Sample records for mrc rt01 isrctn47772397
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NASA Astrophysics Data System (ADS)
Buettner, Florian; Gulliford, Sarah L.; Webb, Steve; Sydes, Matthew R.; Dearnaley, David P.; Partridge, Mike
2009-11-01
Many studies have been performed to assess correlations between measures derived from dose-volume histograms and late rectal toxicities for radiotherapy of prostate cancer. The purpose of this study was to quantify correlations between measures describing the shape and location of the dose distribution and different outcomes. The dose to the rectal wall was projected on a two-dimensional map. In order to characterize the dose distribution, its centre of mass, longitudinal and lateral extent, and eccentricity were calculated at different dose levels. Furthermore, the dose-surface histogram (DSH) was determined. Correlations between these measures and seven clinically relevant rectal-toxicity endpoints were quantified by maximally selected standardized Wilcoxon rank statistics. The analysis was performed using data from the RT01 prostate radiotherapy trial. For some endpoints, the shape of the dose distribution is more strongly correlated with the outcome than simple DSHs. Rectal bleeding was most strongly correlated with the lateral extent of the dose distribution. For loose stools, the strongest correlations were found for longitudinal extent; proctitis was most strongly correlated with DSH. For the other endpoints no statistically significant correlations could be found. The strengths of the correlations between the shape of the dose distribution and outcome differed considerably between the different endpoints. Due to these significant correlations, it is desirable to use shape-based tools in order to assess the quality of a dose distribution.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Syndikus, Isabel; Morgan, Rachel C.; Sydes, Matthew R., E-mail: ms@ctu.mrc.ac.u
2010-07-01
Purpose: In men with localized prostate cancer, dose-escalated conformal radiotherapy (CFRT) improves efficacy outcomes at the cost of increased toxicity. We present a detailed analysis to provide further information about the incidence and prevalence of late gastrointestinal side effects. Methods and Materials: The UK Medical Research Council RT01 trial included 843 men with localized prostate cancer, who were treated for 6 months with neoadjuvant radiotherapy and were randomly assigned to either 64-Gy or 74-Gy CFRT. Toxicity was evaluated before CFRT and during long-term follow-up using Radiation Therapy Oncology Group (RTOG) grading, the Late Effects on Normal Tissue: Subjective, Objective, Managementmore » (LENT/SOM) scale, and Royal Marsden Hospital assessment scores. Patients regularly completed Functional Assessment of Cancer Therapy--Prostate (FACT-P) and University of California, Los Angeles, Prostate Cancer Index (UCLA-PCI) questionnaires. Results: In the dose-escalated group, the hazard ratio (HR) for rectal bleeding (LENT/SOM grade {>=}2) was 1.55 (95% CI, 1.17-2.04); for diarrhea (LENT/SOM grade {>=}2), the HR was 1.79 (95% CI, 1.10-2.94); and for proctitis (RTOG grade {>=}2), the HR was 1.64 (95% CI, 1.20-2.25). Compared to baseline scores, the prevalence of moderate and severe toxicities generally increased up to 3 years and than lessened. At 5 years, the cumulative incidence of patient-reported severe bowel problems was 6% vs. 8% (standard vs. escalated, respectively) and severe distress was 4% vs. 5%, respectively. Conclusions: There is a statistically significant increased risk of various adverse gastrointestinal events with dose-escalated CFRT. This remains at clinically acceptable levels, and overall prevalence ultimately decreases with duration of follow-up.« less
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Mixed raster content (MRC) model for compound image compression
NASA Astrophysics Data System (ADS)
de Queiroz, Ricardo L.; Buckley, Robert R.; Xu, Ming
1998-12-01
This paper will describe the Mixed Raster Content (MRC) method for compressing compound images, containing both binary test and continuous-tone images. A single compression algorithm that simultaneously meets the requirements for both text and image compression has been elusive. MRC takes a different approach. Rather than using a single algorithm, MRC uses a multi-layered imaging model for representing the results of multiple compression algorithms, including ones developed specifically for text and for images. As a result, MRC can combine the best of existing or new compression algorithms and offer different quality-compression ratio tradeoffs. The algorithms used by MRC set the lower bound on its compression performance. Compared to existing algorithms, MRC has some image-processing overhead to manage multiple algorithms and the imaging model. This paper will develop the rationale for the MRC approach by describing the multi-layered imaging model in light of a rate-distortion trade-off. Results will be presented comparing images compressed using MRC, JPEG and state-of-the-art wavelet algorithms such as SPIHT. MRC has been approved or proposed as an architectural model for several standards, including ITU Color Fax, IETF Internet Fax, and JPEG 2000.
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Chang, Cheng-Wei; Chen, Chaang-Ray; Huang, Chao-Ying; Shu, Wun-Yi; Chiang, Chi-Shiun; Hong, Ji-Hong; Hsu, Ian C.
2013-01-01
Simian virus 40 (SV40) transforms cells through the suppression of tumor-suppressive responses by large T and small t antigens; studies on the effects of these two oncoproteins have greatly improved our knowledge of tumorigenesis. Large T antigen promotes cellular transformation by binding and inactivating p53 and pRb tumor suppressor proteins. Previous studies have shown that not all of the tumor-suppressive responses were inactivated in SV40-transformed cells; however, the underlying cause is not fully studied. In this study, we investigated the UVB-responsive transcriptome of an SV40-transformed fibroblast (MRC5CVI) and that of its untransformed counterpart (MRC-5). We found that, in response to UVB irradiation, MRC-5 and MRC5CVI commonly up-regulated the expression of oxidative phosphorylation genes. MRC-5 up-regulated the expressions of chromosome condensation, DNA repair, cell cycle arrest, and apoptotic genes, but MRC5CVI did not. Further cell death assays indicated that MRC5CVI was more sensitive than MRC-5 to UVB-induced cell death with increased caspase-3 activation; combining with the transcriptomic results suggested that MRC5CVI may undergo UVB-induced cell death through mechanisms other than transcriptional regulation. Our study provides a further understanding of the effects of SV40 transformation on cellular stress responses, and emphasizes the value of SV40-transformed cells in the researches of sensitizing neoplastic cells to radiations. PMID:24019915
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Characterization of SWIR cameras by MRC measurements
NASA Astrophysics Data System (ADS)
Gerken, M.; Schlemmer, H.; Haan, Hubertus A.; Siemens, Christofer; Münzberg, M.
2014-05-01
Cameras for the SWIR wavelength range are becoming more and more important because of the better observation range for day-light operation under adverse weather conditions (haze, fog, rain). In order to choose the best suitable SWIR camera or to qualify a camera for a given application, characterization of the camera by means of the Minimum Resolvable Contrast MRC concept is favorable as the MRC comprises all relevant properties of the instrument. With the MRC known for a given camera device the achievable observation range can be calculated for every combination of target size, illumination level or weather conditions. MRC measurements in the SWIR wavelength band can be performed widely along the guidelines of the MRC measurements of a visual camera. Typically measurements are performed with a set of resolution targets (e.g. USAF 1951 target) manufactured with different contrast values from 50% down to less than 1%. For a given illumination level the achievable spatial resolution is then measured for each target. The resulting curve is showing the minimum contrast that is necessary to resolve the structure of a target as a function of spatial frequency. To perform MRC measurements for SWIR cameras at first the irradiation parameters have to be given in radiometric instead of photometric units which are limited in their use to the visible range. In order to do so, SWIR illumination levels for typical daylight and twilight conditions have to be defined. At second, a radiation source is necessary with appropriate emission in the SWIR range (e.g. incandescent lamp) and the irradiance has to be measured in W/m2 instead of Lux = Lumen/m2. At third, the contrast values of the targets have to be calibrated newly for the SWIR range because they typically differ from the values determined for the visual range. Measured MRC values of three cameras are compared to the specified performance data of the devices and the results of a multi-band in-house designed Vis-SWIR camera
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Hayano, Motoshi; Kanoh, Yutaka; Matsumoto, Seiji; Masai, Hisao
2011-01-01
How early- and late-firing origins are selected on eukaryotic chromosomes is largely unknown. Here, we show that Mrc1, a conserved factor required for stabilization of stalled replication forks, selectively binds to the early-firing origins in a manner independent of Cdc45 and Hsk1 kinase in the fission yeast Schizosaccharomyces pombe. In mrc1Δ cells (and in swi1Δ cells to some extent), efficiency of firing is stimulated, and its timing is advanced selectively at those origins that are normally bound by Mrc1. In contrast, the late or inefficient origins which are not bound by Mrc1 are not activated in mrc1Δ cells. The enhanced firing and precocious Cdc45 loading at Mrc1-bound early-firing origins are not observed in a checkpoint mutant of mrc1, suggesting that non-checkpoint function is involved in maintaining the normal program of early-firing origins. We propose that prefiring binding of Mrc1 is an important marker of early-firing origins which are precociously activated by the absence of this protein. PMID:21518960
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Modeling late rectal toxicities based on a parameterized representation of the 3D dose distribution
NASA Astrophysics Data System (ADS)
Buettner, Florian; Gulliford, Sarah L.; Webb, Steve; Partridge, Mike
2011-04-01
Many models exist for predicting toxicities based on dose-volume histograms (DVHs) or dose-surface histograms (DSHs). This approach has several drawbacks as firstly the reduction of the dose distribution to a histogram results in the loss of spatial information and secondly the bins of the histograms are highly correlated with each other. Furthermore, some of the complex nonlinear models proposed in the past lack a direct physical interpretation and the ability to predict probabilities rather than binary outcomes. We propose a parameterized representation of the 3D distribution of the dose to the rectal wall which explicitly includes geometrical information in the form of the eccentricity of the dose distribution as well as its lateral and longitudinal extent. We use a nonlinear kernel-based probabilistic model to predict late rectal toxicity based on the parameterized dose distribution and assessed its predictive power using data from the MRC RT01 trial (ISCTRN 47772397). The endpoints under consideration were rectal bleeding, loose stools, and a global toxicity score. We extract simple rules identifying 3D dose patterns related to a specifically low risk of complication. Normal tissue complication probability (NTCP) models based on parameterized representations of geometrical and volumetric measures resulted in areas under the curve (AUCs) of 0.66, 0.63 and 0.67 for predicting rectal bleeding, loose stools and global toxicity, respectively. In comparison, NTCP models based on standard DVHs performed worse and resulted in AUCs of 0.59 for all three endpoints. In conclusion, we have presented low-dimensional, interpretable and nonlinear NTCP models based on the parameterized representation of the dose to the rectal wall. These models had a higher predictive power than models based on standard DVHs and their low dimensionality allowed for the identification of 3D dose patterns related to a low risk of complication.
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The MRC dyspnoea scale by telephone interview to monitor health status in elderly COPD patients.
Paladini, Luciana; Hodder, Rick; Cecchini, Isabella; Bellia, Vincenzo; Incalzi, Raffaele Antonelli
2010-07-01
Dyspnoea is the most common symptom associated with poor quality of life in patients affected by Chronic Obstructive Pulmonary Disease (COPD). While COPD severity is commonly staged by lung function, the Medical Research Council (MRC) dyspnoea scale has been proposed as a more clinically meaningful method of quantifying disease severity in COPD. We wished to assess whether this scale might also be useful during telephone surveys as a simple surrogate marker of perceived health status in elderly patients with COPD. We conducted a comprehensive health status assessment by telephone survey of 200 elderly patients who had a physician diagnosis of COPD. The telephone survey contained 71 items and explored such domains as educational level, financial status, living arrangements and social contacts, co-morbid illness, and the severity and the impact of COPD on health status. Patients were categorized according to the reported MRC score: mild dyspnoea (MRC scale of 1), moderate dyspnoea (MRC scale of 2 and 3), or severe dyspnoea (MRC of 4 and 5). Deterioration in most of the recorded indicators of health status correlated with an increasingly severe MRC score. This was most evident for instrumental activities of daily living (IADL), perceived health and emotional status, pain-related limitations, limitations in social life, hospital admissions in preceding year and prevalence of most co-morbidities. The MRC dyspnoea scale is a reliable index of disease severity and health status in elderly COPD patients which should prove useful for remote monitoring of COPD and for rating health status for epidemiological purposes.
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The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence
2014-01-01
Background The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. Findings This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. Conclusions In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle. PMID:25379053
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The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence.
Uzunova, Sonya Dimitrova; Zarkov, Alexander Stefanov; Ivanova, Anna Marianova; Stoynov, Stoyno Stefanov; Nedelcheva-Veleva, Marina Nedelcheva
2014-01-01
The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle.
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Alirezaie, Behnam; Taqavian, Mohammad; Aghaiypour, Khosrow; Esna-Ashari, Fatemeh; Shafyi, Abbas
2011-05-01
The cell substrate has a pivotal role in live virus vaccines production. It is necessary to evaluate the effects of the cell substrate on the properties of the propagated viruses, especially in the case of viruses which are unstable genetically such as polioviruses, by monitoring the molecular and phenotypical characteristics of harvested viruses. To investigate the presence/absence of mutation(s), the near full-length genomic sequence of different harvests of the type 3 Sabin strain of poliovirus propagated in MRC-5 cells were determined. The sequences were compared with genomic sequences of different virus seeds, vaccines, and OPV-like isolates. Nearly complete genomic sequencing results, however, revealed no detectable mutations throughout the genome RNA-plaque purified (RSO)-derived monopool of type 3 OPVs manufactured in MRC-5. Thirty-six years of experience in OPV production, trend analysis, and vaccine surveillance also suggest that: (i) different monopools of serotype 3 OPV produced in MRC-5 retained their phenotypic characteristics (temperature sensitivity and neuroattenuation), (ii) MRC-5 cells support the production of acceptable virus yields, (iii) OPV replicated in the MRC-5 cell substrate is a highly efficient and safe vaccine. These results confirm previous reports that MRC-5 is a desirable cell substrate for the production of OPV. Copyright © 2011 Wiley-Liss, Inc.
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Watts, Thomas D; Johanesen, Priscilla A; Lyras, Dena; Rood, Julian I; Adams, Vicki
2017-05-01
Clostridium perfringens produces an extensive repertoire of toxins and extracellular enzymes, many of which are intimately involved in the progression of disease and are encoded by genes on conjugative plasmids. In addition, many C. perfringens strains can carry up to five of these conjugative toxin or antimicrobial resistance plasmids, each of which has a similar 35kb backbone. This conserved backbone includes the tcp conjugation locus and the central control region (CCR), which encodes genes involved in plasmid regulation, replication and partitioning, including a parMRC partitioning locus. Most conjugative plasmids in C. perfringens have a conserved replication protein, raising questions as to how multiple, closely related plasmids are maintained within a single strain. Bioinformatics analysis has highlighted the presence of at least 10 different parMRC partitioning system families (parMRC A-J ) in these plasmids, with differences in amino acid sequence identity between each ParM family ranging from 15% to 54%. No two plasmids that encode genes belonging to the same partitioning family have been observed in a single strain, suggesting that these families represent the basis for plasmid incompatibility. In an attempt to validate the proposed parMRC incompatibility groups, genetically marked C. perfringens plasmids encoding identical parMRC C or parMRC D homologues or different combinations of parMRC A , parMRC C and parMRC D family homologues were introduced into a single strain via conjugation. The stability of each plasmid was determined using an incompatibility assay in which the plasmid profile of each strain was monitored over the course of two days in the absence of direct selection. The results showed that plasmids with identical parMRC C or parMRC D homologues were incompatible and could not coexist in the absence of external selection. By contrast, plasmids that encoded different parMRC homologues were compatible and could coexist in the same cell in the
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de Oña, M; Melón, S; de la Iglesia, P; Hidalgo, F; Verdugo, A F
1995-01-01
Ninety-four pharyngeal swab samples corresponding to 94 patients with suspected influenza virus infection were inoculated in Madin-Darby canine kidney (MDCK) cells, the conventional cell system for the isolation of influenza virus, and in fibroblastic human embryo lung (MRC-5) cells, a cell system less commonly used for this purpose but one frequently used in clinical virology laboratories. Both cell preparations were treated with trypsin. Influenza virus was recovered from 15% of the samples inoculated in MDCK cells and from 18% of those inoculated in MRC-5 cells. The use of MRC-5 cells can simplify the search for respiratory viruses and would assist in the rapid detection of influenza virus during new epidemics. PMID:7665680
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Advanced manufacturing rules check (MRC) for fully automated assessment of complex reticle designs
NASA Astrophysics Data System (ADS)
Gladhill, R.; Aguilar, D.; Buck, P. D.; Dawkins, D.; Nolke, S.; Riddick, J.; Straub, J. A.
2005-11-01
Advanced electronic design automation (EDA) tools, with their simulation, modeling, design rule checking, and optical proximity correction capabilities, have facilitated the improvement of first pass wafer yields. While the data produced by these tools may have been processed for optimal wafer manufacturing, it is possible for the same data to be far from ideal for photomask manufacturing, particularly at lithography and inspection stages, resulting in production delays and increased costs. The same EDA tools used to produce the data can be used to detect potential problems for photomask manufacturing in the data. A production implementation of automated photomask manufacturing rule checking (MRC) is presented and discussed for various photomask lithography and inspection lines. This paper will focus on identifying data which may cause production delays at the mask inspection stage. It will be shown how photomask MRC can be used to discover data related problems prior to inspection, separating jobs which are likely to have problems at inspection from those which are not. Photomask MRC can also be used to identify geometries requiring adjustment of inspection parameters for optimal inspection, and to assist with any special handling or change of routing requirements. With this foreknowledge, steps can be taken to avoid production delays that increase manufacturing costs. Finally, the data flow implemented for MRC can be used as a platform for other photomask data preparation tasks.
