Two improving strategies which were established in digital field and analog field are proposed to conceal the time-delay signature. The proposed scheme has the potential to be used in radar and optical secure communication systems.

Concepts of self-acting circulation loops for downward heat transfer (reverse thermosiphons)

International Nuclear Information System (INIS)

Dobriansky, Y.

2011-01-01

This paper reviews the scientific and technical knowledge related to general self-acting flow loops (thermosiphons and heat pipes) that transmit heat upwards and self-acting reverse flow loops that transmit heat downwards. This paper classifies the heat and mass transfer processes that take place in general flow loops and analyses the nomenclature applied in the literature. It also presents the principles of operation of sixteen reverse flow loops; four of the loops are powered by an external source of energy, while the remaining loops are self-acting. Of the self-acting loops, vapor was used for heat transfer in seven of them and liquid was used in the remaining ones. Based on the available research results, a list of the advantages and disadvantages of both types of loops is presented.

Lineage-Specific Real-Time RT-PCR for Yellow Fever Virus Outbreak Surveillance, Brazil.

Science.gov (United States)

Fischer, Carlo; Torres, Maria C; Patel, Pranav; Moreira-Soto, Andres; Gould, Ernest A; Charrel, Rémi N; de Lamballerie, Xavier; Nogueira, Rita Maria Ribeiro; Sequeira, Patricia C; Rodrigues, Cintia D S; Kümmerer, Beate M; Drosten, Christian; Landt, Olfert; Bispo de Filippis, Ana Maria; Drexler, Jan Felix

2017-11-01

The current yellow fever outbreak in Brazil prompted widespread yellow fever virus (YFV) vaccination campaigns, imposing a responsibility to distinguish between vaccine- and wild-type YFV-associated disease. We developed novel multiplex real-time reverse transcription PCRs that differentiate between vaccine and American wild-type YFV. We validated these highly specific and sensitive assays in an outbreak setting.

Lineage-Specific Real-Time RT-PCR for Yellow Fever Virus Outbreak Surveillance, Brazil

OpenAIRE

Fischer, Carlo; Torres, Maria C.; Patel, Pranav; Moreira-Soto, Andres; Gould, Ernest A.; Charrel, Rémi N.; de Lamballerie, Xavier; Nogueira, Rita Maria Ribeiro; Sequeira, Patricia C.; Rodrigues, Cintia D.S.; Kümmerer, Beate M.; Drosten, Christian; Landt, Olfert; Bispo de Filippis, Ana Maria; Drexler, Jan Felix

2017-01-01

The current yellow fever outbreak in Brazil prompted widespread yellow fever virus (YFV) vaccination campaigns, imposing a responsibility to distinguish between vaccine- and wild-type YFV-associated disease. We developed novel multiplex real-time reverse transcription PCRs that differentiate between vaccine and American wild-type YFV. We validated these highly specific and sensitive assays in an outbreak setting.

Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus

Directory of Open Access Journals (Sweden)

Tandale Babasaheb V

2008-12-01

Full Text Available Abstract Background Chandipura virus (CHPV, a member of family Rhabdoviridae was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55–75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR using TaqMan technology was developed for rapid diagnosis. Methods Primers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [in vivo (mice in ovo (eggs, in vitro (Vero E6, PS, RD and Sand fly cell line] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard. Results Real-time one step RT-PCR was optimized using in vitro transcribed (IVT RNA. Standard curve showed linear relationship for wide range of 102-1010 (r2 = 0.99 with maximum Coefficient of variation (CV = 5.91% for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 × 100 PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 102 PFU/ml. Vero and PS cell-lines (1.2 × 103 PFU/ml were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy

Assessing reference genes for accurate transcript normalization using quantitative real-time PCR in pearl millet [Pennisetum glaucum (L. R. Br].

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Prasenjit Saha

Full Text Available Pearl millet [Pennisetum glaucum (L. R.Br.], a close relative of Panicoideae food crops and bioenergy grasses, offers an ideal system to perform functional genomics studies related to C4 photosynthesis and abiotic stress tolerance. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR provides a sensitive platform to conduct such gene expression analyses. However, the lack of suitable internal control reference genes for accurate transcript normalization during qRT-PCR analysis in pearl millet is the major limitation. Here, we conducted a comprehensive assessment of 18 reference genes on 234 samples which included an array of different developmental tissues, hormone treatments and abiotic stress conditions from three genotypes to determine appropriate reference genes for accurate normalization of qRT-PCR data. Analyses of Ct values using Stability Index, BestKeeper, ΔCt, Normfinder, geNorm and RefFinder programs ranked PP2A, TIP41, UBC2, UBQ5 and ACT as the most reliable reference genes for accurate transcript normalization under different experimental conditions. Furthermore, we validated the specificity of these genes for precise quantification of relative gene expression and provided evidence that a combination of the best reference genes are required to obtain optimal expression patterns for both endogeneous genes as well as transgenes in pearl millet.

Evaluation of carrier-mediated siRNA delivery

DEFF Research Database (Denmark)

Colombo, Stefano; Nielsen, Hanne Mørck; Foged, Camilla

2013-01-01

RNA delivery. An in vitro cell culture model system expressing enhanced green fluorescent protein (EGFP) was used to develop the assay, which was based on the intracellular quantification of a full-length double-stranded Dicer substrate siRNA by stem-loop RT qPCR. The result is a well-documented protocol......RNA delivered by use of carriers remains an analytical challenge. The purpose of the present study was to optimize and validate an analytical protocol based on stem-loop reverse transcription quantitative polymerase chain reaction (RT qPCR) to quantitatively monitor the carrier-mediated intracellular si...

Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses.

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Hamperl, Stephan; Bocek, Michael J; Saldivar, Joshua C; Swigut, Tomek; Cimprich, Karlene A

2017-08-10

Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.).

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Galeano, Esteban; Vasconcelos, Tarcísio Sales; Ramiro, Daniel Alves; De Martin, Valentina de Fátima; Carrer, Helaine

2014-07-22

Teak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak. Identification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes. This study proposes a first broad

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

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NandyMazumdar, Monali; Nedialkov, Yuri; Svetlov, Dmitri; Sevostyanova, Anastasia; Belogurov, Georgiy A; Artsimovitch, Irina

2016-12-27

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

Design of a 1 _s real-time low-noise data acquisition for power converters control loop

CERN Document Server

AUTHOR|(SzGeCERN)712364; Arpaia, Pasquale; Cerqueira Bastos, Miguel; Martino, Michele

2015-01-01

The proof of principle of a real-time data acquisition system to be integrated into a digital control loop for controlling the power converters of the Compact LInear Collider is presented. The system is based on an ultra low noise analogue front-end with 1:1 ppm RMS noise (referred to input), and about 1 _s of real-time delay. After the analogue conditioning, a fully-differential analogue-todigital converter is foreseen. The requirements of this system, directly derived from the accelerator performance, are discussed and translated into design specification. The results obtained by means of Pspice simulations are reported in order to prove that the design is feasible with the proposed architecture. Finally, the results of the experimental validation of the prototype, currently under design, will be included in the final paper.

EWS/FLI mediates transcriptional repression via NKX2.2 during oncogenic transformation in Ewing's sarcoma.

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Leah A Owen

2008-04-01

Full Text Available EWS/FLI is a master regulator of Ewing's sarcoma formation. Gene expression studies in A673 Ewing's sarcoma cells have demonstrated that EWS/FLI downregulates more genes than it upregulates, suggesting that EWS/FLI, and/or its targets, function as transcriptional repressors. One critical EWS/FLI target, NKX2.2, is a transcription factor that contains both transcriptional activation and transcriptional repression domains, raising the possibility that it mediates portions of the EWS/FLI transcriptional signature. We now report that microarray analysis demonstrated that the transcriptional profile of NKX2.2 consists solely of downregulated genes, and overlaps with the EWS/FLI downregulated signature, suggesting that NKX2.2 mediates oncogenic transformation via transcriptional repression. Structure-function analysis revealed that the DNA binding and repressor domains in NKX2.2 are required for oncogenesis in Ewing's sarcoma cells, while the transcriptional activation domain is completely dispensable. Furthermore, blockade of TLE or HDAC function, two protein families thought to mediate the repressive function of NKX2.2, inhibited the transformed phenotype and reversed the NKX2.2 transcriptional profile in Ewing's sarcoma cells. Whole genome localization studies (ChIP-chip revealed that a significant portion of the NKX2.2-repressed gene expression signature was directly mediated by NKX2.2 binding. These data demonstrate that the transcriptional repressive function of NKX2.2 is necessary, and sufficient, for the oncogenic phenotype of Ewing's sarcoma, and suggest a therapeutic approach to this disease.

Robust Real-Time Music Transcription with a Compositional Hierarchical Model.

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Pesek, Matevž; Leonardis, Aleš; Marolt, Matija

2017-01-01

The paper presents a new compositional hierarchical model for robust music transcription. Its main features are unsupervised learning of a hierarchical representation of input data, transparency, which enables insights into the learned representation, as well as robustness and speed which make it suitable for real-world and real-time use. The model consists of multiple layers, each composed of a number of parts. The hierarchical nature of the model corresponds well to hierarchical structures in music. The parts in lower layers correspond to low-level concepts (e.g. tone partials), while the parts in higher layers combine lower-level representations into more complex concepts (tones, chords). The layers are learned in an unsupervised manner from music signals. Parts in each layer are compositions of parts from previous layers based on statistical co-occurrences as the driving force of the learning process. In the paper, we present the model's structure and compare it to other hierarchical approaches in the field of music information retrieval. We evaluate the model's performance for the multiple fundamental frequency estimation. Finally, we elaborate on extensions of the model towards other music information retrieval tasks.

Theta, time reversal and temperature

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Gaiotto, Davide [Perimeter Institute for Theoretical Physics,Waterloo, Ontario, N2L 2Y5 (Canada); Kapustin, Anton [Walter Burke Institute for Theoretical Physics, California Institute of Technology,Pasadena, CA 91125 (United States); Komargodski, Zohar [Department of Particle Physics and Astrophysics, Weizmann Institute of Science,Rehovot 76100 (Israel); Seiberg, Nathan [School of Natural Sciences, Institute for Advanced Study,Princeton, NJ 08540 (United States)

2017-05-17

SU(N) gauge theory is time reversal invariant at θ=0 and θ=π. We show that at θ=π there is a discrete ’t Hooft anomaly involving time reversal and the center symmetry. This anomaly leads to constraints on the vacua of the theory. It follows that at θ=π the vacuum cannot be a trivial non-degenerate gapped state. (By contrast, the vacuum at θ=0 is gapped, non-degenerate, and trivial.) Due to the anomaly, the theory admits nontrivial domain walls supporting lower-dimensional theories. Depending on the nature of the vacuum at θ=π, several phase diagrams are possible. Assuming area law for space-like loops, one arrives at an inequality involving the temperatures at which CP and the center symmetry are restored. We also analyze alternative scenarios for SU(2) gauge theory. The underlying symmetry at θ=π is the dihedral group of 8 elements. If deconfined loops are allowed, one can have two O(2)-symmetric fixed points. It may also be that the four-dimensional theory around θ=π is gapless, e.g. a Coulomb phase could match the underlying anomalies.

Theta, time reversal and temperature

International Nuclear Information System (INIS)

Gaiotto, Davide; Kapustin, Anton; Komargodski, Zohar; Seiberg, Nathan

2017-01-01

SU(N) gauge theory is time reversal invariant at θ=0 and θ=π. We show that at θ=π there is a discrete ’t Hooft anomaly involving time reversal and the center symmetry. This anomaly leads to constraints on the vacua of the theory. It follows that at θ=π the vacuum cannot be a trivial non-degenerate gapped state. (By contrast, the vacuum at θ=0 is gapped, non-degenerate, and trivial.) Due to the anomaly, the theory admits nontrivial domain walls supporting lower-dimensional theories. Depending on the nature of the vacuum at θ=π, several phase diagrams are possible. Assuming area law for space-like loops, one arrives at an inequality involving the temperatures at which CP and the center symmetry are restored. We also analyze alternative scenarios for SU(2) gauge theory. The underlying symmetry at θ=π is the dihedral group of 8 elements. If deconfined loops are allowed, one can have two O(2)-symmetric fixed points. It may also be that the four-dimensional theory around θ=π is gapless, e.g. a Coulomb phase could match the underlying anomalies.

The stomatogastric nervous system as a model for studying sensorimotor interactions in real-time closed-loop conditions

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Nelly eDaur

2012-03-01

Full Text Available The perception of proprioceptive signals that report the internal state of the body is one of the essential tasks of the nervous system and helps to continuously adapt body movements to changing circumstances. Despite the impact of proprioceptive feedback on motor activity it has rarely been studied in conditions in which motor output and sensory activity interact as they do in behaving animals, i.e. in closed-loop conditions. The interaction of motor and sensory activities, however, can create emergent properties that may govern the functional characteristics of the system. We here demonstrate the use of a well-characterized model system for central pattern generation, the stomatogastric nervous system, for studying these properties in vitro. We created a real-time computer model of a single-cell muscle tendon organ in the gastric mill of the crab foregut that uses intracellular current injections to control the activity of the biological proprioceptor. The resulting motor output of a gastric mill motor neuron is then recorded intracellularly and fed into a simple muscle model consisting of a series of low-pass filters. The muscle output is used to activate a one-dimensional Hodgkin-Huxley type model of the muscle tendon organ in real-time, allowing closed-loop conditions. Model properties were either hand-tuned to achieve the best match with data from semi-intact muscle preparations, or an exhaustive search was performed to determine the best set of parameters. We report the real-time capabilities of our models, its performance and its interaction with the biological motor system.

Genetic instability associated with loop or stem–loop structures within transcription units can be independent of nucleotide excision repair

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Burns, John A; Chowdhury, Moinuddin A; Cartularo, Laura; Berens, Christian; Scicchitano, David A

2018-01-01

Abstract Simple sequence repeats (SSRs) are found throughout the genome, and under some conditions can change in length over time. Germline and somatic expansions of trinucleotide repeats are associated with a series of severely disabling illnesses, including Huntington's disease. The underlying mechanisms that effect SSR expansions and contractions have been experimentally elusive, but models suggesting a role for DNA repair have been proposed, in particular the involvement of transcription-coupled nucleotide excision repair (TCNER) that removes transcription-blocking DNA damage from the transcribed strand of actively expressed genes. If the formation of secondary DNA structures that are associated with SSRs were to block RNA polymerase progression, TCNER could be activated, resulting in the removal of the aberrant structure and a concomitant change in the region's length. To test this, TCNER activity in primary human fibroblasts was assessed on defined DNA substrates containing extrahelical DNA loops that lack discernible internal base pairs or DNA stem–loops that contain base pairs within the stem. The results show that both structures impede transcription elongation, but there is no corresponding evidence that nucleotide excision repair (NER) or TCNER operates to remove them. PMID:29474673

Practical Considerations regarding Implementation of Wind Power Applications into Real-Time Hardware-In-The-Loop Framework

DEFF Research Database (Denmark)

Petersen, Lennart; Iov, Florin

2017-01-01

, where the focus is laid on the model development in a real-time simulator. It enables to verify the functionality of developed controls, which is one of the research priorities due to the increased complexity of large wind power plants requiring high level of com-munication between plant control......This paper addresses the system implementation of voltage control architecture in wind power plants into a Real-Time Hardware-In-The-Loop framework. The increasing amount of wind power penetration into the power systems has en-gaged the wind power plants to take over the responsibility for adequate...... control of the node voltages, which has previ-ously been accomplished by conventional generation. Voltage support at the point of common coupling is realized by an overall wind power plant controller which requires high-performance and robust control solution. In most cases the system including all...

An sRNA and Cold Shock Protein Homolog-Based Feedforward Loop Post-transcriptionally Controls Cell Cycle Master Regulator CtrA.

Science.gov (United States)

Robledo, Marta; Schlüter, Jan-Philip; Loehr, Lars O; Linne, Uwe; Albaum, Stefan P; Jiménez-Zurdo, José I; Becker, Anke

2018-01-01

Adjustment of cell cycle progression is crucial for bacterial survival and adaptation under adverse conditions. However, the understanding of modulation of cell cycle control in response to environmental changes is rather incomplete. In α-proteobacteria, the broadly conserved cell cycle master regulator CtrA underlies multiple levels of control, including coupling of cell cycle and cell differentiation. CtrA levels are known to be tightly controlled through diverse transcriptional and post-translational mechanisms. Here, small RNA (sRNA)-mediated post-transcriptional regulation is uncovered as an additional level of CtrA fine-tuning. Computational predictions as well as transcriptome and proteome studies consistently suggested targeting of ctrA and the putative cold shock chaperone cspA5 mRNAs by the trans- encoded sRNA ( trans- sRNA) GspR (formerly SmelC775) in several Sinorhizobium species. GspR strongly accumulated in the stationary growth phase, especially in minimal medium (MM) cultures. Lack of the gspR locus confers a fitness disadvantage in competition with the wild type, while its overproduction hampers cell growth, suggesting that this riboregulator interferes with cell cycle progression. An eGFP-based reporter in vivo assay, involving wild-type and mutant sRNA and mRNA pairs, experimentally confirmed GspR-dependent post-transcriptional down-regulation of ctrA and cspA5 expression, which most likely occurs through base-pairing to the respective mRNA. The energetically favored secondary structure of GspR is predicted to comprise three stem-loop domains, with stem-loop 1 and stem-loop 3 targeting ctrA and cspA5 mRNA, respectively. Moreover, this work reports evidence for post-transcriptional control of ctrA by CspA5. Thus, this regulation and GspR-mediated post-transcriptional repression of ctrA and cspA5 expression constitute a coherent feed-forward loop, which may enhance the negative effect of GspR on CtrA levels. This novel regulatory circuit involving

Rapid genome detection of Schmallenberg virus and bovine viral diarrhea virus by use of isothermal amplification methods and high-speed real-time reverse transcriptase PCR.

Science.gov (United States)

Aebischer, Andrea; Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

2014-06-01

Over the past few years, there has been an increasing demand for rapid and simple diagnostic tools that can be applied outside centralized laboratories by using transportable devices. In veterinary medicine, such mobile test systems would circumvent barriers associated with the transportation of samples and significantly reduce the time to diagnose important infectious animal diseases. Among a wide range of available technologies, high-speed real-time reverse transcriptase quantitative PCR (RT-qPCR) and the two isothermal amplification techniques loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) represent three promising candidates for integration into mobile pen-side tests. The aim of this study was to investigate the performance of these amplification strategies and to evaluate their suitability for field application. In order to enable a valid comparison, novel pathogen-specific assays have been developed for the detection of Schmallenberg virus and bovine viral diarrhea virus. The newly developed assays were evaluated in comparison with established standard RT-qPCR using samples from experimentally or field-infected animals. Even though all assays allowed detection of the target virus in less than 30 min, major differences were revealed concerning sensitivity, specificity, robustness, testing time, and complexity of assay design. These findings indicated that the success of an assay will depend on the integrated amplification technology. Therefore, the application-specific pros and cons of each method that were identified during this study provide very valuable insights for future development and optimization of pen-side tests. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism.

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Reilman, Ewoud; Mars, Ruben A T; van Dijl, Jan Maarten; Denham, Emma L

2014-10-01

Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR.

Science.gov (United States)

Sung, Heungsup; Yong, Dongeun; Ki, Chang Seok; Kim, Jae Seok; Seong, Moon Woo; Lee, Hyukmin; Kim, Mi Na

2016-09-01

Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both Phomogenizing sputum samples prior to RNA extraction.

Dynamic Effects of Topoisomerase I Inhibition on R-Loops and Short Transcripts at Active Promoters.

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Jessica Marinello

Full Text Available Topoisomerase I-DNA-cleavage complexes (Top1cc stabilized by camptothecin (CPT have specific effects at transcriptional levels. We recently reported that Top1cc increase antisense transcript (aRNAs levels at divergent CpG-island promoters and, transiently, DNA/RNA hybrids (R-loop in nuclear and mitochondrial genomes of colon cancer HCT116 cells. However, the relationship between R-loops and aRNAs was not established. Here, we show that aRNAs can form R-loops in N-TERA-2 cells under physiological conditions, and that promoter-associated R-loops are somewhat increased and extended in length immediately upon cell exposure to CPT. In contrast, persistent Top1ccs reduce the majority of R-loops suggesting that CPT-accumulated aRNAs are not commonly involved in R-loops. The enhancement of aRNAs by Top1ccs is present both in human colon cancer HCT116 cells and WI38 fibroblasts suggesting a common response of cancer and normal cells. Although Top1ccs lead to DSB and DDR kinases activation, we do not detect a dependence of aRNA accumulation on ATM or DNA-PK activation. However, we showed that the cell response to persistent Top1ccs can involve an impairment of aRNA turnover rather than a higher synthesis rate. Finally, a genome-wide analysis shows that persistent Top1ccs also determine an accumulation of sense transcripts at 5'-end gene regions suggesting an increased occurrence of truncated transcripts. Taken together, the results indicate that Top1 may regulate transcription initiation by modulating RNA polymerase-generated negative supercoils, which can in turn favor R-loop formation at promoters, and that transcript accumulation at TSS is a response to persistent transcriptional stress by Top1 poisoning.

Gibberellin-regulated gene in the basal region of rice leaf sheath encodes basic helix-loop-helix transcription factor.

Science.gov (United States)

Komatsu, Setsuko; Takasaki, Hironori

2009-07-01

Genes regulated by gibberellin (GA) during leaf sheath elongation in rice seedlings were identified using the transcriptome approach. mRNA from the basal regions of leaf sheaths treated with GA3 was analyzed by high-coverage gene expression profiling. 33,004 peaks were detected, and 30 transcripts showed significant changes in the presence of GA3. Among these, basic helix-loop-helix transcription factor (AK073385) was significantly upregulated. Quantitative PCR analysis confirmed that expression of AK073385 was controlled by GA3 in a time- and dose-dependent manner. Basic helix-loop-helix transcription factor (AK073385) is therefore involved in the regulation of gene expression by GA3.

Mediator and Cohesin Connect Gene Expression and Chromatin Architecture

Science.gov (United States)

Kagey, Michael H.; Newman, Jamie J.; Bilodeau, Steve; Zhan, Ye; Orlando, David A.; van Berkum, Nynke L.; Ebmeier, Christopher C.; Goossens, Jesse; Rahl, Peter B.; Levine, Stuart S.; Taatjes, Dylan J.; Dekker, Job; Young, Richard A.

2010-01-01

Summary Transcription factors control cell specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. We report here that Mediator and Cohesin physically and functionally connect the enhancers and core promoters of active genes in embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with Cohesin, which can form rings that connect two DNA segments. The Cohesin loading factor Nipbl is associated with Mediator/Cohesin complexes, providing a means to load Cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by Mediator and Cohesin. Mediator and Cohesin occupy different promoters in different cells, thus generating cell-type specific DNA loops linked to the gene expression program of each cell. PMID:20720539

Theory on the mechanism of distal action of transcription factors: looping of DNA versus tracking along DNA

Energy Technology Data Exchange (ETDEWEB)

Murugan, R, E-mail: rmurugan@gmail.co [Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036 (India)

2010-10-15

In this paper, we develop a theory on the mechanism of distal action of the transcription factors, which are bound at their respective cis-regulatory enhancer modules on the promoter-RNA polymerase II (PR) complexes to initiate the transcription event in eukaryotes. We consider both the looping and tracking modes of their distal communication and calculate the mean first passage time that is required for the distal interactions of the complex of enhancer and transcription factor with the PR via both these modes. We further investigate how this mean first passage time is dependent on the length of the DNA segment (L, base-pairs) that connects the cis-regulatory binding site and the respective promoter. When the radius of curvature of this connecting segment of DNA is R that was induced upon binding of the transcription factor at the cis-acting element and RNAPII at the promoter in cis-positions, our calculations indicate that the looping mode of distal action will dominate when L is such that L > 2{pi}R and the tracking mode of distal action will be favored when L < 2{pi}R. The time required for the distal action will be minimum when L = 2{pi}R where the typical value of R for the binding of histones will be R {approx} 16 bps and L {approx} 10{sup 2} bps. It seems that the free energy associated with the binding of the transcription factor with its cis-acting element and the distance of this cis-acting element from the corresponding promoter of the gene of interest is negatively correlated. Our results suggest that the looping and tracking modes of distal action are concurrently operating on the transcription activation and the physics that determines the timescales associated with the looping/tracking in the mechanism of action of these transcription factors on the initiation of the transcription event must put a selection pressure on the distribution of the distances of cis-regulatory modules from their respective promoters of the genes. The computational analysis

A simple and rapid identification method for newly emerged porcine Deltacoronavirus with loop-mediated isothermal amplification

Directory of Open Access Journals (Sweden)

Fanfan Zhang

2017-10-01

Full Text Available Abstract Background Porcine Deltacoronavirus (PDCoV is a newly emerged enteropathogenic coronavirus that causes diarrhea and mortality in neonatal piglets. PDCoV has spread to many countries around the world, leading to significant economic losses in the pork industry. Therefore, a rapid and sensitive method for detection of PDCoV in clinical samples is urgently needed. Results In this study, we developed a single-tube one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP assay specific for nucleocapsid gene to diagnose and monitor PDCoV infections. The detection limit of RT-LAMP assay was 1 × 101 copies of PDCoV, which was approximately 100-fold more sensitive than gel-based one-step reverse transcription polymerase chain reaction (RT-PCR. This assay could specifically amplify PDCoV and had no cross amplification with porcine epidemic diarrhea virus (PEDV, transmissible gastroenteritis virus (TGEV, porcine kobuvirus (PKoV, porcine astrovirus (PAstV, porcine reproductive and respiratory syndrome virus (PRRSV, classic swine fever virus (CSFV, and porcine circovirus type 2 (PCV2. By screening a panel of clinical specimens (N = 192, this method presented a similar sensitivity with nested RT-PCR and was 1–2 log more sensitive than conventional RT-PCR in detection of PDCoV. Conclusions The RT-LAMP assay established in this study is a potentially valuable tool, especially in low-resource laboratories and filed settings, for a rapid diagnosis, surveillance, and molecular epidemiology investigation of PDCoV infections. To the best of our knowledge, this is the first work for detection of newly emerged PDCoV with LAMP technology.

Comparison of multiplex reverse transcription-PCR-enzyme ...

African Journals Online (AJOL)

Comparison of multiplex reverse transcription-PCR-enzyme hybridization assay with immunofluorescence techniques for the detection of four viral respiratory pathogens in pediatric community acquired pneumonia.

Theory on the mechanism of distal action of transcription factors: looping of DNA versus tracking along DNA

International Nuclear Information System (INIS)

Murugan, R

2010-01-01

In this paper, we develop a theory on the mechanism of distal action of the transcription factors, which are bound at their respective cis-regulatory enhancer modules on the promoter-RNA polymerase II (PR) complexes to initiate the transcription event in eukaryotes. We consider both the looping and tracking modes of their distal communication and calculate the mean first passage time that is required for the distal interactions of the complex of enhancer and transcription factor with the PR via both these modes. We further investigate how this mean first passage time is dependent on the length of the DNA segment (L, base-pairs) that connects the cis-regulatory binding site and the respective promoter. When the radius of curvature of this connecting segment of DNA is R that was induced upon binding of the transcription factor at the cis-acting element and RNAPII at the promoter in cis-positions, our calculations indicate that the looping mode of distal action will dominate when L is such that L > 2πR and the tracking mode of distal action will be favored when L 2 bps. It seems that the free energy associated with the binding of the transcription factor with its cis-acting element and the distance of this cis-acting element from the corresponding promoter of the gene of interest is negatively correlated. Our results suggest that the looping and tracking modes of distal action are concurrently operating on the transcription activation and the physics that determines the timescales associated with the looping/tracking in the mechanism of action of these transcription factors on the initiation of the transcription event must put a selection pressure on the distribution of the distances of cis-regulatory modules from their respective promoters of the genes. The computational analysis of the upstream sequences of promoters of various genes in the human and mouse genomes for the presence of putative cis-regulatory elements for a set of known transcription factors using

Requirements for DNA strand transfer during reverse transcription in mutant HIV-1 virions

NARCIS (Netherlands)

Berkhout, B.; van Wamel, J.; Klaver, B.

1995-01-01

Retroviruses convert their RNA genome into a DNA form by means of reverse transcription. According to the current model of reverse transcription, two strand transfer reactions are needed to synthesize a full-length DNA genome. Because reverse transcription is initiated close to the 5' end of the RNA

Man-machine interface design of real-time hardware-in-loop simulation system for power regulation of nuclear heating reactor

International Nuclear Information System (INIS)

Ni Xiaoli; Huang Xiaojin; Dong Zhe

2009-01-01

It is necessary to set up real-time hardware-in-loop simulation system for power regulation of nuclear heating reactor (NHR) because it is used in the load following instance such as seawater desalination and energy source. As the experiment data are so large that it is hard to be real-time all in one computer and to save and show the data.With the distributed configuration, the system was set up having a legible and intuitionist man-machine interface, speeding the model calculation computer and meeting the requirements of power regulation of NHR. Screen clear and concise, easy command input and results output make the system easier to verify. (authors)

Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters.

Science.gov (United States)

Grunseich, Christopher; Wang, Isabel X; Watts, Jason A; Burdick, Joshua T; Guber, Robert D; Zhu, Zhengwei; Bruzel, Alan; Lanman, Tyler; Chen, Kelian; Schindler, Alice B; Edwards, Nancy; Ray-Chaudhury, Abhik; Yao, Jianhua; Lehky, Tanya; Piszczek, Grzegorz; Crain, Barbara; Fischbeck, Kenneth H; Cheung, Vivian G

2018-02-01

R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor β (TGF-β), is reduced; that then leads to the activation of the TGF-β pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Noise processing by microRNA-mediated circuits: The Incoherent Feed-Forward Loop, revisited

Directory of Open Access Journals (Sweden)

Silvia Grigolon

2016-04-01

Full Text Available The intrinsic stochasticity of gene expression is usually mitigated in higher eukaryotes by post-transcriptional regulation channels that stabilise the output layer, most notably protein levels. The discovery of small non-coding RNAs (miRNAs in specific motifs of the genetic regulatory network has led to identifying noise buffering as the possible key function they exert in regulation. Recent in vitro and in silico studies have corroborated this hypothesis. It is however also known that miRNA-mediated noise reduction is hampered by transcriptional bursting in simple topologies. Here, using stochastic simulations validated by analytical calculations based on van Kampen's expansion, we revisit the noise-buffering capacity of the miRNA-mediated Incoherent Feed Forward Loop (IFFL, a small module that is widespread in the gene regulatory networks of higher eukaryotes, in order to account for the effects of intermittency in the transcriptional activity of the modulator gene. We show that bursting considerably alters the circuit's ability to control static protein noise. By comparing with other regulatory architectures, we find that direct transcriptional regulation significantly outperforms the IFFL in a broad range of kinetic parameters. This suggests that, under pulsatile inputs, static noise reduction may be less important than dynamical aspects of noise and information processing in characterising the performance of regulatory elements.

Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

Science.gov (United States)

2016-09-07

sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

A Mirnov loop array for field-reversed configurations

International Nuclear Information System (INIS)

Tuszewski, M.

1990-01-01

An array of 64 magnetic pick-up loops has been used for stability studies of large field-reversed configurations in the FRX-C/LSM device. This array proved reliable, could resolve signals of a few Gauss, and allowed the detection of several plasma instabilities. 3 refs., 4 figs

RNA/DNA Hybrid Interactome Identifies DXH9 as a Molecular Player in Transcriptional Termination and R-Loop-Associated DNA Damage.

Science.gov (United States)

Cristini, Agnese; Groh, Matthias; Kristiansen, Maiken S; Gromak, Natalia

2018-05-08

R-loops comprise an RNA/DNA hybrid and displaced single-stranded DNA. They play important biological roles and are implicated in pathology. Even so, proteins recognizing these structures are largely undefined. Using affinity purification with the S9.6 antibody coupled to mass spectrometry, we defined the RNA/DNA hybrid interactome in HeLa cells. This consists of known R-loop-associated factors SRSF1, FACT, and Top1, and yet uncharacterized interactors, including helicases, RNA processing, DNA repair, and chromatin factors. We validate specific examples of these interactors and characterize their involvement in R-loop biology. A top candidate DHX9 helicase promotes R-loop suppression and transcriptional termination. DHX9 interacts with PARP1, and both proteins prevent R-loop-associated DNA damage. DHX9 and other interactome helicases are overexpressed in cancer, linking R-loop-mediated DNA damage and disease. Our RNA/DNA hybrid interactome provides a powerful resource to study R-loop biology in health and disease. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

HIV-1 reverse transcription initiation: a potential target for novel antivirals?

NARCIS (Netherlands)

Abbink, Truus E. M.; Berkhout, Ben

2008-01-01

Reverse transcription is an essential step in the retroviral life cycle, as it converts the genomic RNA into DNA. In this review, we describe recent developments concerning the initiation step of this complex, multi-step reaction. During initiation of reverse transcription, a cellular tRNA primer is

Implementation of a Serial Delay Insertion Type Loop Communication for a Real Time Multitransputer System.

Science.gov (United States)

1985-06-01

just pass the message WAIT NOW AFTER Rlock + timeo t -- if time is out write.screen( TIME IS OUT") MAIN PROGRAM* CHAN linki , link2, link3, link4...PAR D.I.Loop.Interface (link4, linkl,) D.I.Loop.Interface ( linki , link2, 2) D.I.Loop.Interface (link2, link3, 3) JD.I Loop.Interface (link3, link4, 4

The transcription factor Nerfin-1 prevents reversion of neurons into neural stem cells.

Science.gov (United States)

Froldi, Francesca; Szuperak, Milan; Weng, Chen-Fang; Shi, Wei; Papenfuss, Anthony T; Cheng, Louise Y

2015-01-15

Cellular dedifferentiation is the regression of a cell from a specialized state to a more multipotent state and is implicated in cancer. However, the transcriptional network that prevents differentiated cells from reacquiring stem cell fate is so far unclear. Neuroblasts (NBs), the Drosophila neural stem cells, are a model for the regulation of stem cell self-renewal and differentiation. Here we show that the Drosophila zinc finger transcription factor Nervous fingers 1 (Nerfin-1) locks neurons into differentiation, preventing their reversion into NBs. Following Prospero-dependent neuronal specification in the ganglion mother cell (GMC), a Nerfin-1-specific transcriptional program maintains differentiation in the post-mitotic neurons. The loss of Nerfin-1 causes reversion to multipotency and results in tumors in several neural lineages. Both the onset and rate of neuronal dedifferentiation in nerfin-1 mutant lineages are dependent on Myc- and target of rapamycin (Tor)-mediated cellular growth. In addition, Nerfin-1 is required for NB differentiation at the end of neurogenesis. RNA sequencing (RNA-seq) and chromatin immunoprecipitation (ChIP) analysis show that Nerfin-1 administers its function by repression of self-renewing-specific and activation of differentiation-specific genes. Our findings support the model of bidirectional interconvertibility between neural stem cells and their post-mitotic progeny and highlight the importance of the Nerfin-1-regulated transcriptional program in neuronal maintenance. © 2015 Froldi et al.; Published by Cold Spring Harbor Laboratory Press.

The Mediator complex and transcription regulation

Science.gov (United States)

Poss, Zachary C.; Ebmeier, Christopher C.

2013-01-01

The Mediator complex is a multi-subunit assembly that appears to be required for regulating expression of most RNA polymerase II (pol II) transcripts, which include protein-coding and most non-coding RNA genes. Mediator and pol II function within the pre-initiation complex (PIC), which consists of Mediator, pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH and is approximately 4.0 MDa in size. Mediator serves as a central scaffold within the PIC and helps regulate pol II activity in ways that remain poorly understood. Mediator is also generally targeted by sequence-specific, DNA-binding transcription factors (TFs) that work to control gene expression programs in response to developmental or environmental cues. At a basic level, Mediator functions by relaying signals from TFs directly to the pol II enzyme, thereby facilitating TF-dependent regulation of gene expression. Thus, Mediator is essential for converting biological inputs (communicated by TFs) to physiological responses (via changes in gene expression). In this review, we summarize an expansive body of research on the Mediator complex, with an emphasis on yeast and mammalian complexes. We focus on the basics that underlie Mediator function, such as its structure and subunit composition, and describe its broad regulatory influence on gene expression, ranging from chromatin architecture to transcription initiation and elongation, to mRNA processing. We also describe factors that influence Mediator structure and activity, including TFs, non-coding RNAs and the CDK8 module. PMID:24088064

MiR-192-Mediated Positive Feedback Loop Controls the Robustness of Stress-Induced p53 Oscillations in Breast Cancer Cells.