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The Falcon and the Trident: Air Force-Navy Airpower Coordination and the New MRC Model
1994-06-01
other ships from Australia and New Zealand , quickly placed themselves at NavFE’s disposal. At the same time, MacArthur received orders from the JCS...THE FALCON AND THE TRIDENT: AIR FORCE-NAVY AIRPOWER COORDINATION AND THE NEW MRC MODEL MARK S. HOFFMAN, MAJ, USAF...TITLE AND SUBTITLE The Falcon and The Trident: Air Force-Navy Airpower Coordination and The New MRC Model 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c
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Nagano, Yasuhiko; Matsuo, Kenichi; Gorai, Katsuya; Sugimori, Kazuya; Kunisaki, Chikara; Ike, Hideyuki; Tanaka, Katsuaki; Imada, Toshio; Shimada, Hiroshi
2006-01-01
We present a case of a 72-year-old man with a common bile duct cancer, who was initially believed to have multiple liver metastases based on computed tomography findings, and in whom magnetic resonance cholangiography (MRC) revealed a diagnosis of bile duct hamartomas. At exploration for pancreaticoduodenectomy, liver palpation revealed disseminated nodules at the surface of the liver. These nodules showed gray-white nodular lesions of about 0.5 cm in diameter scattered on the surface of both liver lobes, which were looked like multiple liver metastases from bile duct cancer. Frozen section of the liver biopsy disclosed multiple bile ducts with slightly dilated lumens embedded in the collagenous stroma characteristics of multiple bile duct hamartomas (BDHs). Only two reports have described the MRC features of bile duct hamartomas. Of all imaging procedures, MRC provides the most relevant features for the imaging diagnosis of bile duct hamartomas. PMID:16534895
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Role of protein kinase C-η in cigarette smoke extract-induced apoptosis in MRC-5-cells.
Son, E S; Kyung, S Y; Lee, S P; Jeong, S H; Shin, J Y; Ohba, M; Yeo, E J; Park, J W
2015-09-01
Cigarette smoke (CS) is a major risk factor for emphysema, which causes cell death in structural cells of the lung by mechanisms that are still not completely understood. We demonstrated previously that CS extract (CSE) induces caspase activation in MRC-5 human lung fibroblasts, activated protein kinase C-η (PKC-η), and translocated PKC-η from the cytosol to the membrane. The objective of this study was to investigate the involvement of PKC-η activation in a CSE-induced extrinsic apoptotic pathway. We determined that CSE increases expression of caspase 3 and 8 cleavage in MRC-5 cells and overexpression of PKC-η significantly increased expression of caspase 3 and 8 cleavage compared with control LacZ-infected cells. In contrast, dominant negative (dn) PKC-η inhibited apoptosis in MRC-5 cells exposed to CSE and decreased expression of caspase 3 and 8 compared with control cells. Exposure to 10% CSE for >8 h significantly increased lactate dehydrogenase release in PKC-η-infected cells compared with LacZ-infected cells. Additionally, PKC-η-infected cells had an increased number of Hoechst 33342 stained nuclei compared with LacZ-infected cells, while dn PKC-η-infected cells exhibited fewer morphological changes than LacZ-infected cells under phase-contrast microscopy. In conclusion, PKC-η activation plays a pro-apoptotic role in CSE-induced extrinsic apoptotic pathway in MRC-5 cells. These results suggest that modulation of PKC-η may be a useful tool for regulating the extrinsic apoptosis of MRC-5 cells by CSE and may have therapeutic potential in the treatment of CS-induced lung injury. © The Author(s) 2014.
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Melnik, Andre; Wilson-Zbinden, Caroline; Schellhaas, René; Kastner, Lisa; Piwko, Wojciech; Dees, Martina; Picotti, Paola; Maric, Marija; Labib, Karim; Luke, Brian; Peter, Matthias
2016-01-01
Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1’s replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks. PMID:26849847
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ROS-dependent HMGA2 upregulation mediates Cd-induced proliferation in MRC-5 cells.
Xie, Huaying; Wang, Jiayue; Jiang, Liping; Geng, Chengyan; Li, Qiujuan; Mei, Dan; Zhao, Lian; Cao, Jun
2016-08-01
Cadmium (Cd) is a heavy metal widely found in a number of environmental matrices, and the exposure to Cd is increasing nowadays. In this study, the role of high mobility group A2 (HMGA2) in Cd-induced proliferation was investigated in MRC-5 cells. Exposure to Cd (2μM) for 48h significantly enhanced the growth of MRC-5 cells, increased reactive oxygen species (ROS) production, and induced both mRNA and protein expression of HMGA2. Evidence for Cd-induced reduction of the number of G0/G1 phase cells and an increase in the number of cells in S phase and G2/M phase was sought by flow cytometric analysis. Western blot analysis showed that cyclin D1, cyclin B1, and cyclin E were upregulated in Cd-treated cells. Further study revealed that N-acetyl cysteine (NAC) markedly prevented Cd-induced proliferation of MRC-5 cells, ROS generation, and the increasing protein level of HMGA2. Silencing of HMGA2 gene by siRNA blocked Cd-induced cyclin D1, cyclin B1, and cyclin E expression and reduction of the number of G0/G1 phase cells. Combining, our data showed that Cd-induced ROS formation provoked HMGA2 upregulation, caused cell cycle changes, and led to cell proliferation. This suggests that HMGA2 might be an important biomarker in Cd-induced cell proliferation. Copyright © 2016 Elsevier Ltd. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Bowen, S; Lee, E; Miyaoka, R
Purpose: NSCLC patient RT is planned without consideration of spatial heterogeneity in lung function or tumor response, which may have contributed to failed uniform dose escalation in a randomized trial. The feasibility of functional lung avoidance and response-adaptive escalation (FLARE) RT to reduce dose to [{sup 99m}Tc]MAA-SPECT/CT perfused lung while redistributing 74Gy within [{sup 18}F]FDG-PET/CT biological target volumes was assessed. Methods: Eight Stage IIB–IIIB NSCLC patients underwent FDG-PET/CT and MAA-SPECT/CT treatment planning scans. Perfused lung objectives were derived from scatter/collimator/attenuation-corrected MAA-SPECT uptake relative to ITV-subtracted lung to maintain <20Gy mean lung dose (MLD). Prescriptions included 60Gy to PTV and concomitantmore » boost of 74Gy mean to biological target volumes (BTV=GTV+PET margin) scaled to each BTV voxel by relative FDG-PET SUV. Dose-painting-by-numbers prescriptions were integrated into commercial TPS via previously reported ROI discretization. Dose constraints for lung, heart, cord, and esophagus were defined. FLARE RT plans were optimized with VMAT, proton pencil beam scanning (PBS) with 3%-3mm robust optimization, and combination PBS (avoidance) plus VMAT (escalation). Dosimetric differences were evaluated by Friedman non-parametric paired test with multiple sampling correction. Results: PTV and normal tissue objectives were not violated in 24 FLARE RT plans. Population median of mean BTV dose was 73.7Gy (68.5–75.5Gy), mean FDG-PET peak dose was 89.7Gy (73.5–103Gy), MLD was 12.3Gy (7.5–19.6Gy), and perfused MLD was 4.8Gy (0.9–12.1Gy). VMAT achieved higher dose to the FDG-PET peak subvolume (p=0.01), while PBS delivered lower dose to lung (p<0.001). Voxelwise linear correlation between BTV dose and FDG-PET uptake was higher for VMAT (R=0.93) and PBS+VMAT (R=0.94) compared to PBS alone (R=0.89). Conclusion: FLARE RT is feasible with VMAT and PBS. A combination of PBS for functional lung
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Brundage, Michael; Sydes, Matthew R.; Parulekar, Wendy R.; Warde, Padraig; Cowan, Richard; Bezjak, Andrea; Kirkbride, Peter; Parliament, Matthew; Moynihan, Clare; Bahary, Jean-Paul; Parmar, Mahesh K.B.; Sanders, Karen; Chen, Bingshu E.; Mason, Malcolm D.
2015-01-01
Purpose The NCIC CTG PR3/MRC PR07 randomized phase III trial compared androgen-deprivation therapy (ADT) alone versus ADT with radiotherapy (RT) for patients with locally advanced prostate cancer. This article reports the health-related quality-of-life (HRQOL) outcomes of this trial. Patients and Methods A total of 1,205 patients were randomly allocated to either ADT alone or ADT with RT. HRQOL was assessed at baseline and every 6 months thereafter using the European Organisation for Research and Treatment of Cancer Core Questionnaire and a prostate cancer–specific checklist or the Functional Assessment of Cancer Therapy–Prostate questionnaire. Mean changes from baseline scores for five function domains and nine symptom domains were analyzed as those most relevant to ADT and RT. The proportions of patients with improved, stable, or worsened HRQOL scores according to instrument-specific minimal important differences were calculated. Results Baseline questionnaires were completed by 1,028 patients (88%). At 6 months, RT had a statistically significant impact on mean score for bowel symptoms (P = .02), diarrhea (P < .001), urinary function (P = .003), and erectile dysfunction (P = .008); by 3 years, however, there were no significant between-group differences in any domain. Generalized linear mixed modeling revealed no significant between-arm differences in any of the function scales but showed significant deterioration in both arms over time for Functional Assessment of Cancer Therapy–Prostate total score, treatment outcome index, and physical and functional well-being. Conclusion The addition of RT to ADT for patients with locally advanced prostate cancer significantly improved overall survival and had only modest and transient negative impact on relevant domains of HRQOL. PMID:26014295
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A design of an interface board between a MRC thermistor probe and a personal computer.
DOT National Transportation Integrated Search
2013-09-01
The main purpose of this project was to design and build a prototype of an interface board between an MRC temperature probe : (thermistor array) and a personal laptop computer. This interface board replaces and significantly improve the capabilities ...
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NASA Astrophysics Data System (ADS)
Sa'adiyah, Devy; Bangga, Galih; Widodo, Wawan; Ikhwan, Nur
2017-08-01
Tangential fired boiler is one of the methods that can produce more complete combustion. This method applied in Suralaya Power Plant, Indonesia. However, the boiler where supposed to use low rank coal (LRC), but at a given time must be mixed with medium rank coal (MRC) from another unit because of lack of LRC coal. Accordingly to the situation, the study about choosing the right position of LRC and MRC in the burner elevation must be investigated. The composition of coal is 70%LRC / 30%MRC where MRC will be placed at the lower (A & C - Case I)) or higher (E & G - Case II) elevation as the cases in this study. The study is carried out using Computational Fluid Dynamics (CFD) method. The simulation with original case (100%LRC) has a good agreement with the measurement data. As the results, MRC is more recommended at the burner elevation A & C rather than burner elevation E & G because it has closer temperature (880 K) compared with 100%LRC and has smaller local heating area between upper side wall and front wall with the range of temperature 1900 - 2000 K. For emissions, case I has smaller NOx and higher CO2 with 104 ppm and 15,6%. Moreover, it has samller O2 residue with 5,8% due to more complete combustion.
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Outage Probability of MRC for κ-μ Shadowed Fading Channels under Co-Channel Interference.
Chen, Changfang; Shu, Minglei; Wang, Yinglong; Yang, Ming; Zhang, Chongqing
2016-01-01
In this paper, exact closed-form expressions are derived for the outage probability (OP) of the maximal ratio combining (MRC) scheme in the κ-μ shadowed fading channels, in which both the independent and correlated shadowing components are considered. The scenario assumes the received desired signals are corrupted by the independent Rayleigh-faded co-channel interference (CCI) and background white Gaussian noise. To this end, first, the probability density function (PDF) of the κ-μ shadowed fading distribution is obtained in the form of a power series. Then the incomplete generalized moment-generating function (IG-MGF) of the received signal-to-interference-plus-noise ratio (SINR) is derived in the closed form. By using the IG-MGF results, closed-form expressions for the OP of MRC scheme are obtained over the κ-μ shadowed fading channels. Simulation results are included to validate the correctness of the analytical derivations. These new statistical results can be applied to the modeling and analysis of several wireless communication systems, such as body centric communications.
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Outage Probability of MRC for κ-μ Shadowed Fading Channels under Co-Channel Interference
Chen, Changfang; Shu, Minglei; Wang, Yinglong; Yang, Ming; Zhang, Chongqing
2016-01-01
In this paper, exact closed-form expressions are derived for the outage probability (OP) of the maximal ratio combining (MRC) scheme in the κ-μ shadowed fading channels, in which both the independent and correlated shadowing components are considered. The scenario assumes the received desired signals are corrupted by the independent Rayleigh-faded co-channel interference (CCI) and background white Gaussian noise. To this end, first, the probability density function (PDF) of the κ-μ shadowed fading distribution is obtained in the form of a power series. Then the incomplete generalized moment-generating function (IG-MGF) of the received signal-to-interference-plus-noise ratio (SINR) is derived in the closed form. By using the IG-MGF results, closed-form expressions for the OP of MRC scheme are obtained over the κ-μ shadowed fading channels. Simulation results are included to validate the correctness of the analytical derivations. These new statistical results can be applied to the modeling and analysis of several wireless communication systems, such as body centric communications. PMID:27851817
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Validation of the RT3 triaxial accelerometer for the assessment of physical activity.
Rowlands, Ann V; Thomas, Philip W M; Eston, Roger G; Topping, Rodney
2004-03-01
The aims of this study were to assess and compare the validity of the RT3 accelerometer for the assessment of physical activity in boys and men, to compare RT3 and Tritrac accelerometer counts, and to determine count cut-off values for moderate (> or =3 < 6 METs) and vigorous (> or =6 METs) activity. Nineteen boys (age: 9.5 +/- 0.8 yr) and 15 men (age: 20.7 +/- 1.4 yr) walked and ran on a treadmill, kicked a ball to and fro, played hopscotch, and sat quietly. An RT3 was worn on the right hip; boys also wore a Tritrac on the left hip. Oxygen consumption was expressed as a ratio of body mass raised to the power of 0.75 (S VO2). RT3 counts correlated significantly with S VO2 in boys (r = 0.87, P < 0.01) and men (r = 0.85, P < 0.01). However, during treadmill activities, RT3 counts were significantly higher for boys (P < 0.05). RT3 counts corresponding to "moderate" and "vigorous" activity were similar for boys and men for all activities (moderate = 970.2 for boys and 984.0 for men; vigorous = 2333.0 for boys and 2340.8 for men) but approximately 400 counts lower in men when only treadmill activities were considered. Tritrac counts correlated significantly with S VO2 in boys (r = 0.87, P < 0.01), but were significantly lower than RT3 counts across most activities (P < 0.05). The RT3 accelerometer is a good measure of physical activity for boys and men. However, moderate and vigorous intensity count thresholds differ for boys and men when the predominant activities are walking and running. RT3 counts are significantly higher than Tritrac counts for a number of activities. These findings have implications when comparing activity counts between studies using the different instruments.
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Response of Fibroblasts MRC-5 to Flufenamic Acid-Grafted MCM-41 Nanoparticles.
Lara, Giovanna Gomes; Cipreste, Marcelo Fernandes; Andrade, Gracielle Ferreira; Silva, Wellington Marcos da; Sousa, Edésia Martins Barros de
2018-01-09
Recently, flufenamic acid (FFA) was discovered among fenamates as a free radical scavenger and gap junction blocker; however, its effects have only been studied in cancer cells. Normal cells in the surroundings of a tumor also respond to radiation, although they are not hit by it directly. This phenomenon is known as the bystander effect, where response molecules pass from tumor cells to normal ones, through communication channels called gap junctions. The use of the enhanced permeability and retention effect, through which drug-loaded nanoparticles smaller than 200 nm may accumulate around a tumor, can prevent the local side effect upon controlled release of the drug. The present work, aimed at functionalizing MCM-41 (Mobil Composition of Matter No. 41) silica nanoparticles with FFA and determining its biocompatibility with human fibroblasts MRC-5 (Medical Research Council cell strain 5). MCM-41, was synthesized and characterized structurally and chemically, with multiple techniques. The biocompatibility assay was performed by Live/Dead technique, with calcein and propidium-iodide. MRC-5 cells were treated with FFA-grafted MCM-41 for 48 h, and 98% of cells remained viable, without signs of necrosis or morphological changes. The results show the feasibility of MCM-41 functionalization with FFA, and its potential protection of normal cells, in comparison to the role of FFA in cancerous ones.
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Response of Fibroblasts MRC-5 to Flufenamic Acid-Grafted MCM-41 Nanoparticles
Lara, Giovanna Gomes; Andrade, Gracielle Ferreira; da Silva, Wellington Marcos
2018-01-01
Recently, flufenamic acid (FFA) was discovered among fenamates as a free radical scavenger and gap junction blocker; however, its effects have only been studied in cancer cells. Normal cells in the surroundings of a tumor also respond to radiation, although they are not hit by it directly. This phenomenon is known as the bystander effect, where response molecules pass from tumor cells to normal ones, through communication channels called gap junctions. The use of the enhanced permeability and retention effect, through which drug-loaded nanoparticles smaller than 200 nm may accumulate around a tumor, can prevent the local side effect upon controlled release of the drug. The present work, aimed at functionalizing MCM-41 (Mobil Composition of Matter No. 41) silica nanoparticles with FFA and determining its biocompatibility with human fibroblasts MRC-5 (Medical Research Council cell strain 5). MCM-41, was synthesized and characterized structurally and chemically, with multiple techniques. The biocompatibility assay was performed by Live/Dead technique, with calcein and propidium–iodide. MRC-5 cells were treated with FFA-grafted MCM-41 for 48 h, and 98% of cells remained viable, without signs of necrosis or morphological changes. The results show the feasibility of MCM-41 functionalization with FFA, and its potential protection of normal cells, in comparison to the role of FFA in cancerous ones. PMID:29315235
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Huddart, Robert A; Gabe, Rhian; Cafferty, Fay H; Pollock, Philip; White, Jeff D; Shamash, Jonathan; Cullen, Michael H; Stenning, Sally P
2015-03-01
are promising but require confirmation in an international phase 3 trial. In this study we tested a new, more intensive way to deliver a combination of drugs often used to treat men with testicular cancer. We found that response rates were higher but that the CBOP/BEP regimen caused more short-term toxicity. Because most patients are diagnosed when their cancer is less advanced, it took twice as long to complete the trial as expected. Although we plan to carry out a larger trial, we will need international collaboration. ISRCTN53643604; http://www.controlled-trials.com/ISRCTN53643604. Copyright © 2014 European Association of Urology. Published by Elsevier B.V. All rights reserved.
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Protein oxidation and degradation during proliferative senescence of human MRC-5 fibroblasts.
Sitte, N; Merker, K; von Zglinicki, T; Grune, T
2000-03-01
One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The aging process on a cellular level can be treated either as the ongoing proliferation until a certain number of cell divisions is reached (the Hayflick limit) or as the aging of nondividing cells, that is, the age-related changes in cells without proliferation. The present investigation was undertaken to reveal the changes in protein turnover, proteasome activity, and protein oxidation status during proliferative senescence. We were able to demonstrate that the activity of the cytosolic proteasomal system declines dramatically during the proliferative senescence of human MRC-5 fibroblasts. Regardless of the loss in activity, it could be demonstrated that there are no changes in the transcription and translation of proteasomal subunits. This decline in proteasome activity was accompanied by an increased concentration of oxidized proteins. Cells at higher proliferation stages were no longer able to respond with increased degradation of endogenous [(35)S]-Met-radiolabeled proteins after hydrogen peroxide- or quinone-induced oxidative stress. It could be demonstrated that oxidized proteins in senescent human MRC-5 fibroblasts are not as quickly removed as they are in young cells. Therefore, our study demonstrates that the accumulation of oxidized proteins and decline in protein turnover and activity of the proteasomal system are not only a process of postmitotic aging but also occur during proliferative senescence and result in an increased half-life of oxidized proteins.
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TH-E-202-01: Pitfalls and Remedies in PET/CT Imaging for RT Planning
DOE Office of Scientific and Technical Information (OSTI.GOV)
Pan, T.
2016-06-15
PET/CT is a very important imaging tool in the management of oncology patients. PET/CT has been applied for treatment planning and response evaluation in radiation therapy. This educational session will discuss: Pitfalls and remedies in PET/CT imaging for RT planning The use of hypoxia PET imaging for radiotherapy PET for tumor response evaluation The first presentation will address the issue of mis-registration between the CT and PET images in the thorax and the abdomen. We will discuss the challenges of respiratory gating and introduce an average CT technique to improve the registration for dose calculation and image-guidance in radiation therapy.more » The second presentation will discuss the use of hypoxia PET Imaging for radiation therapy. We will discuss various hypoxia radiotracers, the choice of clinical acquisition protocol (in particular a single late static acquisition versus a dynamic acquisition), and the compartmental modeling with different transfer rate constants explained. We will demonstrate applications of hypoxia imaging for dose escalation/de-escalation in clinical trials. The last presentation will discuss the use of PET/CT for tumor response evaluation. We will discuss anatomic response assessment vs. metabolic response assessment, visual evaluation and semi-quantitative evaluation, and limitations of current PET/CT assessment. We will summarize clinical trials using PET response in guiding adaptive radiotherapy. Finally, we will summarize recent advancements in PET/CT radiomics and non-FDG PET tracers for response assessment. Learning Objectives: Identify the causes of mis-registration of CT and PET images in PET/CT, and review the strategies to remedy the issue. Understand the basics of PET imaging of tumor hypoxia (radiotracers, how PET measures the hypoxia selective uptake, imaging protocols, applications in chemo-radiation therapy). Understand the basics of dynamic PET imaging, compartmental modeling and parametric images. Understand
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MO-E-BRC-01: Online Adaptive MR-Guided RT: Workflow and Clinical Implementation
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kashani, R.
Online adaptive radiation therapy has the potential to ensure delivery of optimal treatment to the patient by accounting for anatomical and potentially functional changes that occur from one fraction to the next and over the course of treatment. While on-line adaptive RT (ART) has been a topic of many publications, discussions, and research, it has until very recently remained largely a concept and not a practical implementation. However, recent advances in on-table imaging, use of deformable image registration for contour generation and dose tracking, faster and more efficient plan optimization, as well as fast quality assurance method has enabled themore » implementation of ART in the clinic in the past couple of years. The introduction of these tools into routine clinical use requires many considerations and progressive knowledge to understand how processes that have historically taken hours/days to complete can now be done in less than 30 minutes. This session will discuss considerations to perform real time contouring, planning and patient specific QA, as well as a practical workflow and the required resources. Learning Objectives: To understand the difficulties, challenges and available technologies for online adaptive RT. To understand how to implement online adaptive therapy in a clinical environment and to understand the workflow and resources required. To understand the limitations and sources of uncertainty in the online adaptive process I have research funding from ViewRay Inc. and Philips Medical Systems.; R. Kashani, I have research funding from ViewRay Inc. and Philips Medical Systems.; X. Li, Research supported by Elekta Inc.« less
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Isolation and characterization of trichothecin from corn cultures of Fusarium graminearum MRC 1125.
Combrinck, S; Gelderblom, W C; Spies, H S; Burger, B V; Thiel, P G; Marasas, W F
1988-01-01
Trichothecin was isolated and purified from corn cultures of a toxic strain of Fusarium graminearum. This strain, designated MRC 1125, was obtained from corn in southern Africa. The brine shrimp toxicity assay was used throughout the isolation procedure to monitor the toxicity of the fractions. The compound was characterized by detailed 1H (500-MHz) and 13C (125-MHz) nuclear magnetic resonance spectroscopy and mass spectrometry. This is the first report of the production of trichothecin by a Fusarium species. PMID:3415233
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Pavone, Vito; DE Cristo, Claudia; Testa, Gianluca; Canavese, Federico; Lucenti, Ludovico; Sessa, Giuseppe
2018-04-12
Non-operative treatment with immobilization is the gold-standard for paediatric clavicular fractures. Purpose of this study is to evaluate functional outcomes and efficacy of non-operative treatment of clavicular fractures in a succession of 131 children. Between 2006 and 2012, we treated non-surgically 131 children for a clavicular fracture. All fractures have been classified according to Robinson classification. Clavicle shortening, range of movements and muscular strength through the Medical Research Council (MRC) scale were evaluated. To assess the outcomes, QuickDASH questionnaire, dividing the sample in 3 age-related group, was administered. The average follow-up was 26 months (8-84 months). Clavicle shortening at the time of injury occurred in 18 cases. All fractures reached union. Average time to union was 34 days. Mean time return to activity was 12.6 weeks. No cases of nonunion or delayed union were reported. Complications occurred in 21 cases. A shortening persisted in 2 cases. Only one patient had a slight functional restriction. Average QuickDASH score was 6.2±1.1 (range 4.3-9.4). All patients recovered to a MRC score of 5, except for one patient with a score of 4. Best QuickDASH scores were observed in the group aged under 8 years and in non-comminuted and lateral third fractures of the clavicle. Observing results, clavicle fractures have a satisfactory clinical healing as shown by the good scores at QuickDASH and MRC scale. Younger children under 8 years can achieve the best results with a conservative treatment in terms of bone healing and activity level.
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ERIC Educational Resources Information Center
Robins, Jennifer; Antrim, Patricia
2013-01-01
In 2004 the Individuals with Disabilities Education Act authorized funding for Response to Intervention (RtI) instruction in the United States. By 2011, 71 percent of school districts had adopted RtI (Institute of Education Sciences 2011). The goal of RtI is to provide personalized, just-in-time intervention in reading and math for students who…
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Taylor, Lisa M; James, Alexander; Schuller, Christine E; Brce, Jesena; Lock, Richard B; Mackenzie, Karen L
2004-10-15
Recent investigations, including our own, have shown that specific strains of fibroblasts expressing telomerase reverse transcriptase (hTERT) have an extended lifespan, but are not immortal. We previously demonstrated that hTERT-transduced MRC5 fetal lung fibroblasts (MRC5hTERTs) bypassed senescence but eventually succumbed to a second mortality barrier (crisis). In the present study, 67 MRC5hTERT clones were established by limiting dilution of a mass culture. Whereas 39/67 clones had an extended lifespan, all 39 extended lifespan clones underwent crisis. 11 of 39 clones escaped crisis and were immortalized. There was no apparent relationship between the fate of clones at crisis and the level of telomerase activity. Telomeres were hyperextended in the majority of the clones analyzed. There was no difference in telomere length of pre-crisis compared with post-crisis and immortal clones, indicating that hyperextended telomeres were conducive for immortalization and confirming that crisis was independent of telomere length. Immortalization of MRC5hTERT cells was associated with repression of the cyclin-dependent kinase inhibitor p16INK4a and up-regulation of pRB. However, the regulation of pRB phosphorylation and the response of the p53/p21cip1/waf1 pathway were normal in immortal cells subject to genotoxic stress. Overexpression of oncogenic ras failed to de-repress p16INK4a in immortal cells. Furthermore, expression of ras enforced senescent-like growth arrest in p16INK4a-positive, but not p16INK4a-negative MRC5hTERT cells. Immortal cells expressing ras formed small, infrequent colonies in soft agarose, but were non-tumorigenic. Overall, these results implicate the inactivation of p16INK4a as a critical event for overcoming telomere-independent crisis, immortalizing MRC5 fibroblasts and overcoming ras-induced premature senescence.
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Callens, Etienne; Graba, Sémia; Essalhi, Mohamed; Gillet-Juvin, Karine; Chevalier-Bidaud, Brigitte; Chenu, Romain; Mahut, Bruno; Delclaux, Christophe
2014-09-01
The first objective of our study was to assess whether patients diagnosed with cardio-respiratory disorders report overestimation or underestimation on recall (Medical Research Council (MRC) dyspnea scale) of their true functional capacity (walked distance during a 6-minute walk test (6MWT)). The second objective was to assess whether the measurement of breathlessness at the end of a 6MWT (Borg score) may help to identify dyspneic patients on recall. The 6MWTs of 746 patients aged from 40 to 80 years who were diagnosed with either chronic obstructive pulmonary disease (COPD, n = 355), diffuse parenchymal lung disease (n = 140), pulmonary vascular diseases (n = 188) or congestive heart failure (n = 63) were selected from a prospective Clinical Database Warehouse. The percentage of patients who overestimated (MRC ≤ 2 with distance < lower limit of normal (LLN), 61/746, 8%; 95% confidence interval (CI): 6 to 10%) or underestimated (MRC > 2 with distance ≥LLN, 121/746, 16%; 95%CI: 14 to 19%) on recall their capacity was elevated. The overestimation seemed related to self-limitation, while the underestimation seemed related to patients who "work through" their breathing discomfort. These two latter groups of patients were mainly diagnosed with COPD. A Borg dyspnea score >3 (upper limit of normal) at the end of the 6MWT had 84% specificity for the prediction of a MRC score >1. Almost one fourth of patients suffering from cardio-pulmonary disorders overestimate or underestimate on recall their true functional capacity. An elevated Borg dyspnea score at the end of the 6MWT has a good specificity to predict dyspnea on recall.
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Silica Nanoparticles Induce Oxidative Stress and Autophagy but Not Apoptosis in the MRC-5 Cell Line
Petrache Voicu, Sorina Nicoleta; Dinu, Diana; Sima, Cornelia; Hermenean, Anca; Ardelean, Aurel; Codrici, Elena; Stan, Miruna Silvia; Zărnescu, Otilia; Dinischiotu, Anca
2015-01-01
This study evaluated the in vitro effects of 62.5 µg/mL silica nanoparticles (SiO2 NPs) on MRC-5 human lung fibroblast cells for 24, 48 and 72 h. The nanoparticles’ morphology, composition, and structure were investigated using high resolution transmission electron microscopy, selected area electron diffraction and X-ray diffraction. Our study showed a decreased cell viability and the induction of cellular oxidative stress as evidenced by an increased level of reactive oxygen species (ROS), carbonyl groups, and advanced oxidation protein products after 24, 48, and 72 h, as well as a decreased concentration of glutathione (GSH) and protein sulfhydryl groups. The protein expression of Hsp27, Hsp60, and Hsp90 decreased at all time intervals, while the level of protein Hsp70 remained unchanged during the exposure. Similarly, the expression of p53, MDM2 and Bcl-2 was significantly decreased for all time intervals, while the expression of Bax, a marker for apoptosis, was insignificantly downregulated. These results correlated with the increase of pro-caspase 3 expression. The role of autophagy in cellular response to SiO2 NPs was demonstrated by a fluorescence-labeled method and by an increased level of LC3-II/LC3-I ratio. Taken together, our data suggested that SiO2 NPs induced ROS-mediated autophagy in MRC-5 cells as a possible mechanism of cell survival. PMID:26690408
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Tahk, Hongmin; Lee, Min Hwa; Lee, Kang Bum; Cheon, Doo-Sung; Choi, Changsun
2011-07-01
This study aimed to develop a specific and sensitive duplex reverse transcription polymerase chain reaction enzyme-linked immunosorbent assay (duplex RT-PCR-ELISA) for hepatitis A virus (HAV) and hepatitis E virus (HEV). Duplex RT-PCR-ELISA could detect and differentiate HAV and HEV with specific probes. When ELISA technique was used to detect probe-bound RT-PCR products, duplex RT-PCR-ELISA could detect as little as 0.1 ng/μL HAV and HEV from clinical samples. Human norovirus, enterovirus, poliovirus, murine norovirus and feline calicivirus were used for the specificity test; all were negative. Therefore duplex RT-PCR-ELISA can be used for the simultaneous detection of HAV and HEV in contaminated fecal samples. Copyright © 2011 Elsevier B.V. All rights reserved.
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MZC Gel Inhibits SHIV-RT and HSV-2 in Macaque Vaginal Mucosa and SHIV-RT in Rectal Mucosa.
Calenda, Giulia; Villegas, Guillermo; Barnable, Patrick; Litterst, Claudia; Levendosky, Keith; Gettie, Agegnehu; Cooney, Michael L; Blanchard, James; Fernández-Romero, José A; Zydowsky, Thomas M; Teleshova, Natalia
2017-03-01
The Population Council's microbicide gel MZC (also known as PC-1005) containing MIV-150 and zinc acetate dihydrate (ZA) in carrageenan (CG) has shown promise as a broad-spectrum microbicide against HIV, herpes simplex virus (HSV), and human papillomavirus. Previous data show antiviral activity against these viruses in cell-based assays, prevention of vaginal and rectal simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) infection, and reduction of vaginal HSV shedding in rhesus macaques and also excellent antiviral activity against HSV and human papillomavirus in murine models. Recently, we demonstrated that MZC is safe and effective against SHIV-RT in macaque vaginal explants. Here we established models of ex vivo SHIV-RT/HSV-2 coinfection of vaginal mucosa and SHIV-RT infection of rectal mucosa in macaques (challenge of rectal mucosa with HSV-2 did not result in reproducible tissue infection), evaluated antiviral activity of MZC, and compared quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay readouts for monitoring SHIV-RT infection. MZC (at nontoxic dilutions) significantly inhibited SHIV-RT in vaginal and rectal mucosas and HSV-2 in vaginal mucosa when present during viral challenge. Analysis of SHIV-RT infection and MZC activity by 1-step simian immunodeficiency virus gag quantitative RT-PCR and p27 enzyme-linked immunosorbent assay demonstrated similar virus growth dynamics and MZC activity by both methods and higher sensitivity of quantitative RT-PCR. Our data provide more evidence that MZC is a promising dual compartment multipurpose prevention technology candidate.
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Implementing RtI with Gifted Students
ERIC Educational Resources Information Center
Coleman, Mary Ruth, Ed.; Johnsen, Susan K., Ed.
2012-01-01
"Implementing RtI With Gifted Students" shares how RtI can fit within the framework of gifted education programming models. This edited book will serve as a reference guide for those interested in learning more about RtI and how it might be effectively implemented to meet the needs of all gifted students. Chapters contributed by top gifted…
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Waggoner, Jesse J; Sahadeo, Nikita S D; Brown, Arianne; Mohamed-Hadley, Alisha; Hadley, Dexter; Carrington, Leslie; Carrington, Christine V F; Pinsky, Benjamin A
2015-02-01
Dengue virus (DENV) transmission occurs throughout the Caribbean, though laboratory confirmation and epidemiologic surveillance are limited by the availability of serotype-specific molecular diagnostics. In this study, we show that a serotype-specific DENV multiplex, real-time reverse transcriptase-PCR (RT-PCR) detected DENV RNA in significantly more samples (82/182) than a reference hemi-nested RT-PCR (57/182; P=0.01). Copyright © 2015 Elsevier Inc. All rights reserved.
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Effect of Low Frequency Ultrasound on Combined rt-PA and Eptifibatide Thrombolysis in Human Clots
Meunier, Jason M.; Holland, Christy K.; Pancioli, Arthur M.; Lindsell, Christopher J.; Shaw, George J.
2009-01-01
Introduction Fibrinolytics such as recombinant tissue plasminogen activator (rt-PA) are used to treat thrombotic disease such as acute myocardial infarction (AMI) and ischemic stroke. Interest in increasing efficacy and reducing side effects has led to the study of adjuncts such as GP IIb-IIIa inhibitors and ultrasound (US) enhanced thrombolysis. Currently, GP IIb-IIIa inhibitor and fibrinolytic treatment are often used in AMI, and are under investigation for stroke treatment. However, little is known of the efficacy of combined GP IIb-IIIa inhibitor, fibrinolytic and ultrasound treatment. We measure the lytic efficacy of rt-PA, eptifibatide (Epf) and 120 kHz ultrasound treatment in an in-vitro human clot model. Materials and Methods Blood was drawn from 15 subjects after IRB approval. Clots were made in 20 μL pipettes, and placed in a water tank for microscopic visualization during lytic treatment. Clots were exposed to control, rt-PA (rt-PA), eptifibatide (Epf), or rt-PA+eptifibatide (rt-PA+Epf), with or without ultrasound for 30 minutes at 37°C in human plasma. Clot lysis was measured over time, using a microscopic imaging technique. The fractional clot loss (FCL) and initial lytic rate (LR) were used to quantify lytic efficacy. Results and Conclusions LR values for (−US) treated clots were 0.8±0.1(control), 1.8±0.3 (Epf), 1.5±0.2 (rt-PA), and 1.3±0.4 (rt-PA+Epf) (% clot width/minute) respectively. In comparison, the (+US) group exhibited LR values of 1.6±0.2 (control), 4.3±0.4 (Epf), 6.3±0.4 (rt-PA), and 4.6±0.6 (rt-PA+Epf). For (−US) treated clots, FCL was 6.0±0.8 (control), 9.2±2.5 (Epf), 15.6±1.7 (rt-PA), and 28.0±2.2% (rt-PA+Epf) respectively. FCL for (+US) clots was 13.5±2.4 (control), 20.7±6.4 (Epf), 44.4±3.6 (rt-PA) and 30.3±3.6% (rt-PA+Epf) respectively. Although the addition of eptifibatide enhances the in-vitro lytic efficacy of rt-PA in the absence of ultrasound, the efficacy of ultrasound and rt-PA is greater than that of
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PALATAL DYSMORPHOGENESIS: QUANTITATIVE RT-PCR
ABSTRACT
Palatal Dysmorphogenesis : Quantitative RT-PCR
Gary A. Held and Barbara D. Abbott
Reverse transcription PCR (RT-PCR) is a very sensitive method for detecting mRNA in tissue samples. However, as it is usually performed it is does not yield quantitativ... -
NASA Astrophysics Data System (ADS)
Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen
2008-10-01
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.