Directory of Open Access Journals (Sweden)

Richard Moore

2015-12-01

Full Text Available The p53 tumor suppressor protein plays a critical role in cellular stress and cancer prevention. A number of post-transcriptional regulators, termed microRNAs, are closely connected with the p53-mediated cellular networks. While the molecular interactions among p53 and microRNAs have emerged, a systems-level understanding of the regulatory mechanism and the role of microRNAs-forming feedback loops with the p53 core remains elusive. Here we have identified from literature that there exist three classes of microRNA-mediated feedback loops revolving around p53, all with the nature of positive feedback coincidentally. To explore the relationship between the cellular performance of p53 with the microRNA feedback pathways, we developed a mathematical model of the core p53-MDM2 module coupled with three microRNA-mediated positive feedback loops involving miR-192, miR-34a, and miR-29a. Simulations and bifurcation analysis in relationship to extrinsic noise reproduce the oscillatory behavior of p53 under DNA damage in single cells, and notably show that specific microRNA abrogation can disrupt the wild-type cellular phenotype when the ubiquitous cell-to-cell variability is taken into account. To assess these in silico results we conducted microRNA-perturbation experiments in MCF7 breast cancer cells. Time-lapse microscopy of cell-population behavior in response to DNA double-strand breaks, together with image classification of single-cell phenotypes across a population, confirmed that the cellular p53 oscillations are compromised after miR-192 perturbations, matching well with the model predictions. Our study via modeling in combination with quantitative experiments provides new evidence on the role of microRNA-mediated positive feedback loops in conferring robustness to the system performance of stress-induced response of p53.

Underwater Time Service and Synchronization Based on Time Reversal Technique

Science.gov (United States)

Lu, Hao; Wang, Hai-bin; Aissa-El-Bey, Abdeldjalil; Pyndiah, Ramesh

2010-09-01

Real time service and synchronization are very important to many underwater systems. But the time service and synchronization in existence cannot work well due to the multi-path propagation and random phase fluctuation of signals in the ocean channel. The time reversal mirror technique can realize energy concentration through self-matching of the ocean channel and has very good spatial and temporal focusing properties. Based on the TRM technique, we present the Time Reversal Mirror Real Time service and synchronization (TRMRT) method which can bypass the processing of multi-path on the server side and reduce multi-path contamination on the client side. So TRMRT can improve the accuracy of time service. Furthermore, as an efficient and precise method of time service, TRMRT could be widely used in underwater exploration activities and underwater navigation and positioning systems.

Regulation of TCF ETS-domain transcription factors by helix-loop-helix motifs.

Science.gov (United States)

Stinson, Julie; Inoue, Toshiaki; Yates, Paula; Clancy, Anne; Norton, John D; Sharrocks, Andrew D

2003-08-15

DNA binding by the ternary complex factor (TCF) subfamily of ETS-domain transcription factors is tightly regulated by intramolecular and intermolecular interactions. The helix-loop-helix (HLH)-containing Id proteins are trans-acting negative regulators of DNA binding by the TCFs. In the TCF, SAP-2/Net/ERP, intramolecular inhibition of DNA binding is promoted by the cis-acting NID region that also contains an HLH-like motif. The NID also acts as a transcriptional repression domain. Here, we have studied the role of HLH motifs in regulating DNA binding and transcription by the TCF protein SAP-1 and how Cdk-mediated phosphorylation affects the inhibitory activity of the Id proteins towards the TCFs. We demonstrate that the NID region of SAP-1 is an autoinhibitory motif that acts to inhibit DNA binding and also functions as a transcription repression domain. This region can be functionally replaced by fusion of Id proteins to SAP-1, whereby the Id moiety then acts to repress DNA binding in cis. Phosphorylation of the Ids by cyclin-Cdk complexes results in reduction in protein-protein interactions between the Ids and TCFs and relief of their DNA-binding inhibitory activity. In revealing distinct mechanisms through which HLH motifs modulate the activity of TCFs, our results therefore provide further insight into the role of HLH motifs in regulating TCF function and how the inhibitory properties of the trans-acting Id HLH proteins are themselves regulated by phosphorylation.

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of common genetically modified organisms (GMOs).

Science.gov (United States)

Feng, Jiawang; Tang, Shiming; Liu, Lideng; Kuang, Xiaoshan; Wang, Xiaoyu; Hu, Songnan; You, Shuzhu

2015-03-01

Here, we developed a loop-mediated isothermal amplification (LAMP) assay for 11 common transgenic target DNA in GMOs. Six sets of LAMP primer candidates for each target were designed and their specificity, sensitivity, and reproductivity were evaluated. With the optimized LAMP primers, this LAMP assay was simply run within 45-60 min to detect all these targets in GMOs tested. The sensitivity, specificity, and reproductivity of the LAMP assay were further analyzed in comparison with those of Real-Time PCR. In consistent with real-time PCR, detection of 0.5% GMOs in equivalent background DNA was possible using this LAMP assay for all targets. In comparison with real-time PCR, the LAMP assay showed the same results with simple instruments. Hence, the LAMP assay developed can provide a rapid and simple approach for routine screening as well as specific events detection of many GMOs.

Coupling between feedback loops in autoregulatory networks affects bistability range, open-loop gain and switching times

International Nuclear Information System (INIS)

Tiwari, Abhinav; Igoshin, Oleg A

2012-01-01

Biochemical regulatory networks governing diverse cellular processes such as stress-response, differentiation and cell cycle often contain coupled feedback loops. We aim at understanding how features of feedback architecture, such as the number of loops, the sign of the loops and the type of their coupling, affect network dynamical performance. Specifically, we investigate how bistability range, maximum open-loop gain and switching times of a network with transcriptional positive feedback are affected by additive or multiplicative coupling with another positive- or negative-feedback loop. We show that a network's bistability range is positively correlated with its maximum open-loop gain and that both quantities depend on the sign of the feedback loops and the type of feedback coupling. Moreover, we find that the addition of positive feedback could decrease the bistability range if we control the basal level in the signal-response curves of the two systems. Furthermore, the addition of negative feedback has the capacity to increase the bistability range if its dissociation constant is much lower than that of the positive feedback. We also find that the addition of a positive feedback to a bistable network increases the robustness of its bistability range, whereas the addition of a negative feedback decreases it. Finally, we show that the switching time for a transition from a high to a low steady state increases with the effective fold change in gene regulation. In summary, we show that the effect of coupled feedback loops on the bistability range and switching times depends on the underlying mechanistic details. (paper)

A multimodal instrument for real-time in situ study of ultrasound and cavitation mediated drug delivery.

Science.gov (United States)

Bian, Shuning; Seth, Anjali; Daly, Dan; Carlisle, Robert; Stride, Eleanor

2017-03-01

The development of a multimodal instrument capable of real-time in situ measurements of cavitation activity and effect in tissue mimicking phantoms during ultrasound and cavitation mediated drug delivery experiments is described here. The instrument features an acoustic arm that can expose phantoms to high-intensity focused-ultrasound while measuring cavitation activity and an optical arm that monitors cavitation effect using confocal microscopy. This combination of modalities allows real-time in situ characterisation of drug delivery in tissue and tissue mimicking phantoms during ultrasound and cavitation mediated drug delivery experiments. A representative result, obtained with a tissue mimicking phantom and acoustically activated droplets, is presented here as a demonstration of the instrument's capabilities and potential applications.

Real-time zymography and reverse zymography: a method for detecting activities of matrix metalloproteinases and their inhibitors using FITC-labeled collagen and casein as substrates.

Science.gov (United States)

Hattori, Shunji; Fujisaki, Hitomi; Kiriyama, Tomomi; Yokoyama, Tsukao; Irie, Shinkichi

2002-02-01

Zymography and reverse zymography are widely used techniques for identifying the proteolytic activity of enzymes and the presence of protease inhibitors in polyacrylamide gels. In the current studies, we utilized a fluorescein-isothiocyanate-labeled substrate to develop novel zymographic and reverse zymographic methods for detecting matrix metalloproteinases and tissue inhibitors of the metalloproteinases, respectively. Using a transilluminator, the results can be observed visually without stopping the enzymatic reaction. For this reason, we have named these methods real-time zymography and real-time reverse zymography. These methods have the following advantages compared with conventional protocols: (1) because the reaction can be repeatedly monitored on the polyacrylamide gels, optimization of the incubation time can be achieved without preliminary analyses; (2) higher sensitivity is achieved with a lower amount of substrate than with conventional methods; (3) a semi-quantitative analysis of matrix metalloproteinases is possible. An additional advantage of the real-time reverse zymography is that, because the fluorescence detection is specific for substrate digestion, the inhibitor bands can be easily distinguished from contaminating proteins.

Closed-Loop Real-Time Imaging Enables Fully Automated Cell-Targeted Patch-Clamp Neural Recording In Vivo.

Science.gov (United States)

Suk, Ho-Jun; van Welie, Ingrid; Kodandaramaiah, Suhasa B; Allen, Brian; Forest, Craig R; Boyden, Edward S

2017-08-30

Targeted patch-clamp recording is a powerful method for characterizing visually identified cells in intact neural circuits, but it requires skill to perform. We previously developed an algorithm that automates "blind" patching in vivo, but full automation of visually guided, targeted in vivo patching has not been demonstrated, with currently available approaches requiring human intervention to compensate for cell movement as a patch pipette approaches a targeted neuron. Here we present a closed-loop real-time imaging strategy that automatically compensates for cell movement by tracking cell position and adjusting pipette motion while approaching a target. We demonstrate our system's ability to adaptively patch, under continuous two-photon imaging and real-time analysis, fluorophore-expressing neurons of multiple types in the living mouse cortex, without human intervention, with yields comparable to skilled human experimenters. Our "imagepatching" robot is easy to implement and will help enable scalable characterization of identified cell types in intact neural circuits. Copyright © 2017 Elsevier Inc. All rights reserved.

Auxin-dependent compositional change in Mediator in ARF7- and ARF19-mediated transcription.

Science.gov (United States)

Ito, Jun; Fukaki, Hidehiro; Onoda, Makoto; Li, Lin; Li, Chuanyou; Tasaka, Masao; Furutani, Masahiko

2016-06-07

Mediator is a multiprotein complex that integrates the signals from transcription factors binding to the promoter and transmits them to achieve gene transcription. The subunits of Mediator complex reside in four modules: the head, middle, tail, and dissociable CDK8 kinase module (CKM). The head, middle, and tail modules form the core Mediator complex, and the association of CKM can modify the function of Mediator in transcription. Here, we show genetic and biochemical evidence that CKM-associated Mediator transmits auxin-dependent transcriptional repression in lateral root (LR) formation. The AUXIN/INDOLE 3-ACETIC ACID 14 (Aux/IAA14) transcriptional repressor inhibits the transcriptional activity of its binding partners AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 by making a complex with the CKM-associated Mediator. In addition, TOPLESS (TPL), a transcriptional corepressor, forms a bridge between IAA14 and the CKM component MED13 through the physical interaction. ChIP assays show that auxin induces the dissociation of MED13 but not the tail module component MED25 from the ARF7 binding region upstream of its target gene. These findings indicate that auxin-induced degradation of IAA14 changes the module composition of Mediator interacting with ARF7 and ARF19 in the upstream region of their target genes involved in LR formation. We suggest that this regulation leads to a quick switch of signal transmission from ARFs to target gene expression in response to auxin.

Strand transfer and elongation of HIV-1 reverse transcription is facilitated by cell factors in vitro.

Directory of Open Access Journals (Sweden)

David Warrilow

Full Text Available Recent work suggests a role for multiple host factors in facilitating HIV-1 reverse transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 reverse transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor reverse transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of reverse transcription. Instead, the cell factor(s enhanced first strand transfer and synthesis of late reverse transcription suggesting it stabilized the reverse transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous reverse transcription (ERT system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded reverse transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s suggesting a lack of involvement of the capsid in the in vitro assay. In addition, reverse transcription products were found to be resistant to exogenous DNase I activity when the active fraction was present in the ERT assay. These results indicate that the cell factor(s may improve reverse transcription by facilitating DNA strand transfer and DNA synthesis. It also had a protective function for the reverse transcription products, but it is unclear if this is related to improved DNA synthesis.

Real-Time Hardware-in-the-Loop Laboratory Testing for Multisensor Sense and Avoid Systems

Directory of Open Access Journals (Sweden)

Giancarmine Fasano

2013-01-01

Full Text Available This paper focuses on a hardware-in-the-loop facility aimed at real-time testing of architectures and algorithms of multisensor sense and avoid systems. It was developed within a research project aimed at flight demonstration of autonomous non-cooperative collision avoidance for Unmanned Aircraft Systems. In this framework, an optionally piloted Very Light Aircraft was used as experimental platform. The flight system is based on multiple-sensor data integration and it includes a Ka-band radar, four electro-optical sensors, and two dedicated processing units. The laboratory test system was developed with the primary aim of prototype validation before multi-sensor tracking and collision avoidance flight tests. System concept, hardware/software components, and operating modes are described in the paper. The facility has been built with a modular approach including both flight hardware and simulated systems and can work on the basis of experimentally tested or synthetically generated scenarios. Indeed, hybrid operating modes are also foreseen which enable performance assessment also in the case of alternative sensing architectures and flight scenarios that are hardly reproducible during flight tests. Real-time multisensor tracking results based on flight data are reported, which demonstrate reliability of the laboratory simulation while also showing the effectiveness of radar/electro-optical fusion in a non-cooperative collision avoidance architecture.

Associated technologies ensures complete loop mediated ...

African Journals Online (AJOL)

Loop Mediated Isothermal Amplification (LAMP) assay could be a useful adjunct diagnostic assay along with the conventional methods that would preclude the requirement of continuous maintenance of pure cultures. Moreover, LAMP assay is simple, rapid, specific and sensitive for the detection of pathogens. Having ...

A real-time evaluation and demonstration of strategies for 'Over-The-Loop' ensemble streamflow forecasting in US watersheds

Science.gov (United States)

Wood, Andy; Clark, Elizabeth; Mendoza, Pablo; Nijssen, Bart; Newman, Andy; Clark, Martyn; Nowak, Kenneth; Arnold, Jeffrey

2017-04-01

' (SHARP) to implement, assess and demonstrate real-time over-the-loop ensemble flow forecasts in a range of US watersheds. The system relies on fully ensemble techniques, including: an 100-member ensemble of meteorological model forcings and an ensemble particle filter data assimilation for initializing watershed states; analog/regression-based downscaling of ensemble weather forecasts from GEFS; and statistical post-processing of ensemble forecast outputs, all of which run in real-time within a workflow managed by ECWMF's ecFlow libraries over large US regional domains. We describe SHARP and present early hindcast and verification results for short to seasonal range streamflow forecasts in a number of US case study watersheds.

Development of real-time reverse transcription polymerase chain reaction assays to quantify insulin-like growth factor receptor and insulin receptor expression in equine tissue

Directory of Open Access Journals (Sweden)

Stephen B. Hughes

2013-08-01

Full Text Available The insulin-like growth factor system (insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor and six insulin-like growth factor-binding proteins and insulin are essential to muscle metabolism and most aspects of male and female reproduction. Insulin-like growth factor and insulin play important roles in the regulation of cell growth, differentiation and the maintenance of cell differentiation in mammals. In order to better understand the local factors that regulate equine physiology, such as muscle metabolism and reproduction (e.g., germ cell development and fertilisation, real-time reverse transcription polymerase chain reaction assays for quantification of equine insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were developed. The assays were sensitive: 192 copies/µLand 891 copies/µL for insulin-like growth factor 1 receptor, messenger ribonucleic acid and insulin receptor respectively (95%limit of detection, and efficient: 1.01 for the insulin-like growth factor 1 receptor assay and 0.95 for the insulin receptor assay. The assays had a broad linear range of detection (seven logs for insulin-like growth factor 1 receptor and six logs for insulin receptor. This allowed for analysis of very small amounts of messenger ribonucleic acid. Low concentrations of both insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were detected in endometrium, lung and spleen samples, whilst high concentrations were detected in heart, muscle and kidney samples, this was most likely due to the high level of glucose metabolism and glucose utilisation by these tissues. The assays developed for insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid expression have been shown to work on equine tissue and will contribute to the understanding of insulin and insulin-like growth factor 1

The Mediator Complex: At the Nexus of RNA Polymerase II Transcription.

Science.gov (United States)

Jeronimo, Célia; Robert, François

2017-10-01

Mediator is an essential, large, multisubunit, transcriptional co-activator highly conserved across eukaryotes. Mediator interacts with gene-specific transcription factors at enhancers as well as with the RNA polymerase II (RNAPII) transcription machinery bound at promoters. It also interacts with several other factors involved in various aspects of transcription, chromatin regulation, and mRNA processing. Hence, Mediator is at the nexus of RNAPII transcription, regulating its many steps and connecting transcription with co-transcriptional events. To achieve this flexible role, Mediator, which is divided into several functional modules, reorganizes its conformation and composition while making transient contacts with other components. Here, we review the mechanisms of action of Mediator and propose a unifying model for its function. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quality Management in Reverse Logistics A Broad Look on Quality Issues and Their Interaction with Closed-Loop Supply Chains

CERN Document Server

2013-01-01

The concepts of Quality Management (QM) have been used for a long time by manufacturing companies to ensure customers receive a constant and high quality level of products. But what happens with remanufactured or refurbished products?   Quality Management in Reverse Logistics: A Broad Look on Quality Issues and Their Interaction with Closed-Loop Supply Chains discusses the new, promising area of QM and Reverse Logistics interaction. More specifically, it examines and evaluates a number of QM tools and techniques, which can be applied in practice in order to understand, review and improve any closed-loop supply chain process.   Modeling and quantitative methods that can be used by students, practitioners and academics in the Reverse Logistics industry are described. The readers will broaden their scope of thinking in the area of Reverse Logistics, where Quality Management can be applied.

Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search.

Science.gov (United States)

Piller, Nicolas; Decosterd, Isabelle; Suter, Marc R

2013-07-10

The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated.We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system - specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model. We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process

A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus

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Aline Lavado Tolardo

2016-06-01

Full Text Available Vesiculoviruses (VSV are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections.

Extensive Mutagenesis of the Conserved Box E Motif in Duck Hepatitis B Virus P Protein Reveals Multiple Functions in Replication and a Common Structure with the Primer Grip in HIV-1 Reverse Transcriptase

OpenAIRE

Wang, Yong-Xiang; Luo, Cheng; Zhao, Dan; Beck, Jürgen; Nassal, Michael

2012-01-01

Hepadnaviruses, including the pathogenic hepatitis B virus (HBV), replicate their small DNA genomes through protein-primed reverse transcription, mediated by the terminal protein (TP) domain in their P proteins and an RNA stem-loop, ϵ, on the pregenomic RNA (pgRNA). No direct structural data are available for P proteins, but their reverse transcriptase (RT) domains contain motifs that are conserved in all RTs (box A to box G), implying a similar architecture; however, experimental support for...

Mediator complex cooperatively regulates transcription of retinoic acid target genes with Polycomb Repressive Complex 2 during neuronal differentiation.

Science.gov (United States)

Fukasawa, Rikiya; Iida, Satoshi; Tsutsui, Taiki; Hirose, Yutaka; Ohkuma, Yoshiaki

2015-11-01

The Mediator complex (Mediator) plays key roles in transcription and functions as the nexus for integration of various transcriptional signals. Previously, we screened for Mediator cyclin-dependent kinase (CDK)-interacting factors and identified three proteins related to chromatin regulation. One of them, SUZ12 is required for both stability and activity of Polycomb Repressive Complex 2 (PRC2). PRC2 primarily suppresses gene expression through histone H3 lysine 27 trimethylation, resulting in stem cell maintenance and differentiation; perturbation of this process leads to oncogenesis. Recent work showed that Mediator contributes to the embryonic stem cell state through DNA loop formation, which is strongly associated with chromatin architecture; however, it remains unclear how Mediator regulates gene expression in cooperation with chromatin regulators (i.e. writers, readers and remodelers). We found that Mediator CDKs interact directly with the PRC2 subunit EZH2, as well as SUZ12. Known PRC2 target genes were deregulated by Mediator CDK knockdown during neuronal differentiation, and both Mediator and PRC2 complexes co-occupied the promoters of developmental genes regulated by retinoic acid. Our results provide a mechanistic link between Mediator and PRC2 during neuronal differentiation. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

QUANTIFICATION OF ENTEROVIRUS AND HEPATITIS A VIRUSES IN WELLS AND SPRINGS IN EAST TENNESSEE USING REAL-TIME REVERSE TRANSCIPTION PCR

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This project involves development, validation testing and application of a fast, efficient method of quantitatively measuring occurrence and concentration of common human viral pathogens, enterovirus and hepatitis A virus, in ground water samples using real-time reverse transcrip...

VERSE - Virtual Equivalent Real-time Simulation

Science.gov (United States)

Zheng, Yang; Martin, Bryan J.; Villaume, Nathaniel

2005-01-01

Distributed real-time simulations provide important timing validation and hardware in the- loop results for the spacecraft flight software development cycle. Occasionally, the need for higher fidelity modeling and more comprehensive debugging capabilities - combined with a limited amount of computational resources - calls for a non real-time simulation environment that mimics the real-time environment. By creating a non real-time environment that accommodates simulations and flight software designed for a multi-CPU real-time system, we can save development time, cut mission costs, and reduce the likelihood of errors. This paper presents such a solution: Virtual Equivalent Real-time Simulation Environment (VERSE). VERSE turns the real-time operating system RTAI (Real-time Application Interface) into an event driven simulator that runs in virtual real time. Designed to keep the original RTAI architecture as intact as possible, and therefore inheriting RTAI's many capabilities, VERSE was implemented with remarkably little change to the RTAI source code. This small footprint together with use of the same API allows users to easily run the same application in both real-time and virtual time environments. VERSE has been used to build a workstation testbed for NASA's Space Interferometry Mission (SIM PlanetQuest) instrument flight software. With its flexible simulation controls and inexpensive setup and replication costs, VERSE will become an invaluable tool in future mission development.

Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction.

Science.gov (United States)

Grau, Frederic R; Schroeder, Megan E; Mulhern, Erin L; McIntosh, Michael T; Bounpheng, Mangkey A

2015-03-01

African swine fever (ASF), classical swine fever (CSF), and foot-and-mouth disease (FMD) are highly contagious animal diseases of significant economic importance. Pigs infected with ASF and CSF viruses (ASFV and CSFV) develop clinical signs that may be indistinguishable from other diseases. Likewise, various causes of vesicular disease can mimic clinical signs caused by the FMD virus (FMDV). Early detection is critical to limiting the impact and spread of these disease outbreaks, and the ability to perform herd-level surveillance for all 3 diseases rapidly and cost effectively using a single diagnostic sample and test is highly desirable. This study assessed the feasibility of simultaneous ASFV, CSFV, and FMDV detection by multiplex reverse transcription real-time polymerase chain reaction (mRT-qPCR) in swine oral fluids collected through the use of chewing ropes. Animal groups were experimentally infected independently with each virus, observed for clinical signs, and oral fluids collected and tested throughout the course of infection. All animal groups chewed on the ropes readily before and after onset of clinical signs and before onset of lameness or serious clinical signs. ASFV was detected as early as 3 days postinoculation (dpi), 2-3 days before onset of clinical disease; CSFV was detected at 5 dpi, coincident with onset of clinical disease; and FMDV was detected as early as 1 dpi, 1 day before the onset of clinical disease. Equivalent results were observed in 4 independent studies and demonstrate the feasibility of oral fluids and mRT-qPCR for surveillance of ASF, CSF, and FMD in swine populations. © 2015 The Author(s).

Demonstration of a very inexpensive, turbidimetric, real-time, RT-LAMP detection platform using shrimp Laem-Singh virus (LSNV as a model.

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Narong Arunrut

Full Text Available Rapid and accurate detection of pathogens under field laboratory conditions is necessary for effective control of veterinary pathogens. Here we describe a prototype, portable, pathogen detection device developed for single tube, real-time, reverse transcription, loop-mediated isothermal amplification (RT-LAMP using Laem-Singh virus (LSNV as a model. LSNV is an RNA virus and a component cause of growth retardation in black tiger shrimp. We chose its RNA-dependent RNA polymerase (RdRp gene as the target for our tests. The basis for detection was measurement of turbidity arising from formation of a white, insoluble magnesium pyrophosphate precipitate byproduct upon amplification of the RdRp target sequence from 100 ng template RNA extracted from shrimp. The measurement device consisted of a heating block to maintain constant temperature in the RT-LAMP reaction for 8 Eppindorf sample tubes, a light-emitting diode (LED light source providing red light emission at 650 nm wavelength to pass through sample tubes, a light dependent resistance (LDR photo-detector and a software program to report turbidity events and could potentially be marketed for under US$3000. The device was connected to a computer to display real-time results in a variety of formats. The optimized protocol for LSNV detection consisted of incubation of the sample tubes at 65 °C for 1 h during which turbidity was continuously measured, and quantitative results could be obtained by reaction time measurement. The sensitivity of detection was comparable to that of conventional nested RT-PCR and there was no cross reaction with other common shrimp viruses. The device was used for quantitative measurement of relative copy numbers of LSNV RdRp in 8 shrimp tissues and they were found to be highest in the gills followed in order by the lymphoid organ and hemolymph (p ≤ 0.05. This platform can be easily adapted for detection of other pathogens under field laboratory settings.

Molecular diagnostics for human leptospirosis.

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Waggoner, Jesse J; Pinsky, Benjamin A

2016-10-01

The definitive diagnosis of leptospirosis, which results from infection with spirochetes of the genus Leptospira, currently relies on the use of culture, serological testing (microscopic agglutination testing), and molecular detection. The purpose of this review is to describe new molecular diagnostics for Leptospira and discuss advancements in the use of available methods. Efforts have been focused on improving the clinical sensitivity of Leptospira detection using molecular methods. In this review, we describe a reoptimized pathogenic species-specific real-time PCR (targeting lipL32) that has demonstrated improved sensitivity, findings by two groups that real-time reverse-transcription PCR assays targeting the 16S rrs gene can improve detection, and two new loop-mediated amplification techniques. Quantitation of leptospiremia, detection in different specimen types, and the complementary roles played by molecular detection and microscopic agglutination testing will be discussed. Finally, a protocol for Leptospira strain subtyping using variable number tandem repeat targets and high-resolution melting will be described. Molecular diagnostics have an established role for the diagnosis of leptospirosis and provide an actionable diagnosis in the acute setting. The use of real-time reverse-transcription PCR for testing serum/plasma and cerebrospinal fluid, when available, may improve the detection of Leptospira without decreasing clinical specificity.

Real-Time and Real-Fast Performance of General-Purpose and Real-Time Operating Systems in Multithreaded Physical Simulation of Complex Mechanical Systems

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Carlos Garre

2014-01-01

Full Text Available Physical simulation is a valuable tool in many fields of engineering for the tasks of design, prototyping, and testing. General-purpose operating systems (GPOS are designed for real-fast tasks, such as offline simulation of complex physical models that should finish as soon as possible. Interfacing hardware at a given rate (as in a hardware-in-the-loop test requires instead maximizing time determinism, for which real-time operating systems (RTOS are designed. In this paper, real-fast and real-time performance of RTOS and GPOS are compared when simulating models of high complexity with large time steps. This type of applications is usually present in the automotive industry and requires a good trade-off between real-fast and real-time performance. The performance of an RTOS and a GPOS is compared by running a tire model scalable on the number of degrees-of-freedom and parallel threads. The benchmark shows that the GPOS present better performance in real-fast runs but worse in real-time due to nonexplicit task switches and to the latency associated with interprocess communication (IPC and task switch.

Evaluation of the expression of internal control transcripts by real ...

African Journals Online (AJOL)

Evaluation of the expression of internal control transcripts by real-time RT-PCR analysis during tomato flower abscission. Song Gao, Tao Xu, Mingfang Qi, Yufeng Liu, Hong Li, Shuangshuang Lv, Jinhong Li, Tianlai Li ...

Plant Mediator complex and its critical functions in transcription regulation.

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Yang, Yan; Li, Ling; Qu, Li-Jia

2016-02-01

The Mediator complex is an important component of the eukaryotic transcriptional machinery. As an essential link between transcription factors and RNA polymerase II, the Mediator complex transduces diverse signals to genes involved in different pathways. The plant Mediator complex was recently purified and comprises conserved and specific subunits. It functions in concert with transcription factors to modulate various responses. In this review, we summarize the recent advances in understanding the plant Mediator complex and its diverse roles in plant growth, development, defense, non-coding RNA production, response to abiotic stresses, flowering, genomic stability and metabolic homeostasis. In addition, the transcription factors interacting with the Mediator complex are also highlighted. © 2015 Institute of Botany, Chinese Academy of Sciences.

Fungal mediator tail subunits contain classical transcriptional activation domains.

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Liu, Zhongle; Myers, Lawrence C

2015-04-01

Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters.

Science.gov (United States)

Chen, Liang; Chen, Jia-Yu; Zhang, Xuan; Gu, Ying; Xiao, Rui; Shao, Changwei; Tang, Peng; Qian, Hao; Luo, Daji; Li, Hairi; Zhou, Yu; Zhang, Dong-Er; Fu, Xiang-Dong

2017-11-16

R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome instability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase-H-based approach; this reveals predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end; by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such a free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed. Copyright © 2017 Elsevier Inc. All rights reserved.

A multimodal instrument for real-time in situ study of ultrasound and cavitation mediated drug delivery

OpenAIRE

Bian, S; Seth, A; Daly, D; Carlisle, R; Stride, E

2017-01-01

The development of a multimodal instrument capable of real-time in situ measurements of cavitation activity and effect in tissue mimicking phantoms during ultrasound and cavitation mediated drug delivery experiments is described here. The instrument features an acoustic arm that can expose phantoms to high-intensity focused-ultrasound while measuring cavitation activity and an optical arm that monitors cavitation effect using confocal microscopy. This combination of modalities allows real-tim...

Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems.

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Julia K Bialek

Full Text Available CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs, act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5' long terminal repeat (LTR, for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination.

Plasma density control in real-time on the COMPASS tokamak

Energy Technology Data Exchange (ETDEWEB)

Janky, F., E-mail: filip.janky.work@gmail.com [Institute of Plasma Physics AS CR, v.v.i., Association EURATOM/IPP.CR, Za Slovankou 3, 182 00 Praha 8 (Czech Republic); Department of Surface and Plasma Science, Faculty of Mathematics and Physics, Charles University in Prague, V Holešovičkách 2, 180 00 Praha 8 (Czech Republic); Hron, M. [Institute of Plasma Physics AS CR, v.v.i., Association EURATOM/IPP.CR, Za Slovankou 3, 182 00 Praha 8 (Czech Republic); Havlicek, J. [Institute of Plasma Physics AS CR, v.v.i., Association EURATOM/IPP.CR, Za Slovankou 3, 182 00 Praha 8 (Czech Republic); Department of Surface and Plasma Science, Faculty of Mathematics and Physics, Charles University in Prague, V Holešovičkách 2, 180 00 Praha 8 (Czech Republic); Varavin, M.; Zacek, F.; Seidl, J.; Panek, R. [Institute of Plasma Physics AS CR, v.v.i., Association EURATOM/IPP.CR, Za Slovankou 3, 182 00 Praha 8 (Czech Republic)

2015-10-15

Highlights: • We fitted length of the chord of the interferometry crossing plasma in the different plasma scenarios. • We add correction to the actual length of the chord of the interferometry according to plasma shape and position in real-time code. • We used this correction to control plasma density in real-time. - Abstract: The electron density on COMPASS is measured using 2 mm microwave interferometer. Interferometer signal is used as an input for the feedback control loop, running under the MARTe real-time framework. Two different threads are used to calculate (fast 50 μs thread) and to control (slow 500 μs thread) the electron density. The interferometer measures a line averaged density along a measurement chord. This paper describes an approach to control the line-averaged electron density in a real-time loop, using a correction to the real plasma shape, the plasma position, and non-linear effects of the electron density measurement at high densities. Newly developed real-time electron density control give COMPASS the chance to control the electron density more accurately which is essential for parametric scans for diagnosticians, for physics experiments and also for achieving plasma scenarios with H-mode.

Real-time RT-PCR, a necessary tool to support the diagnosis and surveillance of rotavirus in Mexico.

Science.gov (United States)

De La Cruz Hernández, Sergio Isaac; Anaya Molina, Yazmin; Gómez Santiago, Fabián; Terán Vega, Heidi Lizbeth; Monroy Leyva, Elda; Méndez Pérez, Héctor; García Lozano, Herlinda

2018-04-01

Rotavirus produces diarrhea in children under 5 years old. Most of those conventional methods such as polyacrylamide gel electrophoresis (PAGE) and reverse transcription-polymerase chain reaction (RT-PCR) have been used for rotavirus detection. However, these techniques need a multi-step process to get the results. In comparison with conventional methods, the real-time RT-PCR is a highly sensitive method, which allows getting the results in only one day. In this study a real-time RT-PCR assay was tested using a panel of 440 samples from patients with acute gastroenteritis, and characterized by PAGE and RT-PCR. The results show that the real-time RT-PCR detected rotavirus from 73% of rotavirus-negative samples analyzed by PAGE and RT-PCR; thus, the percentage of rotavirus-positive samples increased to 81%. The results indicate that this real-time RT-PCR should be part of a routine analysis, and as a support of the diagnosis of rotavirus in Mexico. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparison of multiplex RT-PCR and real-time HybProbe assay for serotyping of dengue virus using reference strains and clinical samples from India

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Anita Chakravarti

2016-01-01

Full Text Available Background: Dengue virus serotyping is crucial from clinical management and epidemiological point of view. Aims: To compare efficacy of two molecular detection and typing methods, namely, multiplex reverse transcription polymerase chain reaction (RT-PCR and real-time Hybprobe assay using a panel of known dilution of four reference Dengue virus strains and a panel of sera collected from clinically suspected dengue patients. Settings: This study was conducted at a tertiary-care teaching hospital in Delhi, India. Materials and Methods: Dengue serotype specific virus strains were used as prototypes for serotyping assays. Viral load was quantified by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR. Acute phase serum samples were collected from 79 patients with clinically suspected Dengue fever on their first day of presentation during September-October 2012. Viral RNA from serum and cell culture supernatant was extracted. Reverse transcription was carried out. Quantitative detection of DENV RNA from reference strain culture supernatants and each of the 79 patient samples by real-time PCR was performed using light cycler Taqman master mix kit. Serotyping was done by multiplex RT-PCR assay and Hybprobe assay. Results: The multiplex RT-PCR assay, though found to be 100% specific, couldn't serotype either patient or reference strains with viral load less than 1000 RNA copies/ml. The Hybprobe assay was found to have 100% specificity and had a lower limit of serotype detection of merely 3.54 RNA copies/ml. Conclusions: HybProbe assay has an important role especially in situations where serotyping is to be performed in clinical samples with low viral load.

Detection and differentiation of field and vaccine strains of canine distemper virus using reverse transcription followed by nested real time PCR (RT-nqPCR) and RFLP analysis.