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Gilbert, R E; Pike, K; Kenyon, S L; Tarnow-Mordi, W; Taylor, D J
2005-06-01
We analysed the type of bacteraemia before discharge from Neonatal Intensive Care Units in babies born to women randomised to the MRC ORACLE Trials. There was no evidence for an effect of oral antibiotics given prior to delivery on bacteraemia due to gram negative or enterococci bacteria, but Group B streptococcal (GBS) bacteraemia was significantly reduced in women with preterm prelabour rupture of the membranes (1.58% to 0.55%, relative risk 0.34; 95% CI: 0.17-0.70). There was no detectable effect in women in spontaneous preterm labour with intact membranes as the risk of GBS bacteraemia in their babies was very small regardless of treatment.
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Morgan, Gareth J; Gregory, Walter M; Davies, Faith E; Bell, Sue E; Szubert, Alexander J; Brown, Julia M; Coy, Nuria N; Cook, Gordon; Russell, Nigel H; Rudin, Claudius; Roddie, Huw; Drayson, Mark T; Owen, Roger G; Ross, Fiona M; Jackson, Graham H; Child, J Anthony
2012-01-05
Thalidomide maintenance has the potential to modulate residual multiple myeloma (MM) after an initial response. This trial compared the effect of thalidomide maintenance and no maintenance on progression-free survival (PFS) and overall survival (OS) in MM patients. After intensive or nonintensive induction therapy, 820 newly diagnosed MM patients were randomized to open-label thalidomide maintenance until progression, or no maintenance. Interphase FISH (iFISH) analysis was performed at study entry. Median PFS was significantly longer with thalidomide maintenance (log-rank P < .001). Median OS was similar between regimens (log-rank P = .40). Patients with favorable iFISH showed improved PFS (P = .004) and a trend toward a late survival benefit. Patients with adverse iFISH receiving thalidomide showed no significant PFS benefit and worse OS (P = .009). Effective relapse therapy enhanced survival after progression, translating into a significant OS benefit. Meta-analysis of this and other studies show a significant late OS benefit (P < .001, 7-year difference hazard ratio = 12.3; 95% confidence interval, 5.5-19.0). Thalidomide maintenance significantly improves PFS and can be associated with improved OS. iFISH testing is important in assessing the clinical impact of maintenance therapy. Overview analysis demonstrated that thalidomide maintenance was associated with a significant late OS benefit. This trial was registered at www.isrctn.org as #ISRCTN68454111.
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Anderson, Emma L; Caleyachetty, Rishi; Stafford, Mai; Kuh, Diana; Hardy, Rebecca; Lawlor, Debbie A; Fraser, Abigail; Howe, Laura D
2017-09-07
Studies assessing associations of childhood psychosocial adversity (e.g. sexual abuse, physical neglect, parental death), as opposed to socioeconomic adversity, with cardiovascular disease (CVD) risk factors in adulthood are scarce. We aimed to assess associations of various forms of psychosocial adversity and cumulative adversity in childhood, with multiple CVD risk factors in mid-life. Participants were from the MRC National Survey of Health and Development. Childhood psychosocial risk factors were reported prospectively by parents from 1950-1957, and retrospectively by participants at mean age 43 years in 1989. CVD risk factors were assessed at mean age 60-64 years in 2006-2011. Associations of a summary score of total psychosocial adversity and CVD risk in adulthood were assessed. There was no consistent evidence that cumulative psychosocial adversity, nor any specific form of psychosocial adversity in childhood, was associated with CVD risk factors in late adulthood. There was some evidence that parental death in the first 15 years was associated with higher SBP (Beta: 0.23, 95% confidence interval: 0.06 to 0.40, P=0.01) and DBP (Beta: 0.15, 95% confidence interval: -0.01 to 0.32, P=0.07). We found no evidence that exposure to greater psychosocial adversity, or specific forms of psychosocial adversity during childhood is associated with adult CVD risk factors. Further large population studies are needed to clarify whether parental death is associated with higher systolic and diastolic blood pressure.
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Response to Intervention (RtI) in Secondary Schools: A Comparison of the RtI Service Delivery Model
ERIC Educational Resources Information Center
Epler-Brooks, Pam L.
2011-01-01
This qualitative, collective case study researched how the Response to Intervention (RtI) service delivery model was used within the secondary educational environment in two Ohio schools. Areas researched included the type of professional development used to introduce and sustain RtI, the amount of administrative support, the use of universal…
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NASA'S SERVIR Gulf of Mexico Project: The Gulf of Mexico Regional Collaborative (GoMRC)
NASA Technical Reports Server (NTRS)
Quattrochi, Dale A.; Irwin, Daniel; Presson, Joan; Estes, Maury; Estes, Sue; Judd, Kathleen
2006-01-01
The Gulf of Mexico Regional Collaborative (GoMRC) is a NASA-funded project that has as its goal to develop an integrated, working, prototype IT infrastructure for Earth science data, knowledge and models for the five Gulf U.S. states and Mexico, and to demonstrate its ability to help decision-makers better understand critical Gulf-scale issues. Within this preview, the mission of this project is to provide cross cutting solution network and rapid prototyping capability for the Gulf of Mexico region, in order to demonstrate substantial, collaborative, multi-agency research and transitional capabilities using unique NASA data sets and models to address regional problems. SERVIR Mesoamerica is seen as an excellent existing framework that can be used to integrate observational and GIs data bases, provide a sensor web interface, visualization and interactive analysis tools, archival functions, data dissemination and product generation within a Rapid Prototyping concept to assist decision-makers in better understanding Gulf-scale environmental issues.
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Maughan, Timothy S; Adams, Richard A; Smith, Christopher G; Meade, Angela M; Seymour, Matthew T; Wilson, Richard H; Idziaszczyk, Shelley; Harris, Rebecca; Fisher, David; Kenny, Sarah L; Kay, Edward; Mitchell, Jenna K; Madi, Ayman; Jasani, Bharat; James, Michelle D; Bridgewater, John; Kennedy, M John; Claes, Bart; Lambrechts, Diether; Kaplan, Richard; Cheadle, Jeremy P
2011-01-01
Summary Background In the Medical Research Council (MRC) COIN trial, the epidermal growth factor receptor (EGFR)-targeted antibody cetuximab was added to standard chemotherapy in first-line treatment of advanced colorectal cancer with the aim of assessing effect on overall survival. Methods In this randomised controlled trial, patients who were fit for but had not received previous chemotherapy for advanced colorectal cancer were randomly assigned to oxaliplatin and fluoropyrimidine chemotherapy (arm A), the same combination plus cetuximab (arm B), or intermittent chemotherapy (arm C). The choice of fluoropyrimidine therapy (capecitabine or infused fluouroracil plus leucovorin) was decided before randomisation. Randomisation was done centrally (via telephone) by the MRC Clinical Trials Unit using minimisation. Treatment allocation was not masked. The comparison of arms A and C is described in a companion paper. Here, we present the comparison of arm A and B, for which the primary outcome was overall survival in patients with KRAS wild-type tumours. Analysis was by intention to treat. Further analyses with respect to NRAS, BRAF, and EGFR status were done. The trial is registered, ISRCTN27286448. Findings 1630 patients were randomly assigned to treatment groups (815 to standard therapy and 815 to addition of cetuximab). Tumour samples from 1316 (81%) patients were used for somatic molecular analyses; 565 (43%) had KRAS mutations. In patients with KRAS wild-type tumours (arm A, n=367; arm B, n=362), overall survival did not differ between treatment groups (median survival 17·9 months [IQR 10·3–29·2] in the control group vs 17·0 months [9·4–30·1] in the cetuximab group; HR 1·04, 95% CI 0·87–1·23, p=0·67). Similarly, there was no effect on progression-free survival (8·6 months [IQR 5·0–12·5] in the control group vs 8·6 months [5·1–13·8] in the cetuximab group; HR 0·96, 0·82–1·12, p=0·60). Overall response rate increased from 57% (n=209
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Coudray-Meunier, Coralie; Fraisse, Audrey; Martin-Latil, Sandra; Guillier, Laurent; Delannoy, Sabine; Fach, Patrick; Perelle, Sylvie
2015-05-18
Sensitive and quantitative detection of foodborne enteric viruses is classically achieved by quantitative RT-PCR (RT-qPCR). Recently, digital PCR (dPCR) was described as a novel approach to genome quantification without need for a standard curve. The performance of microfluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR for detecting the main viruses responsible for foodborne outbreaks (human Noroviruses (NoV) and Hepatitis A virus (HAV)) in spiked lettuce and bottled water. Two process controls (Mengovirus and Murine Norovirus) were used and external amplification controls (EAC) were added to examine inhibition of RT-qPCR and RT-dPCR. For detecting viral RNA and cDNA, the sensitivity of the RT-dPCR assays was either comparable to that of RT-qPCR (RNA of HAV, NoV GI, Mengovirus) or slightly (around 1 log10) decreased (NoV GII and MNV-1 RNA and of HAV, NoV GI, NoV GII cDNA). The number of genomic copies determined by dPCR was always from 0.4 to 1.7 log10 lower than the expected numbers of copies calculated by using the standard qPCR curve. Viral recoveries calculated by RT-dPCR were found to be significantly higher than by RT-qPCR for NoV GI, HAV and Mengovirus in water, and for NoV GII and HAV in lettuce samples. The RT-dPCR assay proved to be more tolerant to inhibitory substances present in lettuce samples. This absolute quantitation approach may be useful to standardize quantification of enteric viruses in bottled water and lettuce samples and may be extended to quantifying other human pathogens in food samples. Copyright © 2015 Elsevier B.V. All rights reserved.
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Soltan, Mohamed A; Tsai, Yun-Long; Lee, Pei-Yu A; Tsai, Chuan-Fu; Chang, Hsiao-Fen G; Wang, Hwa-Tang T; Wilkes, Rebecca P
2016-09-01
There is no gold standard for detection of Rotavirus Group A (RVA), one of the main causes of diarrhea in neonatal animals. Sensitive and specific real-time RT-PCR (rtRT-PCR) assays are available for RVA but require submission of the clinical samples to diagnostic laboratories. Patient-side immunoassays for RVA protein detection have shown variable results, particularly with samples from unintended species. A sensitive and specific test for detection of RVA on the farm would facilitate rapid management decisions. The insulated isothermal RT-PCR (RT-iiPCR) assay works in a portable machine to allow sensitive and specific on-site testing. The aim of this investigation was to evaluate a commercially available RT-iiPCR assay for RVA detection in feces from different animal species. This assay was compared to an in-house rtRT-PCR assay and a commercially available rtRT-PCR kit, as well as an ELISA and EM for RVA detection. All three PCR assays targeted the well-conserved NSP5 gene. Clinical fecal samples from 108 diarrheic animals (mainly cattle and horses) were tested. The percentage of positive samples by ELISA, EM, in-house rtRT-PCR, commercial rtRT-PCR, and RT-iiPCR was 29.4%, 31%, 36.7%, 51.4%, 56.9%, respectively. The agreement between different assays was high (81.3-100%) in samples containing high viral loads. The sensitivity of the RT-iiPCR assay appeared to be higher than the commercially available rtRT-PCR assay, with a limit of detection (95% confidence index) of 3-4 copies of in vitro transcribed dsRNA. In conclusion, the user-friendly, field-deployable RT-iiPCR system holds substantial promise for on-site detection of RVA. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.
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Biomarker discovery for colon cancer using a 761 gene RT-PCR assay.
Clark-Langone, Kim M; Wu, Jenny Y; Sangli, Chithra; Chen, Angela; Snable, James L; Nguyen, Anhthu; Hackett, James R; Baker, Joffre; Yothers, Greg; Kim, Chungyeul; Cronin, Maureen T
2007-08-15
Reverse transcription PCR (RT-PCR) is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE) clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco typeDX assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis) and the likelihood of tumor response to standard chemotherapy regimens (prediction). We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application. RNA was extracted from formalin fixed paraffin embedded (FPE) tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery. We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of genes analyzed while maintaining the high specificity
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NASA SPoRT GOES-R Proving Ground Activities
NASA Technical Reports Server (NTRS)
Stano, Geoffrey T.; Fuell, Kevin K.; Jedloec, Gary J.
2010-01-01
The NASA Short-term Prediction Research and Transition (SPoRT) program is a partner with the GOES-R Proving Ground (PG) helping prepare forecasters understand the unique products to come from the GOES-R instrument suite. SPoRT is working collaboratively with other members of the GOES-R PG team and Algorithm Working Group (AWG) scientists to develop and disseminate a suite of proxy products that address specific forecast problems for the WFOs, Regional and National Support Centers, and other NOAA users. These products draw on SPoRT s expertise with the transition and evaluation of products into operations from the MODIS instrument and the North Alabama Lightning Mapping Array (NALMA). The MODIS instrument serves as an excellent proxy for the Advanced Baseline Imager (ABI) that will be aboard GOES-R. SPoRT has transitioned and evaluated several multi-channel MODIS products. The true and false color products are being used in natural hazard detection by several SPoRT partners to provide better observation of land features, such as fires, smoke plumes, and snow cover. Additionally, many of SPoRT s partners are coastal offices and already benefit from the MODIS sea surface temperature composite. This, along with other surface feature observations will be developed into ABI proxy products for diagnostic use in the forecast process as well as assimilation into forecast models. In addition to the MODIS instrument, the NALMA has proven very valuable to WFOs with access to these total lightning data. These data provide situational awareness and enhanced warning decision making to improve lead times for severe thunderstorm and tornado warnings. One effort by SPoRT scientists includes a lightning threat product to create short-term model forecasts of lightning activity. Additionally, SPoRT is working with the AWG to create GLM proxy data from several of the ground based total lightning networks, such as the NALMA. The evaluation will focus on the vastly improved spatial
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Desai, Neil B.; Stein, Nicholas F.; LaQuaglia, Michael P.
2013-01-01
Purpose: Desmoplastic small round cell tumor (DSRCT) is a rare malignancy typically involving the peritoneum in young men. Whole abdominopelvic radiation therapy (WAP-RT) using conventional 2-dimensional (2D) radiation therapy (RT) is used to address local recurrence but has been limited by toxicity. Our objectives were to assess the benefit of intensity modulated radiation therapy (IMRT) on toxicity and to update the largest series on radiation for DSRCT. Methods and Materials: The records of 31 patients with DSRCT treated with WAP-RT (22 with 2D-RT and 9 with IMRT) between 1992 and 2011 were retrospectively reviewed. All received multi-agent chemotherapy and maximalmore » surgical debulking followed by 30 Gy of WAP-RT. A further focal boost of 12 to 24 Gy was used in 12 cases. Boost RT and autologous stem cell transplantation were nearly exclusive to patients treated with 2D-RT. Toxicities were assessed with the Common Terminology Criteria for Adverse Events. Dosimetric analysis compared IMRT and simulated 2D-RT dose distributions. Results: Of 31 patients, 30 completed WAP-RT, with a median follow-up after RT of 19 months. Acute toxicity was reduced with IMRT versus 2D-RT: P=.04 for gastrointestinal toxicity of grade 2 or higher (33% vs 77%); P=.02 for grade 4 hematologic toxicity (33% vs 86%); P=.01 for rates of granulocyte colony-stimulating factor; and P=.04 for rates of platelet transfusion. Post treatment red blood cell and platelet transfusion rates were also reduced (P=.01). IMRT improved target homogeneity ([D05-D95]/D05 of 21% vs 46%) and resulted in a 21% mean bone dose reduction. Small bowel obstruction was the most common late toxicity (23% overall). Updated 3-year overall survival and progression-free survival rates were 50% and 24%, respectively. Overall survival was associated with distant metastasis at diagnosis on multivariate analysis. Most failures remained intraperitoneal (88%). Conclusions: IMRT for consolidative WAP-RT in DSRCT improves
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SlicerRT: radiation therapy research toolkit for 3D Slicer.
Pinter, Csaba; Lasso, Andras; Wang, An; Jaffray, David; Fichtinger, Gabor
2012-10-01
Interest in adaptive radiation therapy research is constantly growing, but software tools available for researchers are mostly either expensive, closed proprietary applications, or free open-source packages with limited scope, extensibility, reliability, or user support. To address these limitations, we propose SlicerRT, a customizable, free, and open-source radiation therapy research toolkit. SlicerRT aspires to be an open-source toolkit for RT research, providing fast computations, convenient workflows for researchers, and a general image-guided therapy infrastructure to assist clinical translation of experimental therapeutic approaches. It is a medium into which RT researchers can integrate their methods and algorithms, and conduct comparative testing. SlicerRT was implemented as an extension for the widely used 3D Slicer medical image visualization and analysis application platform. SlicerRT provides functionality specifically designed for radiation therapy research, in addition to the powerful tools that 3D Slicer offers for visualization, registration, segmentation, and data management. The feature set of SlicerRT was defined through consensus discussions with a large pool of RT researchers, including both radiation oncologists and medical physicists. The development processes used were similar to those of 3D Slicer to ensure software quality. Standardized mechanisms of 3D Slicer were applied for documentation, distribution, and user support. The testing and validation environment was configured to automatically launch a regression test upon each software change and to perform comparison with ground truth results provided by other RT applications. Modules have been created for importing and loading DICOM-RT data, computing and displaying dose volume histograms, creating accumulated dose volumes, comparing dose volumes, and visualizing isodose lines and surfaces. The effectiveness of using 3D Slicer with the proposed SlicerRT extension for radiation therapy
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Wang, Meihua; Cairncross, Gregory; Shaw, Edward; Jenkins, Robert; Scheithauer, Bernd; Brachman, David; Buckner, Jan; Fink, Karen; Souhami, Luis; Laperriere, Normand; Mehta, Minesh; Curran, Walter
2010-07-01
Radiation Therapy Oncology Group 9402 compared procarbazine, lomustine, and vincristine (PCV) chemotherapy plus radiation therapy (PCV + RT) vs. RT alone for anaplastic oligodendroglioma. Here we report longitudinal changes in cognition and quality of life, effects of patient factors and treatments on cognition, quality of life and survival, and prognostic implications of cognition and quality of life. Cognition was assessed by Mini Mental Status Examination (MMSE) and quality of life by Brain-Quality of Life (B-QOL). Scores were analyzed for survivors and within 5 years of death. Shared parameter models evaluated MMSE/B-QOL with survival. For survivors, MMSE and B-QOL scores were similar longitudinally and between treatments. For those who died, MMSE scores remained stable initially, whereas B-QOL slowly declined; both declined rapidly in the last year of life and similarly between arms. In the aggregate, scores decreased over time (p = 0.0413 for MMSE; p = 0.0016 for B-QOL) and were superior with age <50 years (p < 0.001 for MMSE; p = 0.0554 for B-QOL) and Karnofsky Performance Score (KPS) 80-100 (p < 0.001). Younger age and higher KPS were associated with longer survival. After adjusting for patient factors and drop-out, survival was longer after PCV + RT (HR = 0.66, 95% CI = 0.49-0.9, p = 0.0084; HR = 0.74, 95% CI = 0.54-1.01, p = 0.0592) in models with MMSE and B-QOL. In addition, there were no differences in MMSE and B-QOL scores between arms (p = 0.4752 and p = 0.2767, respectively); higher scores predicted longer survival. MMSE and B-QOL scores held steady in the upper range in both arms for survivors. Younger, fitter patients had better MMSE and B-QOL and longer survival. (c) 2010 Elsevier Inc. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Wang Meihua, E-mail: mwang@phila.acr.or; Cairncross, Gregory; Shaw, Edward
2010-07-01
Purpose: Radiation Therapy Oncology Group 9402 compared procarbazine, lomustine, and vincristine (PCV) chemotherapy plus radiation therapy (PCV + RT) vs. RT alone for anaplastic oligodendroglioma. Here we report longitudinal changes in cognition and quality of life, effects of patient factors and treatments on cognition, quality of life and survival, and prognostic implications of cognition and quality of life. Methods and Materials: Cognition was assessed by Mini Mental Status Examination (MMSE) and quality of life by Brain-Quality of Life (B-QOL). Scores were analyzed for survivors and within 5 years of death. Shared parameter models evaluated MMSE/B-QOL with survival. Results: For survivors,more » MMSE and B-QOL scores were similar longitudinally and between treatments. For those who died, MMSE scores remained stable initially, whereas B-QOL slowly declined; both declined rapidly in the last year of life and similarly between arms. In the aggregate, scores decreased over time (p = 0.0413 for MMSE; p = 0.0016 for B-QOL) and were superior with age <50 years (p < 0.001 for MMSE; p = 0.0554 for B-QOL) and Karnofsky Performance Score (KPS) 80-100 (p < 0.001). Younger age and higher KPS were associated with longer survival. After adjusting for patient factors and drop-out, survival was longer after PCV + RT (HR = 0.66, 95% CI = 0.49-0.9, p = 0.0084; HR = 0.74, 95% CI = 0.54-1.01, p = 0.0592) in models with MMSE and B-QOL. In addition, there were no differences in MMSE and B-QOL scores between arms (p = 0.4752 and p = 0.2767, respectively); higher scores predicted longer survival. Conclusion: MMSE and B-QOL scores held steady in the upper range in both arms for survivors. Younger, fitter patients had better MMSE and B-QOL and longer survival.« less
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AWIPS II Application Development, a SPoRT Perspective
NASA Technical Reports Server (NTRS)
Burks, Jason E.; Smith, Matthew; McGrath, Kevin M.
2014-01-01
The National Weather Service (NWS) is deploying its next-generation decision support system, called AWIPS II (Advanced Weather Interactive Processing System II). NASA's Short-term Prediction Research and Transition (SPoRT) Center has developed several software 'plug-ins' to extend the capabilities of AWIPS II. SPoRT aims to continue its mission of improving short-term forecasts by providing NASA and NOAA products on the decision support system used at NWS weather forecast offices (WFOs). These products are not included in the standard Satellite Broadcast Network feed provided to WFOs. SPoRT has had success in providing support to WFOs as they have transitioned to AWIPS II. Specific examples of transitioning SPoRT plug-ins to WFOs with newly deployed AWIPS II systems will be presented. Proving Ground activities (GOES-R and JPSS) will dominate SPoRT's future AWIPS II activities, including tool development as well as enhancements to existing products. In early 2012 SPoRT initiated the Experimental Product Development Team, a group of AWIPS II developers from several institutions supporting NWS forecasters with innovative products. The results of the team's spring and fall 2013 meeting will be presented. Since AWIPS II developers now include employees at WFOs, as well as many other institutions related to weather forecasting, the NWS has dealt with a multitude of software governance issues related to the difficulties of multiple remotely collaborating software developers. This presentation will provide additional examples of Research-to-Operations plugins, as well as an update on how governance issues are being handled in the AWIPS II developer community.
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Predicting Gene Structures from Multiple RT-PCR Tests
NASA Astrophysics Data System (ADS)
Kováč, Jakub; Vinař, Tomáš; Brejová, Broňa
It has been demonstrated that the use of additional information such as ESTs and protein homology can significantly improve accuracy of gene prediction. However, many sources of external information are still being omitted from consideration. Here, we investigate the use of product lengths from RT-PCR experiments in gene finding. We present hardness results and practical algorithms for several variants of the problem and apply our methods to a real RT-PCR data set in the Drosophila genome. We conclude that the use of RT-PCR data can improve the sensitivity of gene prediction and locate novel splicing variants.
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Cytopathic effect of Human cosavirus (HCoSV) on primary cell cultures of human embryonic lung MRC5.
Rezig, Dorra; Touzi, Henda; Meddeb, Zina; Triki, Henda
2014-10-01
Human cosaviruses (HCoSVs) are newly discovered viruses in Picornaviridae family. Until now, most published studies reported HCoSV detection using molecular techniques and genetic characterization of the virus. Nevertheless, no laboratory has yet reported the replication of these viruses in cultured cell lines. In the present work, the propagation of HCoSV strains isolated from human fecal specimens in MRC5 cell line and their induced cytopathic effects (CPE) was studied. The first sign of virus growth was observed 24-48h after inoculation. The cells rounded up and clumped together rapidly; empty areas became visible and, on the third day of CPE, a remarkable decrease in live cells was observed. This represents the first report on in vitro model of HCoSV replication which opens up opportunities for future investigations of these new viruses. Copyright © 2014 Elsevier B.V. All rights reserved.
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Simultaneous detection of three lily viruses using Triplex IC-RT-PCR.
Zhang, Yubao; Wang, Yajun; Xie, Zhongkui; Yang, Guo; Guo, Zhihong; Wang, Le
2017-11-01
Viruses commonly infecting lily (Lilium spp.) include: Lily symptomless virus (LSV), Cucumber mosaic virus (CMV) and Lily mottle virus (LMoV). These viruses usually co-infect lilies causing severe economic losses in terms of quantity and quality of flower and bulb production around the world. Reliable and precise detection systems need to be developed for virus identification. We describe the development of a triplex immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) assay for the simultaneous detection of LSV, CMV and LMoV. The triplex IC-RT-PCR was compared with a quadruplex RT-PCR assay. Relative to the quadruplex RT-PCR, the specificity of the triplex IC-RT-PCR system for LSV, CMV and LMoV was 100% for field samples. The sensitivity of the triplex IC-RT-PCR system was 99.4%, 81.4% and 98.7% for LSV, CMV and LMoV, respectively. Agreement (κ) between the results obtained from the two tests was 0.968, 0.844 and 0.984 for LSV, CMV and LMoV, respectively. This is the first report of the simultaneous detection of LSV, CMV and LMoV in a triplex IC-RT-PCR assay. In particular we believe this convenient and reliable triplex IC-RT-PCR method could be used routinely for large-scale field surveys or crop health monitoring of lily. Copyright © 2017. Published by Elsevier B.V.
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Avian influenza virus detection and quantitation by real-time RT-PCR
USDA-ARS?s Scientific Manuscript database
Real-time RT-PCR (rRT-PCR) has been used for avian influenza virus (AIV) detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of rRT-PCR are: high sensitivity, high specificity, rapid time-to-result, scalability, cost, and its inherentl...
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Using iRT, a normalized retention time for more targeted measurement of peptides
Escher, Claudia; Reiter, Lukas; MacLean, Brendan; Ossola, Reto; Herzog, Franz; Chilton, John; MacCoss, Michael J.; Rinner, Oliver
2014-01-01
Multiple reaction monitoring (MRM) has recently become the method of choice for targeted quantitative measurement of proteins using mass spectrometry. The method, however, is limited in the number of peptides that can be measured in one run. This number can be markedly increased by scheduling the acquisition if the accurate retention time (RT) of each peptide is known. Here we present iRT, an empirically derived dimensionless peptide-specific value that allows for highly accurate RT prediction. The iRT of a peptide is a fixed number relative to a standard set of reference iRT-peptides that can be transferred across laboratories and chromatographic systems. We show that iRT facilitates the setup of multiplexed experiments with acquisition windows more than 4 times smaller compared to in silico RT predictions resulting in improved quantification accuracy. iRTs can be determined by any laboratory and shared transparently. The iRT concept has been implemented in Skyline, the most widely used software for MRM experiments. PMID:22577012
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Profiling structured product labeling with NDF-RT and RxNorm
2012-01-01
Background Structured Product Labeling (SPL) is a document markup standard approved by Health Level Seven (HL7) and adopted by United States Food and Drug Administration (FDA) as a mechanism for exchanging drug product information. The SPL drug labels contain rich information about FDA approved clinical drugs. However, the lack of linkage to standard drug ontologies hinders their meaningful use. NDF-RT (National Drug File Reference Terminology) and NLM RxNorm as standard drug ontology were used to standardize and profile the product labels. Methods In this paper, we present a framework that intends to map SPL drug labels with existing drug ontologies: NDF-RT and RxNorm. We also applied existing categorical annotations from the drug ontologies to classify SPL drug labels into corresponding classes. We established the classification and relevant linkage for SPL drug labels using the following three approaches. First, we retrieved NDF-RT categorical information from the External Pharmacologic Class (EPC) indexing SPLs. Second, we used the RxNorm and NDF-RT mappings to classify and link SPLs with NDF-RT categories. Third, we profiled SPLs using RxNorm term type information. In the implementation process, we employed a Semantic Web technology framework, in which we stored the data sets from NDF-RT and SPLs into a RDF triple store, and executed SPARQL queries to retrieve data from customized SPARQL endpoints. Meanwhile, we imported RxNorm data into MySQL relational database. Results In total, 96.0% SPL drug labels were mapped with NDF-RT categories whereas 97.0% SPL drug labels are linked to RxNorm codes. We found that the majority of SPL drug labels are mapped to chemical ingredient concepts in both drug ontologies whereas a relatively small portion of SPL drug labels are mapped to clinical drug concepts. Conclusions The profiling outcomes produced by this study would provide useful insights on meaningful use of FDA SPL drug labels in clinical applications through
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Profiling structured product labeling with NDF-RT and RxNorm.
Zhu, Qian; Jiang, Guoqian; Chute, Christopher G
2012-12-20
Structured Product Labeling (SPL) is a document markup standard approved by Health Level Seven (HL7) and adopted by United States Food and Drug Administration (FDA) as a mechanism for exchanging drug product information. The SPL drug labels contain rich information about FDA approved clinical drugs. However, the lack of linkage to standard drug ontologies hinders their meaningful use. NDF-RT (National Drug File Reference Terminology) and NLM RxNorm as standard drug ontology were used to standardize and profile the product labels. In this paper, we present a framework that intends to map SPL drug labels with existing drug ontologies: NDF-RT and RxNorm. We also applied existing categorical annotations from the drug ontologies to classify SPL drug labels into corresponding classes. We established the classification and relevant linkage for SPL drug labels using the following three approaches. First, we retrieved NDF-RT categorical information from the External Pharmacologic Class (EPC) indexing SPLs. Second, we used the RxNorm and NDF-RT mappings to classify and link SPLs with NDF-RT categories. Third, we profiled SPLs using RxNorm term type information. In the implementation process, we employed a Semantic Web technology framework, in which we stored the data sets from NDF-RT and SPLs into a RDF triple store, and executed SPARQL queries to retrieve data from customized SPARQL endpoints. Meanwhile, we imported RxNorm data into MySQL relational database. In total, 96.0% SPL drug labels were mapped with NDF-RT categories whereas 97.0% SPL drug labels are linked to RxNorm codes. We found that the majority of SPL drug labels are mapped to chemical ingredient concepts in both drug ontologies whereas a relatively small portion of SPL drug labels are mapped to clinical drug concepts. The profiling outcomes produced by this study would provide useful insights on meaningful use of FDA SPL drug labels in clinical applications through standard drug ontologies such as NDF-RT and
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Evolution of an Expanded Mannose Receptor Gene Family
Staines, Karen; Hunt, Lawrence G.; Young, John R.; Butter, Colin
2014-01-01
Sequences of peptides from a protein specifically immunoprecipitated by an antibody, KUL01, that recognises chicken macrophages, identified a homologue of the mammalian mannose receptor, MRC1, which we called MRC1L-B. Inspection of the genomic environment of the chicken gene revealed an array of five paralogous genes, MRC1L-A to MRC1L-E, located between conserved flanking genes found either side of the single MRC1 gene in mammals. Transcripts of all five genes were detected in RNA from a macrophage cell line and other RNAs, whose sequences allowed the precise definition of spliced exons, confirming or correcting existing bioinformatic annotation. The confirmed gene structures were used to locate orthologues of all five genes in the genomes of two other avian species and of the painted turtle, all with intact coding sequences. The lizard genome had only three genes, one orthologue of MRC1L-A and two orthologues of the MRC1L-B antigen gene resulting from a recent duplication. The Xenopus genome, like that of most mammals, had only a single MRC1-like gene at the corresponding locus. MRC1L-A and MRC1L-B genes had similar cytoplasmic regions that may be indicative of similar subcellular migration and functions. Cytoplasmic regions of the other three genes were very divergent, possibly indicating the evolution of a new functional repertoire for this family of molecules, which might include novel interactions with pathogens. PMID:25390371
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Suryavanshi, Moushumi; Mehta, Anurag; Jaipuria, Jiten; Kumar, Dushyant; Vishwakarma, Gayatri; Panigrahi, Manoj Kumar; Verma, Haristuti; Saifi, Mumtaz; Sharma, Sanjeev; Tandon, Simran; Doval, D C; Das, Bhudev C
2018-02-09
would have benefited from RT-PCR testing if it was used as a first-line test. If RT-PCR would have been used as a second-line test among those with IHC score 2 (n = 43), then only 6 patients would have been assigned a conclusive RT-PCR category (category 1 or 3) translating to a clinical benefit of 14% (6/43) as a second-line test. As a second-line test it had 51% probability to prove more cost-effective than the conventional pathway, provided the cost of RT-PCR was 0.4 times the cost of IHC. Also in a three-step pathway, RT-PCR upfront would have 56% probability of higher cost-benefit provided the cost of RT-PCR was 0.1 times the cost of IHC. RT-PCR results were found to be suboptimal to IHC in terms of discriminative ability and clinical benefit; thus, it is unlikely to replace IHC as a first-line test in the near future.
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Yang, Jie; Yu, Fei; Liu, Hong; An, Hedi; Xiong, Ran; Huang, Dongya
2016-08-11
We sought to assess the safety, effectiveness and cost of 0.6 mg/kg rt-PA treatment for patients with acute mild stroke and to compare that with 0.9 mg/kg. We retrospectively analyzed consecutive acute ischemic stroke patients who had a NIHSS score ≤5 at admission and who were treated with rt-PA within 4.5 hours of symptom onset. The demographic data, clinical outcomes and hospitalization cost were analyzed. A total of 108 patients were included. Forty six patients (42.6%) received a 0.6 mg/kg dosage of rt-PA. The baseline characteristics of the two groups were well matched (p > 0.05). Regarding the safety and effectiveness, the 0.6 mg/kg dosage group had a comparable proportion of symptomatic intracranial hemorrhage (sICH) (0.6 mg/kg, 4.3% vs 0.9 mg/kg, 4.8%; p > 0.05), early neurological deterioration (END) (19.6% vs 17.7%; p > 0.05), in-hospital mortality (4.3% vs 1.6%; p > 0.05), and a similar rate of favorable functional outcome (mRS score 0-1) at 3 months (73.9% vs 71.0%; p > 0.05) to those who received the standard dosage. However, the hospital cost was markedly lower in the 0.6 mg/kg group (0.6 mg/kg, 3,401.7 USD vs 0.9 mg/kg, 4,157.4 USD; p < 0.01). Our study suggest that 0.6 mg/kg rt-PA shared similar effectiveness and safety profile compared with that of 0.9 mg/kg in treating mild stroke, but cost less.
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Using iRT, a normalized retention time for more targeted measurement of peptides.
Escher, Claudia; Reiter, Lukas; MacLean, Brendan; Ossola, Reto; Herzog, Franz; Chilton, John; MacCoss, Michael J; Rinner, Oliver
2012-04-01
Multiple reaction monitoring (MRM) has recently become the method of choice for targeted quantitative measurement of proteins using mass spectrometry. The method, however, is limited in the number of peptides that can be measured in one run. This number can be markedly increased by scheduling the acquisition if the accurate retention time (RT) of each peptide is known. Here we present iRT, an empirically derived dimensionless peptide-specific value that allows for highly accurate RT prediction. The iRT of a peptide is a fixed number relative to a standard set of reference iRT-peptides that can be transferred across laboratories and chromatographic systems. We show that iRT facilitates the setup of multiplexed experiments with acquisition windows more than four times smaller compared to in silico RT predictions resulting in improved quantification accuracy. iRTs can be determined by any laboratory and shared transparently. The iRT concept has been implemented in Skyline, the most widely used software for MRM experiments. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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NASA Technical Reports Server (NTRS)
Goodman, S. J.; Lapenta, W.; Jedlovec, G.; Dodge, J.; Bradshaw, T.
2003-01-01
The NASA Short-term Prediction Research and Transition (SPoRT) Center in Huntsville, Alabama was created to accelerate the infusion of NASA earth science observations, data assimilation and modeling research into NWS forecast operations and decision-making. The principal focus of experimental products is on the regional scale with an emphasis on forecast improvements on a time scale of 0-24 hours. The SPoRT Center research is aligned with the regional prediction objectives of the US Weather Research Program dealing with 0-1 day forecast issues ranging from convective initiation to 24-hr quantitative precipitation forecasting. The SPoRT Center, together with its other interagency partners, universities, and the NASA/NOAA Joint Center for Satellite Data Assimilation, provides a means and a process to effectively transition NASA Earth Science Enterprise observations and technology to National Weather Service operations and decision makers at both the global/national and regional scales. This paper describes the process for the transition of experimental products into forecast operations, current products undergoing assessment by forecasters, and plans for the future.