Science.gov (United States)

Fischer, Cristine Dossin Bastos; Ikuta, Nilo; Canal, Cláudio Wageck; Makiejczuk, Aline; Allgayer, Mariangela da Costa; Cardoso, Cristine Hoffmeister; Lehmann, Fernanda Kieling; Fonseca, André Salvador Kazantzi; Lunge, Vagner Ricardo

2013-12-01

Canine distemper virus (CDV) is the cause of a severe and highly contagious disease in dogs. Practical diagnosis of canine distemper based on clinical signs and laboratory tests are required to confirm CDV infection. The present study aimed to develop a molecular assay to detect and differentiate field and vaccine CDV strains. Reverse transcription followed by nested real time polymerase chain reaction (RT-nqPCR) was developed, which exhibited analytical specificity (all the samples from healthy dogs and other canine infectious agents were not incorrectly detected) and sensitivity (all replicates of a vaccine strain were positive up to the 3125-fold dilution - 10(0.7) TCID50). RT-nqPCR was validated for CDV detection on different clinical samples (blood, urine, rectal and conjunctival swabs) of 103 animals suspected to have distemper. A total of 53 animals were found to be positive based on RT-nqPCR in at least one clinical sample. Blood resulted in more positive samples (50 out of 53, 94.3%), followed by urine (44/53, 83.0%), rectal (38/53, 71%) and conjunctival (27/53, 50.9%) swabs. A commercial immunochromatography (IC) assay had detected CDV in only 30 conjunctival samples of these positive dogs. Nucleoprotein (NC) gene sequencing of 25 samples demonstrated that 23 of them were closer to other Brazilian field strains and the remaining two to vaccine strains. A single nucleotide sequences difference, which creates an Msp I restriction enzyme digestion, was used to differentiate between field and vaccine CDV strains by restriction fragment length polymorphism (RFLP) analysis. The complete assay was more sensitive than was IC for the detection of CDV. Blood was the more frequently positive specimen and the addition of a restriction enzyme step allowed the differentiation of vaccine and Brazilian field strains. Copyright © 2013 Elsevier B.V. All rights reserved.

A closed-loop energy price controlling method for real-time energy balancing in a smart grid energy market

International Nuclear Information System (INIS)

Alagoz, B. Baykant; Kaygusuz, Asim; Akcin, Murat; Alagoz, Serkan

2013-01-01

Future smart grids will require a flexible, observable, and controllable network for reliable and efficient energy delivery under uncertain generation and demand conditions. One of the mechanisms for efficient and reliable energy generation is dynamic demand-responsive generation management based on energy price adjustments that creates a balance in energy markets. This study presents a closed-loop PID (proportional–integral–derivative) controller-based price control method for autonomous and real-time balancing of energy demand and generation in smart grid electricity markets. The PID control system can regulate energy prices online to respond dynamically and instantaneously to the varying energy demands of grid consumers. Independent energy suppliers in the smart grid decide whether to sell their energy to the grid according to the energy prices declared by the closed-loop PID controller system. Energy market simulations demonstrate that PID-controlled energy price regulation can effectively maintain an energy balance for hourly demand fluctuations of consumers. - Highlights: • This study presents a control theoretic approach for management of energy balance. • A closed-loop PID controller-based price controlling method is used in smart grid. • The simulation results demonstrate advantages of PID-based energy price control. • This method is appropriate for demand responsive management of smart grid markets

Self-Regulation of the Primary Auditory Cortex Attention Via Directed Attention Mediated By Real Time fMRI Neurofeedback

Science.gov (United States)

2017-05-05

NELSON FROM: 59 MDW /SGYU SUBJECT: Professional Presentation Approval 1. Your paper, entitled Self - regulation of the Primary Auditory Cortex Attention via...DATE Sherwood - p.1 Self - regulation of the primary auditory cortex attention via directed attention mediated by real-time fMRI neurofeedback M S...auditory cortex hyperactivity by self - regulation of the primary auditory cortex (A 1) based on real-time functional magnetic resonance imaging neurofeedback

Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination.

Science.gov (United States)

Thomas-Claudepierre, Anne-Sophie; Robert, Isabelle; Rocha, Pedro P; Raviram, Ramya; Schiavo, Ebe; Heyer, Vincent; Bonneau, Richard; Luo, Vincent M; Reddy, Janardan K; Borggrefe, Tilman; Skok, Jane A; Reina-San-Martin, Bernardo

2016-03-07

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation. © 2016 Thomas-Claudepierre et al.

Mediator Undergoes a Compositional Change during Transcriptional Activation.

Science.gov (United States)

Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

2016-11-03

Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter. Copyright © 2016 Elsevier Inc. All rights reserved.

Real-time operating system timing jitter and its impact on motor control

Science.gov (United States)

Proctor, Frederick M.; Shackleford, William P.

2001-12-01

General-purpose microprocessors are increasingly being used for control applications due to their widespread availability and software support for non-control functions like networking and operator interfaces. Two classes of real-time operating systems (RTOS) exist for these systems. The traditional RTOS serves as the sole operating system, and provides all OS services. Examples include ETS, LynxOS, QNX, Windows CE and VxWorks. RTOS extensions add real-time scheduling capabilities to non-real-time OSes, and provide minimal services needed for the time-critical portions of an application. Examples include RTAI and RTL for Linux, and HyperKernel, OnTime and RTX for Windows NT. Timing jitter is an issue in these systems, due to hardware effects such as bus locking, caches and pipelines, and software effects from mutual exclusion resource locks, non-preemtible critical sections, disabled interrupts, and multiple code paths in the scheduler. Jitter is typically on the order of a microsecond to a few tens of microseconds for hard real-time operating systems, and ranges from milliseconds to seconds in the worst case for soft real-time operating systems. The question of its significance on the performance of a controller arises. Naturally, the smaller the scheduling period required for a control task, the more significant is the impact of timing jitter. Aside from this intuitive relationship is the greater significance of timing on open-loop control, such as for stepper motors, than for closed-loop control, such as for servo motors. Techniques for measuring timing jitter are discussed, and comparisons between various platforms are presented. Techniques to reduce jitter or mitigate its effects are presented. The impact of jitter on stepper motor control is analyzed.

Reverse engineering of TLX oncogenic transcriptional networks identifies RUNX1 as tumor suppressor in T-ALL.

Science.gov (United States)

Della Gatta, Giusy; Palomero, Teresa; Perez-Garcia, Arianne; Ambesi-Impiombato, Alberto; Bansal, Mukesh; Carpenter, Zachary W; De Keersmaecker, Kim; Sole, Xavier; Xu, Luyao; Paietta, Elisabeth; Racevskis, Janis; Wiernik, Peter H; Rowe, Jacob M; Meijerink, Jules P; Califano, Andrea; Ferrando, Adolfo A

2012-02-26

The TLX1 and TLX3 transcription factor oncogenes have a key role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL). Here we used reverse engineering of global transcriptional networks to decipher the oncogenic regulatory circuit controlled by TLX1 and TLX3. This systems biology analysis defined T cell leukemia homeobox 1 (TLX1) and TLX3 as master regulators of an oncogenic transcriptional circuit governing T-ALL. Notably, a network structure analysis of this hierarchical network identified RUNX1 as a key mediator of the T-ALL induced by TLX1 and TLX3 and predicted a tumor-suppressor role for RUNX1 in T cell transformation. Consistent with these results, we identified recurrent somatic loss-of-function mutations in RUNX1 in human T-ALL. Overall, these results place TLX1 and TLX3 at the top of an oncogenic transcriptional network controlling leukemia development, show the power of network analyses to identify key elements in the regulatory circuits governing human cancer and identify RUNX1 as a tumor-suppressor gene in T-ALL.

Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP) for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals.

Science.gov (United States)

Zhou, Qian-Jin; Wang, Lei; Chen, Jiong; Wang, Rui-Na; Shi, Yu-Hong; Li, Chang-Hong; Zhang, De-Min; Yan, Xiao-Jun; Zhang, Yan-Jun

2014-09-01

Rapid, low-cost, and user-friendly strategies are urgently needed for early disease diagnosis and timely treatment, particularly for on-site screening of pathogens in aquaculture. In this study, we successfully developed a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP), which was capable of simultaneously detecting 10 pathogenic bacteria in aquatic animals, i.e., Nocardia seriolae, Pseudomonas putida, Streptococcus iniae, Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio rotiferianus, and Vibrio vulnificus. The assay provided a nearly-automated approach, with only a single pipetting step per chip for sample dispensing. This technique could achieve limits of detection (LOD) ranging from 0.40 to 6.42pg per 1.414μL reaction in less than 30 min. The robust reproducibility was demonstrated by a little variation among duplications for each bacterium with the coefficient of variation (CV) for time to positive (Tp) value less than 0.10. The clinical sensitivity and specificity of this on-chip LAMP assay in detecting field samples were 96.2% and 93.8% by comparison with conventional microbiological methods. Compared with other well-known techniques, on-chip LAMP assay provides low sample and reagent consumption, ease-of-use, accelerated analysis, multiple bacteria and on-site detection, and high reproducibility, indicating that such a technique would be applicable for on-site detection and routine monitoring of multiple pathogens in aquaculture. Copyright © 2014 Elsevier B.V. All rights reserved.

The reverse transcription inhibitor abacavir shows anticancer activity in prostate cancer cell lines.

Directory of Open Access Journals (Sweden)

Francesca Carlini

Full Text Available BACKGROUND: Transposable Elements (TEs comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1 and Human Endogenous Retroviruses (HERVs that code for their own endogenous reverse transcriptase (RT. Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC, a nucleoside reverse transcription inhibitor (NRTI, on PC3 and LNCaP prostate cancer cell lines. PRINCIPAL FINDINGS: ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. CONCLUSIONS: Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications.

Mediator binds to boundaries of chromosomal interaction domains and to proteins involved in DNA looping, RNA metabolism, chromatin remodeling, and actin assembly.

Science.gov (United States)

Chereji, Razvan V; Bharatula, Vasudha; Elfving, Nils; Blomberg, Jeanette; Larsson, Miriam; Morozov, Alexandre V; Broach, James R; Björklund, Stefan

2017-09-06

Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Saccharomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3'-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Neural network evaluation of tokamak current profiles for real time control

Science.gov (United States)

Wróblewski, Dariusz

1997-02-01

Active feedback control of the current profile, requiring real-time determination of the current profile parameters, is envisioned for tokamaks operating in enhanced confinement regimes. The distribution of toroidal current in a tokamak is now routinely evaluated based on external (magnetic probes, flux loops) and internal (motional Stark effect) measurements of the poloidal magnetic field. However, the analysis involves reconstruction of magnetohydrodynamic equilibrium and is too intensive computationally to be performed in real time. In the present study, a neural network is used to provide a mapping from the magnetic measurements (internal and external) to selected parameters of the safety factor profile. The single-pass, feedforward calculation of output of a trained neural network is very fast, making this approach particularly suitable for real-time applications. The network was trained on a large set of simulated equilibrium data for the DIII-D tokamak. The database encompasses a large variety of current profiles including the hollow current profiles important for reversed central shear operation. The parameters of safety factor profile (a quantity related to the current profile through the magnetic field tilt angle) estimated by the neural network include central safety factor, q0, minimum value of q, qmin, and the location of qmin. Very good performance of the trained neural network both for simulated test data and for experimental datais demonstrated.

Neural network evaluation of tokamak current profiles for real time control

International Nuclear Information System (INIS)

Wroblewski, D.

1997-01-01

Active feedback control of the current profile, requiring real-time determination of the current profile parameters, is envisioned for tokamaks operating in enhanced confinement regimes. The distribution of toroidal current in a tokamak is now routinely evaluated based on external (magnetic probes, flux loops) and internal (motional Stark effect) measurements of the poloidal magnetic field. However, the analysis involves reconstruction of magnetohydrodynamic equilibrium and is too intensive computationally to be performed in real time. In the present study, a neural network is used to provide a mapping from the magnetic measurements (internal and external) to selected parameters of the safety factor profile. The single-pass, feedforward calculation of output of a trained neural network is very fast, making this approach particularly suitable for real-time applications. The network was trained on a large set of simulated equilibrium data for the DIII-D tokamak. The database encompasses a large variety of current profiles including the hollow current profiles important for reversed central shear operation. The parameters of safety factor profile (a quantity related to the current profile through the magnetic field tilt angle) estimated by the neural network include central safety factor, q 0 , minimum value of q, q min , and the location of q min . Very good performance of the trained neural network both for simulated test data and for experimental datais demonstrated. copyright 1997 American Institute of Physics

Reverse engineering a mouse embryonic stem cell-specific transcriptional network reveals a new modulator of neuronal differentiation.

Science.gov (United States)

De Cegli, Rossella; Iacobacci, Simona; Flore, Gemma; Gambardella, Gennaro; Mao, Lei; Cutillo, Luisa; Lauria, Mario; Klose, Joachim; Illingworth, Elizabeth; Banfi, Sandro; di Bernardo, Diego

2013-01-01

Gene expression profiles can be used to infer previously unknown transcriptional regulatory interaction among thousands of genes, via systems biology 'reverse engineering' approaches. We 'reverse engineered' an embryonic stem (ES)-specific transcriptional network from 171 gene expression profiles, measured in ES cells, to identify master regulators of gene expression ('hubs'). We discovered that E130012A19Rik (E13), highly expressed in mouse ES cells as compared with differentiated cells, was a central 'hub' of the network. We demonstrated that E13 is a protein-coding gene implicated in regulating the commitment towards the different neuronal subtypes and glia cells. The overexpression and knock-down of E13 in ES cell lines, undergoing differentiation into neurons and glia cells, caused a strong up-regulation of the glutamatergic neurons marker Vglut2 and a strong down-regulation of the GABAergic neurons marker GAD65 and of the radial glia marker Blbp. We confirmed E13 expression in the cerebral cortex of adult mice and during development. By immuno-based affinity purification, we characterized protein partners of E13, involved in the Polycomb complex. Our results suggest a role of E13 in regulating the division between glutamatergic projection neurons and GABAergic interneurons and glia cells possibly by epigenetic-mediated transcriptional regulation.

Closing the Loop : Reverse supply chain management andproduct return processes in electronics retailing

OpenAIRE

Gorskova, Julija; Ortega, Edrion

2012-01-01

Abstract Problem There is a gap in the knowledge concerning reverse product flows due to a lack of research and empirical data in the field of reverse supply chain management in general. Furthermore, more research is needed to investigate the factors influencing the decision making process regarding the right reverse supply chain recovery option choice for companies in order to close the supply chain loop. Processing product returns has become a critical activity for organisations as the volu...

Real-time control of electron density in a capacitively coupled plasma

International Nuclear Information System (INIS)

Keville, Bernard; Gaman, Cezar; Turner, Miles M.; Zhang Yang; Daniels, Stephen; Holohan, Anthony M.

2013-01-01

Reactive ion etching (RIE) is sensitive to changes in chamber conditions, such as wall seasoning, which have a deleterious effect on process reproducibility. The application of real time, closed loop control to RIE may reduce this sensitivity and facilitate production with tighter tolerances. The real-time, closed loop control of plasma density with RF power in a capacitively coupled argon plasma using a hairpin resonance probe as a sensor is described. Elementary control analysis shows that an integral controller provides stable and effective set point tracking and disturbance attenuation. The trade off between performance and robustness may be quantified in terms of one parameter, namely the position of the closed loop pole. Experimental results are presented, which are consistent with the theoretical analysis.

Intersection local times, loop soups and permanental Wick powers

CERN Document Server

Jan, Yves Le; Rosen, Jay

2017-01-01

Several stochastic processes related to transient Lévy processes with potential densities u(x,y)=u(y-x), that need not be symmetric nor bounded on the diagonal, are defined and studied. They are real valued processes on a space of measures \\mathcal{V} endowed with a metric d. Sufficient conditions are obtained for the continuity of these processes on (\\mathcal{V},d). The processes include n-fold self-intersection local times of transient Lévy processes and permanental chaoses, which are `loop soup n-fold self-intersection local times' constructed from the loop soup of the Lévy process. Loop soups are also used to define permanental Wick powers, which generalizes standard Wick powers, a class of n-th order Gaussian chaoses. Dynkin type isomorphism theorems are obtained that relate the various processes. Poisson chaos processes are defined and permanental Wick powers are shown to have a Poisson chaos decomposition. Additional properties of Poisson chaos processes are studied and a martingale extension is obt...

Strong transcription blockage mediated by R-loop formation within a G-rich homopurine-homopyrimidine sequence localized in the vicinity of the promoter.

Science.gov (United States)

Belotserkovskii, Boris P; Soo Shin, Jane Hae; Hanawalt, Philip C

2017-06-20

Guanine-rich (G-rich) homopurine-homopyrimidine nucleotide sequences can block transcription with an efficiency that depends upon their orientation, composition and length, as well as the presence of negative supercoiling or breaks in the non-template DNA strand. We report that a G-rich sequence in the non-template strand reduces the yield of T7 RNA polymerase transcription by more than an order of magnitude when positioned close (9 bp) to the promoter, in comparison to that for a distal (∼250 bp) location of the same sequence. This transcription blockage is much less pronounced for a C-rich sequence, and is not significant for an A-rich sequence. Remarkably, the blockage is not pronounced if transcription is performed in the presence of RNase H, which specifically digests the RNA strands within RNA-DNA hybrids. The blockage also becomes less pronounced upon reduced RNA polymerase concentration. Based upon these observations and those from control experiments, we conclude that the blockage is primarily due to the formation of stable RNA-DNA hybrids (R-loops), which inhibit successive rounds of transcription. Our results could be relevant to transcription dynamics in vivo (e.g. transcription 'bursting') and may also have practical implications for the design of expression vectors. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Automatic dataflow model extraction from modal real-time stream processing applications

NARCIS (Netherlands)

Geuns, S.J.; Hausmans, J.P.H.M.; Bekooij, Marco Jan Gerrit

2013-01-01

Many real-time stream processing applications are initially described as a sequential application containing while-loops, which execute for an unknown number of iterations. These modal applications have to be executed in parallel on an MPSoC system in order to meet their real-time throughput

GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR.

Science.gov (United States)

Gubelmann, Carine; Gattiker, Alexandre; Massouras, Andreas; Hens, Korneel; David, Fabrice; Decouttere, Frederik; Rougemont, Jacques; Deplancke, Bart

2011-01-01

The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/. Database URL: http://deplanckelab.epfl.ch.

[Colorimetric detection of HPV6 and HPV16 by loop mediated isothermal amplification].

Science.gov (United States)

Lu, Chun-bin; Luo, Le; Yang, Meng-jie; Nie, Kai; Wang, Miao; Ma, Xue-Jun

2011-01-01

A simple, rapid and sensitive colorimetric loop mediated isothermal amplification (LAMP) method was established to detect HPV6 and HPV 16 respectively. The method employed a set of four specially designed primers that recognized six distinct sequences of HPV6-E6 or HPV16-E7 for amplification of nucleic acid under isothermal conditions at 63 degrees C for one hour. The amplification process of LAMP was monitored by the addition of HNB (hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by real-time turbidimeter and agarose electrophoresis. Thirteen cervical swab samples having single infection with 13 different HPV genotypes were examined to evaluate the specificity. A serial dilution of a cloned plasmid containing HPV-E6 or HPV-E7 gene was examined to evaluate the sensitivity. The results showed that no cross-reaction with other HPV genotypes was observed. The colorimetric LAMP assay could achieve a sensitivity of 1000 copies, 10-20 times lower than that of real-time PCR. The assay was further evaluated with 62 clinical specimens and consistent results were obtained compared with the detection using Kai Pu HPV Genotyping Kit. We concluded that this colorimetric LAMP assay had potential usefulness for the rapid screening of the HPV6 or HPV16 infection in the laboratories and hospitals of provincial and municipal region in China.

Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction.

Science.gov (United States)

Wilkes, Rebecca P; Tsai, Yun-Long; Lee, Pei-Yu; Lee, Fu-Chun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

2014-09-09

Canine distemper virus (CDV) has been associated with outbreaks of canine infectious respiratory disease in shelters and boarding kennel environments. POCKITTM Nucleic Acid Analyzer is a field-deployable device capable of generating automatically interpreted insulated isothermal polymerase chain reaction (iiPCR) results from extracted nucleic acid within one hour. In this study, reverse transcription iiPCR (RT-iiPCR) was developed to facilitate point-of-need diagnosis of CDV infection. Analytical sensitivity (limit of detection 95%) of the established CDV RT-iiPCR was about 11 copies of in vitro transcribed RNA per reaction. CDV RT-iiPCR generated positive signals from CDV, but not Bordetella bronchiseptica, canine parvovirus, canine herpesvirus, canine adenovirus 2, canine influenza virus (subtype H3N8), canine parainfluenza virus, and canine respiratory coronavirus. To evaluate accuracy of the established reaction in canine distemper clinical diagnosis, 110 specimens from dogs, raccoons, and foxes suspected with CDV infection were tested simultaneously by CDV RT-iiPCR and real-time RT-PCR. CDV RT-iiPCR demonstrated excellent sensitivity (100%) and specificity (100%), compared to real-time RT-PCR. The results indicated an excellent correlation between RT-iiPCR and a reference real time RT-PCR method. Working in a lyophilized format, the established method has great potential to be used for point-of-care diagnosis of canine distemper in animals, especially in resource-limited facilities.

Real Time Synchronization for Creativity in Distributed Innovation Teams

DEFF Research Database (Denmark)

Peitersen, Dennis Kjaersgaard; Dolog, Peter; Pedersen, Esben Staunsbjerg

2009-01-01

In this paper we introduce a synchronization approach for real time collaborative sketching for creativity in distributed innovation teams. We base our approach on reverse AJAX. This way we ensure scalable solution for real time drawing and sketching important in creativity settings....

The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.

Directory of Open Access Journals (Sweden)

Clément Monot

2013-05-01

Full Text Available L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP first uses its endonuclease (EN to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A tail, a process known as target-primed reverse transcription (TPRT. Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5'-TTTT/A-3' sites and frequently preceded by imperfect T-tracts. However, it is currently unclear whether--and to which degree--the liberated 3'-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3' end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3' overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.

Science.gov (United States)

Monot, Clément; Kuciak, Monika; Viollet, Sébastien; Mir, Ashfaq Ali; Gabus, Caroline; Darlix, Jean-Luc; Cristofari, Gaël

2013-05-01

L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A) tail, a process known as target-primed reverse transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5'-TTTT/A-3' sites) and frequently preceded by imperfect T-tracts. However, it is currently unclear whether--and to which degree--the liberated 3'-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3' end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3' overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

Real-time PCR (qPCR) primer design using free online software.

Science.gov (United States)

Thornton, Brenda; Basu, Chhandak

2011-01-01

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software. Copyright © 2010 Wiley Periodicals, Inc.

Real-time DNA Amplification and Detection System Based on a CMOS Image Sensor.

Science.gov (United States)

Wang, Tiantian; Devadhasan, Jasmine Pramila; Lee, Do Young; Kim, Sanghyo

2016-01-01

In the present study, we developed a polypropylene well-integrated complementary metal oxide semiconductor (CMOS) platform to perform the loop mediated isothermal amplification (LAMP) technique for real-time DNA amplification and detection simultaneously. An amplification-coupled detection system directly measures the photon number changes based on the generation of magnesium pyrophosphate and color changes. The photon number decreases during the amplification process. The CMOS image sensor observes the photons and converts into digital units with the aid of an analog-to-digital converter (ADC). In addition, UV-spectral studies, optical color intensity detection, pH analysis, and electrophoresis detection were carried out to prove the efficiency of the CMOS sensor based the LAMP system. Moreover, Clostridium perfringens was utilized as proof-of-concept detection for the new system. We anticipate that this CMOS image sensor-based LAMP method will enable the creation of cost-effective, label-free, optical, real-time and portable molecular diagnostic devices.

Water soluble contrast enema examination of the integrity of the rectal anastomosis prior to loop ileostomy reversal may be superfluous.

Science.gov (United States)

Larsson, Anna; Lindmark, Gudrun; Syk, Ingvar; Buchwald, Pamela

2015-03-01

Defunctioning loop ileostomy in low anterior resection (LAR) is routinely used to reduce consequences of anastomotic leakage. The purpose of this study was to analyze which examination technique is optimal for evaluating the integrity of the anastomosis prior to loop ileostomy reversal. Retrospective analysis of 95 patients who had been subjected to LAR at Helsingborg Hospital and Skåne University Hospital, Sweden, was undertaken between January 2007 and June 2009. The examination techniques of the rectal anastomosis prior to reversal and the clinical outcome after reversal were studied. Radiologic anastomosis control using water soluble contrast enema, digital rectal examination (DRE), and rectoscopy were performed in 53 % (50/95), 98 % (93/95), and 69 % (66/95), respectively. In two patients, no control of the anastomosis was performed before reversal. Fifty-two percent (49/95) of the patients were examined using all techniques. Six patients demonstrated leakage detected before reversal of which two were only radiological leakages. These two patients underwent loop ileostomy reversal after delay without complications. They were the only ones where the three examination techniques did not prove coherence. Four patients had symptomatic leakage; these were detected with rectoscopy and DRE and verified with enema. Three patients developed anastomotic leakage after loop ileostomy reversal despite normal preoperative examinations. Two of these patients had rectovaginal fistulas (AVFs). This retrospective study indicates that contrast enema does not provide additional information if rectoscopy and DRE are normal. Despite negative examinations, three of nine leakages were diagnosed after loop ileostomy reversal. Especially, AVFs seem difficult to diagnose.

Loop-mediated isothermal amplification for rapid and convenient detection of Mycoplasma hyopneumoniae.

Science.gov (United States)

Li, Jiahe; Minion, F Chris; Petersen, Andrew C; Jiang, Fei; Yang, Sheng; Guo, Panpan; Li, Jinxiang; Wu, Wenxue

2013-04-01

Loop-mediated isothermal amplification (LAMP), a novel method of gene amplification, was employed in this study for detecting Mycoplasma hyopneumoniae in the respiratory tract or lungs of swine. The pathogen can be detected in LAMP reactions containing as few as 10 fg purified target DNA (10 copies of M. hyopneumoniae genome) within 30 min, which was comparable to real-time PCR. After 30-min reaction at 63 °C, the addition of a certain amount of dye (SYBR Green I and hydroxyl naphthol blue at a proper ratio) into the LAMP reaction system makes the results easily determined as positive or negative by visual inspection. In addition, the LAMP was able to distinguish between M. hyopneumoniae and other closely-related mycoplasma strains, indicating a high degree of specificity. The LAMP assay was more simple and cheap, since the reaction could be completed under isothermal conditions and less laboratorial infrastructure are required. And, it was proven reliable for M. hyopneumoniae diagnosis of nasal swab and lung samples from the field.

Collection effort and reverse channel choices in a closed-loop supply chain

DEFF Research Database (Denmark)

Govindan, Kannan; Liu, Liwen; Wang, Zongjun

2017-01-01

We investigate the pricing and reverse channel choice decision issues in a closed-loop supply chain, within which the original equipment manufacturer (OEM) sells the new and remanufactured products through a retailer, while collects used products (cores) via dual recycling channels. We consider...

MARTe: A Multiplatform Real-Time Framework

Science.gov (United States)

Neto, André C.; Sartori, Filippo; Piccolo, Fabio; Vitelli, Riccardo; De Tommasi, Gianmaria; Zabeo, Luca; Barbalace, Antonio; Fernandes, Horacio; Valcarcel, Daniel F.; Batista, Antonio J. N.

2010-04-01

Development of real-time applications is usually associated with nonportable code targeted at specific real-time operating systems. The boundary between hardware drivers, system services, and user code is commonly not well defined, making the development in the target host significantly difficult. The Multithreaded Application Real-Time executor (MARTe) is a framework built over a multiplatform library that allows the execution of the same code in different operating systems. The framework provides the high-level interfaces with hardware, external configuration programs, and user interfaces, assuring at the same time hard real-time performances. End-users of the framework are required to define and implement algorithms inside a well-defined block of software, named Generic Application Module (GAM), that is executed by the real-time scheduler. Each GAM is reconfigurable with a set of predefined configuration meta-parameters and interchanges information using a set of data pipes that are provided as inputs and required as output. Using these connections, different GAMs can be chained either in series or parallel. GAMs can be developed and debugged in a non-real-time system and, only once the robustness of the code and correctness of the algorithm are verified, deployed to the real-time system. The software also supplies a large set of utilities that greatly ease the interaction and debugging of a running system. Among the most useful are a highly efficient real-time logger, HTTP introspection of real-time objects, and HTTP remote configuration. MARTe is currently being used to successfully drive the plasma vertical stabilization controller on the largest magnetic confinement fusion device in the world, with a control loop cycle of 50 ?s and a jitter under 1 ?s. In this particular project, MARTe is used with the Real-Time Application Interface (RTAI)/Linux operating system exploiting the new ?86 multicore processors technology.

A real-time BWR stability measurement system

International Nuclear Information System (INIS)

March-Leuba, J.; King, W.T.

1988-01-01

This paper describes the characteristics of a portable, real-time system used for nonperturbational measurements of stability in boiling water reactors. The algorithm used in this system estimates the closed-loop asymptotic decay ratio using only the naturally occurring neutron noise and it is based on the univariate autoregressive methodology. (author)

Neural network evaluation of tokamak current profiles for real time control (abstract)

Science.gov (United States)

Wróblewski, Dariusz

1997-01-01

Active feedback control of the current profile, requiring real-time determination of the current profile parameters, is envisioned for tokamaks operating in enhanced confinement regimes. The distribution of toroidal current in a tokamak is now routinely evaluated based on external (magnetic probes, flux loops) and internal (motional Stark effect) measurements of the poloidal magnetic field. However, the analysis involves reconstruction of magnetohydrodynamic equilibrium and is too intensive computationally to be performed in real time. In the present study, a neural network is used to provide a mapping from the magnetic measurements (internal and external) to selected parameters of the safety factor profile. The single-pass, feedforward calculation of output of a trained neural network is very fast, making this approach particularly suitable for real-time applications. The network was trained on a large set of simulated equilibrium data for the DIII-D tokamak. The database encompasses a large variety of current profiles including the hollow current profiles important for reversed central shear operation. The parameters of safety factor profile (a quantity related to the current profile through the magnetic field tilt angle) estimated by the neural network include central safety factor, q0, minimum value of q, qmin, and the location of qmin. Very good performance of the trained neural network both for simulated test data and for experimental data is demonstrated.

Neural network evaluation of tokamak current profiles for real time control (abstract)

International Nuclear Information System (INIS)

Wroblewski, D.

1997-01-01

Active feedback control of the current profile, requiring real-time determination of the current profile parameters, is envisioned for tokamaks operating in enhanced confinement regimes. The distribution of toroidal current in a tokamak is now routinely evaluated based on external (magnetic probes, flux loops) and internal (motional Stark effect) measurements of the poloidal magnetic field. However, the analysis involves reconstruction of magnetohydrodynamic equilibrium and is too intensive computationally to be performed in real time. In the present study, a neural network is used to provide a mapping from the magnetic measurements (internal and external) to selected parameters of the safety factor profile. The single-pass, feedforward calculation of output of a trained neural network is very fast, making this approach particularly suitable for real-time applications. The network was trained on a large set of simulated equilibrium data for the DIII-D tokamak. The database encompasses a large variety of current profiles including the hollow current profiles important for reversed central shear operation. The parameters of safety factor profile (a quantity related to the current profile through the magnetic field tilt angle) estimated by the neural network include central safety factor, q 0 , minimum value of q, q min , and the location of q min . Very good performance of the trained neural network both for simulated test data and for experimental data is demonstrated. copyright 1997 American Institute of Physics

Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.

Science.gov (United States)

Abbasi, Ibrahim; Kirstein, Oscar D; Hailu, Asrat; Warburg, Alon

2016-10-01

Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Subunit architecture and functional modular rearrangements of the transcriptional mediator complex.

Science.gov (United States)

Tsai, Kuang-Lei; Tomomori-Sato, Chieri; Sato, Shigeo; Conaway, Ronald C; Conaway, Joan W; Asturias, Francisco J

2014-06-05

The multisubunit Mediator, comprising ∼30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA-binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism. Copyright © 2014 Elsevier Inc. All rights reserved.

Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA

DEFF Research Database (Denmark)

Stangegaard, Michael; Dufva, I.H.; Dufva, Hans Martin

2006-01-01

oligonucleotides (pentadecamers) consistently, yielded at least 2 fold as much cDNA as did random hexamers using either-poly(A) RNA or an amplified version of messenger RNA (aRNA) as a template. The cDNA generated using pentadecamers did not differ in size distribution or the amount of incorporated label compared...... with cDNA generated with random hexamers. The increased efficiency of priming using random pentadecamers resulted in reverse transcription of > 80% of the template aRNA, while random hexamers induced reverse transcription of only 40% of the template aRNA. This suggests a better coverage...... that random pentadecamers can replace random hexamers in reverse transcription reactions on both poly(A) RNA and amplified RNA, resulting in higher cDNA yields and quality....

Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes.

Science.gov (United States)

Treder, Krzysztof; Chołuj, Joanna; Zacharzewska, Bogumiła; Babujee, Lavanya; Mielczarek, Mateusz; Burzyński, Adam; Rakotondrafara, Aurélie M

2018-02-01

Potato virus Y (PVY) infection has been a global challenge for potato production and the leading cause of downgrading and rejection of seed crops for certification. Accurate and timely diagnosis is a key for effective disease control. Here, we have optimized a reverse transcription loop-mediated amplification (RT-LAMP) assay to differentiate the PVY O and N serotypes. The RT-LAMP assay is based on isothermal autocyclic strand displacement during DNA synthesis. The high specificity of this method relies heavily on the primer sets designed for the amplification of the targeted regions. We designed specific primer sets targeting a region within the coat protein gene that contains nucleotide signatures typical for O and N coat protein types, and these primers differ in their annealing temperature. Combining this assay with total RNA extraction by magnetic capture, we have established a highly sensitive, simplified and shortened RT-LAMP procedure as an alternative to conventional nucleic acid assays for diagnosis. This optimized procedure for virus detection may be used as a preliminary test for identifying the viral serotype prior to investing time and effort in multiplex RT-PCR tests when a specific strain is needed.

Activation of TGF-β1-CD147 positive feedback loop in hepatic stellate cells promotes liver fibrosis.

Science.gov (United States)

Li, Hai-Yan; Ju, Di; Zhang, Da-Wei; Li, Hao; Kong, Ling-Min; Guo, Yanhai; Li, Can; Wang, Xi-Long; Chen, Zhi-Nan; Bian, Huijie

2015-11-12

Activation of hepatic stellate cells (HSCs) by transforming growth factor-β1 (TGF-β1) initiates HBV-associated fibrogenesis. The mechanism of TGF-β1 modulating HSC activation is not fully uncovered. We hypothesized a positive feedback signaling loop of TGF-β1-CD147 promoting liver fibrogenesis by activation of HSCs. Human HSC cell line LX-2 and spontaneous liver fibrosis model derived from HBV transgenic mice were used to evaluate the activation of molecules in the signaling loop. Wound healing and cell contraction assay were performed to detect the CD147-overexpressed HSC migration and contraction. The transcriptional regulation of CD147 by TGF-β1/Smad4 was determined using dual-luciferase reporter assay and chromatin immunoprecipitation. We found that a positive reciprocal regulation between TGF-β1 and CD147 mediated HSC activation. CD147 over-expression promoted HSC migration and accelerated TGF-β1-induced cell contraction. Phosphorylation of Smad2 and Smad3 in cooperation with Smad4 mediated the TGF-β1-regulated CD147 expression. Smad4 activated the transcription by direct interaction with CD147 promoter. Meanwhile, CD147 modulated the activated phenotype of HSCs through the ERK1/2 and Sp1 which up-regulated α-SMA, collagen I, and TGF-β1 synthesis. These findings indicate that TGF-β1-CD147 loop plays a key role in regulating the HSC activation and combination of TGF-β receptor inhibitor and anti-CD147 antibody might be promised to reverse fibrogenesis.

Transcription on lampbrush chromosome loops in the absence of U2 snRNA.

OpenAIRE

Tsvetkov, A; Jantsch, M; Wu, Z; Murphy, C; Gall, J G

1992-01-01

The five small nuclear RNAs (snRNAs) involved in splicing occur on the loops of amphibian lampbrush chromosomes and in hundreds to thousands of extrachromosomal granules called B snurposomes. To assess the role of these snRNAs during transcription and to explore possible relationships between the loops and B snurposomes, we injected single-stranded antisense oligodeoxynucleotides (oligos) against U1 and U2 snRNA into toad and newt oocytes. As shown before, antisense U1 and U2 oligos caused tr...

The role of incoherent microRNA-mediated feedforward loops in noise buffering.

Directory of Open Access Journals (Sweden)

Matteo Osella

2011-03-01

Full Text Available MicroRNAs are endogenous non-coding RNAs which negatively regulate the expression of protein-coding genes in plants and animals. They are known to play an important role in several biological processes and, together with transcription factors, form a complex and highly interconnected regulatory network. Looking at the structure of this network, it is possible to recognize a few overrepresented motifs which are expected to perform important elementary regulatory functions. Among them, a special role is played by the microRNA-mediated feedforward loop in which a master transcription factor regulates a microRNA and, together with it, a set of target genes. In this paper we show analytically and through simulations that the incoherent version of this motif can couple the fine-tuning of a target protein level with an efficient noise control, thus conferring precision and stability to the overall gene expression program, especially in the presence of fluctuations in upstream regulators. Among the other results, a nontrivial prediction of our model is that the optimal attenuation of fluctuations coincides with a modest repression of the target expression. This feature is coherent with the expected fine-tuning function and in agreement with experimental observations of the actual impact of a wide class of microRNAs on the protein output of their targets. Finally, we describe the impact on noise-buffering efficiency of the cross-talk between microRNA targets that can naturally arise if the microRNA-mediated circuit is not considered as isolated, but embedded in a larger network of regulations.

Chemical-looping combustion in a reverse-flow fixed bed reactor

International Nuclear Information System (INIS)

Han, Lu; Bollas, George M.

2016-01-01

A reverse-flow fixed bed reactor concept for CLC (chemical-looping combustion) is explored. The limitations of conventional fixed bed reactors, as applied to CLC, are overcome by reversing the gas flow direction periodically to enhance the mixing characteristics of the bed, thus improving oxygen carrier utilization and energy efficiency with respect to power generation. The reverse-flow reactor is simulated by a dusty-gas model and compared with an equivalent fixed bed reactor without flow reversal. Dynamic optimization is used to calculate conditions at which each reactor operates at maximum energy efficiency. Several cases studies illustrate the benefits of reverse-flow operation for the CLC with CuO and NiO oxygen carriers and methane and syngas fuels. The results show that periodic reversal of the flow during reduction improves the contact between the fuel and unconverted oxygen carrier, enabling the system to suppress unwanted catalytic reactions and axial temperature and conversion gradients. The operational scheme presented reduces the fluctuations of temperature during oxidation and increases the high-temperature heat produced by the process. CLC in a reverse-flow reactor has the potential to achieve higher energy efficiency than conventional fixed bed CLC reactors, when integrated with a downstream gas turbine of a combined cycle power plant. - Highlights: • Reverse-flow fixed bed CLC reactors for combined cycle power systems. • Dynamic optimization tunes operation of batch and transient CLC systems. • The reverse-flow CLC system provides stable turbine-ready gas stream. • Reverse-flow CLC fixed bed reactor has superior CO 2 capture and thermal efficiency.

IGBT Switching Characteristic Curve Embedded Half-Bridge MMC Modelling and Real Time Simulation Realization

Science.gov (United States)

Zhengang, Lu; Hongyang, Yu; Xi, Yang

2017-05-01

The Modular Multilevel Converter (MMC) is one of the most attractive topologies in recent years for medium or high voltage industrial applications, such as high voltage dc transmission (HVDC) and medium voltage varying speed motor drive. The wide adoption of MMCs in industry is mainly due to its flexible expandability, transformer-less configuration, common dc bus, high reliability from redundancy, and so on. But, when the sub module number of MMC is more, the test of MMC controller will cost more time and effort. Hardware in the loop test based on real time simulator will save a lot of time and money caused by the MMC test. And due to the flexible of HIL, it becomes more and more popular in the industry area. The MMC modelling method remains an important issue for the MMC HIL test. Specifically, the VSC model should realistically reflect the nonlinear device switching characteristics, switching and conduction losses, tailing current, and diode reverse recovery behaviour of a realistic converter. In this paper, an IGBT switching characteristic curve embedded half-bridge MMC modelling method is proposed. This method is based on the switching curve referring and sample circuit calculation, and it is sample for implementation. Based on the proposed method, a FPGA real time simulation is carried out with 200ns sample time. The real time simulation results show the proposed method is correct.

APOBEC3G inhibits elongation of HIV-1 reverse transcripts.

Directory of Open Access Journals (Sweden)

Kate N Bishop

2008-12-01

Full Text Available APOBEC3G (A3G is a host cytidine deaminase that, in the absence of Vif, restricts HIV-1 replication and reduces the amount of viral DNA that accumulates in cells. Initial studies determined that A3G induces extensive mutation of nascent HIV-1 cDNA during reverse transcription. It has been proposed that this triggers the degradation of the viral DNA, but there is now mounting evidence that this mechanism may not be correct. Here, we use a natural endogenous reverse transcriptase assay to show that, in cell-free virus particles, A3G is able to inhibit HIV-1 cDNA accumulation not only in the absence of hypermutation but also without the apparent need for any target cell factors. We find that although reverse transcription initiates in the presence of A3G, elongation of the cDNA product is impeded. These data support the model that A3G reduces HIV-1 cDNA levels by inhibiting synthesis rather than by inducing degradation.

A Novel Real-Time Path Servo Control of a Hardware-in-the-Loop for a Large-Stroke Asymmetric Rod-Less Pneumatic System under Variable Loads

Directory of Open Access Journals (Sweden)

Hao-Ting Lin

2017-06-01

Full Text Available This project aims to develop a novel large stroke asymmetric pneumatic servo system of a hardware-in-the-loop for path tracking control under variable loads based on the MATLAB Simulink real-time system. High pressure compressed air provided by the air compressor is utilized for the pneumatic proportional servo valve to drive the large stroke asymmetric rod-less pneumatic actuator. Due to the pressure differences between two chambers, the pneumatic actuator will operate. The highly nonlinear mathematical models of the large stroke asymmetric pneumatic system were analyzed and developed. The functional approximation technique based on the sliding mode controller (FASC is developed as a controller to solve the uncertain time-varying nonlinear system. The MATLAB Simulink real-time system was a main control unit of a hardware-in-the-loop system proposed to establish driver blocks for analog and digital I/O, a linear encoder, a CPU and a large stroke asymmetric pneumatic rod-less system. By the position sensor, the position signals of the cylinder will be measured immediately. The measured signals will be viewed as the feedback signals of the pneumatic servo system for the study of real-time positioning control and path tracking control. Finally, real-time control of a large stroke asymmetric pneumatic servo system with measuring system, a large stroke asymmetric pneumatic servo system, data acquisition system and the control strategy software will be implemented. Thus, upgrading the high position precision and the trajectory tracking performance of the large stroke asymmetric pneumatic servo system will be realized to promote the high position precision and path tracking capability. Experimental results show that fifth order paths in various strokes and the sine wave path are successfully implemented in the test rig. Also, results of variable loads under the different angle were implemented experimentally.

A Novel Real-Time Path Servo Control of a Hardware-in-the-Loop for a Large-Stroke Asymmetric Rod-Less Pneumatic System under Variable Loads.

Science.gov (United States)

Lin, Hao-Ting

2017-06-04

This project aims to develop a novel large stroke asymmetric pneumatic servo system of a hardware-in-the-loop for path tracking control under variable loads based on the MATLAB Simulink real-time system. High pressure compressed air provided by the air compressor is utilized for the pneumatic proportional servo valve to drive the large stroke asymmetric rod-less pneumatic actuator. Due to the pressure differences between two chambers, the pneumatic actuator will operate. The highly nonlinear mathematical models of the large stroke asymmetric pneumatic system were analyzed and developed. The functional approximation technique based on the sliding mode controller (FASC) is developed as a controller to solve the uncertain time-varying nonlinear system. The MATLAB Simulink real-time system was a main control unit of a hardware-in-the-loop system proposed to establish driver blocks for analog and digital I/O, a linear encoder, a CPU and a large stroke asymmetric pneumatic rod-less system. By the position sensor, the position signals of the cylinder will be measured immediately. The measured signals will be viewed as the feedback signals of the pneumatic servo system for the study of real-time positioning control and path tracking control. Finally, real-time control of a large stroke asymmetric pneumatic servo system with measuring system, a large stroke asymmetric pneumatic servo system, data acquisition system and the control strategy software will be implemented. Thus, upgrading the high position precision and the trajectory tracking performance of the large stroke asymmetric pneumatic servo system will be realized to promote the high position precision and path tracking capability. Experimental results show that fifth order paths in various strokes and the sine wave path are successfully implemented in the test rig. Also, results of variable loads under the different angle were implemented experimentally.

Eigenvalue sensitivity of sampled time systems operating in closed loop

Science.gov (United States)

Bernal, Dionisio

2018-05-01

The use of feedback to create closed-loop eigenstructures with high sensitivity has received some attention in the Structural Health Monitoring field. Although practical implementation is necessarily digital, and thus in sampled time, work thus far has center on the continuous time framework, both in design and in checking performance. It is shown in this paper that the performance in discrete time, at typical sampling rates, can differ notably from that anticipated in the continuous time formulation and that discrepancies can be particularly large on the real part of the eigenvalue sensitivities; a consequence being important error on the (linear estimate) of the level of damage at which closed-loop stability is lost. As one anticipates, explicit consideration of the sampling rate poses no special difficulties in the closed-loop eigenstructure design and the relevant expressions are developed in the paper, including a formula for the efficient evaluation of the derivative of the matrix exponential based on the theory of complex perturbations. The paper presents an easily reproduced numerical example showing the level of error that can result when the discrete time implementation of the controller is not considered.

DNA damage mediated transcription arrest: Step back to go forward.

Science.gov (United States)

Mullenders, Leon

2015-12-01

The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored. Copyright © 2015 Elsevier B.V. All rights reserved.

Development and application of a loop-mediated isothermal ...

African Journals Online (AJOL)

Haemophilus parasuis is the causative agent of Glässer's disease that has received much attention recently, due to the increasing economic losses this disease inflicts upon the pig industry worldwide. In this study, loop-mediated isothermal amplification method (LAMP) methodology was designed for diagnosing H.

Adenovirus DNA binding protein inhibits SrCap-activated CBP and CREB-mediated transcription

International Nuclear Information System (INIS)

Xu Xiequn; Tarakanova, Vera; Chrivia, John; Yaciuk, Peter

2003-01-01

The SNF2-related CBP activator protein (SrCap) is a potent activator of transcription mediated by CBP and CREB. We have previously demonstrated that the Adenovirus 2 DNA Binding Protein (DBP) binds to SrCap and inhibits the transcription mediated by the carboxyl-terminal region of SrCap (amino acids 1275-2971). We report here that DBP inhibits the ability of full-length SrCap (1-2971) to activate transcription mediated by Gal-CREB and Gal-CBP. In addition, DBP also inhibits the ability of SrCap to enhance Protein Kinase A (PKA) activated transcription of the enkaphalin promoter. DBP was found to dramatically inhibit transcription of a mammalian two-hybrid system that was dependent on the interaction of SrCap and CBP binding domains. We also found that DBP has no effect on transcription mediated by a transcriptional activator that is not related to SrCap, indicating that our reported transcriptional inhibition is specific for SrCap and not due to nonspecific effects of DBP's DNA binding activity on the CAT reporter plasmid. Taken together, these results suggest a model in which DBP inhibits cellular transcription mediated by the interaction between SrCap and CBP

A regulatory transcriptional loop controls proliferation and differentiation in Drosophila neural stem cells.

Directory of Open Access Journals (Sweden)

Tetsuo Yasugi

Full Text Available Neurogenesis is initiated by a set of basic Helix-Loop-Helix (bHLH transcription factors that specify neural progenitors and allow them to generate neurons in multiple rounds of asymmetric cell division. The Drosophila Daughterless (Da protein and its mammalian counterparts (E12/E47 act as heterodimerization factors for proneural genes and are therefore critically required for neurogenesis. Here, we demonstrate that Da can also be an inhibitor of the neural progenitor fate whose absence leads to stem cell overproliferation and tumor formation. We explain this paradox by demonstrating that Da induces the differentiation factor Prospero (Pros whose asymmetric segregation is essential for differentiation in one of the two daughter cells. Da co-operates with the bHLH transcription factor Asense, whereas the other proneural genes are dispensible. After mitosis, Pros terminates Asense expression in one of the two daughter cells. In da mutants, pros is not expressed, leading to the formation of lethal transplantable brain tumors. Our results define a transcriptional feedback loop that regulates the balance between self-renewal and differentiation in Drosophila optic lobe neuroblasts. They indicate that initiation of a neural differentiation program in stem cells is essential to prevent tumorigenesis.

Frequent dual initiation of reverse transcription in murine leukemia virus-based vectors containing two primer-binding sites

International Nuclear Information System (INIS)

Voronin, Yegor A.; Pathak, Vinay K.

2003-01-01

Retroviruses package two copies of viral RNA into each virion. Although each RNA contains a primer-binding site for initiation of DNA synthesis, it is unknown whether reverse transcription is initiated on both RNAs. To determine whether a single virion is capable of initiating reverse transcription more than once, we constructed a murine leukemia virus-based vector containing a second primer-binding site (PBS) derived from spleen necrosis virus and inserted the green fluorescent protein gene (GFP) between the two PBSs. Initiation of reverse transcription at either PBS results in a provirus that expresses GFP. However, initiation at both PBSs can result in the deletion of GFP, which can be detected by flow cytometry and Southern blotting analysis. Approximately 22-29% of the proviruses formed deleted the GFP in a single replication cycle, indicating the minimum proportion of virions that initiated reverse transcription on both PBSs. These results show that a significant proportion of MLV-based vectors containing two PBSs have the capacity to initiate reverse transcription more than once

A novel method of multiple nucleic acid detection: Real-time RT-PCR coupled with probe-melting curve analysis.

Science.gov (United States)

Han, Yang; Hou, Shao-Yang; Ji, Shang-Zhi; Cheng, Juan; Zhang, Meng-Yue; He, Li-Juan; Ye, Xiang-Zhong; Li, Yi-Min; Zhang, Yi-Xuan

2017-11-15

A novel method, real-time reverse transcription PCR (real-time RT-PCR) coupled with probe-melting curve analysis, has been established to detect two kinds of samples within one fluorescence channel. Besides a conventional TaqMan probe, this method employs another specially designed melting-probe with a 5' terminus modification which meets the same label with the same fluorescent group. By using an asymmetric PCR method, the melting-probe is able to detect an extra sample in the melting stage effectively while it almost has little influence on the amplification detection. Thus, this method allows the availability of united employment of both amplification stage and melting stage for detecting samples in one reaction. The further demonstration by simultaneous detection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in one channel as a model system is presented in this essay. The sensitivity of detection by real-time RT-PCR coupled with probe-melting analysis was proved to be equal to that detected by conventional real-time RT-PCR. Because real-time RT-PCR coupled with probe-melting analysis can double the detection throughputs within one fluorescence channel, it is expected to be a good solution for the problem of low-throughput in current real-time PCR. Copyright © 2017 Elsevier Inc. All rights reserved.

Microchip capillary electrophoresis with laser-induced fluorescence combined with one-step duplex reverse-transcription polymerase chain reaction for the rapid detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens.

Science.gov (United States)

Jia, Ruan; Chengjun, Sun; Heng, Chen; Chen, Zhou; Yuanqian, Li; Yongxin, Li

2015-07-01

Enterovirus 71 and Coxsackievirus A16 are the main pathogens causing hand-foot-mouth disease. In this paper, microchip capillary electrophoresis with laser-induced fluorescence combined with one-step duplex reverse transcript-polymerase chain reaction has been developed for the detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens. The specific reverse transcription-polymerase chain reaction amplicons labeled with SYBR Orange were separated by microchip capillary electrophoresis and detected by laser induced fluorescence detector within 7 min. The intraday and interday relative standard deviation of migration time for DNA Marker was in the range of 1.36-2.94 and 2.78-3.96%, respectively. The detection limits were as low as 2.06 × 10(3) copies/mL for Enterovirus 71 and 5 × 10(3) copies/mL for Coxsackievirus A16. No cross-reactivity was observed with rotavirus, astrovirus, norovirus, and adenovirus, which showed good specificity of the method. This assay was validated using 100 throat swab specimens that were detected by real-time reverse-transcript polymerase chain reaction in parallel and the two methods produced the same results. This study provided a rapid, sensitive and specific method for the detection of Enterovirus 71 and Coxsackievirus A16, which make a contribution to significant time and cost saving for the identification and treatment of patients. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Real-time numerical simulation with high efficiency for an experimental reactor system

International Nuclear Information System (INIS)

Ding Shuling; Li Fu; Li Sifeng; Chu Xinyuan

2006-01-01

The paper presents a systematic and efficient method for numerical real-time simulation of an experimental reactor. The reactor models were built based on the physical characteristics of the experimental reactor, and several real-time simulation approaches were discussed and compared in the paper. How to implement the real-time reactor simulation system in Windows platform for the sake of hardware-in-loop experiment for the reactor power control system was discussed. (authors)

Validated reverse transcription droplet digital PCR serves as a higher order method for absolute quantification of Potato virus Y strains.

Science.gov (United States)

Mehle, Nataša; Dobnik, David; Ravnikar, Maja; Pompe Novak, Maruša

2018-05-03

RNA viruses have a great potential for high genetic variability and rapid evolution that is generated by mutation and recombination under selection pressure. This is also the case of Potato virus Y (PVY), which comprises a high diversity of different recombinant and non-recombinant strains. Consequently, it is hard to develop reverse transcription real-time quantitative PCR (RT-qPCR) with the same amplification efficiencies for all PVY strains which would enable their equilibrate quantification; this is specially needed in mixed infections and other studies of pathogenesis. To achieve this, we initially transferred the PVY universal RT-qPCR assay to a reverse transcription droplet digital PCR (RT-ddPCR) format. RT-ddPCR is an absolute quantification method, where a calibration curve is not needed, and it is less prone to inhibitors. The RT-ddPCR developed and validated in this study achieved a dynamic range of quantification over five orders of magnitude, and in terms of its sensitivity, it was comparable to, or even better than, RT-qPCR. RT-ddPCR showed lower measurement variability. We have shown that RT-ddPCR can be used as a reference tool for the evaluation of different RT-qPCR assays. In addition, it can be used for quantification of RNA based on in-house reference materials that can then be used as calibrators in diagnostic laboratories.

Detection of ROS1 Gene Rearrangement in Lung Adenocarcinoma: Comparison of IHC, FISH and Real-Time RT-PCR

OpenAIRE

Shan, Ling; Lian, Fang; Guo, Lei; Qiu, Tian; Ling, Yun; Ying, Jianming; Lin, Dongmei

2015-01-01

Aims To compare fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and quantitative real-time reverse transcription-PCR (qRT-PCR) assays for detection of ROS1 fusion in a large number of ROS1-positive lung adenocatcinoma (ADC) patients. Methods Using IHC analysis, sixty lung ADCs including 16 cases with ROS1 protein expression and 44 cases without ROS1 expression were selected for this study. The ROS1 fusion status was examined by FISH and qRT-PCR assay. Results Among 60 ca...

Histone acetyltransferase (HAT) activity of p300 modulates human T lymphotropic virus type 1 p30II-mediated repression of LTR transcriptional activity

International Nuclear Information System (INIS)

Michael, Bindhu; Nair, Amrithraj M.; Datta, Antara; Hiraragi, Hajime; Ratner, Lee; Lairmore, Michael D.

2006-01-01

Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T cell leukemia/lymphoma, and is implicated in a variety of lymphocyte-mediated inflammatory disorders. HTLV-1 provirus has regulatory and accessory genes in four pX open reading frames. HTLV-1 pX ORF-II encodes two proteins, p13 II and p30 II , which are incompletely defined in virus replication or pathogenesis. We have demonstrated that pX ORF-II mutations block virus replication in vivo and that ORF-II encoded p30 II , a nuclear-localizing protein that binds with CREB-binding protein (CBP)/p300, represses CREB and Tax responsive element (TRE)-mediated transcription. Herein, we have identified p30 II motifs important for p300 binding and in regulating TRE-mediated transcription in the absence and presence of HTLV-1 provirus. Within amino acids 100-179 of p30 II , a region important for repression of LTR-mediated transcription, we identified a single lysine residue at amino acid 106 (K3) that significantly modulates the ability of p30 II to repress TRE-mediated transcription. Exogenous p300, in a dose-responsive manner, reverses p30 II -dependent repression of TRE-mediated transcription, in the absence or presence of the provirus, In contrast to wild type p300, p300 HAT mutants (defective in histone acetyltransferase activity) only partially rescued p30 II -mediated LTR repression. Deacetylation by histone deacetylase-1 (HDAC-1) enhanced p30 II -mediated LTR repression, while inhibition of deacetylation by trichostatin A decreases p30 II -mediated LTR repression. Collectively, our data indicate that HTLV-1 p30 II modulates viral gene expression in a cooperative manner with p300-mediated acetylation

Mediator subunit18 controls flowering time and floral organ identity in Arabidopsis.

Directory of Open Access Journals (Sweden)

Zhengui Zheng

Full Text Available Mediator is a conserved multi-protein complex that plays an important role in regulating transcription by mediating interactions between transcriptional activator proteins and RNA polymerase II. Much evidence exists that Mediator plays a constitutive role in the transcription of all genes transcribed by RNA polymerase II. However, evidence is mounting that specific Mediator subunits may control the developmental regulation of specific subsets of RNA polymerase II-dependent genes. Although the Mediator complex has been extensively studied in yeast and mammals, only a few reports on Mediator function in flowering time control of plants, little is known about Mediator function in floral organ identity. Here we show that in Arabidopsis thaliana, MEDIATOR SUBUNIT 18 (MED18 affects flowering time and floral organ formation through FLOWERING LOCUS C (FLC and AGAMOUS (AG. A MED18 loss-of-function mutant showed a remarkable syndrome of later flowering and altered floral organ number. We show that FLC and AG mRNA levels and AG expression patterns are altered in the mutant. Our results support parallels between the regulation of FLC and AG and demonstrate a developmental role for Mediator in plants.

Normalization of Reverse Transcription Quantitative PCR Data During Ageing in Distinct Cerebral Structures.

Science.gov (United States)

Bruckert, G; Vivien, D; Docagne, F; Roussel, B D

2016-04-01

Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a routine method in many laboratories. Normalization of data from experimental conditions is critical for data processing and is usually achieved by the use of a single reference gene. Nevertheless, as pointed by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, several reference genes should be used for reliable normalization. Ageing is a physiological process that results in a decline of many expressed genes. Reliable normalization of RT-qPCR data becomes crucial when studying ageing. Here, we propose a RT-qPCR study from four mouse brain regions (cortex, hippocampus, striatum and cerebellum) at different ages (from 8 weeks to 22 months) in which we studied the expression of nine commonly used reference genes. With the use of two different algorithms, we found that all brain structures need at least two genes for a good normalization step. We propose specific pairs of gene for efficient data normalization in the four brain regions studied. These results underline the importance of reliable reference genes for specific brain regions in ageing.

CRY Drives Cyclic CK2-Mediated BMAL1 Phosphorylation to Control the Mammalian Circadian Clock.

Directory of Open Access Journals (Sweden)

Teruya Tamaru

Full Text Available Intracellular circadian clocks, composed of clock genes that act in transcription-translation feedback loops, drive global rhythmic expression of the mammalian transcriptome and allow an organism to anticipate to the momentum of the day. Using a novel clock-perturbing peptide, we established a pivotal role for casein kinase (CK-2-mediated circadian BMAL1-Ser90 phosphorylation (BMAL1-P in regulating central and peripheral core clocks. Subsequent analysis of the underlying mechanism showed a novel role of CRY as a repressor for protein kinase. Co-immunoprecipitation experiments and real-time monitoring of protein-protein interactions revealed that CRY-mediated periodic binding of CK2β to BMAL1 inhibits BMAL1-Ser90 phosphorylation by CK2α. The FAD binding domain of CRY1, two C-terminal BMAL1 domains, and particularly BMAL1-Lys537 acetylation/deacetylation by CLOCK/SIRT1, were shown to be critical for CRY-mediated BMAL1-CK2β binding. Reciprocally, BMAL1-Ser90 phosphorylation is prerequisite for BMAL1-Lys537 acetylation. We propose a dual negative-feedback model in which a CRY-dependent CK2-driven posttranslational BMAL1-P-BMAL1 loop is an integral part of the core clock oscillator.

Triptolide inhibits transcription of hTERT through down-regulation of transcription factor specificity protein 1 in primary effusion lymphoma cells

Energy Technology Data Exchange (ETDEWEB)

Long, Cong; Wang, Jingchao [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071 (China); Guo, Wei [Department of Pathology and Physiology, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071 (China); Wang, Huan; Wang, Chao; Liu, Yu [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071 (China); Sun, Xiaoping, E-mail: xsun6@whu.edu.cn [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071 (China); State Key Laboratory of Virology, Wuhan University, Wuhan, 430072 (China)

2016-01-01

Primary effusion lymphoma (PEL) is a rare and aggressive non-Hodgkin's lymphoma. Human telomerase reverse transcriptase (hTERT), a key component responsible for the regulation of telomerase activity, plays important roles in cellular immortalization and cancer development. Triptolide purified from Tripterygium extracts displays a broad-spectrum bioactivity profile, including immunosuppressive, anti-inflammatory, and anti-tumor. In this study, it is investigated whether triptolide reduces hTERT expression and suppresses its activity in PEL cells. The mRNA and protein levels of hTERT were examined by real time-PCR and Western blotting, respectively. The activity of hTERT promoter was determined by Dual luciferase reporter assay. Our results demonstrated that triptolide decreased expression of hTERT at both mRNA and protein levels. Further gene sequence analysis indicated that the activity of hTERT promoter was suppressed by triptolide. Triptolide also reduced the half-time of hTERT. Additionally, triptolide inhibited the expression of transcription factor specificity protein 1(Sp1) in PEL cells. Furthermore, knock-down of Sp1 by using specific shRNAs resulted in down-regulation of hTERT transcription and protein expression levels. Inhibition of Sp1 by specific shRNAs enhanced triptolide-induced cell growth inhibition and apoptosis. Collectively, our results demonstrate that the inhibitory effect of triptolide on hTERT transcription is possibly mediated by inhibition of transcription factor Sp1 in PEL cells. - Highlights: • Triptolide reduces expression of hTERT by decreasing its transcription level. • Triptolide reduces promoter activity and stability of hTERT. • Triptolide down-regulates expression of Sp1. • Special Sp1 shRNAs inhibit transcription and protein expression of hTERT. • Triptolide and Sp1 shRNA2 induce cell proliferation inhibition and apoptosis.

A novel CMOS image sensor system for quantitative loop-mediated isothermal amplification assays to detect food-borne pathogens.

Science.gov (United States)

Wang, Tiantian; Kim, Sanghyo; An, Jeong Ho

2017-02-01

Loop-mediated isothermal amplification (LAMP) is considered as one of the alternatives to the conventional PCR and it is an inexpensive portable diagnostic system with minimal power consumption. The present work describes the application of LAMP in real-time photon detection and quantitative analysis of nucleic acids integrated with a disposable complementary-metal-oxide semiconductor (CMOS) image sensor. This novel system works as an amplification-coupled detection platform, relying on a CMOS image sensor, with the aid of a computerized circuitry controller for the temperature and light sources. The CMOS image sensor captures the light which is passing through the sensor surface and converts into digital units using an analog-to-digital converter (ADC). This new system monitors the real-time photon variation, caused by the color changes during amplification. Escherichia coli O157 was used as a proof-of-concept target for quantitative analysis, and compared with the results for Staphylococcus aureus and Salmonella enterica to confirm the efficiency of the system. The system detected various DNA concentrations of E. coli O157 in a short time (45min), with a detection limit of 10fg/μL. The low-cost, simple, and compact design, with low power consumption, represents a significant advance in the development of a portable, sensitive, user-friendly, real-time, and quantitative analytic tools for point-of-care diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Soft Real-Time PID Control on a VME Computer

Science.gov (United States)

Karayan, Vahag; Sander, Stanley; Cageao, Richard

2007-01-01

microPID (uPID) is a computer program for real-time proportional + integral + derivative (PID) control of a translation stage in a Fourier-transform ultraviolet spectrometer. microPID implements a PID control loop over a position profile at sampling rate of 8 kHz (sampling period 125microseconds). The software runs in a strippeddown Linux operating system on a VersaModule Eurocard (VME) computer operating in real-time priority queue using an embedded controller, a 16-bit digital-to-analog converter (D/A) board, and a laser-positioning board (LPB). microPID consists of three main parts: (1) VME device-driver routines, (2) software that administers a custom protocol for serial communication with a control computer, and (3) a loop section that obtains the current position from an LPB-driver routine, calculates the ideal position from the profile, and calculates a new voltage command by use of an embedded PID routine all within each sampling period. The voltage command is sent to the D/A board to control the stage. microPID uses special kernel headers to obtain microsecond timing resolution. Inasmuch as microPID implements a single-threaded process and all other processes are disabled, the Linux operating system acts as a soft real-time system.

Real-time horizontal position control for Aditya-upgrade tokamak

International Nuclear Information System (INIS)

Kumar, Rohit; Ghosh, Joydeep; Tanna, Rakesh L.

2015-01-01

Position of plasma column is required to be controlled in real time for improved operation of any tokamak. A PID based system for real-time horizontal plasma position control has been designed for Aditya Upgrade tokamak. Modelling of transfer functions of actuators, plasma and diagnostic system are carried out for ADITYA-U tokamak. The PID controller is optimized using MATLAB-SIMULINK for horizontal position control. Further feed-forward loop is implemented where disturbance due to density variation is suppressed, which results in improved performance as compared to conventional PID operation. In this paper the detailed design of the whole system for real time control of plasma horizontal position in Aditya Upgrade tokamak is presented. (author)

A digital, constant-frequency pulsed phase-locked-loop instrument for real-time, absolute ultrasonic phase measurements

Science.gov (United States)

Haldren, H. A.; Perey, D. F.; Yost, W. T.; Cramer, K. E.; Gupta, M. C.

2018-05-01

A digitally controlled instrument for conducting single-frequency and swept-frequency ultrasonic phase measurements has been developed based on a constant-frequency pulsed phase-locked-loop (CFPPLL) design. This instrument uses a pair of direct digital synthesizers to generate an ultrasonically transceived tone-burst and an internal reference wave for phase comparison. Real-time, constant-frequency phase tracking in an interrogated specimen is possible with a resolution of 0.000 38 rad (0.022°), and swept-frequency phase measurements can be obtained. Using phase measurements, an absolute thickness in borosilicate glass is presented to show the instrument's efficacy, and these results are compared to conventional ultrasonic pulse-echo time-of-flight (ToF) measurements. The newly developed instrument predicted the thickness with a mean error of -0.04 μm and a standard deviation of error of 1.35 μm. Additionally, the CFPPLL instrument shows a lower measured phase error in the absence of changing temperature and couplant thickness than high-resolution cross-correlation ToF measurements at a similar signal-to-noise ratio. By showing higher accuracy and precision than conventional pulse-echo ToF measurements and lower phase errors than cross-correlation ToF measurements, the new digitally controlled CFPPLL instrument provides high-resolution absolute ultrasonic velocity or path-length measurements in solids or liquids, as well as tracking of material property changes with high sensitivity. The ability to obtain absolute phase measurements allows for many new applications than possible with previous ultrasonic pulsed phase-locked loop instruments. In addition to improved resolution, swept-frequency phase measurements add useful capability in measuring properties of layered structures, such as bonded joints, or materials which exhibit non-linear frequency-dependent behavior, such as dispersive media.

Time reversal imaging, Inverse problems and Adjoint Tomography}

Science.gov (United States)

Montagner, J.; Larmat, C. S.; Capdeville, Y.; Kawakatsu, H.; Fink, M.

2010-12-01

With the increasing power of computers and numerical techniques (such as spectral element methods), it is possible to address a new class of seismological problems. The propagation of seismic waves in heterogeneous media is simulated more and more accurately and new applications developed, in particular time reversal methods and adjoint tomography in the three-dimensional Earth. Since the pioneering work of J. Claerbout, theorized by A. Tarantola, many similarities were found between time-reversal methods, cross-correlations techniques, inverse problems and adjoint tomography. By using normal mode theory, we generalize the scalar approach of Draeger and Fink (1999) and Lobkis and Weaver (2001) to the 3D- elastic Earth, for theoretically understanding time-reversal method on global scale. It is shown how to relate time-reversal methods on one hand, with auto-correlations of seismograms for source imaging and on the other hand, with cross-correlations between receivers for structural imaging and retrieving Green function. Time-reversal methods were successfully applied in the past to acoustic waves in many fields such as medical imaging, underwater acoustics, non destructive testing and to seismic waves in seismology for earthquake imaging. In the case of source imaging, time reversal techniques make it possible an automatic location in time and space as well as the retrieval of focal mechanism of earthquakes or unknown environmental sources . We present here some applications at the global scale of these techniques on synthetic tests and on real data, such as Sumatra-Andaman (Dec. 2004), Haiti (Jan. 2010), as well as glacial earthquakes and seismic hum.

A Classification of Basic Helix-Loop-Helix Transcription Factors of Soybean

Directory of Open Access Journals (Sweden)

Karen A. Hudson

2015-01-01

Full Text Available The complete genome sequence of soybean allows an unprecedented opportunity for the discovery of the genes controlling important traits. In particular, the potential functions of regulatory genes are a priority for analysis. The basic helix-loop-helix (bHLH family of transcription factors is known to be involved in controlling a wide range of systems critical for crop adaptation and quality, including photosynthesis, light signalling, pigment biosynthesis, and seed pod development. Using a hidden Markov model search algorithm, 319 genes with basic helix-loop-helix transcription factor domains were identified within the soybean genome sequence. These were classified with respect to their predicted DNA binding potential, intron/exon structure, and the phylogeny of the bHLH domain. Evidence is presented that the vast majority (281 of these 319 soybean bHLH genes are expressed at the mRNA level. Of these soybean bHLH genes, 67% were found to exist in two or more homeologous copies. This dataset provides a framework for future studies on bHLH gene function in soybean. The challenge for future research remains to define functions for the bHLH factors encoded in the soybean genome, which may allow greater flexibility for genetic selection of growth and environmental adaptation in this widely grown crop.

Mediator phosphorylation prevents stress response transcription during non-stress conditions.

Science.gov (United States)

Miller, Christian; Matic, Ivan; Maier, Kerstin C; Schwalb, Björn; Roether, Susanne; Strässer, Katja; Tresch, Achim; Mann, Matthias; Cramer, Patrick

2012-12-28

The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an end point of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here, we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast Saccharomyces cerevisiae is phosphorylated at multiple sites of 17 of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of cells to high salt concentrations. Phosphorylated sites in the Mediator tail subunit Med15 are required for suppression of stress-induced changes in gene expression under non-stress conditions. Thus dynamic and differential Mediator phosphorylation contributes to gene regulation in eukaryotic cells.

Loop-mediated isothermal amplification (LAMP) based detection of ...

African Journals Online (AJOL)

SAM

2014-05-07

May 7, 2014 ... 2 months for growing in a culture. Therefore, to control .... The LAMP reaction is carried out in a 25 µL reaction mixture containing ..... J. Fish Dis. 32(6):491-497. Goto M, Honda E, Ogura A, Nomoto A, Hanaki K (2009). Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy ...

Suppression of Thyroid Hormone Receptor-Mediated Transcription ...

African Journals Online (AJOL)

TH)-induced TR-mediated transcription. We further examined the effects of methamidophos on TR-thyroid hormone response element (TRE) binding using the liquid chemiluminescent DNA pull-down assay (LCDPA), and found no dissociation of ...

Malleable machines in transcription regulation: the mediator complex.

Directory of Open Access Journals (Sweden)

Agnes Tóth-Petróczy

2008-12-01

Full Text Available The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II. The complex has a modular architecture (Head, Middle, and Tail and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs distinguished by a high protein-protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the

Open loop, auto reversing liquid nitrogen circulation thermal system for thermo vacuum chamber

International Nuclear Information System (INIS)

Naidu, M C A; Nolakha, Dinesh; Saharkar, B S; Kavani, K M; Patel, D R

2012-01-01

In a thermo vacuum chamber, attaining and controlling low and high temperatures (-100 Deg. C to +120 Deg. C) is a very important task. This paper describes the development of 'Open loop, auto reversing liquid nitrogen based thermal system'. System specifications, features, open loop auto reversing system, liquid nitrogen flow paths etc. are discussed in this paper. This thermal system consists of solenoid operated cryogenic valves, double embossed thermal plate (shroud), heating elements, temperature sensors and PLC. Bulky items like blowers, heating chambers, liquid nitrogen injection chambers, huge pipe lines and valves were not used. This entire thermal system is very simple to operate and PLC based, fully auto system with auto tuned to given set temperatures. This system requires a very nominal amount of liquid nitrogen (approx. 80 liters / hour) while conducting thermo vacuum tests. This system was integrated to 1.2m dia thermo vacuum chamber, as a part of its augmentation, to conduct extreme temperature cycling tests on passive antenna reflectors of satellites.