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Estimating Energy Expenditure with the RT3 Triaxial Accelerometer
ERIC Educational Resources Information Center
Maddison, Ralph; Jiang, Yannan; Vander Hoorn, Stephen; Mhurchu, Cliona Ni; Lawes, Carlene M. M.; Rodgers, Anthony; Rush, Elaine
2009-01-01
The RT3 is a relatively new triaxial accelerometer that has replaced the TriTrac. The aim of this study was to validate the RT3 against doubly labeled water (DLW) in a free-living, mixed weight sample of adults. Total energy expenditure (TEE) was measured over a 15-day period using DLW. Activity-related energy expenditure (AEE) was estimated by…
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Phosri, Santi; Jangpromma, Nisachon; Patramanon, Rina; Kongyingyoes, Bunkerd; Mahakunakorn, Pramote; Klaynongsruang, Sompong
2017-02-01
A putative protective effect of cHb and cWb against H 2 O 2 -induced oxidative damage was evaluated in detail using MRC-5 cells. In addition, the carrageenan (Carr)-induced mouse paw edema model and the cotton pellet-induced granuloma model were employed to examine the in vivo anti-inflammatory activity of cHb and cWb in mice. It was demonstrated that both cHb and cWb treatments significantly increased cell viability and inhibited morphology alterations in MRC-5 cells exposed to H 2 O 2 . Orally administered cHb and cWb significantly reduced Carr-induced paw edema volume and cotton pellet-induced granuloma formation. Moreover, cHb and cWb decreased the expression levels of important pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α), while only cWb was found to increase the expression of the anti-inflammatory cytokine IL-10 significantly. Finally, the activity of antioxidant enzymes (SOD, CAT, and GPx) in the liver improved after cHb and cWb treatment under acute and chronic inflammation. Taken collectively, the results of this study suggest that both cHb and cWb protect against hydrogen peroxide-induced damage in fibroblast cells. Moreover, cHb and cWb were found to exhibit anti-inflammatory activity in both the acute and chronic stages of inflammation and appear to enhance antioxidant enzyme activity and decrease lipid peroxidation in the livers of mice. Therefore, this study indicates that cHb and cWb have great potential to be used in the development of dietary supplements for the prevention of oxidative stress related to inflammatory disorders.
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ERIC Educational Resources Information Center
Murin, Tricia M.
2016-01-01
Providing equitable education for all students is the responsibility of administrators, teachers, and parents. Even though the MTSS/RtII Framework has evolved from the RtI and RtII models, the basis is the same: intervening and identifying students' needs and analyzing data and programming instruction to meet all students' needs. Even though in…
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Niu, Peihua; Qi, Shunxiang; Yu, Benzhang; Zhang, Chen; Wang, Ji; Li, Qi; Ma, Xuejun
2016-11-01
Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease (HFMD). A commercial TaqMan probe-based real-time PCR assay has been widely used for the differential detection of EV71 despite its relatively high cost and failure to detect samples with a low viral load (Ct value > 35). In this study, a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of EV71 in HFMD was developed. The sensitivity and specificity of this assay were evaluated using a reference EV71 stock and a panel of controls consisting of coxsackievirus A16 (CVA16) and common respiratory viruses, respectively. The clinical performance of this assay was evaluated and compared with those of a commercial TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional two-step nested RT-PCR assay. The limit of detection for the RTN RT-PCR assay was 0.01 TCID50/ml, with a Ct value of 38.3, which was the same as that of the traditional two-step nested RT-PCR assay and approximately tenfold lower than that of the qRT-PCR assay. When testing the reference strain EV71, this assay showed favorable detection reproducibility and no obvious cross-reactivity. The testing results of 100 clinical throat swabs from HFMD-suspected patients revealed that 41 samples were positive for EV71 by both RTN RT-PCR and traditional two-step nested RT-PCR assays, whereas only 29 were EV71 positive by qRT-PCR assay.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
NONE
2016-06-15
In recent years, steady progress has been made towards the implementation of MRI in external beam radiation therapy for processes ranging from treatment simulation to in-room guidance. Novel procedures relying mostly on MR data are currently implemented in the clinic. This session will cover topics such as (a) commissioning and quality control of the MR in-room imagers and simulators specific to RT, (b) treatment planning requirements, constraints and challenges when dealing with various MR data, (c) quantification of organ motion with an emphasis on treatment delivery guidance, and (d) MR-driven strategies for adaptive RT workflows. The content of the sessionmore » was chosen to address both educational and practical key aspects of MR guidance. Learning Objectives: Good understanding of MR testing recommended for in-room MR imaging as well as image data validation for RT chain (e.g. image transfer, filtering for consistency, spatial accuracy, manipulation for task specific); Familiarity with MR-based planning procedures: motivation, core workflow requirements, current status, challenges; Overview of the current methods for the quantification of organ motion; Discussion on approaches for adaptive treatment planning and delivery. T. Stanescu - License agreement with Modus Medical Devices to develop a phantom for the quantification of MR image system-related distortions.; T. Stanescu, N/A.« less
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Effect of low frequency ultrasound on combined rt-PA and eptifibatide thrombolysis in human clots.
Meunier, Jason M; Holland, Christy K; Pancioli, Arthur M; Lindsell, Christopher J; Shaw, George J
2009-01-01
Fibrinolytics such as recombinant tissue plasminogen activator (rt-PA) are used to treat thrombotic disease such as acute myocardial infarction (AMI) and ischemic stroke. Interest in increasing efficacy and reducing side effects has led to the study of adjuncts such as GP IIb-IIIa inhibitors and ultrasound (US) enhanced thrombolysis. Currently, GP IIb-IIIa inhibitor and fibrinolytic treatment are often used in AMI, and are under investigation for stroke treatment. However, little is known of the efficacy of combined GP IIb-IIIa inhibitor, fibrinolytic and ultrasound treatment. We measure the lytic efficacy of rt-PA, eptifibatide (Epf) and 120 kHz ultrasound treatment in an in-vitro human clot model. Blood was drawn from 15 subjects after IRB approval. Clots were made in 20 microL pipettes, and placed in a water tank for microscopic visualization during lytic treatment. Clots were exposed to control, rt-PA (rt-PA), eptifibatide (Epf), or rt-PA+eptifibatide (rt-PA + Epf), with (+US) or without (-US) ultrasound for 30 minutes at 37 degrees C in human plasma. Clot lysis was measured over time, using a microscopic imaging technique. The fractional clot loss (FCL) and initial lytic rate (LR) were used to quantify lytic efficacy. LR values for (- US) treated clots were 0.8+/-0.1(control), 1.8+/-0.3 (Epf), 1.5+/-0.2 (rt-PA), and 1.3+/-0.4 (rt-PA + Epf) (% clot width/minute) respectively. In comparison, the (+ US) group exhibited LR values of 1.6+/-0.2 (control), 4.3+/-0.4 (Epf), 6.3+/-0.4 (rt-PA), and 4.6+/-0.6 (rt-PA + Epf). For (- US) treated clots, FCL was 6.0+/-0.8 (control), 9.2+/-2.5 (Epf), 15.6+/-1.7 (rt-PA), and 28.0+/-2.2% (rt-PA + Epf) respectively. FCL for (+ US) clots was 13.5+/-2.4 (control), 20.7+/-6.4 (Epf), 44.4+/-3.6 (rt-PA) and 30.3+/-3.6% (rt-PA + Epf) respectively. Although the addition of eptifibatide enhances the in-vitro lytic efficacy of rt-PA in the absence of ultrasound, the efficacy of ultrasound and rt-PA is greater than that of combined
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High-Throughput RT-PCR for small-molecule screening assays
Bittker, Joshua A.
2012-01-01
Quantitative measurement of the levels of mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real-time PCR analysis as well as the commercial availability of start-to-finish kits for RT-PCR has enabled the use of this method for high-throughput small-molecule screening on a scale comparable to traditional high-throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT-PCR as a primary small-molecule screening assay, including the different methods available for mRNA isolation and analysis. PMID:23487248
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Ye, Fei; Ning, Jing; Liu, Xinguo; Jin, Xiaodong; Wang, Tieshan; Li, Qiang
2016-01-01
Low-dose hyper-radiosensitivity (LDHRS) is a hot topic in normal tissue radiation protection. However, the primary causes for LDHRS still remain unclear. In this study, the impact of non-DNA-targeted effects (NTEs) on high-LET radiation–induced LDHRS was investigated. Human normal lung fibroblast MRC-5 cells were irradiated with high-LET carbon ions, and low-dose biological effects (in terms of various bio-endpoints, including colony formation, DNA damage and micronuclei formation) were detected under conditions with and without gap junctional intercellular communication (GJIC) inhibition. LDHRS was observed when the radiation dose was <0.2 Gy for all bio-endpoints under investigation, but vanished when the GJIC was suppressed. Based on the probability of cells being hit and micro-dose per cell calculation, we deduced that the LDHRS phenomenon came from the combined action of direct hits and NTEs. We concluded that GJIC definitely plays an important role in cytotoxic substance spreading in high-LET carbon ion–induced LDHRS. PMID:26559335
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The SPoRT-WRF: Evaluating the Impact of NASA Datasets on Convective Forecasts
NASA Technical Reports Server (NTRS)
Zavodsky, Bradley; Kozlowski, Danielle; Case, Jonathan; Molthan, Andrew
2012-01-01
Short-term Prediction Research and Transition (SPoRT) seeks to improve short-term, regional weather forecasts using unique NASA products and capabilities SPoRT has developed a unique, real-time configuration of the NASA Unified Weather Research and Forecasting (WRF)WRF (ARW) that integrates all SPoRT modeling research data: (1) 2-km SPoRT Sea Surface Temperature (SST) Composite, (2) 3-km LIS with 1-km Greenness Vegetation Fraction (GVFs) (3) 45-km AIRS retrieved profiles. Transitioned this real-time forecast to NOAA's Hazardous Weather Testbed (HWT) as deterministic model at Experimental Forecast Program (EFP). Feedback from forecasters/participants and internal evaluation of SPoRT-WRF shows a cool, dry bias that appears to suppress convection likely related to methodology for assimilation of AIRS profiles Version 2 of the SPoRT-WRF will premier at the 2012 EFP and include NASA physics, cycling data assimilation methodology, better coverage of precipitation forcing, and new GVFs
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Pretreatment to avoid positive RT-PCR results with inactivated viruses.
Nuanualsuwan, Suphachai; Cliver, Dean O
2002-07-01
Enteric viruses that are important causes of human disease must often be detected by reverse transcription-polymerase chain reaction (RT-PCR), a method that commonly yields positive results with samples that contain only inactivated virus. This study was intended to develop a pretreatment for samples, so that inactivated viruses would not be detected by the RT-PCR procedure. Model viruses were human hepatitis A virus, vaccine poliovirus 1 and feline calicivirus as a surrogate for the Norwalk-like viruses. Each virus was inactivated (from an initial titer of approximately 10(3) PFU/ml) by ultraviolet light, hypochlorite or heating at 72 degrees C. Inactivated viruses, that were treated with proteinase K and ribonuclease for 30 min at 37 degrees C before RT-PCR, gave a negative result, which is to say that no amplicon was detected after the reaction was completed. This antecedent to the RT-PCR method may be applicable to other types of viruses, to viruses inactivated in other ways and to other molecular methods of virus detection.
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Wu, Xiujuan; Li, Chunrong; Zhang, Bing; Shen, Donghui; Li, Ting; Liu, Kangding; Zhang, Hong-Liang
2015-09-02
Guillain-Barré syndrome (GBS) is an immune-mediated disorder of the peripheral nervous system. Respiratory failure requiring mechanical ventilation (MV) is a serious complication of GBS. Identification of modifiable risk factors for MV and poor short-term prognosis in mechanically ventilated patients with GBS may contribute to the individualized management and may help improve the outcome of the patients. We retrospectively analyzed the clinical data of 541 patients who were diagnosed with GBS from 2003 to 2014. Independent predictors for MV and short-term prognosis in mechanically ventilated patients were identified via multivariate logistic regression analysis. The mean age was 41.6 years with a male predilection (61.2%). Eighty patients (14.8%) required MV. Multivariate analysis revealed that shorter interval from onset to admission (p < 0.05), facial nerve palsy (p < 0.01), glossopharyngeal and vagal nerve deficits (p < 0.01) and lower Medical Research Council (MRC) sum score at nadir (p < 0.01) were risk factors for MV; disease occurrence in summer (p < 0.01) was a protective factor. As to prognostic factors, absence of antecedent infections (p < 0.01) and lower MRC sum score at nadir (p < 0.01) were predictors of poor short-term prognosis in mechanically ventilated patients regardless of treatment modality. We further investigated the predictors of poor short-term prognosis in patients requiring MV with different nadir MRC sum scores. Combined use of intravenous corticosteroids with intravenous immunoglobulin (odds ratio 10.200, 95% confidence interval 1.068-97.407, p < 0.05) was an independent predictor of poor short-term prognosis in mechanically ventilated patients with a nadir MRC sum score from 0 to 12 points, regardless of existence of antecedent infection. Clinical predictors of MV and poor short-term prognosis in mechanically ventilated GBS patients were distinct. Add-on use of intravenous corticosteroids was a risk factor for poor short
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ScriptingRT: A Software Library for Collecting Response Latencies in Online Studies of Cognition
Schubert, Thomas W.; Murteira, Carla; Collins, Elizabeth C.; Lopes, Diniz
2013-01-01
ScriptingRT is a new open source tool to collect response latencies in online studies of human cognition. ScriptingRT studies run as Flash applets in enabled browsers. ScriptingRT provides the building blocks of response latency studies, which are then combined with generic Apache Flex programming. Six studies evaluate the performance of ScriptingRT empirically. Studies 1–3 use specialized hardware to measure variance of response time measurement and stimulus presentation timing. Studies 4–6 implement a Stroop paradigm and run it both online and in the laboratory, comparing ScriptingRT to other response latency software. Altogether, the studies show that Flash programs developed in ScriptingRT show a small lag and an increased variance in response latencies. However, this did not significantly influence measured effects: The Stroop effect was reliably replicated in all studies, and the found effects did not depend on the software used. We conclude that ScriptingRT can be used to test response latency effects online. PMID:23805326
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Temperature affects thrombolytic efficacy using rt-PA and eptifibatide, an in vitro study.
Meunier, Jason M; Chang, Wan-Tsu W; Bluett, Brent; Wenker, Evan; Lindsell, Christopher J; Shaw, George J
2012-09-01
The potential for hypothermia as a neuroprotectant during stroke has led to its increase in clinical use. At the same time, combination pharmaceutical therapies for ischemic stroke using recombinant tissue plasminogen activator (rt-PA), and GP IIb-IIIa inhibitors, such as Eptifibatide (Epf ), are under study. However, there is little data on how the reactions triggered by these agents are impacted by temperature. Here, clot lysis during exposure to the combination of rt-PA and Epf is measured in an in vitro human clot model at hypothermic temperatures. The hypothesis is that lytic efficacy of rt-PA and Epf decreases with decreasing temperature. Whole blood clots from 31 volunteers were exposed to rt-PA (0.5 μg/mL) and Epf (0.63 μg/mL) in human fresh-frozen plasma (rt-PA+Epf ), rt-PA alone in plasma (rt-PA Alone), or to plasma alone (Control), at temperatures from 30°C to 37°C, for 30 minutes. Clot lysis was measured using a microscopic imaging technique; the mean fractional clot loss (FCL) at 30 minutes was used to determine lytic efficacy. Temperature had a significant impact on FCL in clots exposed to rt-PA+Epf, with the FCL being lower at 30°C to 36°C than at 37°C. The FCL remained significantly higher for rt-PA+Epf–treated clots than Controls regardless of temperature, with the exception of measurements made at 30°C when no significant differences in the FCL were observed between groups. The use of hypothermia as a neuroprotectant may negatively impact the therapeutic benefit of thrombolytic agents.
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Temperature Affects Thrombolytic Efficacy Using rt-PA and Eptifibatide, an In Vitro Study
Chang, Wan-Tsu W.; Bluett, Brent; Wenker, Evan; Lindsell, Christopher J.; Shaw, George J.
2012-01-01
The potential for hypothermia as a neuroprotectant during stroke has led to its increase in clinical use. At the same time, combination pharmaceutical therapies for ischemic stroke using recombinant tissue plasminogen activator (rt-PA), and GP IIb-IIIa inhibitors, such as Eptifibatide (Epf ), are under study. However, there is little data on how the reactions triggered by these agents are impacted by temperature. Here, clot lysis during exposure to the combination of rt-PA and Epf is measured in an in vitro human clot model at hypothermic temperatures. The hypothesis is that lytic efficacy of rt-PA and Epf decreases with decreasing temperature. Whole blood clots from 31 volunteers were exposed to rt-PA (0.5 μg/mL) and Epf (0.63 μg/mL) in human fresh-frozen plasma (rt-PA+Epf ), rt-PA alone in plasma (rt-PA Alone), or to plasma alone (Control), at temperatures from 30°C to 37°C, for 30 minutes. Clot lysis was measured using a microscopic imaging technique; the mean fractional clot loss (FCL) at 30 minutes was used to determine lytic efficacy. Temperature had a significant impact on FCL in clots exposed to rt-PA+Epf, with the FCL being lower at 30°C to 36°C than at 37°C. The FCL remained significantly higher for rt-PA+Epf–treated clots than Controls regardless of temperature, with the exception of measurements made at 30°C when no significant differences in the FCL were observed between groups. The use of hypothermia as a neuroprotectant may negatively impact the therapeutic benefit of thrombolytic agents. PMID:23667777
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The role of RT carry-over for congruence sequence effects in masked priming.