Hybrid automata models of cardiac ventricular electrophysiology for real-time computational applications.

Science.gov (United States)

Andalam, Sidharta; Ramanna, Harshavardhan; Malik, Avinash; Roop, Parthasarathi; Patel, Nitish; Trew, Mark L

2016-08-01

Virtual heart models have been proposed for closed loop validation of safety-critical embedded medical devices, such as pacemakers. These models must react in real-time to off-the-shelf medical devices. Real-time performance can be obtained by implementing models in computer hardware, and methods of compiling classes of Hybrid Automata (HA) onto FPGA have been developed. Models of ventricular cardiac cell electrophysiology have been described using HA which capture the complex nonlinear behavior of biological systems. However, many models that have been used for closed-loop validation of pacemakers are highly abstract and do not capture important characteristics of the dynamic rate response. We developed a new HA model of cardiac cells which captures dynamic behavior and we implemented the model in hardware. This potentially enables modeling the heart with over 1 million dynamic cells, making the approach ideal for closed loop testing of medical devices.

The Mediator complex: a master coordinator of transcription and cell lineage development.

Science.gov (United States)

Yin, Jing-wen; Wang, Gang

2014-03-01

Mediator is a multiprotein complex that is required for gene transcription by RNA polymerase II. Multiple subunits of the complex show specificity in relaying information from signals and transcription factors to the RNA polymerase II machinery, thus enabling control of the expression of specific genes. Recent studies have also provided novel mechanistic insights into the roles of Mediator in epigenetic regulation, transcriptional elongation, termination, mRNA processing, noncoding RNA activation and super enhancer formation. Based on these specific roles in gene regulation, Mediator has emerged as a master coordinator of development and cell lineage determination. Here, we describe the most recent advances in understanding the mechanisms of Mediator function, with an emphasis on its role during development and disease.

Development of a real-time RT-PCR and Reverse Line probe Hybridisation assay for the routine detection and genotyping of Noroviruses in Ireland.

LENUS (Irish Health Repository)

Menton, John F

2007-01-01

BACKGROUND: Noroviruses are the most common cause of non-bacterial gastroenteritis. Improved detection methods have seen a large increase in the number of human NoV genotypes in the last ten years. The objective of this study was to develop a fast method to detect, quantify and genotype positive NoV samples from Irish hospitals. RESULTS: A real-time RT-PCR assay and a Reverse Line Blot Hybridisation assay were developed based on the ORF1-ORF2 region. The sensitivity and reactivity of the two assays used was validated using a reference stool panel containing 14 NoV genotypes. The assays were then used to investigate two outbreaks of gastroenteritis in two Irish hospitals. 56 samples were screened for NoV using a real-time RT-PCR assay and 26 samples were found to be positive. Genotyping of these positive samples found that all positives belonged to the GII\\/4 variant of NoV. CONCLUSION: The combination of the Real-time assay and the reverse line blot hybridisation assay provided a fast and accurate method to investigate a NoV associated outbreak. It was concluded that the predominant genotype circulating in these Irish hospitals was GII\\/4 which has been associated with the majority of NoV outbreaks worldwide. The assays developed in this study are useful tools for investigating NoV infection.

Multiplex, Quantitative, Reverse Transcription PCR Detection of Influenza Viruses Using Droplet Microfluidic Technology

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Ravi Prakash

2014-12-01

Full Text Available Quantitative, reverse transcription, polymerase chain reaction (qRT-PCR is facilitated by leveraging droplet microfluidic (DMF system, which due to its precision dispensing and sample handling capabilities at microliter and lower volumes has emerged as a popular method for miniaturization of the PCR platform. This work substantially improves and extends the functional capabilities of our previously demonstrated single qRT-PCR micro-chip, which utilized a combination of electrostatic and electrowetting droplet actuation. In the reported work we illustrate a spatially multiplexed micro-device that is capable of conducting up to eight parallel, real-time PCR reactions per usage, with adjustable control on the PCR thermal cycling parameters (both process time and temperature set-points. This micro-device has been utilized to detect and quantify the presence of two clinically relevant respiratory viruses, Influenza A and Influenza B, in human samples (nasopharyngeal swabs, throat swabs. The device performed accurate detection and quantification of the two respiratory viruses, over several orders of RNA copy counts, in unknown (blind panels of extracted patient samples with acceptably high PCR efficiency (>94%. The multi-stage qRT-PCR assays on eight panel patient samples were accomplished within 35–40 min, with a detection limit for the target Influenza virus RNAs estimated to be less than 10 RNA copies per reaction.

Strategies for real-time position control of a single atom in cavity QED

International Nuclear Information System (INIS)

Lynn, T W; Birnbaum, K; Kimble, H J

2005-01-01

Recent realizations of single-atom trapping and tracking in cavity QED open the door for feedback schemes which actively stabilize the motion of a single atom in real time. We present feedback algorithms for cooling the radial component of motion for a single atom trapped by strong coupling to single-photon fields in an optical cavity. Performance of various algorithms is studied through simulations of single-atom trajectories, with full dynamical and measurement noise included. Closed loop feedback algorithms compare favourably to open loop 'switching' analogues, demonstrating the importance of applying actual position information in real time. The high optical information rate in current experiments enables real-time tracking that approaches the standard quantum limit for broadband position measurements, suggesting that realistic active feedback schemes may reach a regime where measurement backaction appreciably alters the motional dynamics

Development of loop-mediated isothermal amplification (LAMP assay for rapid and sensitive identification of ostrich meat.

Directory of Open Access Journals (Sweden)

Amir Abdulmawjood

Full Text Available Animal species identification is one of the primary duties of official food control. Since ostrich meat is difficult to be differentiated macroscopically from beef, therefore new analytical methods are needed. To enforce labeling regulations for the authentication of ostrich meat, it might be of importance to develop and evaluate a rapid and reliable assay. In the present study, a loop-mediated isothermal amplification (LAMP assay based on the cytochrome b gene of the mitochondrial DNA of the species Struthio camelus was developed. The LAMP assay was used in combination with a real-time fluorometer. The developed system allowed the detection of 0.01% ostrich meat products. In parallel, a direct swab method without nucleic acid extraction using the HYPLEX LPTV buffer was also evaluated. This rapid processing method allowed detection of ostrich meat without major incubation steps. In summary, the LAMP assay had excellent sensitivity and specificity for detecting ostrich meat and could provide a sampling-to-result identification-time of 15 to 20 minutes.

Collider study on the loop-induced dark matter mediation

International Nuclear Information System (INIS)

Tsai, Yuhsin

2016-01-01

Collider experiments are one of the most promising ways to constrain Dark Matter (DM) interactions. For DM couplings involving light mediators, especially for the loop-mediated interactions, a meaningful interpretation of the results requires to go beyond effective field theory. In this note we discuss the study of the magnetic dipole interacting DM, focusing on a model with anarchic dark flavor structure. By including the momentum-dependent form factors that mediate the coupling – given by the Dark Penguin – in collider processes, we study bounds from monophoton, diphoton, and non-pointing photon searches at the LHC. We also compare our results to constraints from the direct detection experiments.

Collider study on the loop-induced dark matter mediation

Energy Technology Data Exchange (ETDEWEB)

Tsai, Yuhsin, E-mail: yhtsai@umd.edu [Maryland Center for Fundamental Physics, University of Maryland, College Park, MD 20742 (United States)

2016-06-21

Collider experiments are one of the most promising ways to constrain Dark Matter (DM) interactions. For DM couplings involving light mediators, especially for the loop-mediated interactions, a meaningful interpretation of the results requires to go beyond effective field theory. In this note we discuss the study of the magnetic dipole interacting DM, focusing on a model with anarchic dark flavor structure. By including the momentum-dependent form factors that mediate the coupling – given by the Dark Penguin – in collider processes, we study bounds from monophoton, diphoton, and non-pointing photon searches at the LHC. We also compare our results to constraints from the direct detection experiments.

Portando uma aplicação de sistema embarcado com arquitetura super Loop para operar com sistema operacional em tempo real

Directory of Open Access Journals (Sweden)

Edielson Prevato Frigieri

2014-08-01

Full Text Available Atualmente, a utilização dos sistemas operacionais, principalmente os sistemas de tempo real (RTOS, tem sido de grande importância no desenvolvimento de sistemas embarcados. Cada vez mais, diferentes e variadas funcionalidades são requisitos dos projetos de sistemas embarcados, como interfaces de comunicação com e sem fio, interfaces com o usuário através de displays, interfaces de acesso de dado como USB, serial, etc. Com o aumento da complexidade, fica extremamente difícil construir um sistema que seja estável e que atenda aos requisitos de tempo a partir de estruturas de programas comuns como o Super Loop. A fim de demonstrar os problemas inerentes deste tipo de arquitetura, foi desenvolvida uma aplicação utilizando o Super Loop e a mesma foi portada para uma solução baseada em RTOS demonstrando os principais conceitos que envolvem um sistema operacional, como utilizá-lo, e os benefícios obtidos com a sua utilização, como estabilidade do sistema, sincronização entre tarefas e cumprimento dos requisitos de tempo. Palavras-chave: Sistemas operacionais de tempo real. Sistemas embarcados. Escalonamento.Porting an embedded system application with super loop architecture to operate with real-time operating systemCurrently, the use of operating systems, especially real-time systems (RTOS, has been of great importance in the development of embedded systems. Increasingly, different and varied features are requirements for embedded system projects, as wired and wireless communication interfaces; user interfaces through displays; data access interfaces such as USB, serial, etc. With the increase in complexity, it becomes extremely difficult to build a system that is stable and that meets the time requirements from common program structures like Super Loop. In order to demonstrate the inherent problems of this type of architecture, an application was developed using Super Loop and it was ported to a solution based on RTOS

The mediating effect of sustainability control system on reverse ...

African Journals Online (AJOL)

This paper analyses part of the viability of green supply chain management practices created for fisheries industry to implement sustainability control system adoption as mediating on reverse logistics innovation and customer environmental collaboration towards sustainability performance. It examines reverse logistics ...

Interlinked dual-time feedback loops can enhance robustness to stochasticity and persistence of memory.

Science.gov (United States)

Smolen, Paul; Baxter, Douglas A; Byrne, John H

2009-03-01

Multiple interlinked positive feedback loops shape the stimulus responses of various biochemical systems, such as the cell cycle or intracellular Ca2+ release. Recent studies with simplified models have identified two advantages of coupling fast and slow feedback loops. This dual-time structure enables a fast response while enhancing resistances of responses and bistability to stimulus noise. We now find that (1) the dual-time structure similarly confers resistance to internal noise due to molecule number fluctuations, and (2) model variants with altered coupling, which better represent some specific biochemical systems, share all the above advantages. We also develop a similar bistable model with coupling of a fast autoactivation loop to a slow loop. This model's topology was suggested by positive feedback proposed to play a role in long-term synaptic potentiation (LTP). The advantages of fast response and noise resistance are also present in this autoactivation model. Empirically, LTP develops resistance to reversal over approximately 1h . The model suggests this resistance may result from increased amounts of synaptic kinases involved in positive feedback.

Structures of transcription pre-initiation complex with TFIIH and Mediator.

Science.gov (United States)

Schilbach, S; Hantsche, M; Tegunov, D; Dienemann, C; Wigge, C; Urlaub, H; Cramer, P

2017-11-09

For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC-core Mediator complex at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the 'E-bridge' helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC-core Mediator complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II.

Remote Whispering Applying Time Reversal

Energy Technology Data Exchange (ETDEWEB)

Anderson, Brian Eric [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

2015-07-16

The purpose of this project was to explore the use of time reversal technologies as a means for communication to a targeted individual or location. The idea is to have the privacy of whispering in one’s ear, but to do this remotely from loudspeakers not located near the target. Applications of this work include communicating with hostages and survivors in rescue operations, communicating imaging and operational conditions in deep drilling operations, monitoring storage of spent nuclear fuel in storage casks without wires, or clandestine activities requiring signaling between specific points. This technology provides a solution in any application where wires and radio communications are not possible or not desired. It also may be configured to self calibrate on a regular basis to adjust for changing conditions. These communications allow two people to converse with one another in real time, converse in an inaudible frequency range or medium (i.e. using ultrasonic frequencies and/or sending vibrations through a structure), or send information for a system to interpret (even allowing remote control of a system using sound). The time reversal process allows one to focus energy to a specific location in space and to send a clean transmission of a selected signal only to that location. In order for the time reversal process to work, a calibration signal must be obtained. This signal may be obtained experimentally using an impulsive sound, a known chirp signal, or other known signals. It may also be determined from a numerical model of a known environment in which the focusing is desired or from passive listening over time to ambient noise.

Time Course of Detection of Human Male DNA from Stained Blood Sample on Various Surfaces by Loop Mediated Isothermal Amplification and Polymerase Chain Reaction

OpenAIRE

Panan Kanchanaphum

2018-01-01

This study explores determining the sex of humans from blood stains taken from different surfaces and compares the time course of detection with the conventional PCR, Conventional Loop Mediated Isothermal Amplification (LAMP), and LAMP-Lateral Flow Dipstick (LFD). For the DNA templates, 7 male and 7 female blood stained samples were extracted and added to LAMP and PCR reaction solution to amplify the SRY gene. The DNA samples were extracted from the following blood stained materials: cloth, w...

Chromosomal loop/nuclear matrix organization of transcriptionally active and inactive RNA polymerases in HeLa nuclei.

Science.gov (United States)

Roberge, M; Dahmus, M E; Bradbury, E M

1988-06-05

The relative distribution of transcriptionally active and inactive RNA polymerases I and II between the nuclear matrix/scaffold and chromosomal loops of HeLa cells was determined. Total RNA polymerase was assessed by immunoblotting and transcribing RNA polymerase by a photoaffinity labeling technique in isolated nuclei. Nuclear matrix/scaffold was isolated by three methods using high-salt, intermediate-salt or low-salt extraction. The distribution of RNA polymerases I and II were very similar within each of the methods, but considerable differences in distributions were found between the different preparation methods. Either intermediate-salt or high-salt treatment of DNase I-digested nuclei showed significant association of RNA polymerases with the nuclear matrix. However, intermediate-salt followed by high-salt treatment released all transcribing and non-transcribing RNA polymerases. Nuclear scaffolds isolated with lithium diiodosalicylate (low-salt) contained very little of the RNA polymerases. This treatment, however, caused the dissociation of RNA polymerase II transcription complexes. These results show unambiguously that RNA polymerases, both in their active and inactive forms, are not nuclear matrix proteins. The data support models in which the transcriptional machinery moves around DNA loops during transcription.

Kalman filters for real-time magnetic island phase tracking

NARCIS (Netherlands)

Borgers, D. P.; Lauret, M.; M.R. de Baar,

2013-01-01

For control of neoclassical tearing modes (NTMs) and the resulting rotating magnetic islands in tokamak plasmas, the frequency and phase of the magnetic islands need to be accurately tracked in real-time. In previous experiments on TEXTOR, this was achieved using a phase-locked loop (PLL). For ASDEX

Reference genes for reverse transcription quantitative PCR in canine brain tissue

NARCIS (Netherlands)

Stassen, Quirine E M; Riemers, Frank M; Reijmerink, Hannah; Leegwater, Peter A J; Penning, Louis C

2015-01-01

BACKGROUND: In the last decade canine models have been used extensively to study genetic causes of neurological disorders such as epilepsy and Alzheimer's disease and unravel their pathophysiological pathways. Reverse transcription quantitative polymerase chain reaction is a sensitive and

Imaging gene expression in real-time using aptamers

Energy Technology Data Exchange (ETDEWEB)

Shin, Il Chung [Iowa State Univ., Ames, IA (United States)

2011-01-01

Signal transduction pathways are usually activated by external stimuli and are transient. The downstream changes such as transcription of the activated genes are also transient. Real-time detection of promoter activity is useful for understanding changes in gene expression, especially during cell differentiation and in development. A simple and reliable method for viewing gene expression in real time is not yet available. Reporter proteins such as fluorescent proteins and luciferase allow for non-invasive detection of the products of gene expression in living cells. However, current reporter systems do not provide for real-time imaging of promoter activity in living cells. This is because of the long time period after transcription required for fluorescent protein synthesis and maturation. We have developed an RNA reporter system for imaging in real-time to detect changes in promoter activity as they occur. The RNA reporter uses strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags), which can be expressed from a promoter of choice. The tobramycin, neomycin and PDC RNA aptamers have been utilized for this system and expressed in yeast from the GAL1 promoter. The IMAGEtag RNA kinetics were quantified by RT-qPCR. In yeast precultured in raffinose containing media the GAL1 promoter responded faster than in yeast precultured in glucose containing media. IMAGEtag RNA has relatively short half-life (5.5 min) in yeast. For imaging, the yeast cells are incubated with their ligands that are labeled with fluorescent dyes. To increase signal to noise, ligands have been separately conjugated with the FRET (Förster resonance energy transfer) pairs, Cy3 and Cy5. With these constructs, the transcribed aptamers can be imaged after activation of the promoter by galactose. FRET was confirmed with three different approaches, which were sensitized emission, acceptor photobleaching and donor lifetime by FLIM (fluorescence lifetime imaging

Imaging gene expression in real-time using aptamers

Energy Technology Data Exchange (ETDEWEB)

Shin, Ilchung [Iowa State Univ., Ames, IA (United States)

2012-01-01

Signal transduction pathways are usually activated by external stimuli and are transient. The downstream changes such as transcription of the activated genes are also transient. Real-time detection of promoter activity is useful for understanding changes in gene expression, especially during cell differentiation and in development. A simple and reliable method for viewing gene expression in real time is not yet available. Reporter proteins such as fluorescent proteins and luciferase allow for non-invasive detection of the products of gene expression in living cells. However, current reporter systems do not provide for real-time imaging of promoter activity in living cells. This is because of the long time period after transcription required for fluorescent protein synthesis and maturation. We have developed an RNA reporter system for imaging in real-time to detect changes in promoter activity as they occur. The RNA reporter uses strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags), which can be expressed from a promoter of choice. The tobramycin, neomycin and PDC RNA aptamers have been utilized for this system and expressed in yeast from the GAL1 promoter. The IMAGEtag RNA kinetics were quantified by RT-qPCR. In yeast precultured in raffinose containing media the GAL1 promoter responded faster than in yeast precultured in glucose containing media. IMAGEtag RNA has relatively short half-life (5.5 min) in yeast. For imaging, the yeast cells are incubated with their ligands that are labeled with fluorescent dyes. To increase signal to noise, ligands have been separately conjugated with the FRET (Förster resonance energy transfer) pairs, Cy3 and Cy5. With these constructs, the transcribed aptamers can be imaged after activation of the promoter by galactose. FRET was confirmed with three different approaches, which were sensitized emission, acceptor photobleaching and donor lifetime by FLIM (fluorescence lifetime imaging

Gen2 RFID-Based System Framework for Resource Circulation in Closed-Loop Supply Chains

Directory of Open Access Journals (Sweden)

Young-woo Kim

2017-10-01

Full Text Available Product recycling has become a mandatory activity under extended producer responsibility. Therefore, it is important to operate a closed-loop supply chain that integrates sourcing, production, delivery, and recycling to achieve not only environmental sustainability, but also economic benefits. However, this goal is affected by chronic problems caused by uncertainties relating to the return timing, quantity, and quality of returned items. Many studies proved that information visibility could solve these problems. In this context, a system framework for closed-loop supply chain management is proposed that gathers real-time information within a supply chain and product lifecycle by using the Internet-of-Things, including radio frequency identification (RFID. Specifically, the most recent Gen2 RFID protocol, which provides new features to create new positive effects, is considered. Additionally, an information system is designed, including RFID tag encoding, which supports the operation of the proposed system. Finally, the lifecycle benefits are examined, such as counterfeit prevention, real-time monitoring and maintenance in the middle-of-life phase, and reverse process streamlining. The ultimate aim is to design a system that facilitates the profitable and environmentally friendly operation of the closed-loop supply chain.

Upgrade of the COMPASS tokamak real-time control system

Energy Technology Data Exchange (ETDEWEB)

Janky, F., E-mail: filip.janky.work@gmail.com [Institute of Plasma Physics, AS CR, v.v.i., Association EURATOM/IPP.CR, Za Slovankou 3, 18200 Prague (Czech Republic); Charles University in Prague, Faculty of Mathematics and Physics, V Holesovickach 2, 18000 Prague (Czech Republic); Havlicek, J. [Institute of Plasma Physics, AS CR, v.v.i., Association EURATOM/IPP.CR, Za Slovankou 3, 18200 Prague (Czech Republic); Charles University in Prague, Faculty of Mathematics and Physics, V Holesovickach 2, 18000 Prague (Czech Republic); Batista, A.J.N. [Associação EURATOM/IST, Instituto de Plasmas e Fusão Nuclear, Instituto Superior Técnico, Universidade Técnica de Lisboa, 1049-001 Lisboa (Portugal); Kudlacek, O.; Seidl, J. [Institute of Plasma Physics, AS CR, v.v.i., Association EURATOM/IPP.CR, Za Slovankou 3, 18200 Prague (Czech Republic); Neto, A.C. [Associação EURATOM/IST, Instituto de Plasmas e Fusão Nuclear, Instituto Superior Técnico, Universidade Técnica de Lisboa, 1049-001 Lisboa (Portugal); Pipek, J.; Hron, M. [Institute of Plasma Physics, AS CR, v.v.i., Association EURATOM/IPP.CR, Za Slovankou 3, 18200 Prague (Czech Republic); Mikulin, O. [Institute of Plasma Physics, AS CR, v.v.i., Association EURATOM/IPP.CR, Za Slovankou 3, 18200 Prague (Czech Republic); Czech Technical University, Faculty of Nuclear Sciences and Physical Engineering, V Holesovickach 2, 18000 Prague (Czech Republic); and others

2014-03-15

Highlights: • An upgrade of the COMPASS real-time system has been made to generally improve the plasma performance. • Stability of discharges in SNT configuration has been increased. • Plasma flat-top phase length has been extended. • Central solenoid protection has been developed. • Plasma position estimation has been improved. - Abstract: The COMPASS plasma control system is based on the MARTe real-time framework. Thanks to MARTe modularity and flexibility new algorithms have been developed for plasma diagnostic (plasma position calculation), control (shaping field control), and protection systems (central solenoid protection). Moreover, the MARTe framework itself was modified to broaden the communication capabilities via Aurora. This paper presents the recent upgrades and improvements made to the COMPASS real-time plasma control system, focusing on the issues related to precision of the real-time calculations, and discussing the improvements in terms of discharge parameters and stability. In particular, the new real-time system has given the possibility to analyze and to minimize the transport delays of each control loop.

Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment.

Science.gov (United States)

Eyboulet, Fanny; Cibot, Camille; Eychenne, Thomas; Neil, Helen; Alibert, Olivier; Werner, Michel; Soutourina, Julie

2013-12-01

Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3' endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes.

Demonstration of real-time control for poloidal beta in KSTAR

Energy Technology Data Exchange (ETDEWEB)

Han, Hyunsun, E-mail: hyunsun@nfri.re.kr [National Fusion Research Institute, Daejeon 305-806 (Korea, Republic of); Hahn, S.H.; Bak, J.G. [National Fusion Research Institute, Daejeon 305-806 (Korea, Republic of); Hyatt, A.; Johnson, R. [General Atomics, San Diego, CA (United States); Woo, M.H.; Kim, J.S.; Bae, Y.S. [National Fusion Research Institute, Daejeon 305-806 (Korea, Republic of)

2015-06-15

Highlights: • Real time control system for poloidal beta has been designed in KSTAR. • Poloidal beta has been calculated based on the diamagnetic loop signals. • The neutral beam Injector plays a role as the actuator. • The control system has been validated in the KSTAR experiments. - Abstract: Sustaining the plasma in a stable and a high performance condition is one of the important control issues for future steady state tokamaks. In the 2014 KSTAR campaign, we have developed a real-time poloidal beta (β{sub p}) control technique and carried out preliminary experiments to identify its feasibility. In the control system, the β{sub p} is calculated in real time using the measured diamagnetic loop signal, and compared with the target value leading to the change of the neutral beam (NB) heating power using a feedback PID control algorithm. To match the requested power of NB which is operated with constant voltage, the on-time periods of the intervals were adjusted as the ratio of the required power to the maximum achievable one. This paper will present the overall procedures of the β{sub p} control, the β{sub p} estimation process and NB operation scheme implemented in the plasma control system (PCS), and the analysis on the preliminary experimental results.

Demonstration of real-time control for poloidal beta in KSTAR

International Nuclear Information System (INIS)

Han, Hyunsun; Hahn, S.H.; Bak, J.G.; Hyatt, A.; Johnson, R.; Woo, M.H.; Kim, J.S.; Bae, Y.S.

2015-01-01

Highlights: • Real time control system for poloidal beta has been designed in KSTAR. • Poloidal beta has been calculated based on the diamagnetic loop signals. • The neutral beam Injector plays a role as the actuator. • The control system has been validated in the KSTAR experiments. - Abstract: Sustaining the plasma in a stable and a high performance condition is one of the important control issues for future steady state tokamaks. In the 2014 KSTAR campaign, we have developed a real-time poloidal beta (β p ) control technique and carried out preliminary experiments to identify its feasibility. In the control system, the β p is calculated in real time using the measured diamagnetic loop signal, and compared with the target value leading to the change of the neutral beam (NB) heating power using a feedback PID control algorithm. To match the requested power of NB which is operated with constant voltage, the on-time periods of the intervals were adjusted as the ratio of the required power to the maximum achievable one. This paper will present the overall procedures of the β p control, the β p estimation process and NB operation scheme implemented in the plasma control system (PCS), and the analysis on the preliminary experimental results

Real-time applications of neural nets

International Nuclear Information System (INIS)

Spencer, J.E.

1989-05-01

Producing, accelerating and colliding very high power, low emittance beams for long periods is a formidable problem in real-time control. As energy has grown exponentially in time so has the complexity of the machines and their control systems. Similar growth rates have occurred in many areas, e.g., improved integrated circuits have been paid for with comparable increases in complexity. However, in this case, reliability, capability and cost have improved due to reduced size, high production and increased integration which allow various kinds of feedback. In contrast, most large complex systems (LCS) are perceived to lack such possibilities because only one copy is made. Neural nets, as a metaphor for LCS, suggest ways to circumvent such limitations. It is argued that they are logically equivalent to multi-loop feedback/forward control of faulty systems. While complimentary to AI, they mesh nicely with characteristics desired for real-time systems. Such issues are considered, examples given and possibilities discussed. 21 refs., 6 figs

Real-time applications of neural nets

International Nuclear Information System (INIS)

Spencer, J.E.

1989-01-01

Producing, accelerating and colliding very high power, low emittance beams for long periods is a formidable problem in real-time control. As energy has grown exponentially in time so has the complexity of the machines and their control systems. Similar growth rates have occurred in many areas e.g. improved integrated circuits have been paid for with comparable increases in complexity. However, in this case, reliability, capability and cost have improved due to reduced size, high production and increased integration which allow various kinds of feedback. In contrast, most large complex systems (LCS) are perceived to lack such possibilities because only one copy is made. Neural nets, as a metaphor for LCS, suggest ways to circumvent such limitations. It is argued that they are logically equivalent to multi-loop feedback/forward control of faulty systems. While complimentary to AI, they mesh nicely with characteristics desired for real-time systems. In this paper, such issues are considered, examples given and possibilities discussed

Real-time applications of neural nets

Energy Technology Data Exchange (ETDEWEB)

Spencer, J.E.

1989-05-01

Producing, accelerating and colliding very high power, low emittance beams for long periods is a formidable problem in real-time control. As energy has grown exponentially in time so has the complexity of the machines and their control systems. Similar growth rates have occurred in many areas, e.g., improved integrated circuits have been paid for with comparable increases in complexity. However, in this case, reliability, capability and cost have improved due to reduced size, high production and increased integration which allow various kinds of feedback. In contrast, most large complex systems (LCS) are perceived to lack such possibilities because only one copy is made. Neural nets, as a metaphor for LCS, suggest ways to circumvent such limitations. It is argued that they are logically equivalent to multi-loop feedback/forward control of faulty systems. While complimentary to AI, they mesh nicely with characteristics desired for real-time systems. Such issues are considered, examples given and possibilities discussed. 21 refs., 6 figs.

Dynamic, in vivo, real-time detection of retinal oxidative status in a model of elevated intraocular pressure using a novel, reversibly responsive, profluorescent nitroxide probe.

Science.gov (United States)

Rayner, Cassie L; Gole, Glen A; Bottle, Steven E; Barnett, Nigel L

2014-12-01

Changes to the redox status of biological systems have been implicated in the pathogenesis of a wide variety of disorders including cancer, Ischemia-reperfusion (I/R) injury and neurodegeneration. In times of metabolic stress e.g. ischaemia/reperfusion, reactive oxygen species (ROS) production overwhelms the intrinsic antioxidant capacity of the cell, damaging vital cellular components. The ability to quantify ROS changes in vivo, is therefore essential to understanding their biological role. Here we evaluate the suitability of a novel reversible profluorescent probe containing a redox-sensitive nitroxide moiety (methyl ester tetraethylrhodamine nitroxide, ME-TRN), as an in vivo, real-time reporter of retinal oxidative status. The reversible nature of the probe's response offers the unique advantage of being able to monitor redox changes in both oxidizing and reducing directions in real time. After intravitreal administration of the ME-TRN probe, we induced ROS production in rat retina using an established model of complete, acute retinal ischaemia followed by reperfusion. After restoration of blood flow, retinas were imaged using a Micron III rodent fundus fluorescence imaging system, to quantify the redox-response of the probe. Fluorescent intensity declined during the first 60 min of reperfusion. The ROS-induced change in probe fluorescence was ameliorated with the retinal antioxidant, lutein. Fluorescence intensity in non-Ischemia eyes did not change significantly. This new probe and imaging technology provide a reversible and real-time response to oxidative changes and may allow the in vivo testing of antioxidant therapies of potential benefit to a range of diseases linked to oxidative stress. Copyright © 2014 Elsevier Ltd. All rights reserved.

Human GW182 Paralogs Are the Central Organizers for RNA-Mediated Control of Transcription.

Science.gov (United States)

Hicks, Jessica A; Li, Liande; Matsui, Masayuki; Chu, Yongjun; Volkov, Oleg; Johnson, Krystal C; Corey, David R

2017-08-15

In the cytoplasm, small RNAs can control mammalian translation by regulating the stability of mRNA. In the nucleus, small RNAs can also control transcription and splicing. The mechanisms for RNA-mediated nuclear regulation are not understood and remain controversial, hindering the effective application of nuclear RNAi and investigation of its natural regulatory roles. Here, we reveal that the human GW182 paralogs TNRC6A/B/C are central organizing factors critical to RNA-mediated transcriptional activation. Mass spectrometry of purified nuclear lysates followed by experimental validation demonstrates that TNRC6A interacts with proteins involved in protein degradation, RNAi, the CCR4-NOT complex, the mediator complex, and histone-modifying complexes. Functional analysis implicates TNRC6A, NAT10, MED14, and WDR5 in RNA-mediated transcriptional activation. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Real-time simulation of aeroelastic rotor loads for horizontal axis wind turbines

International Nuclear Information System (INIS)

Marnett, M; Wellenberg, S; Schröder, W

2014-01-01

Wind turbine drivetrain research and test facilities with hardware-in-the-loop capabilities require a robust and accurate aeroelastic real-time rotor simulation environment. Recent simulation environments do not guarantee a computational response at real-time. Which is why a novel simulation tool has been developed. It resolves the physical time domain of the turbulent wind spectra and the operational response of the turbine at real-time conditions. Therefore, there is a trade-off between accuracy of the physical models and the computational costs. However, the study shows the possibility to preserve the necessary computational accuracy while simultaneously granting dynamic interaction with the aeroelastic rotor simulation environment. The achieved computational costs allow a complete aeroelastic rotor simulation at a resolution frequency of 100 Hz on standard computer platforms. Results obtained for the 5-MW reference wind turbine by the National Renewable Energy Laboratory (NREL) are discussed and compared to NREL's fatigue, aerodynamics, structures, and turbulence (FAST)- Code. The rotor loads show a convincing match. The novel simulation tool is applied to the wind turbine drivetrain test facility at the Center for Wind Power Drives (CWD), RWTH Aachen University to show the real-time hardware-in-the-loop capabilities

BabelFish-Tools for IEEE C37.118.2-compliant real-time synchrophasor data mediation

Science.gov (United States)

Almas, M. S.; Vanfretti, L.; Baudette, M.

BabelFish (BF) is a real-time data mediator for development and fast prototyping of synchrophasor applications. BF is compliant with the synchrophasor data transmission IEEE Std C37.118.2-2011. BF establishes a TCP/IP connection with any Phasor Measurement Unit (PMU) or Phasor Data Concentrator (PDC) stream and parses the IEEE Std C37.118.2-2011 frames in real-time to provide access to raw numerical data in the LabVIEW environment. Furthermore, BF allows the user to select "data-of-interest" and transmit it to either a local or remote application using the User Datagram Protocol (UDP) in order to support both unicast and multicast communication. In the power systems Wide Area Monitoring Protection and Control (WAMPAC) domain, BF provides the first Free/Libre and Open Source Software (FLOSS) for the purpose of giving the users tools for fast prototyping of new applications processing PMU measurements in their chosen environment, thus liberating them of time consuming synchrophasor data handling and allowing them to develop applications in a modular fashion, without a need of a large and monolithic synchrophasor software environment.

Survey and visual detection of Zaire ebolavirus in clinical samples targeting the nucleoprotein gene in Sierra Leone

Directory of Open Access Journals (Sweden)

Jing Yuan

2015-12-01

Full Text Available Ebola virus (EBOV can lead to severe hemorrhagic fever with a high risk of death in humans and other primates. To guide treatment and prevent spread of the viral infection, a rapid and sensitive detection method is required for clinical samples. Here, we described and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP method to detect Zaire ebolavirus using the nucleoprotein gene (NP as a target sequence. Two different techniques were used, a calcein/Mn2+ complex chromogenic method and real-time turbidity monitoring. The RT-LAMP assay detected the NP target sequence with a limit of 4.56 copies/μL within 45 min under 61°C, a similar even or increase in sensitivity than that of real-time reverse transcription-polymerase chain reaction (RT-PCR. Additionally, all pseudoviral particles or non- Zaire EBOV genomes were negative for LAMP detection, indicating that the assay was highly specific for EBOV. To appraise the availability of the RT-LAMP method for use in clinical diagnosis of EBOV, of 417 blood or swab samples collected from patients with clinically suspected infections in Sierra Leone, 307 were identified for RT-LAMP-based surveillance of EBOV. Therefore, the highly specific and sensitive RT-LAMP method allows the rapid detection of EBOV, and is a suitable tool for clinical screening, diagnosis, and primary quarantine purposes.

Comparison of polymerase chain reaction (PCR) and loop-mediated ...

African Journals Online (AJOL)

Comparison of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) for diagnosis of Fusarium solani in human immunodeficiency virus (HIV) positive patients. ... The test was carried out in 1 h reaction at 65°C in a heater block. The specificity of the test was 100% and its sensitivity was a ...

Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA

Directory of Open Access Journals (Sweden)

Eric R. Gamache

2017-04-01

Full Text Available The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5′ terminus of Ty1 RNA harbors cis-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT. To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotides of Ty1 RNA form a pseudoknot with a 7-bp stem (S1, a 1-nucleotide interhelical loop and an 8-bp stem (S2 that delineate two long, structured loops. Nucleotide substitutions that disrupt either pseudoknot stem greatly reduced helper-Ty1-mediated retrotransposition of a mini-Ty1, but only mutations in S2 destabilized mini-Ty1 RNA in cis and helper-Ty1 RNA in trans. Nested in different loops of the pseudoknot are two hairpins with complementary 7-nucleotide motifs at their apices. Nucleotide substitutions in either motif also reduced retrotransposition and destabilized mini- and helper-Ty1 RNA. Compensatory mutations that restore base-pairing in the S2 stem or between the hairpins rescued retrotransposition and RNA stability in cis and trans. These data inform a model whereby a Ty1 RNA kissing complex with two intermolecular kissing-loop interactions initiates dimerization and packaging.

Development of a Real-Time Resistance Measurement for Vibrio parahaemolyticus Detection by the Lecithin-Dependent Hemolysin Gene

Science.gov (United States)

Xiang, Guiming; Pu, Xiaoyun; Jiang, Dongneng; Liu, Linlin; Liu, Chang; Liu, Xiaobo

2013-01-01

The marine bacterium Vibrio parahaemolyticus (V. parahaemolyticus) causes gastroenteritis in humans via the ingestion of raw or undercooked contaminated seafood, and early diagnosis and prompt treatment are important for the prevention of V. parahaemolyticus-related diseases. In this study, a real-time resistance measurement based on loop-mediated isothermal amplification (LAMP), electrochemical ion bonding (Crystal violet and Mg2+), real-time monitoring, and derivative analysis was developed. V. parahaemolyticus DNA was first amplified by LAMP, and the products (DNA and pyrophosphate) represented two types of negative ions that could combine with a positive dye (Crystal violet) and positive ions (Mg2+) to increase the resistance of the reaction liquid. This resistance was measured in real-time using a specially designed resistance electrode, thus permitting the quantitative detection of V. parahaemolyticus. The results were obtained in 1–2 hours, with a minimum bacterial density of 10 CFU.mL−1 and high levels of accuracy (97%), sensitivity (96.08%), and specificity (97.96%) when compared to cultivation methods. Therefore, this simple and rapid method has a potential application in the detection of V. parahaemolyticus on a gene chip or in point-of-care testing. PMID:23991096

Indoor Localization of a Quadrotor Based on WSN: A Real-Time Application

Directory of Open Access Journals (Sweden)

Jose L. Rullan-Lara

2013-01-01

Full Text Available A real-time localization algorithm is presented in this paper. The algorithm presented here uses an extended Kalman filter and is based on Time Difference Of Arrivals (TDOA measurements of radio signal. The position and velocity of an Unmanned Aerial Vehicle (UAV are successfully estimated in closed-loop in real-time, both in hover and path following flights. Relatively small position errors obtained from the experiments prove the good performance of the proposed algorithm.

Use of a novel virus inactivation method for a multicenter avian influenza real-time reverse transcriptase-polymerase chain reaction proficiency study.

Science.gov (United States)

Spackman, Erica; Suarez, David L

2005-01-01

Proficiency assessments are important elements in quality control for diagnostic laboratories. Traditionally, proficiency testing for polymerase chain reaction (PCR)-based assays has involved the use of clinical samples, samples "spiked" with live agents or DNA plasmids. Because of government regulations and biosecurity concerns, distribution of live high-consequence pathogens of livestock and poultry, such as avian influenza, is not possible, and DNA plasmids are not technically suitable for evaluating RNA virus detection. Therefore, a proficiency testing panel using whole avian influenza in a diluent containing a phenolic disinfectant that inactivates the virus while preserving the RNA for at least 8 weeks at -70 C was developed and used in a multicenter proficiency assessment for a type A influenza real-time reverse transcriptase (RT)-PCR test. The test, which was highly standardized, except for variation in the real-time RT-PCR equipment used, was shown to be highly reproducible by proficiency testing in 12 laboratories in the United States, Canada, and Hong Kong. Variation in cycle threshold values among 35 data sets and 490 samples was minimal (CV = 5.19%), and sample identifications were highly accurate (96.7% correct identifications) regardless of real-time PCR instrumentation.

Atomic force microscope cantilever as an encoding sensor for real-time displacement measurement

International Nuclear Information System (INIS)

Chen, Xiaomei; Koenders, Ludger; Wolff, Helmut; Haertig, Frank; Schilling, Meinhard

2010-01-01

A tuning fork-based atomic force microscope cantilever has been investigated for application as an encoding sensor for real-time displacement measurement. The algorithm used to encode the displacement is based on the direct count of the integer pitches of a known grating, and the calculation of the fractional parts of a pitch at the beginning and during displacement. A cross-correlation technique has been adopted and applied to the real-time signal filtering process for the determination of the pitch during scanning by using a half sinusoidal waveform template. For the first investigation, a 1D sinusoidal grating with the pitch of 300 nm is used. The repeatability of displacement measurements over a distance of 70 µm is better than 2.2 nm. As the first application, the real-time displacement of a scanning stage is measured by the new encoding principle as it is moved in an open-loop mode and closed-loop mode based on its built-in capacitance sensor

Real time MHD mode control using ECCD in KSTAR: Plan and requirements

Energy Technology Data Exchange (ETDEWEB)

Joung, M.; Woo, M. H.; Jeong, J. H.; Hahn, S. H.; Yun, S. W.; Lee, W. R.; Bae, Y. S.; Oh, Y. K.; Kwak, J. G.; Yang, H. L. [National Fusion Research Institute, 52 Eoeun-dong, Yuseong-gu, Daejeon (Korea, Republic of); Namkung, W.; Park, H.; Cho, M. H. [Department of Physics, POSTECH, Hyoja-dong, Nam-gu, Pohang, Gyeongangbuk-do (Korea, Republic of); Kim, M. H.; Kim, K. J.; Na, Y. S. [Department of Nuclear Engineering, Seoul National University, Daehak-dong, Gwanak-gu, Seoul (Korea, Republic of); Hosea, J.; Ellis, R. [Princeton Plasma Physics Laboratory, Princeton (United States)

2014-02-12

For a high-performance, advanced tokamak mode in KSTAR, we have been developing a real-time control system of MHD modes such as sawtooth and Neo-classical Tearing Mode (NTM) by ECH/ECCD. The active feedback control loop will be also added to the mirror position and the real-time detection of the mode position. In this year, for the stabilization of NTM that is crucial to plasma performance we have implemented open-loop ECH antenna control system in KSTAR Plasma Control System (PCS) for ECH mirror movement during a single plasma discharge. KSTAR 170 GHz ECH launcher which was designed and fabricated by collaboration with PPPL and POSTECH has a final mirror of a poloidally and toroidally steerable mirror. The poloidal steering motion is only controlled in the real-time NTM control system and its maximum steering speed is 10 degree/sec by DC motor. However, the latency of the mirror control system and the return period of ECH antenna mirror angle are not fast because the existing launcher mirror control system is based on PLC which is connected to the KSTAR machine network through serial to LAN converter. In this paper, we present the design of real time NTM control system, ECH requirements, and the upgrade plan.

Loop-mediated isothermal amplification (LAMP) shield for Arduino DNA detection

NARCIS (Netherlands)

Velders, Aldrik H.; Schoen, Cor; Saggiomo, Vittorio

2018-01-01

Objective: Loop-mediated isothermal amplification (LAMP) of DNA is gaining relevance as a method to detect nucleic acids, as it is easier, faster, and more powerful than conventional Polymerase Chain Reaction. However, LAMP is still mostly used in laboratory settings, because of the lack of a cheap

Semi-Nested Real-Time Reverse Transcription Polymerase Chain Reaction Methods for the Successful Quantitation of Cytokeratin mRNA Expression Levels for the Subtyping of Non-Small-Cell Lung Carcinoma Using Paraffin-Embedded and Microdissected Lung Biopsy Specimens

International Nuclear Information System (INIS)

Nakanishi, Yoko; Shimizu, Tetsuo; Tsujino, Ichiro; Obana, Yukari; Seki, Toshimi; Fuchinoue, Fumi; Ohni, Sumie; Oinuma, Toshinori; Kusumi, Yoshiaki; Yamada, Tsutomu; Takahashi, Noriaki; Hashimoto, Shu; Nemoto, Norimichi

2013-01-01

In patients with inoperable advanced non-small cell lung carcinomas (NSCLCs), histological subtyping using small-mount biopsy specimens was often required to decide the indications for drug treatment. The aim of this study was to assess the utility of highly sensitive mRNA quantitation for the subtyping of advanced NSCLC using small formalin fixing and paraffin embedding (FFPE) biopsy samples. Cytokeratin (CK) 6, CK7, CK14, CK18, and thyroid transcription factor (TTF)-1 mRNA expression levels were measured using semi-nested real-time quantitative (snq) reverse-transcribed polymerase chain reaction (RT-PCR) in microdissected tumor cells collected from 52 lung biopsies. Our results using the present snqRT-PCR method showed an improvement in mRNA quantitation from small FFPE samples, and the mRNA expression level using snqRT-PCR was correlated with the immunohistochemical protein expression level. CK7, CK18, and TTF-1 mRNA were expressed at significantly higher levels (P<0.05) in adenocarcinoma (AD) than in squamous cell carcinoma (SQ), while CK6 and CK14 mRNA expression was significantly higher (P<0.05) in SQ than in AD. Each histology-specific CK, particularly CK18 in AD and CK6 in SQ, were shown to be correlated with a poor prognosis (P=0.02, 0.02, respectively). Our results demonstrated that a quantitative CK subtype mRNA analysis from lung biopsy samples can be useful for predicting the histology subtype and prognosis of advanced NSCLC

Testing the causality of Hawkes processes with time reversal

Science.gov (United States)

Cordi, Marcus; Challet, Damien; Muni Toke, Ioane

2018-03-01

We show that univariate and symmetric multivariate Hawkes processes are only weakly causal: the true log-likelihoods of real and reversed event time vectors are almost equal, thus parameter estimation via maximum likelihood only weakly depends on the direction of the arrow of time. In ideal (synthetic) conditions, tests of goodness of parametric fit unambiguously reject backward event times, which implies that inferring kernels from time-symmetric quantities, such as the autocovariance of the event rate, only rarely produce statistically significant fits. Finally, we find that fitting financial data with many-parameter kernels may yield significant fits for both arrows of time for the same event time vector, sometimes favouring the backward time direction. This goes to show that a significant fit of Hawkes processes to real data with flexible kernels does not imply a definite arrow of time unless one tests it.

A real-time BWR [boiling water reactor] stability measurement system

International Nuclear Information System (INIS)

March-Leuba, J.; King, W.T.

1987-01-01

This paper describes the characteristics of a portable, real-time system used for nonperturbational measurements of stability in boiling water reactors. The algorithm used in this system estimates the closed-loop asymptotic decay ratio using only the naturally occurring neutron noise and it is based on the univariate autoregressive methodology

TALE-mediated modulation of transcriptional enhancers in vivo.

Science.gov (United States)

Crocker, Justin; Stern, David L

2013-08-01

We tested whether transcription activator-like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.

Brassinosteroid-Induced Transcriptional Repression and Dephosphorylation-Dependent Protein Degradation Negatively Regulate BIN2-Interacting AIF2 (a BR Signaling-Negative Regulator) bHLH Transcription Factor.

Science.gov (United States)

Kim, Yoon; Song, Ji-Hye; Park, Seon-U; Jeong, You-Seung; Kim, Soo-Hwan

2017-02-01

Brassinosteroids (BRs) are plant polyhydroxy-steroids that play important roles in plant growth and development via extensive signal integration through direct interactions between regulatory components of different signaling pathways. Recent studies have shown that diverse helix-loop-helix/basic helix-loop-helix (HLH/bHLH) family proteins are actively involved in control of BR signaling pathways and interact with other signaling pathways. In this study, we show that ATBS1-INTERACTING FACTOR 2 (AIF2), a nuclear-localized atypical bHLH transcription factor, specifically interacts with BRASSINOSTEROID-INSENSITIVE 2 (BIN2) among other BR signaling molecules. Overexpression of AIF2 down-regulated transcript expression of growth-promoting genes, thus resulting in retardation of growth. AIF2 renders plants hyposensitive to BR-induced root growth inhibition, but shows little effects on BR-promoted hypocotyl elongation. Notably, AIF2 was dephosphorylated by BR, and the dephosphorylated AIF2 was subject to proteasome-mediated degradation. AIF2 degradation was greatly induced by BR and ABA, but relatively slightly by other hormones such as auxin, gibberellin, cytokinin and ethylene. Moreover, AIF2 transcription was significantly suppressed by a BRI1/BZR1-mediated BR signaling pathway through a direct binding of BRASSINAZOLE RESISTANT 1 (BZR1) to the BR response element (BRRE) region of the AIF2 promoter. In conclusion, our study suggests that BIN2-driven AIF2 phosphorylation could augment the BIN2/AIF2-mediated negative circuit of BR signaling pathways, and the BR-induced transcriptional repression and protein degradation negatively regulate AIF2 transcription factor, reinforcing the BZR1/BES1-mediated positive BR signaling pathway. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

International Nuclear Information System (INIS)

Huang, S-H; Tsai, M-H; Lin, C-W; Yang, T-C; Chuang, P-H; Tsai, I-S; Lu, H-C; Wan Lei; Lin, Y-J; Lai, C-H

2008-01-01

Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples

The Mediator Complex MED15 Subunit Mediates Activation of Downstream Lipid-Related Genes by the WRINKLED1 Transcription Factor.

Science.gov (United States)

Kim, Mi Jung; Jang, In-Cheol; Chua, Nam-Hai

2016-07-01

The Mediator complex is known to be a master coordinator of transcription by RNA polymerase II, and this complex is recruited by transcription factors (TFs) to target promoters for gene activation or repression. The plant-specific TF WRINKLED1 (WRI1) activates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. However, no Mediator subunit has yet been identified that mediates WRI1 transcriptional activity. Promoter-β-glucuronidase fusion experiments showed that MEDIATOR15 (MED15) is expressed in the same cells in the embryo as WRI1. We found that the Arabidopsis (Arabidopsis thaliana) MED15 subunit of the Mediator complex interacts directly with WRI1 in the nucleus. Overexpression of MED15 or WRI1 increased transcript levels of WRI1 target genes involved in glycolysis and fatty acid biosynthesis; these genes were down-regulated in wild-type or WRI1-overexpressing plants by silencing of MED15 However, overexpression of MED15 in the wri1 mutant also increased transcript levels of WRI1 target genes, suggesting that MED15 also may act with other TFs to activate downstream lipid-related genes. Chromatin immunoprecipitation assays confirmed the association of MED15 with six WRI1 target gene promoters. Additionally, silencing of MED15 resulted in reduced fatty acid content in seedlings and mature seeds, whereas MED15 overexpression increased fatty acid content in both developmental stages. Similar results were found in wri1 mutant and WRI1 overexpression lines. Together, our results indicate that the WRI1/MED15 complex transcriptionally regulates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

Issues in visual support to real-time space system simulation solved in the Systems Engineering Simulator

Science.gov (United States)

Yuen, Vincent K.

1989-01-01

The Systems Engineering Simulator has addressed the major issues in providing visual data to its real-time man-in-the-loop simulations. Out-the-window views and CCTV views are provided by three scene systems to give the astronauts their real-world views. To expand the window coverage for the Space Station Freedom workstation a rotating optics system is used to provide the widest field of view possible. To provide video signals to as many viewpoints as possible, windows and CCTVs, with a limited amount of hardware, a video distribution system has been developed to time-share the video channels among viewpoints at the selection of the simulation users. These solutions have provided the visual simulation facility for real-time man-in-the-loop simulations for the NASA space program.

Development of a primer–probe energy transfer based real-time PCR for the detection of Swine influenza virus

DEFF Research Database (Denmark)

Kowalczyk, Andrzej; Markowska-Daniel, Iwona; Rasmussen, Thomas Bruun

2013-01-01

Swine influenza virus (SIV) causes a contagious and requiring official notification disease of pigs and humans. In this study, a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay based on primer–probe energy transfer (PriProET) for the detection of SIV RNA was developed...... of the specific product amplification. The assay is specific for influenza virus with a sensitivity of detection limit of approximately 10 copies of RNA by PCR. Based on serial dilutions of SIV, the detection limit of the assay was approximately 0.003 TCID50/ml for H1N1 A/Swine/Poland/KPR9/2004 virus. The Pri...

Rapid Genome Detection of Schmallenberg Virus and Bovine Viral Diarrhea Virus by Use of Isothermal Amplification Methods and High-Speed Real-Time Reverse Transcriptase PCR

OpenAIRE

Aebischer, Andrea; Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

2014-01-01

Over the past few years, there has been an increasing demand for rapid and simple diagnostic tools that can be applied outside centralized laboratories by using transportable devices. In veterinary medicine, such mobile test systems would circumvent barriers associated with the transportation of samples and significantly reduce the time to diagnose important infectious animal diseases. Among a wide range of available technologies, high-speed real-time reverse transcriptase quantitative PCR (R...

Reversal of the lattice structure in SrCoOx epitaxial thin films studied by real-time optical spectroscopy and first-principles calculations

OpenAIRE

Choi, Woo Seok; Jeen, Hyoungjeen; Lee, Jun Hee; Seo, S. S. Ambrose; Cooper, Valentino R.; Rabe, Karin M.; Lee, Ho Nyung

2013-01-01

Using real-time spectroscopic ellipsometry, we directly observed a reversible lattice and electronic structure evolution in SrCoOx (x = 2.5 - 3) epitaxial thin films. Drastically different electronic ground states, which are extremely susceptible to the oxygen content x, are found in the two topotactic phases, i.e. the brownmillerite SrCoO2.5 and the perovskite SrCoO3. First principles calculations confirmed substantial differences in the electronic structure, including a metal-insulator tran...

Mediation Analyses in the Real World

DEFF Research Database (Denmark)

Lange, Theis; Starkopf, Liis

2016-01-01

The paper by Nguyen et al.1 published in this issue of Epidemiology presents a comparison of the recently suggested inverse odds ratio approach for addressing mediation and a more conventional Baron and Kenny-inspired method. Interestingly, the comparison is not done through a discussion of restr......The paper by Nguyen et al.1 published in this issue of Epidemiology presents a comparison of the recently suggested inverse odds ratio approach for addressing mediation and a more conventional Baron and Kenny-inspired method. Interestingly, the comparison is not done through a discussion...... it simultaneously ensures that the comparison is based on properties, which matter in actual applications, and makes the comparison accessible for a broader audience. In a wider context, the choice to stay close to real-life problems mirrors a general trend within the literature on mediation analysis namely to put...... applications using the inverse odds ration approach, as it simply has not had enough time to move from theoretical concept to published applied paper, we do expect to be able to judge the willingness of authors and journals to employ the causal inference-based approach to mediation analyses. Our hope...

Improvements for real-time magnetic equilibrium reconstruction on ASDEX Upgrade

International Nuclear Information System (INIS)

Giannone, L.; Fischer, R.; McCarthy, P.J.; Odstrcil, T.; Zammuto, I.; Bock, A.; Conway, G.; Fuchs, J.C.; Gude, A.; Igochine, V.; Kallenbach, A.; Lackner, K.; Maraschek, M.; Rapson, C.; Ruan, Q.; Schuhbeck, K.H.; Suttrop, W.; Wenzel, L.

2015-01-01

Highlights: • Spline basis current functions with second-order linear regularisation. • Perturbations of magnetic probe measurements due to ferromagnetic tiles on the inner wall and from oscillations in the fast position coil current are corrected. • A constraint of the safety factor on the magnetic axis is introduced. Soft X-ray tomography is used to assess the quality of the real-time magnetic equilibrium reconstruction. • External loop voltage measurements and magnetic probe pairs inside and outside the vessel wall were used to measure the vacuum vessel wall resistivity. - Abstract: Real-time magnetic equilibria are needed for NTM stabilization and disruption avoidance experiments on ASDEX Upgrade. Five improvements to real-time magnetic equilibrium reconstruction on ASDEX Upgrade have been investigated. The aim is to include as many features of the offline magnetic equilibrium reconstruction code in the real-time equilibrium reconstruction code. Firstly, spline current density basis functions with regularization are used in the offline equilibrium reconstruction code, CLISTE [1]. It is now possible to have the same number of spline basis functions in the real-time code. Secondly, in the presence of edge localized modes, (ELM's), it is found to be necessary to include the low pass filter effect of the vacuum vessel on the fast position control coil currents to correctly compensate the magnetic probes for current oscillations in these coils. Thirdly, the introduction of ferromagnetic tiles in ASDEX Upgrade means that a real-time algorithm for including the perturbations of the magnetic equilibrium generated by these tiles is required. A methodology based on tile surface currents is described. Fourthly, during current ramps it was seen that the difference between fitted and measured magnetic measurements in the equilibrium reconstruction were larger than in the constant current phase. External loop voltage measurements and magnetic probe pairs inside and

Improvements for real-time magnetic equilibrium reconstruction on ASDEX Upgrade

Energy Technology Data Exchange (ETDEWEB)

Giannone, L.; Fischer, R. [Max Planck Institute for Plasma Physics, 85748 Garching (Germany); McCarthy, P.J. [Department of Physics, University College Cork, Cork (Ireland); Odstrcil, T.; Zammuto, I.; Bock, A.; Conway, G.; Fuchs, J.C.; Gude, A.; Igochine, V.; Kallenbach, A.; Lackner, K.; Maraschek, M.; Rapson, C. [Max Planck Institute for Plasma Physics, 85748 Garching (Germany); Ruan, Q. [National Instruments, Austin, TX 78759-3504 (United States); Schuhbeck, K.H.; Suttrop, W. [Max Planck Institute for Plasma Physics, 85748 Garching (Germany); Wenzel, L. [National Instruments, Austin, TX 78759-3504 (United States)

2015-11-15

Highlights: • Spline basis current functions with second-order linear regularisation. • Perturbations of magnetic probe measurements due to ferromagnetic tiles on the inner wall and from oscillations in the fast position coil current are corrected. • A constraint of the safety factor on the magnetic axis is introduced. Soft X-ray tomography is used to assess the quality of the real-time magnetic equilibrium reconstruction. • External loop voltage measurements and magnetic probe pairs inside and outside the vessel wall were used to measure the vacuum vessel wall resistivity. - Abstract: Real-time magnetic equilibria are needed for NTM stabilization and disruption avoidance experiments on ASDEX Upgrade. Five improvements to real-time magnetic equilibrium reconstruction on ASDEX Upgrade have been investigated. The aim is to include as many features of the offline magnetic equilibrium reconstruction code in the real-time equilibrium reconstruction code. Firstly, spline current density basis functions with regularization are used in the offline equilibrium reconstruction code, CLISTE [1]. It is now possible to have the same number of spline basis functions in the real-time code. Secondly, in the presence of edge localized modes, (ELM's), it is found to be necessary to include the low pass filter effect of the vacuum vessel on the fast position control coil currents to correctly compensate the magnetic probes for current oscillations in these coils. Thirdly, the introduction of ferromagnetic tiles in ASDEX Upgrade means that a real-time algorithm for including the perturbations of the magnetic equilibrium generated by these tiles is required. A methodology based on tile surface currents is described. Fourthly, during current ramps it was seen that the difference between fitted and measured magnetic measurements in the equilibrium reconstruction were larger than in the constant current phase. External loop voltage measurements and magnetic probe pairs inside

An FPGA Platform for Real-Time Simulation of Spiking Neuronal Networks.

Science.gov (United States)

Pani, Danilo; Meloni, Paolo; Tuveri, Giuseppe; Palumbo, Francesca; Massobrio, Paolo; Raffo, Luigi

2017-01-01

In the last years, the idea to dynamically interface biological neurons with artificial ones has become more and more urgent. The reason is essentially due to the design of innovative neuroprostheses where biological cell assemblies of the brain can be substituted by artificial ones. For closed-loop experiments with biological neuronal networks interfaced with in silico modeled networks, several technological challenges need to be faced, from the low-level interfacing between the living tissue and the computational model to the implementation of the latter in a suitable form for real-time processing. Field programmable gate arrays (FPGAs) can improve flexibility when simple neuronal models are required, obtaining good accuracy, real-time performance, and the possibility to create a hybrid system without any custom hardware, just programming the hardware to achieve the required functionality. In this paper, this possibility is explored presenting a modular and efficient FPGA design of an in silico spiking neural network exploiting the Izhikevich model. The proposed system, prototypically implemented on a Xilinx Virtex 6 device, is able to simulate a fully connected network counting up to 1,440 neurons, in real-time, at a sampling rate of 10 kHz, which is reasonable for small to medium scale extra-cellular closed-loop experiments.

R-loops cause genomic instability in T helper lymphocytes from patients with Wiskott-Aldrich syndrome.

Science.gov (United States)

Sarkar, Koustav; Han, Seong-Su; Wen, Kuo-Kuang; Ochs, Hans D; Dupré, Loïc; Seidman, Michael M; Vyas, Yatin M

2017-12-15

Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia, which are caused by WAS mutations affecting Wiskott-Aldrich syndrome protein (WASp) expression or activity, manifest in immunodeficiency, autoimmunity, genomic instability, and lymphoid and other cancers. WASp supports filamentous actin formation in the cytoplasm and gene transcription in the nucleus. Although the genetic basis for XLT/WAS has been clarified, the relationships between mutant forms of WASp and the diverse features of these disorders remain ill-defined. We sought to define how dysfunctional gene transcription is causally linked to the degree of T H cell deficiency and genomic instability in the XLT/WAS clinical spectrum. In human T H 1- or T H 2-skewing cell culture systems, cotranscriptional R-loops (RNA/DNA duplex and displaced single-stranded DNA) and DNA double-strand breaks (DSBs) were monitored in multiple samples from patients with XLT and WAS and in normal T cells depleted of WASp. WASp deficiency provokes increased R-loops and R-loop-mediated DSBs in T H 1 cells relative to T H 2 cells. Mechanistically, chromatin occupancy of serine 2-unphosphorylated RNA polymerase II is increased, and that of topoisomerase 1, an R-loop preventing factor, is decreased at R-loop-enriched regions of IFNG and TBX21 (T H 1 genes) in T H 1 cells. These aberrations accompany increased unspliced (intron-retained) and decreased spliced mRNA of IFNG and TBX21 but not IL13 (T H 2 gene). Significantly, increased cellular load of R-loops and DSBs, which are normalized on RNaseH1-mediated suppression of ectopic R-loops, inversely correlates with disease severity scores. Transcriptional R-loop imbalance is a novel molecular defect causative in T H 1 immunodeficiency and genomic instability in patients with WAS. The study proposes that cellular R-loop load could be used as a potential biomarker for monitoring symptom severity and prognostic outcome in the XLT-WAS clinical spectrum

NSTX-U Advances in Real-Time C++11 on Linux

Science.gov (United States)

Erickson, Keith G.

2015-08-01

Programming languages like C and Ada combined with proprietary embedded operating systems have dominated the real-time application space for decades. The new C++11 standard includes native, language-level support for concurrency, a required feature for any nontrivial event-oriented real-time software. Threads, Locks, and Atomics now exist to provide the necessary tools to build the structures that make up the foundation of a complex real-time system. The National Spherical Torus Experiment Upgrade (NSTX-U) at the Princeton Plasma Physics Laboratory (PPPL) is breaking new ground with the language as applied to the needs of fusion devices. A new Digital Coil Protection System (DCPS) will serve as the main protection mechanism for the magnetic coils, and it is written entirely in C++11 running on Concurrent Computer Corporation's real-time operating system, RedHawk Linux. It runs over 600 algorithms in a 5 kHz control loop that determine whether or not to shut down operations before physical damage occurs. To accomplish this, NSTX-U engineers developed software tools that do not currently exist elsewhere, including real-time atomic synchronization, real-time containers, and a real-time logging framework. Together with a recent (and carefully configured) version of the GCC compiler, these tools enable data acquisition, processing, and output using a conventional operating system to meet a hard real-time deadline (that is, missing one periodic is a failure) of 200 microseconds.

NSTX-U Advances in Real-Time C++11 on Linux

International Nuclear Information System (INIS)

Erickson, Keith G.

2015-01-01

Programming languages like C and Ada combined with proprietary embedded operating systems have dominated the real-time application space for decades. The new C++11standard includes native, language-level support for concurrency, a required feature for any nontrivial event-oriented real-time software. Threads, Locks, and Atomics now exist to provide the necessary tools to build the structures that make up the foundation of a complex real-time system. The National Spherical Torus Experiment Upgrade (NSTX-U) at the Princeton Plasma Physics Laboratory (PPPL) is breaking new ground with the language as applied to the needs of fusion devices. A new Digital Coil Protection System (DCPS) will serve as the main protection mechanism for the magnetic coils, and it is written entirely in C++11 running on Concurrent Computer Corporation's real-time operating system, RedHawk Linux. It runs over 600 algorithms in a 5 kHz control loop that determine whether or not to shut down operations before physical damage occurs. To accomplish this, NSTX-U engineers developed software tools that do not currently exist elsewhere, including real-time atomic synchronization, real-time containers, and a real-time logging framework. Together with a recent (and carefully configured) version of the GCC compiler, these tools enable data acquisition, processing, and output using a conventional operating system to meet a hard real-time deadline (that is, missing one periodic is a failure) of 200 microseconds

Loop-Mediated Isothermal Amplification for Detection of Endogenous Sad1 Gene in Cotton: An Internal Control for Rapid Onsite GMO Testing.

Science.gov (United States)

Singh, Monika; Bhoge, Rajesh K; Randhawa, Gurinderjit

2018-04-20

Background : Confirming the integrity of seed samples in powdered form is important priorto conducting a genetically modified organism (GMO) test. Rapid onsite methods may provide a technological solution to check for genetically modified (GM) events at ports of entry. In India, Bt cotton is the commercialized GM crop with four approved GM events; however, 59 GM events have been approved globally. GMO screening is required to test for authorized GM events. The identity and amplifiability of test samples could be ensured first by employing endogenous genes as an internal control. Objective : A rapid onsite detection method was developed for an endogenous reference gene, stearoyl acyl carrier protein desaturase ( Sad1 ) of cotton, employing visual and real-time loop-mediated isothermal amplification (LAMP). Methods : The assays were performed at a constant temperature of 63°C for 30 min for visual LAMP and 62ºC for 40 min for real-time LAMP. Positive amplification was visualized as a change in color from orange to green on addition of SYBR ® Green or detected as real-time amplification curves. Results : Specificity of LAMP assays was confirmed using a set of 10 samples. LOD for visual LAMP was up to 0.1%, detecting 40 target copies, and for real-time LAMP up to 0.05%, detecting 20 target copies. Conclusions : The developed methods could be utilized to confirm the integrity of seed powder prior to conducting a GMO test for specific GM events of cotton. Highlights : LAMP assays for the endogenous Sad1 gene of cotton have been developed to be used as an internal control for onsite GMO testing in cotton.

Detection of Zika virus using reverse-transcription LAMP coupled with reverse dot blot analysis in saliva.

Directory of Open Access Journals (Sweden)

Maite Sabalza

Full Text Available In recent years, there have been increasing numbers of infectious disease outbreaks that spread rapidly to population centers resulting from global travel, population vulnerabilities, environmental factors, and ecological disasters such as floods and earthquakes. Some examples of the recent outbreaks are the Ebola epidemic in West Africa, Middle East respiratory syndrome coronavirus (MERS-Co in the Middle East, and the Zika outbreak through the Americas. We have created a generic protocol for detection of pathogen RNA and/or DNA using loop-mediated isothermal amplification (LAMP and reverse dot-blot for detection (RDB and processed automatically in a microfluidic device. In particular, we describe how a microfluidic assay to detect HIV viral RNA was converted to detect Zika virus (ZIKV RNA. We first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device. Then we implemented the assay in a microfluidic device that will allow analyzing 24 samples simultaneously and automatically from sample introduction to detection by RDB technique. Preliminary data using saliva samples spiked with ZIKV showed that our diagnostic system detects ZIKV RNA in saliva. These results will be validated in further experiments with well-characterized ZIKV human specimens of saliva. The described strategy and methodology to convert the HIV diagnostic assay and platform to a ZIKV RNA detection assay provides a model that can be readily utilized for detection of the next emerging or re-emerging infectious disease.

Digital gene expression analysis of male and female bud transition in Metasequoia reveals high activity of MADS-box transcription factors and hormone-mediated sugar pathways.

Science.gov (United States)

Zhao, Ying; Liang, Haiying; Li, Lan; Tang, Sha; Han, Xiao; Wang, Congpeng; Xia, Xinli; Yin, Weilun

2015-01-01

Metasequoia glyptostroboides is a famous redwood tree of ecological and economic importance, and requires more than 20 years of juvenile-to-adult transition before producing female and male cones. Previously, we induced reproductive buds using a hormone solution in juvenile Metasequoia trees as young as 5-to-7 years old. In the current study, hormone-treated shoots found in female and male buds were used to identify candidate genes involved in reproductive bud transition in Metasequoia. Samples from hormone-treated cone reproductive shoots and naturally occurring non-cone setting shoots were analyzed using 24 digital gene expression (DGE) tag profiles using Illumina, generating a total of 69,520 putative transcripts. Next, 32 differentially and specifically expressed transcripts were determined using quantitative real-time polymerase chain reaction, including the upregulation of MADS-box transcription factors involved in male bud transition and flowering time control proteins involved in female bud transition. These differentially expressed transcripts were associated with 243 KEGG pathways. Among the significantly changed pathways, sugar pathways were mediated by hormone signals during the vegetative-to-reproductive phase transition, including glycolysis/gluconeogenesis and sucrose and starch metabolism pathways. Key enzymes were identified in these pathways, including alcohol dehydrogenase (NAD) and glutathione dehydrogenase for the glycolysis/gluconeogenesis pathway, and glucanphosphorylase for sucrose and starch metabolism pathways. Our results increase our understanding of the reproductive bud transition in gymnosperms. In addition, these studies on hormone-mediated sugar pathways increase our understanding of the relationship between sugar and hormone signaling during female and male bud initiation in Metasequoia.

Digital gene expression analysis of male and female bud transition in Metasequoia reveals high activity of MADS-box transcription factors and hormone-mediated sugar pathways

Directory of Open Access Journals (Sweden)

Ying eZhao

2015-06-01

Full Text Available Metasequoiaglyptostroboidies is a famous redwood tree of ecological and economic importance, and requires more than 20 years of juvenile-to-adult transition before producing female and male cones. Previously, we induced reproductive buds using a hormone solution in juvenile Metasequoia trees as young as5-to-7years old. In the current study, hormone-treated shoots found in female and male buds were used to identify candidate genes involved in reproductive bud transition in Metasequoia. Samples from hormone-treated cone reproductive shoots and naturally occurring non-cone setting shoots were analyzed using 24 digital gene expression (DGE tag profiles using Illumina, generating a total of 69,520 putative transcripts. Next, 32 differentially and specifically expressed transcripts were determined using quantitative real-time polymerase chain reaction, including the upregulation of MADS-box transcription factors involved in male bud transition and flowering time control proteins involved in female bud transition. These differentially expressed transcripts were associated with 243 KEGG pathways. Among the significantly changed pathways, sugar pathways were mediated by hormone signals during the vegetative-to-reproductive phase transition, including glycolysis/gluconeogenesis and sucrose and starch metabolism pathways. Key enzymes were identified in these pathways, including alcohol dehydrogenase (NAD and glutathione dehydrogenase for the glycolysis/gluconeogenesis pathway, and glucanphosphorylase for sucrose and starch metabolism pathways. Our results increase our understanding of the reproductive bud transition in gymnosperms. In addition, these studies on hormone-mediated sugar pathways increase our understanding of the relationship between sugar and hormone signaling during female and male bud initiation in Metasequoia.

Piles of dislocation loops in real crystals. 2. Evolution of dislocation piles under irradiation

International Nuclear Information System (INIS)

Dubinko, V.I.; Turkin, A.A.; Yanovskij, V.V.

1985-01-01

The given paper considers evolution of piles in a real molybdenum crystal under neutron irradiation. Obtained was a stability criterium, when meeting it interstitial piles (one-dimensional periodical structures of interstitial loops) in the crystal tend to stationary state under the irradiation and, when disturbing the criterium, they disintegrate into rapidly growing interstitial isolated loops. It was also shown that the generation of dense vacancy piles results in the formation of an ordering structure of isolated vacancy loops. Theoretical results agree good with experimental data

Real-time position reconstruction with hippocampal place cells.