Huber-Huber, Christoph; Ansorge, Ulrich
2017-05-01
The present study disentangles 2 sources of the congruence sequence effect with masked primes: congruence and response time of the previous trial (reaction time [RT] carry-over). Using arrows as primes and targets and a metacontrast masking procedure we found congruence as well as congruence sequence effects. In addition, congruence sequence effects decreased when RT carry-over was accounted for in a mixed model analysis, suggesting that RT carry-over contributes to congruence sequence effects in masked priming. Crucially, effects of previous trial congruence were not cancelled out completely indicating that RT carry-over and previous trial congruence are 2 sources feeding into the congruence sequence effect. A secondary task requiring response speed judgments demonstrated general awareness of response speed (Experiments 1), but removing this secondary task (Experiment 2) showed that RT carry-over effects were also present in single-task conditions. During (dual-task) prime-awareness test parts of both experiments, however, RT carry-over failed to modulate congruence effects, suggesting that some task sets of the participants can prevent the effect. The basic RT carry-over effects are consistent with the conflict adaptation account, with the adaptation to the statistics of the environment (ASE) model, and possibly with the temporal learning explanation. Additionally considering the task-dependence of RT carry-over, the results are most compatible with the conflict adaptation account. (PsycINFO Database Record (c) 2017 APA, all rights reserved).
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Synthesis, structural and electron paramagnetic resonance studies on Pb0.9Bi0.1Fe0.7W0.3O3 ceramic
NASA Astrophysics Data System (ADS)
Shivaraja, I.; Matteppanvar, Shidaling; Dadami, Sunanda T.; Rayaprol, Sudhindra; Angadi, Basavaraj
2018-04-01
A single phase Pb0.9Bi0.1Fe0.7W0.3O3 (0.9Pb(Fe2/3W1/3)O3 - 0.1BiFeO3 or PBFW) polycrystalline ceramic was synthesized by the two step solid state reaction method, with low-temperature sintering at 800°C for 30 mins and slow cooling to room temperature (RT). Detailed studies of RT X-ray diffraction (XRD) and Raman spectroscopy measurements confirm the formation of high symmetry cubic structure with Pm-3m space group. The Rietveld refinement was carried out on RT XRD data and the obtained structural parameters are a = b = c = 3.97563(6) Å and unit cell volume = 62.837 (2) Å3. Scanning Electron Microscopy (SEM) images show the uniform distribution of grains with some agglomerated nature. RT Raman spectroscopy reveals the main broad peak at 770 cm-1, related to the A1g mode, which confirms the formation of cubic (ABO3 perovskite) structure. The single symmetric electron paramagnetic resonance (EPR) line shape with g = 2.13985 observed in PBFW was identified to be due to Fe3+ ions.
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The SPoRT-WRF: Evaluating the Impact of NASA Datasets on Convective Forecasts
NASA Technical Reports Server (NTRS)
Zavodsky, Bradley; Case, Jonathan; Kozlowski, Danielle; Molthan, Andrew
2012-01-01
The Short-term Prediction Research and Transition Center (SPoRT) is a collaborative partnership between NASA and operational forecasting entities, including a number of National Weather Service offices. SPoRT transitions real-time NASA products and capabilities to its partners to address specific operational forecast challenges. One challenge that forecasters face is applying convection-allowing numerical models to predict mesoscale convective weather. In order to address this specific forecast challenge, SPoRT produces real-time mesoscale model forecasts using the Weather Research and Forecasting (WRF) model that includes unique NASA products and capabilities. Currently, the SPoRT configuration of the WRF model (SPoRT-WRF) incorporates the 4-km Land Information System (LIS) land surface data, 1-km SPoRT sea surface temperature analysis and 1-km Moderate resolution Imaging Spectroradiometer (MODIS) greenness vegetation fraction (GVF) analysis, and retrieved thermodynamic profiles from the Atmospheric Infrared Sounder (AIRS). The LIS, SST, and GVF data are all integrated into the SPoRT-WRF through adjustments to the initial and boundary conditions, and the AIRS data are assimilated into a 9-hour SPoRT WRF forecast each day at 0900 UTC. This study dissects the overall impact of the NASA datasets and the individual surface and atmospheric component datasets on daily mesoscale forecasts. A case study covering the super tornado outbreak across the Ce ntral and Southeastern United States during 25-27 April 2011 is examined. Three different forecasts are analyzed including the SPoRT-WRF (NASA surface and atmospheric data), the SPoRT WRF without AIRS (NASA surface data only), and the operational National Severe Storms Laboratory (NSSL) WRF (control with no NASA data). The forecasts are compared qualitatively by examining simulated versus observed radar reflectivity. Differences between the simulated reflectivity are further investigated using convective parameters along
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Variance to mean ratio, R(t), for poisson processes on phylogenetic trees.
Goldman, N
1994-09-01
The ratio of expected variance to mean, R(t), of numbers of DNA base substitutions for contemporary sequences related by a "star" phylogeny is widely seen as a measure of the adherence of the sequences' evolution to a Poisson process with a molecular clock, as predicted by the "neutral theory" of molecular evolution under certain conditions. A number of estimators of R(t) have been proposed, all predicted to have mean 1 and distributions based on the chi 2. Various genes have previously been analyzed and found to have values of R(t) far in excess of 1, calling into question important aspects of the neutral theory. In this paper, I use Monte Carlo simulation to show that the previously suggested means and distributions of estimators of R(t) are highly inaccurate. The analysis is applied to star phylogenies and to general phylogenetic trees, and well-known gene sequences are reanalyzed. For star phylogenies the results show that Kimura's estimators ("The Neutral Theory of Molecular Evolution," Cambridge Univ. Press, Cambridge, 1983) are unsatisfactory for statistical testing of R(t), but confirm the accuracy of Bulmer's correction factor (Genetics 123: 615-619, 1989). For all three nonstar phylogenies studied, attained values of all three estimators of R(t), although larger than 1, are within their true confidence limits under simple Poisson process models. This shows that lineage effects can be responsible for high estimates of R(t), restoring some limited confidence in the molecular clock and showing that the distinction between lineage and molecular clock effects is vital.(ABSTRACT TRUNCATED AT 250 WORDS)
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RT-PCR for detection of all seven genotypes of Lyssavirus genus.
Vázquez-Morón, S; Avellón, A; Echevarría, J E
2006-08-01
The Lyssavirus genus includes seven species or genotypes named 1-7. Rabies genotypes correlate with geographical distribution and specific hosts. Co-circulation of different lyssaviruses, imported cases, and the presence of unknown viruses, such as Aravan, Khujand, Irkut and West Caucasian Bat Virus, make it necessary to use generic methods able to detect all lyssaviruses. Primer sequences were chosen from conserved regions in all genotypes in order to optimise a generic RT-PCR. Serial dilutions of 12 RNA extracts from all seven Lyssavirus genotypes were examined to compare the sensitivity of the RT-PCR standardised in this study with a published RT-PCR optimised for EBLV1 detection and capable of amplifying RNA from all seven lyssaviruses. All seven genotypes were detected by both RT-PCRs, however, the sensitivity was higher with the new version of the test. Twenty samples submitted for rabies diagnosis were tested by the new RT-PCR. Eight out of 20 samples from six dogs, one horse and one bat were found positive, in agreement with immunofluorescence results. Seven samples from terrestrial mammals were genotype 1 and one from a bat was genotype 5. In conclusion, this method can be used to complement immunofluorescence for the diagnosis of rabies, enabling the detection of unexpected lyssaviruses during rabies surveillance.
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Evaluation of the RT-LAMP and LAMP methods for detection of Mycobacterium tuberculosis.
Wu, Dandan; Kang, Jiwen; Li, Baosheng; Sun, Dianxing
2018-05-01
The current methods for detecting Mycobacterium tuberculosis (Mtb) are not clinically optimal. Standard culture methods (SCMs) are slow, costly, or unreliable, and loop-mediated isothermal amplification (LAMP) cannot differentiate live Mtb. This study compared reverse transcription (RT)-LAMP, LAMP, and an SCM for detecting Mtb. A first experiment tested the sensitivity and specificity of primers for 9 species of Mycobacterium (H37Rv, M. intracellulare, M. marinum, M. kansasii, M. avium, M. flavescens, M. smegmatis, M. fortuitum, and M. chelonae); and 3 non-Mycobacterium species (Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae). A second experiment tested sputum specimens for the presence of Mtb, from 100 patients with tuberculosis (clinical) and 22 from patients without tuberculosis (control), using Roche solid culture (SCM), LAMP, and RT-LAMP. In the clinical samples. The rates of positivity for Mtb of the SCM, LAMP, and RT-LAMP methods were 88%, 92%, and 100%, respectively. The difference in detection rate was significant between RT-LAMP and SCM, but RT-LAMP and LAMP were comparable. In the control group, the detection rates were nil for all three methods. The specificities of the methods were similar. The sensitivity of RT-LAMP was ~10-fold higher than that of LAMP for detecting Mtb. Unlike LAMP, RT-LAMP could identify viable bacteria, and was able to detect a single copy of Mtb. Among SCM, LAMP, and RT-LAMP, the latter is the most suitable for wide use in the lower-level hospitals and clinics of China for detecting Mtb in sputum samples. © 2017 Wiley Periodicals, Inc.
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Bester, Rachelle; Jooste, Anna E C; Maree, Hans J; Burger, Johan T
2012-09-27
Grapevine leafroll-associated virus 3 (GLRaV-3) is the main contributing agent of leafroll disease worldwide. Four of the six GLRaV-3 variant groups known have been found in South Africa, but their individual contribution to leafroll disease is unknown. In order to study the pathogenesis of leafroll disease, a sensitive and accurate diagnostic assay is required that can detect different variant groups of GLRaV-3. In this study, a one-step real-time RT-PCR, followed by high-resolution melting (HRM) curve analysis for the simultaneous detection and identification of GLRaV-3 variants of groups I, II, III and VI, was developed. A melting point confidence interval for each variant group was calculated to include at least 90% of all melting points observed. A multiplex RT-PCR protocol was developed to these four variant groups in order to assess the efficacy of the real-time RT-PCR HRM assay. A universal primer set for GLRaV-3 targeting the heat shock protein 70 homologue (Hsp70h) gene of GLRaV-3 was designed that is able to detect GLRaV-3 variant groups I, II, III and VI and differentiate between them with high-resolution melting curve analysis. The real-time RT-PCR HRM and the multiplex RT-PCR were optimized using 121 GLRaV-3 positive samples. Due to a considerable variation in melting profile observed within each GLRaV-3 group, a confidence interval of above 90% was calculated for each variant group, based on the range and distribution of melting points. The intervals of groups I and II could not be distinguished and a 95% joint confidence interval was calculated for simultaneous detection of group I and II variants. An additional primer pair targeting GLRaV-3 ORF1a was developed that can be used in a subsequent real-time RT-PCR HRM to differentiate between variants of groups I and II. Additionally, the multiplex RT-PCR successfully validated 94.64% of the infections detected with the real-time RT-PCR HRM. The real-time RT-PCR HRM provides a sensitive, automated and
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Development of RT-components for the M-3 Strawberry Harvesting Robot
NASA Astrophysics Data System (ADS)
Yamashita, Tomoki; Tanaka, Motomasa; Yamamoto, Satoshi; Hayashi, Shigehiko; Saito, Sadafumi; Sugano, Shigeki
We are now developing the strawberry harvest robot called “M-3” prototype robot system under the 4th urgent project of MAFF. In order to develop the control software of the M-3 robot more efficiently, we innovated the RT-middleware “OpenRTM-aist” software platform. In this system, we developed 9 kind of RT-Components (RTC): Robot task sequence player RTC, Proxy RTC for image processing software, DC motor controller RTC, Arm kinematics RTC, and so on. In this paper, we discuss advantages of RT-middleware developing system and problems about operating the RTC-configured robotic system by end-users.
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Melikian, George L; Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W Jeffrey; Kaufman, David; Towner, William; Troia-Cancio, Paolo V; Zolopa, Andrew; Robbins, Gregory K; Kagan, Ron; Israelski, Dennis; Shafer, Robert W
2012-05-01
Determining the phenotypic impacts of reverse transcriptase (RT) mutations on individual nucleoside RT inhibitors (NRTIs) has remained a statistical challenge because clinical NRTI-resistant HIV-1 isolates usually contain multiple mutations, often in complex patterns, complicating the task of determining the relative contribution of each mutation to HIV drug resistance. Furthermore, the NRTIs have highly variable dynamic susceptibility ranges, making it difficult to determine the relative effect of an RT mutation on susceptibility to different NRTIs. In this study, we analyzed 1,273 genotyped HIV-1 isolates for which phenotypic results were obtained using the PhenoSense assay (Monogram, South San Francisco, CA). We used a parsimonious feature selection algorithm, LASSO, to assess the possible contributions of 177 mutations that occurred in 10 or more isolates in our data set. We then used least-squares regression to quantify the impact of each LASSO-selected mutation on each NRTI. Our study provides a comprehensive view of the most common NRTI resistance mutations. Because our results were standardized, the study provides the first analysis that quantifies the relative phenotypic effects of NRTI resistance mutations on each of the NRTIs. In addition, the study contains new findings on the relative impacts of thymidine analog mutations (TAMs) on susceptibility to abacavir and tenofovir; the impacts of several known but incompletely characterized mutations, including E40F, V75T, Y115F, and K219R; and a tentative role in reduced NRTI susceptibility for K64H, a novel NRTI resistance mutation.
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Kenyon, Sara; Brocklehurst, Peter; Jones, David; Marlow, Neil; Salt, Alison; Taylor, David
2008-04-24
The Medical Research Council (MRC) ORACLE trial evaluated the use of co-amoxiclav 375 mg and/or erythromycin 250 mg in women presenting with preterm rupture of membranes (PROM) ORACLE I or in spontaneous preterm labour (SPL) ORACLE II using a factorial design. The results showed that for women with a singleton baby with PROM the prescription of erythromycin is associated with improvements in short term neonatal outcomes, although co-amoxiclav is associated with prolongation of pregnancy, a significantly higher rate of neonatal necrotising enterocolitis was found in these babies. Prescription of erythromycin is now established practice for women with PROM. For women with SPL antibiotics demonstrated no improvements in short term neonatal outcomes and are not recommended treatment. There is evidence that both these conditions are associated with subclinical infection so perinatal antibiotic administration may reduce the risk of later disabilities, including cerebral palsy, although the risk may be increased through exposure to inflammatory cytokines, so assessment of longer term functional and educational outcomes is appropriate. The MRC ORACLE Children's Study will follow up UK children at age 7 years born to 4809 women with PROM and the 4266 women with SPL enrolled in the earlier ORACLE trials. We will use a parental questionnaire including validated tools to assess disability and behaviour. We will collect the frequency of specific medical conditions: cerebral palsy, epilepsy, respiratory illness including asthma, diabetes, admission to hospital in last year and other diseases, as reported by parents. National standard test results will be collected to assess educational attainment at Key Stage 1 for children in England. This study is designed to investigate whether or not peripartum antibiotics improve health and disability for children at 7 years of age. The ORACLE Trial and Children Study is registered in the Current Controlled Trials registry. ISCRTN 52995660.
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Kenyon, Sara; Brocklehurst, Peter; Jones, David; Marlow, Neil; Salt, Alison; Taylor, David
2008-01-01
Background The Medical Research Council (MRC) ORACLE trial evaluated the use of co-amoxiclav 375 mg and/or erythromycin 250 mg in women presenting with preterm rupture of membranes (PROM) ORACLE I or in spontaneous preterm labour (SPL) ORACLE II using a factorial design. The results showed that for women with a singleton baby with PROM the prescription of erythromycin is associated with improvements in short term neonatal outcomes, although co-amoxiclav is associated with prolongation of pregnancy, a significantly higher rate of neonatal necrotising enterocolitis was found in these babies. Prescription of erythromycin is now established practice for women with PROM. For women with SPL antibiotics demonstrated no improvements in short term neonatal outcomes and are not recommended treatment. There is evidence that both these conditions are associated with subclinical infection so perinatal antibiotic administration may reduce the risk of later disabilities, including cerebral palsy, although the risk may be increased through exposure to inflammatory cytokines, so assessment of longer term functional and educational outcomes is appropriate. Methods The MRC ORACLE Children's Study will follow up UK children at age 7 years born to 4809 women with PROM and the 4266 women with SPL enrolled in the earlier ORACLE trials. We will use a parental questionnaire including validated tools to assess disability and behaviour. We will collect the frequency of specific medical conditions: cerebral palsy, epilepsy, respiratory illness including asthma, diabetes, admission to hospital in last year and other diseases, as reported by parents. National standard test results will be collected to assess educational attainment at Key Stage 1 for children in England. Discussion This study is designed to investigate whether or not peripartum antibiotics improve health and disability for children at 7 years of age. Trial registration The ORACLE Trial and Children Study is registered in the
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Potential Utility of the Real-Time TMPA-RT Precipitation Estimates in Streamflow Prediction
NASA Technical Reports Server (NTRS)
Su, Fengge; Gao, Huilin; Huffman, George J.; Lettenmaier, Dennis P.
2010-01-01
We investigate the potential utility of the real-time Tropical Rainfall Measuring Mission (TRMM) Multi-satellite Precipitation Analysis (TMPA-RT) data for streamflow prediction, both through direct comparisons of TMPA-RT estimates with a gridded gauge product, and through evaluation of streamflow simulations over four tributaries of La Plata Basin (LPB) in South America using the two precipitation products. Our assessments indicate that the relative accuracy and the hydrologic performance of TMPA-RT-based streamflow simulations generally improved after February 2005. The improvements in TMPA-RT since 2005 are closely related to upgrades in the TMPA-RT algorithm in early February, 2005 which include use of additional microwave sensors (AMSR-E and AMSU-B) and implementation of different calibration schemes. Our work suggests considerable potential for hydrologic prediction using purely satellite-derived precipitation estimates (no adjustments by in situ gauges) in parts of the globe where in situ observations are sparse.
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Modeling qRT-PCR dynamics with application to cancer biomarker quantification.
Chervoneva, Inna; Freydin, Boris; Hyslop, Terry; Waldman, Scott A
2017-01-01
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used for molecular diagnostics and evaluating prognosis in cancer. The utility of mRNA expression biomarkers relies heavily on the accuracy and precision of quantification, which is still challenging for low abundance transcripts. The critical step for quantification is accurate estimation of efficiency needed for computing a relative qRT-PCR expression. We propose a new approach to estimating qRT-PCR efficiency based on modeling dynamics of polymerase chain reaction amplification. In contrast, only models for fluorescence intensity as a function of polymerase chain reaction cycle have been used so far for quantification. The dynamics of qRT-PCR efficiency is modeled using an ordinary differential equation model, and the fitted ordinary differential equation model is used to obtain effective polymerase chain reaction efficiency estimates needed for efficiency-adjusted quantification. The proposed new qRT-PCR efficiency estimates were used to quantify GUCY2C (Guanylate Cyclase 2C) mRNA expression in the blood of colorectal cancer patients. Time to recurrence and GUCY2C expression ratios were analyzed in a joint model for survival and longitudinal outcomes. The joint model with GUCY2C quantified using the proposed polymerase chain reaction efficiency estimates provided clinically meaningful results for association between time to recurrence and longitudinal trends in GUCY2C expression.