Science.gov (United States)

Guger, Christoph; Gener, Thomas; Pennartz, Cyriel M A; Brotons-Mas, Jorge R; Edlinger, Günter; Bermúdez I Badia, S; Verschure, Paul; Schaffelhofer, Stefan; Sanchez-Vives, Maria V

2011-01-01

Brain-computer interfaces (BCI) are using the electroencephalogram, the electrocorticogram and trains of action potentials as inputs to analyze brain activity for communication purposes and/or the control of external devices. Thus far it is not known whether a BCI system can be developed that utilizes the states of brain structures that are situated well below the cortical surface, such as the hippocampus. In order to address this question we used the activity of hippocampal place cells (PCs) to predict the position of an rodent in real-time. First, spike activity was recorded from the hippocampus during foraging and analyzed off-line to optimize the spike sorting and position reconstruction algorithm of rats. Then the spike activity was recorded and analyzed in real-time. The rat was running in a box of 80 cm × 80 cm and its locomotor movement was captured with a video tracking system. Data were acquired to calculate the rat's trajectories and to identify place fields. Then a Bayesian classifier was trained to predict the position of the rat given its neural activity. This information was used in subsequent trials to predict the rat's position in real-time. The real-time experiments were successfully performed and yielded an error between 12.2 and 17.4% using 5-6 neurons. It must be noted here that the encoding step was done with data recorded before the real-time experiment and comparable accuracies between off-line (mean error of 15.9% for three rats) and real-time experiments (mean error of 14.7%) were achieved. The experiment shows proof of principle that position reconstruction can be done in real-time, that PCs were stable and spike sorting was robust enough to generalize from the training run to the real-time reconstruction phase of the experiment. Real-time reconstruction may be used for a variety of purposes, including creating behavioral-neuronal feedback loops or for implementing neuroprosthetic control.

Detection of live Salmonella enterica in fresh-cut vegetables by a TaqMan-based one-step reverse transcription real-time PCR.

Science.gov (United States)

Miao, Y J; Xiong, G T; Bai, M Y; Ge, Y; Wu, Z F

2018-05-01

Fresh-cut produce is at greater risk of Salmonella contamination. Detection and early warning systems play an important role in reducing the dissemination of contaminated products. One-step Reverse Transcription Polymerase Chain Reaction (RT-qPCR) targeting Salmonella tmRNA with or without a 6-h enrichment was evaluated for the detection of Salmonella in fresh-cut vegetables after 6-h storage. LOD of one-step RT-qPCR was 1·0 CFU per ml (about 100 copies tmRNA per ml) by assessed 10-fold serially diluted RNA from 10 6 CFU per ml bacteria culture. Then, one-step RT-qPCR assay was applied to detect viable Salmonella cells in 14 fresh-cut vegetables after 6-h storage. Without enrichment, this assay could detect 10 CFU per g for fresh-cut lettuce, cilantro, spinach, cabbage, Chinese cabbage and bell pepper, and 10 2 CFU per g for other vegetables. With a 6-h enrichment, this assay could detect 10 CFU per g for all fresh-cut vegetables used in this study. Moreover, this assay was able to discriminate viable cells from dead cells. This rapid detection assay may provide potential processing control and early warning method in fresh-cut vegetable processing to strengthen food safety assurance. Significance and Impact of the Study: Fresh-cut produce is at greater risk of Salmonella contamination. Rapid detection methods play an important role in reducing the dissemination of contaminated products. One-step RT-qPCR assay used in this study could detect 10 CFU per g Salmonella for 14 fresh-cut vegetables with a 6-h short enrichment. Moreover, this assay was able to discriminate viable cells from dead cells. This rapid detection assay may provide potential processing control and early warning method in fresh-cut vegetable processing to strengthen food safety assurance. © 2018 The Society for Applied Microbiology.

Real-time frequency-to-time mapping based on spectrally-discrete chromatic dispersion.

Science.gov (United States)

Dai, Yitang; Li, Jilong; Zhang, Ziping; Yin, Feifei; Li, Wangzhe; Xu, Kun

2017-07-10

Traditional photonics-assisted real-time Fourier transform (RTFT) usually suffers from limited chromatic dispersion, huge volume, or large time delay and attendant loss. In this paper we propose frequency-to-time mapping (FTM) by spectrally-discrete dispersion to increase frequency sensitivity greatly. The novel media has periodic ON/OFF intensity frequency response while quadratic phase distribution along disconnected channels, which de-chirps matched optical input to repeated Fourier-transform-limited output. Real-time FTM is then obtained within each period. Since only discrete phase retardation rather than continuously-changed true time delay is required, huge equivalent dispersion is then available by compact device. Such FTM is theoretically analyzed, and implementation by cascaded optical ring resonators is proposed. After a numerical example, our theory is demonstrated by a proof-of-concept experiment, where a single loop containing 0.5-meters-long fiber is used. FTM under 400-MHz unambiguous bandwidth and 25-MHz resolution is reported. Highly-sensitive and linear mapping is achieved with 6.25 ps/MHz, equivalent to ~4.6 × 10 4 -km standard single mode fiber. Extended instantaneous bandwidth is expected by ring cascading. Our proposal may provide a promising method for real-time, low-latency Fourier transform.

A real-time sub-μrad laser beam tracking system

Science.gov (United States)

Buske, Ivo; Schragner, Ralph; Riede, Wolfgang

2007-10-01

We present a rugged and reliable real-time laser beam tracking system operating with a high speed, high resolution piezo-electric tip/tilt mirror. Characteristics of the piezo mirror and position sensor are investigated. An industrial programmable automation controller is used to develop a real-time digital PID controller. The controller provides a one million field programmable gate array (FPGA) to realize a high closed-loop frequency of 50 kHz. Beam tracking with a root-mean-squared accuracy better than 0.15 μrad has been laboratory confirmed. The system is intended as an add-on module for established mechanical mrad tracking systems.

Real-time systems

OpenAIRE

Badr, Salah M.; Bruztman, Donald P.; Nelson, Michael L.; Byrnes, Ronald Benton

1992-01-01

This paper presents an introduction to the basic issues involved in real-time systems. Both real-time operating sys and real-time programming languages are explored. Concurrent programming and process synchronization and communication are also discussed. The real-time requirements of the Naval Postgraduate School Autonomous Under Vehicle (AUV) are then examined. Autonomous underwater vehicle (AUV), hard real-time system, real-time operating system, real-time programming language, real-time sy...

Network Reduction Algorithm for Developing Distribution Feeders for Real-Time Simulators: Preprint

Energy Technology Data Exchange (ETDEWEB)

Nagarajan, Adarsh; Nelson, Austin; Prabakar, Kumaraguru; Hoke, Andy; Asano, Marc; Ueda, Reid; Nepal, Shaili

2017-06-15

As advanced grid-support functions (AGF) become more widely used in grid-connected photovoltaic (PV) inverters, utilities are increasingly interested in their impacts when implemented in the field. These effects can be understood by modeling feeders in real-time systems and testing PV inverters using power hardware-in-the-loop (PHIL) techniques. This paper presents a novel feeder model reduction algorithm using a Monte Carlo method that enables large feeders to be solved and operated on real-time computing platforms. Two Hawaiian Electric feeder models in Synergi Electric's load flow software were converted to reduced order models in OpenDSS, and subsequently implemented in the OPAL-RT real-time digital testing platform. Smart PV inverters were added to the real-time model with AGF responses modeled after characterizing commercially available hardware inverters. Finally, hardware inverters were tested in conjunction with the real-time model using PHIL techniques so that the effects of AGFs on the choice feeders could be analyzed.

Real time urbanism

Directory of Open Access Journals (Sweden)

Ana Ruiz Varona

2012-12-01

Full Text Available Nowadays, given the technological revolution of the society of information, the administrative management of the cities faces a new problem not as related to the projection of the urban space as to the capacity of controlling and measuring the process of direct and centralized production of the cities by part of some non-homogeneous social multitudes, in a hyper-accelerated time towards instantaneity. Against libertarian apologies of the new “participative urbanisms”, the article puts forward a discourse that shows the lost associated to the new problem of temporal instantaneity. In this regard we claim new process of mediation that allow administrations and urbanist monitoring the production of the city. To that end, a previous and necessary step will be the redefinition of the role of a new real time urbanist.

Identification of basic/helix-loop-helix transcription factors reveals candidate genes involved in anthocyanin biosynthesis from the strawberry white-flesh mutant

OpenAIRE

Zhao, Fengli; Li, Gang; Hu, Panpan; Zhao, Xia; Li, Liangjie; Wei, Wei; Feng, Jiayue; Zhou, Houcheng

2018-01-01

As the second largest transcription factor family in plant, the basic helix-loop-helix (bHLH) transcription factor family, characterized by the conserved bHLH domain, plays a central regulatory role in many biological process. However, the bHLH transcription factor family of strawberry has not been systematically identified, especially for the anthocyanin biosynthesis. Here, we identified a total of 113 bHLH transcription factors and described their chromosomal distribution and bioinformatics...

Reversal of the Lattice Structure in SrCoOx Epitaxial Thin Films Studied by Real-Time Optical Spectroscopy and First-Principles Calculations

Science.gov (United States)

Choi, Woo Seok; Jeen, Hyoungjeen; Lee, Jun Hee; Seo, S. S. Ambrose; Cooper, Valentino R.; Rabe, Karin M.; Lee, Ho Nyung

2013-08-01

Using real-time spectroscopic ellipsometry, we directly observed a reversible lattice and electronic structure evolution in SrCoOx (x=2.5-3) epitaxial thin films. Drastically different electronic ground states, which are extremely susceptible to the oxygen content x, are found in the two topotactic phases: i.e., the brownmillerite SrCoO2.5 and the perovskite SrCoO3. First-principles calculations confirmed substantial differences in the electronic structure, including a metal-insulator transition, which originate from the modification in the Co valence states and crystallographic structures. More interestingly, the two phases can be reversibly controlled by changing the ambient pressure at greatly reduced temperatures. Our finding provides an important pathway to understanding the novel oxygen-content-dependent phase transition uniquely found in multivalent transition metal oxides.

Control bandwidth improvements in GRAVITY fringe tracker by switching to a synchronous real time computer architecture

Science.gov (United States)

Abuter, Roberto; Dembet, Roderick; Lacour, Sylvestre; di Lieto, Nicola; Woillez, Julien; Eisenhauer, Frank; Fedou, Pierre; Phan Duc, Than

2016-08-01

The new VLTI (Very Large Telescope Interferometer) 1 instrument GRAVITY5, 22, 23 is equipped with a fringe tracker16 able to stabilize the K-band fringes on six baselines at the same time. It has been designed to achieve a performance for average seeing conditions of a residual OPD (Optical Path Difference) lower than 300 nm with objects brighter than K = 10. The control loop implementing the tracking is composed of a four stage real time computer system compromising: a sensor where the detector pixels are read in and the OPD and GD (Group Delay) are calculated; a controller receiving the computed sensor quantities and producing commands for the piezo actuators; a concentrator which combines both the OPD commands with the real time tip/tilt corrections offloading them to the piezo actuator; and finally a Kalman15 parameter estimator. This last stage is used to monitor current measurements over a window of few seconds and estimate new values for the main Kalman15 control loop parameters. The hardware and software implementation of this design runs asynchronously and communicates the four computers for data transfer via the Reflective Memory Network3. With the purpose of improving the performance of the GRAVITY5, 23 fringe tracking16, 22 control loop, a deviation from the standard asynchronous communication mechanism has been proposed and implemented. This new scheme operates the four independent real time computers involved in the tracking loop synchronously using the Reflective Memory Interrupts2 as the coordination signal. This synchronous mechanism had the effect of reducing the total pure delay of the loop from 3.5 [ms] to 2.0 [ms] which then translates on a better stabilization of the fringes as the bandwidth of the system is substantially improved. This paper will explain in detail the real time architecture of the fringe tracker in both is synchronous and synchronous implementation. The achieved improvements on reducing the delay via this mechanism will be

Human Galectin-9 Is a Potent Mediator of HIV Transcription and Reactivation.

Directory of Open Access Journals (Sweden)

Mohamed Abdel-Mohsen

2016-06-01

Full Text Available Identifying host immune determinants governing HIV transcription, latency and infectivity in vivo is critical to developing an HIV cure. Based on our recent finding that the host factor p21 regulates HIV transcription during antiretroviral therapy (ART, and published data demonstrating that the human carbohydrate-binding immunomodulatory protein galectin-9 regulates p21, we hypothesized that galectin-9 modulates HIV transcription. We report that the administration of a recombinant, stable form of galectin-9 (rGal-9 potently reverses HIV latency in vitro in the J-Lat HIV latency model. Furthermore, rGal-9 reverses HIV latency ex vivo in primary CD4+ T cells from HIV-infected, ART-suppressed individuals (p = 0.002, more potently than vorinostat (p = 0.02. rGal-9 co-administration with the latency reversal agent "JQ1", a bromodomain inhibitor, exhibits synergistic activity (p<0.05. rGal-9 signals through N-linked oligosaccharides and O-linked hexasaccharides on the T cell surface, modulating the gene expression levels of key transcription initiation, promoter proximal-pausing, and chromatin remodeling factors that regulate HIV latency. Beyond latent viral reactivation, rGal-9 induces robust expression of the host antiviral deaminase APOBEC3G in vitro and ex vivo (FDR<0.006 and significantly reduces infectivity of progeny virus, decreasing the probability that the HIV reservoir will be replenished when latency is reversed therapeutically. Lastly, endogenous levels of soluble galectin-9 in the plasma of 72 HIV-infected ART-suppressed individuals were associated with levels of HIV RNA in CD4+ T cells (p<0.02 and with the quantity and binding avidity of circulating anti-HIV antibodies (p<0.009, suggesting a role of galectin-9 in regulating HIV transcription and viral production in vivo during therapy. Our data suggest that galectin-9 and the host glycosylation machinery should be explored as foundations for novel HIV cure strategies.

Comprehensive analysis of the transcriptional profile of the Mediator complex across human cancer types.

Science.gov (United States)

Syring, Isabella; Klümper, Niklas; Offermann, Anne; Braun, Martin; Deng, Mario; Boehm, Diana; Queisser, Angela; von Mässenhausen, Anne; Brägelmann, Johannes; Vogel, Wenzel; Schmidt, Doris; Majores, Michael; Schindler, Anne; Kristiansen, Glen; Müller, Stefan C; Ellinger, Jörg; Shaikhibrahim, Zaki; Perner, Sven

2016-04-26

The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking.We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.

The RNA binding protein HuR does not interact directly with HIV-1 reverse transcriptase and does not affect reverse transcription in vitro

Directory of Open Access Journals (Sweden)

Gronenborn Angela M

2010-05-01

Full Text Available Abstract Background Lemay et al recently reported that the RNA binding protein HuR directly interacts with the ribonuclease H (RNase H domain of HIV-1 reverse transcriptase (RT and influences the efficiency of viral reverse transcription (Lemay et al., 2008, Retrovirology 5:47. HuR is a member of the embryonic lethal abnormal vision protein family and contains 3 RNA recognition motifs (RRMs that bind AU-rich elements (AREs. To define the structural determinants of the HuR-RT interaction and to elucidate the mechanism(s by which HuR influences HIV-1 reverse transcription activity in vitro, we cloned and purified full-length HuR as well as three additional protein constructs that contained the N-terminal and internal RRMs, the internal and C-terminal RRMs, or the C-terminal RRM only. Results All four HuR proteins were purified and characterized by biophysical methods. They are well structured and exist as monomers in solution. No direct protein-protein interaction between HuR and HIV-1 RT was detected using NMR titrations with 15N labeled HuR variants or the 15N labeled RNase H domain of HIV-1 RT. Furthermore, HuR did not significantly affect the kinetics of HIV-1 reverse transcription in vitro, even on RNA templates that contain AREs. Conclusions Our results suggest that HuR does not impact HIV-1 replication through a direct protein-protein interaction with the viral RT.

Criteria for saturated magnetization loop

International Nuclear Information System (INIS)

Harres, A.; Mikhov, M.; Skumryev, V.; Andrade, A.M.H. de; Schmidt, J.E.; Geshev, J.

2016-01-01

Proper estimation of magnetization curve parameters is vital in studying magnetic systems. In the present article, criteria for discrimination non-saturated (minor) from saturated (major) hysteresis loops are proposed. These employ the analysis of (i) derivatives of both ascending and descending branches of the loop, (ii) remanent magnetization curves, and (iii) thermomagnetic curves. Computational simulations are used in order to demonstrate their validity. Examples illustrating the applicability of these criteria to well-known real systems, namely Fe_3O_4 and Ni fine particles, are provided. We demonstrate that the anisotropy-field value estimated from a visual examination of an only apparently major hysteresis loop could be more than two times lower than the real one. - Highlights: • Proper estimation of hysteresis-loop parameters is vital in magnetic studies. • We propose criteria for discrimination minor from major hysteresis loops. • The criteria analyze magnetization, remanence and ZFC/FC curves and/or their derivatives. • Examples of their application on real nanoparticles systems are given. • Using the criteria could avoid twofold or bigger saturation-field underestimation errors.

Criteria for saturated magnetization loop

Energy Technology Data Exchange (ETDEWEB)

Harres, A. [Departamento de Física, UFSM, Santa Maria, 97105-900 Rio Grande do Sul (Brazil); Mikhov, M. [Faculty of Physics, University of Sofia, 1164 Sofia (Bulgaria); Skumryev, V. [Institució Catalana de Recerca i Estudis Avançats, 08010 Barcelona (Spain); Departament de Física, Universitat Autònoma de Barcelona, 08193 Barcelona (Spain); Andrade, A.M.H. de; Schmidt, J.E. [Instituto de Física, UFRGS, Porto Alegre, 91501-970 Rio Grande do Sul (Brazil); Geshev, J., E-mail: julian@if.ufrgs.br [Departament de Física, Universitat Autònoma de Barcelona, 08193 Barcelona (Spain); Instituto de Física, UFRGS, Porto Alegre, 91501-970 Rio Grande do Sul (Brazil)

2016-03-15

Proper estimation of magnetization curve parameters is vital in studying magnetic systems. In the present article, criteria for discrimination non-saturated (minor) from saturated (major) hysteresis loops are proposed. These employ the analysis of (i) derivatives of both ascending and descending branches of the loop, (ii) remanent magnetization curves, and (iii) thermomagnetic curves. Computational simulations are used in order to demonstrate their validity. Examples illustrating the applicability of these criteria to well-known real systems, namely Fe{sub 3}O{sub 4} and Ni fine particles, are provided. We demonstrate that the anisotropy-field value estimated from a visual examination of an only apparently major hysteresis loop could be more than two times lower than the real one. - Highlights: • Proper estimation of hysteresis-loop parameters is vital in magnetic studies. • We propose criteria for discrimination minor from major hysteresis loops. • The criteria analyze magnetization, remanence and ZFC/FC curves and/or their derivatives. • Examples of their application on real nanoparticles systems are given. • Using the criteria could avoid twofold or bigger saturation-field underestimation errors.

SOSPO-SP: Secure Operation of Sustainable Power Systems Simulation Platform for Real-Time System State Evaluation and Control

DEFF Research Database (Denmark)

Morais, Hugo; Vancraeyveld, Pieter; Pedersen, Allan Henning Birger

2014-01-01

Measurement Units (PMUs) provides more information and enables wide-area monitoring with accurate timing. One of the challenges in the near future is converting the high quantity and quality of information provided by PMUs into useful knowledge about operational state of a global system. The use of real-time...... simulation in closed-loop is essential to develop and validate new real-time applications of wide-area PMU data. This paper presents a simulation platform developed within the research project Secure Operation of Sustainable Power Systems (SOSPO). The SOSPO simulation platform (SOSPO-SP) functions...... in a closed-loop, integrating new real-time assessment methods to provide useful information to operators in power system control centers and to develop new control methodologies that handle emergency situations and avoid power system blackouts....

Targeting miR-423-5p Reverses Exercise Training-Induced HCN4 Channel Remodeling and Sinus Bradycardia

DEFF Research Database (Denmark)

D'Souza, Alicia; Pearman, Charles M.; Wang, Yanwen

2017-01-01

-generation sequencing and quantitative real-time reverse transcription polymerase chain reaction showed remodeling of miRs in the sinus node of swim-trained mice. Computational predictions highlighted a prominent role for miR-423-5p. Interaction between miR-423-5p and HCN4 was confirmed by a dose-dependent reduction...

Lateral Root Development in Potato Is Mediated by Stu-mi164 Regulation of NAC Transcription Factor

Directory of Open Access Journals (Sweden)

Li Zhang

2018-03-01

Full Text Available The NAC designation is derived from petunia (Petunia hybrida gene NO APICAL MERISTEM (NAM and Arabidopsis genes ATAF1/ATAF2 and CUP-SHAPED COTYLEDON2 (CUC2, which belongs to the family of plant-specific transcription factors (TFs, and plays important role in plant development processes, such as response to biotic and abiotic stress, and hormone signaling. MicroRNAs (miRNAs are a class of small, non-coding endogenous RNAs which play versatile and significant role in plant stress response and development via negatively affecting gene expression at a post-transcriptional level. Here, we showed that Stu-mi164 had a complementary sequence in the CDS sequence of potato NAC TFs, and that NAC expression exhibited significant differences under osmotic stress. We measured expression levels of the Stu-mi164 target gene StNAC262 between control and PEG-treated plants using real-time PCR, and the results demonstrated that they had inverse relationship. We suggested that Stu-miR164 might drive overexpression of NAC gene under osmotic stress in potato. To confirm the regulation of NAC TFs by Stu-mi164, we developed transgenic plants, using Agrobacterium tumefaciens–mediated transformation, of the potato cultivars “Gannongshu 2” and “Kexin 3” overexpressing the Stu-mi164 or the TF StNAC262. Real-time PCR analysis of transgenic potato plants under osmotic (PEG stress, showed that potato plants overexpressing Stu-mi164 had reduced expression of StNAC262 and their osmotic resistance decreased. Furthermore, these plants had low number of lateral roots although the same length as the control. Our findings support the regulatory role of Stu-miRNAs in controlling plant response to osmotic stress via StNAC262.

High performance real-time flight simulation at NASA Langley

Science.gov (United States)

Cleveland, Jeff I., II

1994-01-01

In order to meet the stringent time-critical requirements for real-time man-in-the-loop flight simulation, computer processing operations must be deterministic and be completed in as short a time as possible. This includes simulation mathematical model computational and data input/output to the simulators. In 1986, in response to increased demands for flight simulation performance, personnel at NASA's Langley Research Center (LaRC), working with the contractor, developed extensions to a standard input/output system to provide for high bandwidth, low latency data acquisition and distribution. The Computer Automated Measurement and Control technology (IEEE standard 595) was extended to meet the performance requirements for real-time simulation. This technology extension increased the effective bandwidth by a factor of ten and increased the performance of modules necessary for simulator communications. This technology is being used by more than 80 leading technological developers in the United States, Canada, and Europe. Included among the commercial applications of this technology are nuclear process control, power grid analysis, process monitoring, real-time simulation, and radar data acquisition. Personnel at LaRC have completed the development of the use of supercomputers for simulation mathematical model computational to support real-time flight simulation. This includes the development of a real-time operating system and the development of specialized software and hardware for the CAMAC simulator network. This work, coupled with the use of an open systems software architecture, has advanced the state of the art in real time flight simulation. The data acquisition technology innovation and experience with recent developments in this technology are described.

Decoding the Mobility and Time Scales of Protein Loops.

Science.gov (United States)

Gu, Yina; Li, Da-Wei; Brüschweiler, Rafael

2015-03-10

The flexible nature of protein loops and the time scales of their dynamics are critical for many biologically important events at the molecular level, such as protein interaction and recognition processes. In order to obtain a predictive understanding of the dynamic properties of loops, 500 ns molecular dynamics (MD) computer simulations of 38 different proteins were performed and validated using NMR chemical shifts. A total of 169 loops were analyzed and classified into three types, namely fast loops with correlation times Web server (http://spin.ccic.ohio-state.edu/index.php/loop). The results demonstrate that loop dynamics with their time scales can be predicted rapidly with reasonable accuracy, which will allow the screening of average protein structures to help better understand the various roles loops can play in the context of protein-protein interactions and binding.

Sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction detecting feline coronavirus mutations in effusion and serum/plasma of cats to diagnose feline infectious peritonitis

NARCIS (Netherlands)

Felten, Sandra; Leutenegger, Christian M.; Balzer, Hans Joerg; Pantchev, Nikola; Matiasek, Kaspar; Wess, Gerhard; Egberink, Herman|info:eu-repo/dai/nl/089740890; Hartmann, Katrin

2017-01-01

Background: Feline coronavirus (FCoV) exists as two pathotypes, and FCoV spike gene mutations are considered responsible for the pathotypic switch in feline infectious peritonitis (FIP) pathogenesis. The aim of this study was to evaluate sensitivity and specificity of a real-time reverse

The convergence of quantum-dot-mediated fluorescence resonance energy transfer and microfluidics for monitoring DNA polyplex self-assembly in real time

International Nuclear Information System (INIS)

Ho Yiping; Wang, T-H; Chen, Hunter H; Leong, Kam W

2009-01-01

We present a novel convergence of quantum-dot-mediated fluorescence resonance energy transfer (QD-FRET) and microfluidics, through which molecular interactions were precisely controlled and monitored using highly sensitive quantum-dot-mediated FRET. We demonstrate its potential in studying the kinetics of self-assembly of DNA polyplexes under laminar flow in real time with millisecond resolution. The integration of nanophotonics and microfluidics offers a powerful tool for elucidating the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

Real-Time Human in the Loop MBS Simulation in the Fraunhofer Robot-Based Driving Simulator

Directory of Open Access Journals (Sweden)

Kleer Michael

2014-08-01

Full Text Available The paper encompasses the overview of hardware architecture and the systems characteristics of the Fraunhofer driving simulator. First, the requirements of the real-time model and the real-time calculation hardware are defined and discussed in detail. Aspects like transport delay and the parallel computation of complex real-time models are presented. In addition, the interfacing of the models with the simulator system is shown. Two simulator driving tests, including a fully interactive rough terrain driving with a wheeled excavator and a test drive with a passenger car, are set to demonstrate system characteristics. Furthermore, the simulator characteristics of practical significance, such as simulator response time delay, simulator acceleration signal bandwidth obtained from artificial excitation and from the simulator driving test, will be presented and discussed.

In situ reverse transcription-PCR for monitoring gene expression in individual Methanosarcina mazei S-6 cells

DEFF Research Database (Denmark)

Lange, Marianne; Tolker-Nielsen, Tim; Molin, Søren

2000-01-01

An in situ reverse transcription-PCR protocol for detecting specific mRNA in Methanosarcina mazei S-6 is described. This method allowed us to detect heat shock-induced increases in the intracellular levels of the transcript of the universal stress gene dnaK. The cell walls of paraformaldehyde...

Cell cycle regulation by feed-forward loops coupling transcription and phosphorylation

DEFF Research Database (Denmark)

Csikász-Nagy, Attila; Kapuy, Orsolya; Tóth, Attila

2009-01-01

of these EPs. From genome-scale data sets of budding yeast, we identify 126 EPs that are regulated by Cdk1 both through direct phosphorylation of the EP and through phosphorylation of the transcription factors that control expression of the EP, so that each of these EPs is regulated by a feed-forward loop (FFL......) from Cdk1. By mathematical modelling, we show that such FFLs can activate EPs at different phases of the cell cycle depending of the effective signs (+ or -) of the regulatory steps of the FFL. We provide several case studies of EPs that are controlled by FFLs exactly as our models predict. The signal...

Differential active site loop conformations mediate promiscuous activities in the lactonase SsoPox.

Directory of Open Access Journals (Sweden)

Julien Hiblot

Full Text Available Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263 that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability.

Hepatitis B virus nuclear export elements: RNA stem-loop α and β, key parts of the HBV post-transcriptional regulatory element.

Science.gov (United States)

Lim, Chun Shen; Brown, Chris M

2016-09-01

Many viruses contain RNA elements that modulate splicing and/or promote nuclear export of their RNAs. The RNAs of the major human pathogen, hepatitis B virus (HBV) contain a large (~600 bases) composite cis-acting 'post-transcriptional regulatory element' (PRE). This element promotes expression from these naturally intronless transcripts. Indeed, the related woodchuck hepadnavirus PRE (WPRE) is used to enhance expression in gene therapy and other expression vectors. These PRE are likely to act through a combination of mechanisms, including promotion of RNA nuclear export. Functional components of both the HBV PRE and WPRE are 2 conserved RNA cis-acting stem-loop (SL) structures, SLα and SLβ. They are within the coding regions of polymerase (P) gene, and both P and X genes, respectively. Based on previous studies using mutagenesis and/or nuclear magnetic resonance (NMR), here we propose 2 covariance models for SLα and SLβ. The model for the 30-nucleotide SLα contains a G-bulge and a CNGG(U) apical loop of which the first and the fourth loop residues form a CG pair and the fifth loop residue is bulged out, as observed in the NMR structure. The model for the 23-nucleotide SLβ contains a 7-base-pair stem and a 9-nucleotide loop. Comparison of the models with other RNA structural elements, as well as similarity searches of human transcriptome and viral genomes demonstrate that SLα and SLβ are specific to HBV transcripts. However, they are well conserved among the hepadnaviruses of non-human primates, the woodchuck and ground squirrel.

A Novel Supervisory Control Algorithm to Improve the Performance of a Real-Time PV Power-Hardware-In-Loop Simulator with Non-RTDS

Directory of Open Access Journals (Sweden)

Dae-Jin Kim

2017-10-01

Full Text Available A programmable direct current (DC power supply with Real-time Digital Simulator (RTDS-based photovoltaic (PV Power Hardware-In-the-Loop (PHIL simulators has been used to improve the control algorithm and reliability of a PV inverter. This paper proposes a supervisory control algorithm for a PV PHIL simulator with a non-RTDS device that is an alternative solution to a high-cost PHIL simulator. However, when such a simulator with the conventional algorithm which is used in an RTDS is connected to a PV inverter, the output is in the transient state and it makes it impossible to evaluate the performance of the PV inverter. Therefore, the proposed algorithm controls the voltage and current target values according to constant voltage (CV and constant current (CC modes to overcome the limitation of the Computing Unit and DC power supply, and it also uses a multi-rate system to account for the characteristics of each component of the simulator. A mathematical model of a PV system, programmable DC power supply, isolated DC measurement device, and Computing Unit are integrated to form a real-time processing simulator. Performance tests are carried out with a commercial PV inverter and prove the superiority of this proposed algorithm against the conventional algorithm.

Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25.

Science.gov (United States)

Xu, Junyi; Zheng, Qiuyue; Yu, Ling; Liu, Ran; Zhao, Xin; Wang, Gang; Wang, Qinghua; Cao, Jijuan

2013-11-01

The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. We developed and evaluated the specificity and sensitivity of the LAMP method for rapid detection of the genetically modified (GM) maize T25. A set of six specific primers was successfully designed to recognize six distinct sequences on the target gene, including a pair of inner primers, a pair of outer primers, and a pair of loop primers. The optimum reaction temperature and time were verified to be 65°C and 45 min, respectively. The detection limit of this LAMP assay was 5 g kg(-1) GMO component. Comparative experiments showed that the LAMP assay was a simple, rapid, accurate, and specific method for detecting the GM maize T25.

Rapid Identification of Dengue Virus by Reverse Transcription-Polymerase Chain Reaction Using Field-Deployable Instrumentation

National Research Council Canada - National Science Library

McAvin, James C; Escamilla, Elizabeth M; Blow, James A; Turell, Micahel J; Quintana, Miguel; Bowles, David E; Swaby, James A; Barnes, William J; Huff, William B; Lahman, Kenton L

2005-01-01

...) reverse transcription-polymerase chain reaction assays were developed for screening and seroype identification of infected mosquito vectors and human sera using a field-deployable, fluorometric thermocycler...

Bridging FPGA and GPU technologies for AO real-time control

Science.gov (United States)

Perret, Denis; Lainé, Maxime; Bernard, Julien; Gratadour, Damien; Sevin, Arnaud

2016-07-01

Our team has developed a common environment for high performance simulations and real-time control of AO systems based on the use of Graphics Processors Units in the context of the COMPASS project. Such a solution, based on the ability of the real time core in the simulation to provide adequate computing performance, limits the cost of developing AO RTC systems and makes them more scalable. A code developed and validated in the context of the simulation may be injected directly into the system and tested on sky. Furthermore, the use of relatively low cost components also offers significant advantages for the system hardware platform. However, the use of GPUs in an AO loop comes with drawbacks: the traditional way of offloading computation from CPU to GPUs - involving multiple copies and unacceptable overhead in kernel launching - is not well suited in a real time context. This last application requires the implementation of a solution enabling direct memory access (DMA) to the GPU memory from a third party device, bypassing the operating system. This allows this device to communicate directly with the real-time core of the simulation feeding it with the WFS camera pixel stream. We show that DMA between a custom FPGA-based frame-grabber and a computation unit (GPU, FPGA, or Coprocessor such as Xeon-phi) across PCIe allows us to get latencies compatible with what will be needed on ELTs. As a fine-grained synchronization mechanism is not yet made available by GPU vendors, we propose the use of memory polling to avoid interrupts handling and involvement of a CPU. Network and Vision protocols are handled by the FPGA-based Network Interface Card (NIC). We present the results we obtained on a complete AO loop using camera and deformable mirror simulators.

Evaluation of a multiplex real-time PCR assay for the detection of respiratory viruses in clinical specimens.

Science.gov (United States)

Rheem, Insoo; Park, Joowon; Kim, Tae-Hyun; Kim, Jong Wan

2012-11-01

In this study, we evaluated the analytical performance and clinical potential of a one-step multiplex real-time PCR assay for the simultaneous detection of 14 types of respiratory viruses using the AdvanSure RV real-time PCR Kit (LG Life Sciences, Korea). Three hundred and twenty clinical specimens were tested with the AdvanSure RV real-time PCR Kit and conventional multiplex reverse transcription (RT)-PCR assay. The assay results were analyzed and the one-step AdvanSure RV real-time PCR Kit was compared with the conventional multiplex RT-PCR assay with respect to the sensitivity and specificity of the detection of respiratory viruses. The limit of detection (LOD) was 1.31 plaque-forming units (PFU)/mL for human rhinoviruses (hRVs), 4.93 PFU/mL for human coronavirus HCoV-229E/NL63, 2.67 PFU/mL for human coronavirus HCoV-OC43, 18.20 PFU/mL for parainfluenza virus 1 (PIV)-1, 24.57 PFU/mL for PIV-2, 1.73 PFU/mL for PIV-3, 1.79 PFU/mL for influenza virus group (Flu) A, 59.51 PFU/mL for FluB, 5.46 PFU/mL for human respiratory syncytial virus (hRSV)-A, 17.23 PFU/mL for hRSV-B, 9.99 PFU/mL for human adenovirus (ADVs). The cross-reactivity test for this assay against 23 types of non-respiratory viruses showed negative results for all viruses tested. The agreement between the one-step AdvanSure multiplex real-time PCR assay and the conventional multiplex RT-PCR assay was 98%. The one-step AdvanSure RV multiplex real-time PCR assay is a simple assay with high potential for specific, rapid and sensitive laboratory diagnosis of respiratory viruses compared to conventional multiplex RT-PCR.

MARTe framework; a middle-ware for real-time applications development

International Nuclear Information System (INIS)

Neto, A.; Alves, D.; Carvalho, B.B.; Carvalho, P.J.; Fernandes, H.; Valcarcel, D.F.; Sartori, F.; Barbalace, A.; Manduchi, G.; Boncagni, L.; Tommasi, G. de; McCullen, P.; Stephen, A.; Vitelli, R.; Zabeo, L.

2012-01-01

The Multi-threaded Application Real-Time executor (MARTe) is a C++ framework that provides a development environment for the design and deployment of real-time applications, e.g. control systems. The kernel of MARTe comprises a set of data-driven independent blocks, connected using a shared bus. This modular design enforces a clear boundary between algorithms, hardware interaction and system configuration. The architecture, being multi-platform, facilitates the test and commissioning of new systems, enabling the execution of plant models in offline environments and with the hardware-in-the-loop, whilst also providing a set of non-intrusive introspection and logging facilities. Furthermore, applications can be developed in non real-time environments and deployed in a real-time operating system, using exactly the same code and configuration data. The framework is already being used in several fusion experiments, with control cycles ranging from 50 microseconds to 10 milliseconds exhibiting jitters of less than 2%, using VxWorks R , RTAI or Linux. Codes can also be developed and executed in Microsoft Windows R and Solaris R . This paper discusses the main design concepts of MARTe, in particular the architectural choices which enabled the combination of real-time accuracy, performance and robustness with complex and modular data driven applications. (authors)

The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation.

Science.gov (United States)

Malik, Sohail; Roeder, Robert G

2010-11-01

The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action of numerous other co-activators and co-repressors, including those acting at the level of chromatin. These interactions ultimately allow the Mediator to deliver outputs that range from maximal activation of genes to modulation of basal transcription to long-term epigenetic silencing.

Mediator MED23 regulates basal transcription in vivo via an interaction with P-TEFb.

Science.gov (United States)

Wang, Wei; Yao, Xiao; Huang, Yan; Hu, Xiangming; Liu, Runzhong; Hou, Dongming; Chen, Ruichuan; Wang, Gang

2013-01-01

The Mediator is a multi-subunit complex that transduces regulatory information from transcription regulators to the RNA polymerase II apparatus. Growing evidence suggests that Mediator plays roles in multiple stages of eukaryotic transcription, including elongation. However, the detailed mechanism by which Mediator regulates elongation remains elusive. In this study, we demonstrate that Mediator MED23 subunit controls a basal level of transcription by recruiting elongation factor P-TEFb, via an interaction with its CDK9 subunit. The mRNA level of Egr1, a MED23-controlled model gene, is reduced 4-5 fold in Med23 (-/-) ES cells under an unstimulated condition, but Med23-deficiency does not alter the occupancies of RNAP II, GTFs, Mediator complex, or activator ELK1 at the Egr1 promoter. Instead, Med23 depletion results in a significant decrease in P-TEFb and RNAP II (Ser2P) binding at the coding region, but no changes for several other elongation regulators, such as DSIF and NELF. ChIP-seq revealed that Med23-deficiency partially reduced the P-TEFb occupancy at a set of MED23-regulated gene promoters. Further, we demonstrate that MED23 interacts with CDK9 in vivo and in vitro. Collectively, these results provide the mechanistic insight into how Mediator promotes RNAP II into transcription elongation.

A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay.

Science.gov (United States)

Babu, Binoy; Washburn, Brian K; Ertek, Tülin Sarigül; Miller, Steven H; Riddle, Charles B; Knox, Gary W; Ochoa-Corona, Francisco M; Olson, Jennifer; Katırcıoğlu, Yakup Zekai; Paret, Mathews L

2017-09-01

Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early detection and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and are inconsistent in detecting the virus from symptomatic plants. Real-time RT-qPCR assay is highly sensitive for detection of RRV, but it is expensive and requires well-equipped laboratories. Both the RT-PCR and RT-qPCR cannot be used in a field-based testing for RRV. Hence a novel probe based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, using primer/probe designed based on the nucleocapsid gene of the RRV has been developed. The assay is highly specific and did not give a positive reaction to other viruses infecting roses belonging to both inclusive and exclusive genus. Dilution assays using the in vitro transcript showed that the primer/probe set is highly sensitive, with a detection limit of 1 fg/μl. In addition, a rapid technique for the extraction of viral RNA (rose varieties, collected from different states in the U.S. The entire process, including the extraction can be completed in 25min, with less sophisticated equipments. The developed assay can be used with high efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscapes. Copyright © 2017 Elsevier B.V. All rights reserved.

Hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice.

Science.gov (United States)

De Domenico, Ivana; Zhang, Tian Y; Koening, Curry L; Branch, Ryan W; London, Nyall; Lo, Eric; Daynes, Raymond A; Kushner, James P; Li, Dean; Ward, Diane M; Kaplan, Jerry

2010-07-01

Hepcidin is a peptide hormone that regulates iron homeostasis and acts as an antimicrobial peptide. It is expressed and secreted by a variety of cell types in response to iron loading and inflammation. Hepcidin mediates iron homeostasis by binding to the iron exporter ferroportin, inducing its internalization and degradation via activation of the protein kinase Jak2 and the subsequent phosphorylation of ferroportin. Here we have shown that hepcidin-activated Jak2 also phosphorylates the transcription factor Stat3, resulting in a transcriptional response. Hepcidin treatment of ferroportin-expressing mouse macrophages showed changes in mRNA expression levels of a wide variety of genes. The changes in transcript levels for half of these genes were a direct effect of hepcidin, as shown by cycloheximide insensitivity, and dependent on the presence of Stat3. Hepcidin-mediated transcriptional changes modulated LPS-induced transcription in both cultured macrophages and in vivo mouse models, as demonstrated by suppression of IL-6 and TNF-alpha transcript and secreted protein. Hepcidin-mediated transcription in mice also suppressed toxicity and morbidity due to single doses of LPS, poly(I:C), and turpentine, which is used to model chronic inflammatory disease. Most notably, we demonstrated that hepcidin pretreatment protected mice from a lethal dose of LPS and that hepcidin-knockout mice could be rescued from LPS toxicity by injection of hepcidin. The results of our study suggest a new function for hepcidin in modulating acute inflammatory responses.

CAN LARGE TIME DELAYS OBSERVED IN LIGHT CURVES OF CORONAL LOOPS BE EXPLAINED IN IMPULSIVE HEATING?

International Nuclear Information System (INIS)

Lionello, Roberto; Linker, Jon A.; Mikić, Zoran; Alexander, Caroline E.; Winebarger, Amy R.

2016-01-01

The light curves of solar coronal loops often peak first in channels associated with higher temperatures and then in those associated with lower temperatures. The delay times between the different narrowband EUV channels have been measured for many individual loops and recently for every pixel of an active region observation. The time delays between channels for an active region exhibit a wide range of values. The maximum time delay in each channel pair can be quite large, i.e., >5000 s. These large time delays make-up 3%–26% (depending on the channel pair) of the pixels where a trustworthy, positive time delay is measured. It has been suggested that these time delays can be explained by simple impulsive heating, i.e., a short burst of energy that heats the plasma to a high temperature, after which the plasma is allowed to cool through radiation and conduction back to its original state. In this paper, we investigate whether the largest observed time delays can be explained by this hypothesis by simulating a series of coronal loops with different heating rates, loop lengths, abundances, and geometries to determine the range of expected time delays between a set of four EUV channels. We find that impulsive heating cannot address the largest time delays observed in two of the channel pairs and that the majority of the large time delays can only be explained by long, expanding loops with photospheric abundances. Additional observations may rule out these simulations as an explanation for the long time delays. We suggest that either the time delays found in this manner may not be representative of real loop evolution, or that the impulsive heating and cooling scenario may be too simple to explain the observations, and other potential heating scenarios must be explored

Reversal of the lattice structure in SrCoO(x) epitaxial thin films studied by real-time optical spectroscopy and first-principles calculations.

Science.gov (United States)

Choi, Woo Seok; Jeen, Hyoungjeen; Lee, Jun Hee; Seo, S S Ambrose; Cooper, Valentino R; Rabe, Karin M; Lee, Ho Nyung

2013-08-30

Using real-time spectroscopic ellipsometry, we directly observed a reversible lattice and electronic structure evolution in SrCoO(x) (x=2.5-3) epitaxial thin films. Drastically different electronic ground states, which are extremely susceptible to the oxygen content x, are found in the two topotactic phases: i.e., the brownmillerite SrCoO2.5 and the perovskite SrCoO3. First-principles calculations confirmed substantial differences in the electronic structure, including a metal-insulator transition, which originate from the modification in the Co valence states and crystallographic structures. More interestingly, the two phases can be reversibly controlled by changing the ambient pressure at greatly reduced temperatures. Our finding provides an important pathway to understanding the novel oxygen-content-dependent phase transition uniquely found in multivalent transition metal oxides.

Evaluating viral interference between Influenza virus and Newcastle disease virus using real-time reverse transcription–polymerase chain reaction in chicken eggs

Directory of Open Access Journals (Sweden)

Ge Shengqiang

2012-07-01

Full Text Available Abstract Background Simultaneous and sequential allantoic cavity inoculations of Specific-pathogen-free (SPF chicken eggs with Influenza virus (AIV and Newcastle disease virus (NDV demonstrated that the interaction of AIV and NDV during co-infection was variable. Our research revisited the replication interference potential of AIV and NDV using real-time reverse transcription–polymerase chain reaction (real-time RT-PCR for AIV and NDV to specifically detect the viral genomes in mixed infections. Results Data from this survey showed that when different doses of NDV (Lasota or F48E8 and AIV (F98 or H5N1 were simultaneously inoculated into embryonating chicken eggs (ECE, interference with the growth of NDV occurred, while interference with the growth of AIV did not occur. When equal amount of the two viruses were sequentially employed, the degree of interference was dependent upon the time of superinfection and the virulence of virus. Conclusion AIV have a negative impact on NDV growth if they are inoculated simultaneously or sequentially and that the degree of interference depended upon the quantity and relative virulence of the virus strains used; however, interference with AIV was not observed. Only if NDV were inoculated at an earlier time will NDV able to interfere with the growth of AIV.

Mediator binding to UASs is broadly uncoupled from transcription and cooperative with TFIID recruitment to promoters.

Science.gov (United States)

Grünberg, Sebastian; Henikoff, Steven; Hahn, Steven; Zentner, Gabriel E

2016-11-15

Mediator is a conserved, essential transcriptional coactivator complex, but its in vivo functions have remained unclear due to conflicting data regarding its genome-wide binding pattern obtained by genome-wide ChIP Here, we used ChEC-seq, a method orthogonal to ChIP, to generate a high-resolution map of Mediator binding to the yeast genome. We find that Mediator associates with upstream activating sequences (UASs) rather than the core promoter or gene body under all conditions tested. Mediator occupancy is surprisingly correlated with transcription levels at only a small fraction of genes. Using the same approach to map TFIID, we find that TFIID is associated with both TFIID- and SAGA-dependent genes and that TFIID and Mediator occupancy is cooperative. Our results clarify Mediator recruitment and binding to the genome, showing that Mediator binding to UASs is widespread, partially uncoupled from transcription, and mediated in part by TFIID. © 2016 The Authors.

Web-based ground loop supervision system for the TJ-II Stellarator

International Nuclear Information System (INIS)

Pena, A. de la; Lapayese, F.; Pacios, L.; Carrasco, R.

2005-01-01

To minimize electromagnetic interferences in diagnostic and control signals, and to guarantee safe operation of TJ-II, ground loops must be avoided. In order to meet this goal, the whole grounding system of the TJ-II was split into multiple single branches that are connected at a single earth point located near the TJ-II structure in the torus hall. A real-time ground loop supervision system (GLSS) has been designed, manufactured and tested by the TJ-II control group for detecting unintentional short circuits between isolated grounded parts. A web server running on the real-time operating system OS-9 provides remote access to the real-time ground loops measurement. Ground loops monitoring and different operation modes can be configured via any web browser. This paper gives the detailed design of the whole TJ-II ground loop supervision system and its results during its operation

Web-based ground loop supervision system for the TJ-II Stellarator

Energy Technology Data Exchange (ETDEWEB)

Pena, A. de la [Asociacion EURATOM-CIEMAT Para Fusion, Avd. Complutense 22, 28040 Madrid (Spain)]. E-mail: a.delapena@ciemat.es; Lapayese, F. [Asociacion EURATOM-CIEMAT Para Fusion, Avd. Complutense 22, 28040 Madrid (Spain); Pacios, L. [Asociacion EURATOM-CIEMAT Para Fusion, Avd. Complutense 22, 28040 Madrid (Spain); Carrasco, R. [Asociacion EURATOM-CIEMAT Para Fusion, Avd. Complutense 22, 28040 Madrid (Spain)

2005-11-15

To minimize electromagnetic interferences in diagnostic and control signals, and to guarantee safe operation of TJ-II, ground loops must be avoided. In order to meet this goal, the whole grounding system of the TJ-II was split into multiple single branches that are connected at a single earth point located near the TJ-II structure in the torus hall. A real-time ground loop supervision system (GLSS) has been designed, manufactured and tested by the TJ-II control group for detecting unintentional short circuits between isolated grounded parts. A web server running on the real-time operating system OS-9 provides remote access to the real-time ground loops measurement. Ground loops monitoring and different operation modes can be configured via any web browser. This paper gives the detailed design of the whole TJ-II ground loop supervision system and its results during its operation.

Reverse Transcription Errors and RNA-DNA Differences at Short Tandem Repeats.

Science.gov (United States)

Fungtammasan, Arkarachai; Tomaszkiewicz, Marta; Campos-Sánchez, Rebeca; Eckert, Kristin A; DeGiorgio, Michael; Makova, Kateryna D

2016-10-01

Transcript variation has important implications for organismal function in health and disease. Most transcriptome studies focus on assessing variation in gene expression levels and isoform representation. Variation at the level of transcript sequence is caused by RNA editing and transcription errors, and leads to nongenetically encoded transcript variants, or RNA-DNA differences (RDDs). Such variation has been understudied, in part because its detection is obscured by reverse transcription (RT) and sequencing errors. It has only been evaluated for intertranscript base substitution differences. Here, we investigated transcript sequence variation for short tandem repeats (STRs). We developed the first maximum-likelihood estimator (MLE) to infer RT error and RDD rates, taking next generation sequencing error rates into account. Using the MLE, we empirically evaluated RT error and RDD rates for STRs in a large-scale DNA and RNA replicated sequencing experiment conducted in a primate species. The RT error rates increased exponentially with STR length and were biased toward expansions. The RDD rates were approximately 1 order of magnitude lower than the RT error rates. The RT error rates estimated with the MLE from a primate data set were concordant with those estimated with an independent method, barcoded RNA sequencing, from a Caenorhabditis elegans data set. Our results have important implications for medical genomics, as STR allelic variation is associated with >40 diseases. STR nonallelic transcript variation can also contribute to disease phenotype. The MLE and empirical rates presented here can be used to evaluate the probability of disease-associated transcripts arising due to RDD. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

MODELING OF POWER SYSTEMS AND TESTING OF RELAY PROTECTION DEVICES IN REAL AND MODEL TIME

Directory of Open Access Journals (Sweden)

I. V. Novash

2017-01-01

Full Text Available The methods of modelling of power system modes and of testing of relay protection devices with the aid the simulation complexes in real time and with the help of computer software systems that enables the simulation of virtual time scale are considered. Information input protection signals in the simulation of the virtual model time are being obtained in the computational experiment, whereas the tests of protective devices are carried out with the help of hardware and software test systems with the use of estimated input signals. Study of power system stability when modes of generating and consuming electrical equipment and conditions of devices of relay protection are being changed requires testing with the use of digital simulators in a mode of a closed loop. Herewith feedbacks between a model of the power system operating in a real time and external devices or their models must be determined (modelled. Modelling in real time and the analysis of international experience in the use of digital simulation power systems for real-time simulation (RTDS simulator have been fulfilled. Examples are given of the use of RTDS systems by foreign energy companies to test relay protection systems and control, to test the equipment and devices of automatic control, analysis of cyber security and evaluation of the operation of energy systems under different scenarios of occurrence of emergency situations. Some quantitative data on the distribution of RTDS in different countries and Russia are presented. It is noted that the leading energy universities of Russia use the real-time simulation not only to solve scientific and technical problems, but also to conduct training and laboratory classes on modelling of electric networks and anti-emergency automatic equipment with the students. In order to check serviceability of devices of relay protection without taking into account the reaction of the power system tests can be performed in an open loop mode with the

Determination of the plasma position for its real-time control in the COMPASS tokamak

International Nuclear Information System (INIS)

Janky, F.; Havlicek, J.; Valcarcel, D.; Hron, M.; Horacek, J.; Kudlacek, O.; Panek, R.; Carvalho, B.B.

2011-01-01

An efficient horizontal and vertical stabilization of the plasma column position are essential for a reliable tokamak operation. Plasma position is generally determined by plasma current, plasma pressure and external vertical and horizontal magnetic fields. Such fields are generated by poloidal field coils and proper algorithm for the current control have to by applied, namely, in case of fast feedback loops. This paper presents a real-time plasma position reconstruction algorithms developed for the COMPASS tokamak. Further, its implementation in the MARTe (Multithreaded Application Real-Time executor) is described and the first results from test of the algorithm for real-time control of horizontal plasma positions are presented.

Determination of the plasma position for its real-time control in the COMPASS tokamak

Energy Technology Data Exchange (ETDEWEB)

Janky, F., E-mail: jankyf@ipp.cas.cz [Institute of Plasma Physics, AS CR, v.v.i., Association EURATOM/IPP.CR, Za Slovankou 3, 182 00 Prague (Czech Republic); Charles University in Prague, Faculty of Mathematics and Physics, V Holesovickach 2, CZ-18000 Prague (Czech Republic); Havlicek, J. [Institute of Plasma Physics, AS CR, v.v.i., Association EURATOM/IPP.CR, Za Slovankou 3, 182 00 Prague (Czech Republic); Charles University in Prague, Faculty of Mathematics and Physics, V Holesovickach 2, CZ-18000 Prague (Czech Republic); Valcarcel, D. [Associacao EURATOM/IST, Instituto de Plasmas e Fusao Nuclear - Laboratorio Associado, Instituto Superior Tecnico, P1049-001 Lisboa (Portugal); Hron, M.; Horacek, J. [Institute of Plasma Physics, AS CR, v.v.i., Association EURATOM/IPP.CR, Za Slovankou 3, 182 00 Prague (Czech Republic); Kudlacek, O. [Czech Technical University, Faculty of Nuclear Sciences and Physical Engineering, Technicka 2, 166 27 Prague (Czech Republic); Panek, R. [Institute of Plasma Physics, AS CR, v.v.i., Association EURATOM/IPP.CR, Za Slovankou 3, 182 00 Prague (Czech Republic); Carvalho, B.B. [Associacao EURATOM/IST, Instituto de Plasmas e Fusao Nuclear - Laboratorio Associado, Instituto Superior Tecnico, P1049-001 Lisboa (Portugal)

2011-10-15

An efficient horizontal and vertical stabilization of the plasma column position are essential for a reliable tokamak operation. Plasma position is generally determined by plasma current, plasma pressure and external vertical and horizontal magnetic fields. Such fields are generated by poloidal field coils and proper algorithm for the current control have to by applied, namely, in case of fast feedback loops. This paper presents a real-time plasma position reconstruction algorithms developed for the COMPASS tokamak. Further, its implementation in the MARTe (Multithreaded Application Real-Time executor) is described and the first results from test of the algorithm for real-time control of horizontal plasma positions are presented.

Time-Reversal MUSIC Imaging with Time-Domain Gating Technique

Science.gov (United States)

Choi, Heedong; Ogawa, Yasutaka; Nishimura, Toshihiko; Ohgane, Takeo

A time-reversal (TR) approach with multiple signal classification (MUSIC) provides super-resolution for detection and localization using multistatic data collected from an array antenna system. The theory of TR-MUSIC assumes that the number of antenna elements is greater than that of scatterers (targets). Furthermore, it requires many sets of frequency-domain data (snapshots) in seriously noisy environments. Unfortunately, these conditions are not practical for real environments due to the restriction of a reasonable antenna structure as well as limited measurement time. We propose an approach that treats both noise reduction and relaxation of the transceiver restriction by using a time-domain gating technique accompanied with the Fourier transform before applying the TR-MUSIC imaging algorithm. Instead of utilizing the conventional multistatic data matrix (MDM), we employ a modified MDM obtained from the gating technique. The resulting imaging functions yield more reliable images with only a few snapshots regardless of the limitation of the antenna arrays.

Digital video timing analyzer for the evaluation of PC-based real-time simulation systems

Science.gov (United States)

Jones, Shawn R.; Crosby, Jay L.; Terry, John E., Jr.

2009-05-01

Due to the rapid acceleration in technology and the drop in costs, the use of commercial off-the-shelf (COTS) PC-based hardware and software components for digital and hardware-in-the-loop (HWIL) simulations has increased. However, the increase in PC-based components creates new challenges for HWIL test facilities such as cost-effective hardware and software selection, system configuration and integration, performance testing, and simulation verification/validation. This paper will discuss how the Digital Video Timing Analyzer (DiViTA) installed in the Aviation and Missile Research, Development and Engineering Center (AMRDEC) provides quantitative characterization data for PC-based real-time scene generation systems. An overview of the DiViTA is provided followed by details on measurement techniques, applications, and real-world examples of system benefits.

A lab-on-a-chip system with integrated sample preparation and loop-mediated isothermal amplification for rapid and quantitative detection of Salmonella spp. in food samples

DEFF Research Database (Denmark)

Sun, Yi; Than Linh, Quyen; Hung, Tran Quang

2015-01-01

was capable to detect Salmonella at concentration of 50 cells per test within 40 min. The simple design, together with high level of integration, isothermal amplification, and quantitative analysis of multiple samples in short time will greatly enhance the practical applicability of the LOC system for rapid...... amplification (LAMP) for rapid and quantitative detection of Salmonella spp. in food samples. The whole diagnostic procedures including DNA isolation, isothermal amplification, and real-time detection were accomplished in a single chamber. Up to eight samples could be handled simultaneously and the system...... and usually take a few hours to days to complete. In response to the demand for rapid on line or at site detection of pathogens, in this study, we describe for the first time an eight-chamber lab-on-a-chip (LOC) system with integrated magnetic beads-based sample preparation and loop-mediated isothermal...

Selection of suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using quantitative real-time polymerase chain reaction

DEFF Research Database (Denmark)

Zornhagen, K. W.; Kristensen, A. T.; Hansen, Anders Elias

2015-01-01

Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a sensitive technique for quantifying gene expression. Stably expressed reference genes are necessary for normalization of RT-qPCR data. Only a few articles have been published on reference genes in canine tumours....... The objective of this study was to demonstrate how to identify suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using RT-qPCR. Primer pairs for 17 potential reference genes were designed and tested in archival tumour biopsies from six dogs. The geNorm algorithm...

A real-time view of the TAR:Tat:P-TEFb complex at HIV-1 transcription sites

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Knezevich Anna

2007-05-01

Full Text Available Abstract HIV-1 transcription is tightly regulated: silent in long-term latency and highly active in acutely-infected cells. Transcription is activated by the viral protein Tat, which recruits the elongation factor P-TEFb by binding the TAR sequence present in nascent HIV-1 RNAs. In this study, we analyzed the dynamic of the TAR:Tat:P-TEFb complex in living cells, by performing FRAP experiments at HIV-1 transcription sites. Our results indicate that a large fraction of Tat present at these sites is recruited by Cyclin T1. We found that in the presence of Tat, Cdk9 remained bound to nascent HIV-1 RNAs for 71s. In contrast, when transcription was activated by PMA/ionomycin, in the absence of Tat, Cdk9 turned-over rapidly and resided on the HIV-1 promoter for only 11s. Thus, the mechanism of trans-activation determines the residency time of P-TEFb at the HIV-1 gene, possibly explaining why Tat is such a potent transcriptional activator. In addition, we observed that Tat occupied HIV-1 transcription sites for 55s, suggesting that the TAR:Tat:P-TEFb complex dissociates from the polymerase following transcription initiation, and undergoes subsequent cycles of association/dissociation.

LHX2 Mediates the FGF-to-SHH Regulatory Loop during Limb Development

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Billy A. Watson

2018-06-01

Full Text Available During limb development, fibroblast growth factors (Fgfs govern proximal–distal outgrowth and patterning. FGFs also synchronize developmental patterning between the proximal–distal and anterior–posterior axes by maintaining Sonic hedgehog (Shh expression in cells of the zone of polarizing activity (ZPA in the distal posterior mesoderm. Shh, in turn, maintains Fgfs in the apical ectodermal ridge (AER that caps the distal tip of the limb bud. Crosstalk between Fgf and Shh signaling is critical for patterned limb development, but the mechanisms underlying this feedback loop are not well-characterized. Implantation of Fgf beads in the proximal posterior limb bud can maintain SHH expression in the former ZPA domain (evident 3 h after application, while prolonged exposure (24 h can induce SHH outside of this domain. Although temporally and spatially disparate, comparative analysis of transcriptome data from these different populations accentuated genes involved in SHH regulation. Comparative analysis identified 25 candidates common to both treatments, with eight linked to SHH expression or function. Furthermore, we demonstrated that LHX2, a LIM Homeodomain transcription factor, is an intermediate in the FGF-mediated regulation of SHH. Our data suggest that LHX2 acts as a competency factor maintaining distal posterior SHH expression subjacent to the AER.

Set1/COMPASS and Mediator are repurposed to promote epigenetic transcriptional memory.

Science.gov (United States)

D'Urso, Agustina; Takahashi, Yoh-Hei; Xiong, Bin; Marone, Jessica; Coukos, Robert; Randise-Hinchliff, Carlo; Wang, Ji-Ping; Shilatifard, Ali; Brickner, Jason H

2016-06-23

In yeast and humans, previous experiences can lead to epigenetic transcriptional memory: repressed genes that exhibit mitotically heritable changes in chromatin structure and promoter recruitment of poised RNA polymerase II preinitiation complex (RNAPII PIC), which enhances future reactivation. Here, we show that INO1 memory in yeast is initiated by binding of the Sfl1 transcription factor to the cis-acting Memory Recruitment Sequence, targeting INO1 to the nuclear periphery. Memory requires a remodeled form of the Set1/COMPASS methyltransferase lacking Spp1, which dimethylates histone H3 lysine 4 (H3K4me2). H3K4me2 recruits the SET3C complex, which plays an essential role in maintaining this mark. Finally, while active INO1 is associated with Cdk8(-) Mediator, during memory, Cdk8(+) Mediator recruits poised RNAPII PIC lacking the Kin28 CTD kinase. Aspects of this mechanism are generalizable to yeast and conserved in human cells. Thus, COMPASS and Mediator are repurposed to promote epigenetic transcriptional poising by a highly conserved mechanism.

DHT selectively reverses Smad3-mediated/TGF-beta-induced responses through transcriptional down-regulation of Smad3 in prostate epithelial cells.

Science.gov (United States)

Song, Kyung; Wang, Hui; Krebs, Tracy L; Wang, Bingcheng; Kelley, Thomas J; Danielpour, David

2010-10-01

Androgens suppress TGF-β responses in the prostate through mechanisms that are not fully explored. We have recently reported that 5α-dihydrotestosterone (DHT) suppresses the ability of TGF-β to inhibit proliferation and induce apoptosis of prostatic epithelial cells and provided evidence that such suppression was fueled by transcriptional down-regulation of TGF-β receptor II (ΤβRII). We now show that androgen receptor (AR) activated by DHT suppresses the TGF-β-induced phosphorylation of Sma- and Mad-related protein (Smad)3 in LNCaP cells overexpressing TβRII under the control of a cytomegalovirus promoter, which is not regulated by DHT, suggesting that transcriptional repression of TβRII alone does not fully account for the impact of DHT on TGF-β responses. Instead, we demonstrate that such suppression occurs through loss of total Smad3, resulting from transcriptional suppression of Smad3. We provide evidence that DHT down-regulates the promoter activity of Smad3 in various prostate cancer cell lines, including NRP-154+AR, DU145+AR, LNCaP, and VCaP, at least partly through androgen-dependent inactivation of Sp1. Moreover, we show that overexpression of Smad3 reverses the ability of DHT to protect against TGF-β-induced apoptosis in NRP-154+AR, supporting our model that loss of Smad3 by DHT is involved in the protection against TGF-β-induced apoptosis. Together, these findings suggest that deregulated/enhanced expression and activation of AR in prostate carcinomas may intercept the tumor suppressor function of TGF-β through transcriptional suppression of Smad3, thereby providing new mechanistic insight into the development of castration-resistant prostate cancer.

Test of feasibility of a novel high precision test of time reversal invariance

International Nuclear Information System (INIS)

Samuel, Deepak

2007-01-01

The first results of a feasibility test of a novel high precision test of time reversal invariance are reported. The Time Reversal Invariance test at COSY (TRIC) was planned to measure the time reversal violating observable A y,xz with an accuracy of 10 -6 in proton-deuteron (p-d) scattering. A novel technique for measuring total cross sections is introduced and the achievable precision of this measuring technique is tested. The correlation coefficient A y,y in p-d scattering fakes a time-reversal violating effect. This work reports the feasibility test of the novel method in the measurement of A y,y in p-p scattering. The first step in the experimental design was the development of a hard real-time data acquisition system. To meet stringent latency requirements, the capabilities of Windows XP had to be augmented with a real-time subsystem. The remote control feature of the data acquisition enables users to operate it from any place via an internet connection. The data acquisition proved its reliability in several beam times without any failures. The analysis of the data showed the presence of 1/f noise which substantially limits the quality of our measurements. The origin of 1/f noise was traced and found to be the Barkhausen noise from the ferrite core of the beam current transformer (BCT). A global weighted fitting technique based on a modified Wiener-Khinchin method was developed and used to suppress the influence of 1/f noise, which increased the error bar of the results by a factor 3. This is the only deviation from our expectations. The results are presented and discussed. (orig.)

Test of feasibility of a novel high precision test of time reversal invariance

Energy Technology Data Exchange (ETDEWEB)

Samuel, Deepak

2007-07-01

The first results of a feasibility test of a novel high precision test of time reversal invariance are reported. The Time Reversal Invariance test at COSY (TRIC) was planned to measure the time reversal violating observable A{sub y,xz} with an accuracy of 10{sup -6} in proton-deuteron (p-d) scattering. A novel technique for measuring total cross sections is introduced and the achievable precision of this measuring technique is tested. The correlation coefficient A{sub y,y} in p-d scattering fakes a time-reversal violating effect. This work reports the feasibility test of the novel method in the measurement of A{sub y,y} in p-p scattering. The first step in the experimental design was the development of a hard real-time data acquisition system. To meet stringent latency requirements, the capabilities of Windows XP had to be augmented with a real-time subsystem. The remote control feature of the data acquisition enables users to operate it from any place via an internet connection. The data acquisition proved its reliability in several beam times without any failures. The analysis of the data showed the presence of 1/f noise which substantially limits the quality of our measurements. The origin of 1/f noise was traced and found to be the Barkhausen noise from the ferrite core of the beam current transformer (BCT). A global weighted fitting technique based on a modified Wiener-Khinchin method was developed and used to suppress the influence of 1/f noise, which increased the error bar of the results by a factor 3. This is the only deviation from our expectations. The results are presented and discussed. (orig.)

Strategies to regulate transcription factor-mediated gene positioning and interchromosomal clustering at the nuclear periphery.

Science.gov (United States)

Randise-Hinchliff, Carlo; Coukos, Robert; Sood, Varun; Sumner, Michael Chas; Zdraljevic, Stefan; Meldi Sholl, Lauren; Garvey Brickner, Donna; Ahmed, Sara; Watchmaker, Lauren; Brickner, Jason H

2016-03-14

In budding yeast, targeting of active genes to the nuclear pore complex (NPC) and interchromosomal clustering is mediated by transcription factor (TF) binding sites in the gene promoters. For example, the binding sites for the TFs Put3, Ste12, and Gcn4 are necessary and sufficient to promote positioning at the nuclear periphery and interchromosomal clustering. However, in all three cases, gene positioning and interchromosomal clustering are regulated. Under uninducing conditions, local recruitment of the Rpd3(L) histone deacetylase by transcriptional repressors blocks Put3 DNA binding. This is a general function of yeast repressors: 16 of 21 repressors blocked Put3-mediated subnuclear positioning; 11 of these required Rpd3. In contrast, Ste12-mediated gene positioning is regulated independently of DNA binding by mitogen-activated protein kinase phosphorylation of the Dig2 inhibitor, and Gcn4-dependent targeting is up-regulated by increasing Gcn4 protein levels. These different regulatory strategies provide either qualitative switch-like control or quantitative control of gene positioning over different time scales. © 2016 Randise-Hinchliff et al.

An artemisinin-mediated ROS evolving and dual protease light-up nanocapsule for real-time imaging of lysosomal tumor cell death.

Science.gov (United States)

Huang, Liwei; Luo, Yingping; Sun, Xian; Ju, Huangxian; Tian, Jiangwei; Yu, Bo-Yang

2017-06-15

Lysosomes are critical organelles for cellular homeostasis and can be used as potential targets to kill tumor cells from inside. Many photo-therapeutic methods have been developed to overproduce reactive oxygen species (ROS) to trigger lysosomal membrane permeabilization (LMP)-associated cell death pathway. However, these technologies rely on extra irradiation to activate the photosensitizers, which limits the applications in treating deep seated tumors and widespread metastatic lesions. This work reports a multifunctional nanocapsule to achieve targeted lysosomal tumor cell death without irradiation and real-time monitoring of drug effect through encapsulating artemisinin and dual protease light-up nanoprobe in a folate-functionalized liposome. The nanocapsule can be specifically uptaken by tumor cells via folate receptor-mediated endocytosis to enter lysosomes, in which artemisinin reacts with ferrous to generate ROS for LMP-associated cell death. By virtue of confocal fluorescence imaging, the artemisinin location in lysosome, ROS-triggered LMP and ultimate cell apoptosis can be visualized with the cathepsin B and caspase-3 activatable nanoprobe. Notably, the artemisinin-mediated ROS evolving for tumor therapy and real-time therapeutic monitoring were successfully implemented by living imaging in tumor-bearing mice, which broaden the nanocapsule for in vivo theranostics and may offer new opportunities for precise medicine. Copyright © 2016 Elsevier B.V. All rights reserved.

Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest.

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Sindhu Subramaniam

Full Text Available Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2. Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation. Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for

Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest.

Science.gov (United States)

Subramaniam, Sindhu; Sreenivas, Prethish; Cheedipudi, Sirisha; Reddy, Vatrapu Rami; Shashidhara, Lingadahalli Subrahmanya; Chilukoti, Ravi Kumar; Mylavarapu, Madhavi; Dhawan, Jyotsna

2014-01-01

Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological

Novel isoforms of the TFIID subunit TAF4 modulate nuclear receptor-mediated transcriptional activity

International Nuclear Information System (INIS)

Brunkhorst, Adrian; Neuman, Toomas; Hall, Anita; Arenas, Ernest; Bartfai, Tamas; Hermanson, Ola; Metsis, Madis

2004-01-01

The transcription factor TFIID consists of TATA-binding protein (TBP) and TBP-associated factors (TAFs). TAFs are essential for modulation of transcriptional activity but the regulation of TAFs is complex and many important aspects remain unclear. In this study, we have identified and characterized five novel truncated forms of the TFIID subunit TAF4 (TAF II 135). Analysis of the mouse gene structure revealed that all truncations were the results of alternative splicing and resulted in the loss of domains or parts of domains implicated in TAF4 functional interactions. Results from transcriptional assays showed that several of the TAF4 isoforms exerted dominant negative effects on TAF4 activity in nuclear receptor-mediated transcriptional activation. In addition, alternative TAF4 isoforms could be detected in specific cell types. Our results indicate an additional level of complexity in TAF4-mediated regulation of transcription and suggest context-specific roles for these new TAF4 isoforms in transcriptional regulation in vivo

Introduction to time reversal theory

International Nuclear Information System (INIS)

Henley, E.M.

1987-01-01

Theory and reaction mechanisms relevant to time reversal invariance are reviewed. Consequences of time reversal invariance are presented under the headings of CP tests, electromagnetic moments, weak emissions or absorptions, and scattering reactions. 8 refs., 4 figs

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics.

Science.gov (United States)

Zambenedetti, Miriam Ribas; Pavoni, Daniela Parada; Dallabona, Andreia Cristine; Dominguez, Alejandro Correa; Poersch, Celina de Oliveira; Fragoso, Stenio Perdigão; Krieger, Marco Aurélio

2017-05-01

Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

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Miriam Ribas Zambenedetti

Full Text Available BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV, hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+ plasmid, generating pET47b(+-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

Inhibition of both HIV-1 reverse transcription and gene expression by a cyclic peptide that binds the Tat-transactivating response element (TAR RNA.

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