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Randomized, clinical trial of RT001: Early signals of efficacy in Friedreich's ataxia.
Zesiewicz, Theresa; Heerinckx, Frederic; De Jager, Robert; Omidvar, Omid; Kilpatrick, Marcus; Shaw, Jessica; Shchepinov, Mikhail S
2018-04-06
RT001 is a deuterated ethyl linoleate that inhibits lipid peroxidation and is hypothesized to reduce cellular damage and recover mitochondrial function in degenerative diseases such as Friedreich's ataxia. To evaluate the safety, pharmacokinetics, and preliminary efficacy of RT001 in Friedreich's ataxia patients. We conducted a phase I/II double-blind, comparator-controlled trial with 2 doses of RT001 in Friedreich's ataxia patients (9 subjects each cohort). Subjects were randomized 2:1 to receive either RT001 (1.8 or 9.0 g/day), or a matching dose of nondeuterated ethyl linoleate as comparator for 28 days. The primary endpoints were safety, tolerability, and pharmacokinetic analysis. Secondary endpoints included cardiopulmonary exercise testing and timed 25-foot walk. Nineteen patients enrolled in the trial, and 18 completed all safety and efficacy measurements. RT001 was found to be safe and tolerable, with plasma levels approaching saturation by 28 days. One subject with a low body mass index experienced steatorrhea taking a high dose and discontinued the study. Deuterated arachidonic acid (a brain-penetrant metabolite of RT001) was found to be present in plasma on day 28. There was an improvement in peak workload in the drug group compared to placebo (0.16 watts/kg; P = 0.008), as well as an improvement trend in peak oxygen consumption (change of 0.16 L/min; P = 0.116), and in stride speed (P = 0.15). RT001 was found to be safe and tolerable over 28 days, and improved peak workload. Further research into the effect of RT001 in Friedreich's ataxia is warranted. © 2018 International Parkinson and Movement Disorder Society. © 2018 International Parkinson and Movement Disorder Society.
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Klungthong, Chonticha; Manasatienkij, Wudtichai; Phonpakobsin, Thipwipha; Chinnawirotpisan, Piyawan; Rodpradit, Prinyada; Hussem, Kittinun; Thaisomboonsuk, Butsaya; Ong-ajchaowlerd, Prapapun; Nisalak, Ananda; Kalayanarooj, Siripen; Buddhari, Darunee; Gibbons, Robert V; Jarman, Richard G; Yoon, In-Kyu; Fernandez, Stefan
2015-02-01
AFRIMS longitudinal dengue surveillance in Thailand depends on the nested RT-PCR and the dengue IgM/IgG ELISA. To examine and improve the sensitivity of the nested RT-PCR using a panel of archived samples collected during dengue surveillance. A retrospective analysis of 16,454 dengue IgM/IgG ELISA positive cases collected between 2000 and 2013 was done to investigate the sensitivity of the nested RT-PCR. From these cases, 318 acute serum specimens or extracted RNA, previously found to be negative by the nested RT-PCR, were tested using TaqMan real-time RT-PCR (TaqMan rRT-PCR). To improve the sensitivity of nested RT-PCR, we designed a new primer based on nucleotide sequences from contemporary strains found to be positive by the TaqMan rRT-PCR. Sensitivity of the new nested PCR was calculated using a panel of 87 samples collected during 2011-2013. The percentage of dengue IgM/IgG ELISA positive cases that were negative by the nested RT-PCR varied from 17% to 42% for all serotypes depending on the year. Using TaqMan rRT-PCR, dengue RNA was detected in 194 (61%) of the 318 acute sera or extracted RNA previously found to be negative by the nested RT-PCR. The newly designed DENV-1 specific primer increased the sensitivity of DENV-1 detection by the nested RT-PCR from 48% to 88%, and of all 4 serotypes from 73% to 87%. These findings demonstrate the impact of genetic diversity and signal erosion on the sensitivity of PCR-based methods. Published by Elsevier B.V.
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US geographic distribution of rt-PA utilization by hospital for acute ischemic stroke.
Kleindorfer, Dawn; Xu, Yingying; Moomaw, Charles J; Khatri, Pooja; Adeoye, Opeolu; Hornung, Richard
2009-11-01
Previously, we have estimated US national rates of recombinant tissue plasminogen activator (rt-PA) use to be 1.8% to 3.0% of all ischemic stroke patients. However, we hypothesized that the rate of rt-PA use may vary widely depending on regional variation, and that a large percentage of the US population likely does not have access to hospitals using rt-PA regularly. We describe the US geographic distribution of hospitals using rt-PA for acute ischemic stroke. This analysis used the MEDPAR database, which is a claims-based dataset that contains every fee-for-service Medicare-eligible hospital discharge in the US. Cases potentially eligible for rt-PA treatment based on diagnosis were defined as those with a hospital DRG code of 14, 15, or 559, and that also had an ICD-9 code of 433, 434, or 436. Thrombolysis use was defined as an ICD-9 code of 99.1. Study interval was July 1, 2005 to June 30, 2007. Hospital locations were mapped using ArcView software; population densities and regions of the US are based on US Census 2000. There were 4750 hospitals in the MEDPAR database, which included 495 186 ischemic stroke admissions during the study period. Of these hospitals, 64% had no reported treatments with rt-PA for ischemic stroke, and 0.9% reported >10% treatment rates within the MEDPAR dataset. Bed size, rural or underserved designation, and population density were significantly associated with reported rt-PA treatment rates, and remained significant in the multivariable regression. Approximately 162 million US citizens reside in counties containing a hospital reporting a >or=2.4% treatment rate within the MEDPAR dataset. We report the first description of US hospital rt-PA treatment rates by hospital. Unfortunately, we found that 64% of US hospitals did not report giving rt-PA at all within the MEDPAR database within a 2-year period. These tended to be hospitals that were smaller (average bed size of 95), located in less densely populated areas, or located in the
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Pancioli, Arthur M; Broderick, Joseph; Brott, Thomas; Tomsick, Thomas; Khoury, Jane; Bean, Judy; del Zoppo, Gregory; Kleindorfer, Dawn; Woo, Daniel; Khatri, Pooja; Castaldo, John; Frey, James; Gebel, James; Kasner, Scott; Kidwell, Chelsea; Kwiatkowski, Thomas; Libman, Richard; Mackenzie, Richard; Scott, Phillip; Starkman, Sidney; Thurman, R Jason
2008-12-01
Multiple approaches are being studied to enhance the rate of thrombolysis for acute ischemic stroke. Treatment of myocardial infarction with a combination of a reduced-dose fibrinolytic agent and a glycoprotein (GP) IIb/IIIa receptor antagonist has been shown to improve the rate of recanalization versus fibrinolysis alone. The combined approach to lysis utilizing eptifibatide and recombinant tissue-type plasminogen activator (rt-PA) (CLEAR) stroke trial assessed the safety of treating acute ischemic stroke patients within 3 hours of symptom onset with this combination. The CLEAR trial was a National Institutes of Health/National Institute of Neurological Disorders and Stroke-funded multicenter, double-blind, randomized, dose-escalation and safety study. Patients were randomized 3:1 to either low-dose rt-PA (tier 1=0.3 mg/kg, tier 2=0.45 mg/kg) plus eptifibatide (75 microg/kg bolus followed by 0.75 microg/kg per min infusion for 2 hours) or standard-dose rt-PA (0.9 mg/kg). The primary safety end point was the incidence of symptomatic intracerebral hemorrhage within 36 hours. Secondary analyses were performed regarding clinical efficacy. Ninety-four patients (40 in tier 1 and 54 in tier 2) were enrolled. The combination group of the 2 dose tiers (n=69) had a median age of 71 years and a median baseline National Institutes of Health Stroke Scale (NIHSS) score of 14, and the standard-dose rt-PA group (n=25) had a median age of 61 years and a median baseline NIHSS score of 10 (P=0.01 for NIHSS score). Fifty-two (75%) of the combination treatment group and 24 (96%) of the standard treatment group had a baseline modified Rankin scale score of 0 (P=0.04). There was 1 (1.4%; 95% CI, 0% to 4.3%) symptomatic intracranial hemorrhage in the combination group and 2 (8.0%; 95% CI, 0% to 19.2%) in the rt-PA-only arm (P=0.17). During randomization in tier 2, a review by the independent data safety monitoring board demonstrated that the safety profile of combination therapy at the
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NASA Astrophysics Data System (ADS)
Lai, Xiaochun; Meng, Ling-Jian
2018-02-01
In this paper, we present simulation studies for the second-generation MRI compatible SPECT system, MRC-SPECT-II, based on an inverted compound eye (ICE) gamma camera concept. The MRC-SPECT-II system consists of a total of 1536 independent micro-pinhole-camera-elements (MCEs) distributed in a ring with an inner diameter of 6 cm. This system provides a FOV of 1 cm diameter and a peak geometrical efficiency of approximately 1.3% (the typical levels of 0.1%-0.01% found in modern pre-clinical SPECT instrumentations), while maintaining a sub-500 μm spatial resolution. Compared to the first-generation MRC-SPECT system (MRC-SPECT-I) (Cai 2014 Nucl. Instrum. Methods Phys. Res. A 734 147-51) developed in our lab, the MRC-SPECT-II system offers a similar resolution with dramatically improved sensitivity and greatly reduced physical dimension. The latter should allow the system to be placed inside most clinical and pre-clinical MRI scanners for high-performance simultaneous MRI and SPECT imaging.
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SPoRT - An End-to-End R2O Activity
NASA Technical Reports Server (NTRS)
Jedlovec, Gary J.
2009-01-01
Established in 2002 to demonstrate the weather and forecasting application of real-time EOS measurements, the Short-term Prediction Research and Transition (SPoRT) program has grown to be an end-to-end research to operations activity focused on the use of advanced NASA modeling and data assimilation approaches, nowcasting techniques, and unique high-resolution multispectral observational data applications from EOS satellites to improve short-term weather forecasts on a regional and local scale. SPoRT currently partners with several universities and other government agencies for access to real-time data and products, and works collaboratively with them and operational end users at 13 WFOs to develop and test the new products and capabilities in a "test-bed" mode. The test-bed simulates key aspects of the operational environment without putting constraints on the forecaster workload. Products and capabilities which show utility in the test-bed environment are then transitioned experimentally into the operational environment for further evaluation and assessment. SPoRT focuses on a suite of data and products from MODIS, AMSR-E, and AIRS on the NASA Terra and Aqua satellites, and total lightning measurements from ground-based networks. Some of the observations are assimilated into or used with various versions of the WRF model to provide supplemental forecast guidance to operational end users. SPoRT is enhancing partnerships with NOAA / NESDIS for new product development and data access to exploit the remote sensing capabilities of instruments on the NPOESS satellites to address short term weather forecasting problems. The VIIRS and CrIS instruments on the NPP and follow-on NPOESS satellites provide similar observing capabilities to the MODIS and AIRS instruments on Terra and Aqua. SPoRT will be transitioning existing and new capabilities into the AWIIPS II environment to continue the continuity of its activities.
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NASA Technical Reports Server (NTRS)
Zavodsky, Bradley
2012-01-01
The Short-term Prediction Research and Transition (SPoRT) Center located at NASA Marshall Space Flight Center has been conducting testbed activities aimed at transitioning satellite products to National Weather Service operational end users for the last 10 years. SPoRT is a NASA/NOAA funded project that has set the bar for transition of products to operational end users through a paradigm of understanding forecast challenges and forecaster needs, displaying products in end users decision support systems, actively assessing the operational impact of these products, and improving products based on forecaster feedback. Aiming for quality partnerships rather than a large quantity of data users, SPoRT has become a community leader in training operational forecasters on the use of up-and-coming satellite data through the use of legacy instruments and proxy data. Traditionally, SPoRT has supplied satellite imagery and products from NASA instruments such as the Moderate-resolution Imaging Spectroradiometer (MODIS) and the Atmospheric Infrared Sounder (AIRS). However, recently, SPoRT has been funded by the GOES-R and Joint Polar Satellite System (JPSS) Proving Grounds to accelerate the transition of selected imagery and products to help improve forecaster awareness of upcoming operational data from the Visible Infrared Imager Radiometer Suite (VIIRS), Cross-track Infrared Sounder (CrIS), Advanced Baseline Imager (ABI), and Geostationary Lightning Mapper (GLM). This presentation provides background on the SPoRT Center, the SPoRT paradigm, and some example products that SPoRT is excited to work with forecasters to evaluate.
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A DICOM-RT based ePR radiation therapy information system for managing brain tumor patients
NASA Astrophysics Data System (ADS)
Liu, Brent J.; Law, Maria; Huang, H. K.; Zee, C. S.; Chan, Lawrence
2005-04-01
The need for comprehensive clinical image data and relevant information in image-guided Radiation Therapy (RT) is becoming steadily apparent. Multiple standalone systems utilizing the most technological advancements in imaging, therapeutic radiation, and computerized treatment planning systems acquire key data during the RT treatment course of a patient. One example are patients treated for brain tumors of greater sizes and irregular shapes that utilize state-of-the-art RT technology to deliver pinpoint accurate radiation doses. One such system, the Cyberknife, is a radiation treatment system that utilizes image-guided information to control a multi-jointed, six degrees of freedom, robotic arm to deliver precise and required radiation dose to the tumor site of a cancer patient. The image-guided system is capable of tracking the lesion orientations with respect to the patient"s position throughout the treatment process. This is done by correlating live radiographic images with pre-operative, CT and MR imaging information to determine relative patient and tumor position repeatedly over the course of the treatment. The disparate and complex data generated by the Cyberknife system along with related data is scattered throughout the RT department compromising an efficient clinical workflow since the data crucial for a clinical decision may be time-consuming to retrieve, temporarily missing, or even lost. To address these shortcomings, the ACR-NEMA Standards Committee extended its DICOM (Digital Imaging & Communications in Medicine) Standard from Radiology to RT by ratifying seven DICOM RT objects starting in 1997. However, they are rarely used by the RT community in daily clinical operations. In the past, the research focus of an RT department has primarily been developing new protocols and devices to improve treatment process and outcomes of cancer patients with minimal effort dedicated to integration of imaging and information systems. Our research, tightly
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Ring trial 2016 for Bluetongue virus detection by real-time RT-PCR in France.
Sailleau, Corinne; Viarouge, Cyril; Breard, Emmanuel; Vitour, Damien; Zientara, Stephan
2017-05-01
Since the unexpected emergence of BTV-8 in Northern Europe and the incursion of BTV-8 and 1 in France in 2006-2007, molecular diagnosis has considerably evolved. Several real-time RT-PCR (rtRT-PCR) methods have been developed and published, and are currently being used in many countries across Europe for BTV detection and typing. In France, the national reference laboratory (NRL) for orbiviruses develops and validates 'ready-to-use' kits with private companies for viral RNA detection. The regional laboratories network that was set up to deal with a heavy demand for analyses has used these available kits. From 2007, ring tests were organized to monitor the performance of the French laboratories. This study presents the results of 63 regional laboratories in the ring trial organized in 2016. Blood samples were sent to the laboratories. Participants were asked to use the rtRT-PCR methods in place in their laboratory, for detection of all BTV serotypes and specifically BTV-8. The French regional laboratories are able to detect and genotype BTV in affected animals. Despite the use of several methods (i.e. RNA extraction and different commercial rtRT-PCRs), the network is homogeneous. The ring trial demonstrated that the French regional veterinary laboratories have reliable and robust BTV diagnostic tools for BTV genome detection.
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PpRT1: the first complete gypsy-like retrotransposon isolated in Pinus pinaster.
Rocheta, Margarida; Cordeiro, Jorge; Oliveira, M; Miguel, Célia
2007-02-01
We have isolated and characterized a complete retrotransposon sequence, named PpRT1, from the genome of Pinus pinaster. PpRT1 is 5,966 bp long and is closely related to IFG7 gypsy retrotransposon from Pinus radiata. The long terminal repeats (LTRs) have 333 bp each and show a 5.4% sequence divergence between them. In addition to the characteristic polypurine tract (PPT) and the primer binding site (PBS), PpRT1 carries internal regions with homology to retroviral genes gag and pol. The pol region contains sequence motifs related to the enzymes protease, reverse transcriptase, RNAseH and integrase in the same typical order known for Ty3/gypsy-like retrotransposons. PpRT1 was extended from an EST database sequence indicating that its transcription is occurring in pine tissues. Southern blot analyses indicate however, that PpRT1 is present in a unique or a low number of copies in the P. pinaster genome. The differences in nucleotide sequence found between PpRT1 and IFG7 may explain the strikingly different copy number in the two pine species genome. Based on the homologies observed when comparing LTR region among different gypsy elements we propose that the highly conserved LTR regions may be useful to amplify other retrotransposon sequences of the same or close retrotransposon family.
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Comparative lytic efficacy of rt-PA and ultrasound in porcine versus human clots.
Huang, Shenwen; Shekhar, Himanshu; Holland, Christy K
2017-01-01
Porcine thrombi are employed routinely in preclinical models of ischemic stroke. In this study, we examined the differential lytic susceptibility of porcine and human whole blood clots with and without the use of microbubbles and ultrasound (US) as an adjuvant. An in vitro system equipped with time-lapse microscopy was used to evaluate recombinant tissue-plasminogen activator (rt-PA) lysis of porcine and human clots in the same species or cross species plasma. Human and porcine whole blood clots were treated with rt-PA and an echo contrast agent, Definity®, and exposed to intermittent 120 kHz US. The rt-PA lytic efficacy observed for porcine clots in porcine plasma was 22 times lower than for human clots in human plasma reported previously. Further, porcine clots did not exhibit increased lysis with adjuvant Definity® and US exposure. However, the rt-PA lytic susceptibility of the porcine clots in human plasma was similar to that of human clots in human plasma. Human clots perfused with porcine plasma did not respond to rt-PA, but adjuvant use of Definity® and US enhanced lysis. These results reveal considerable differences in lytic susceptibility of porcine clots and human clots to rt-PA. The use of porcine clot models to test new human thrombolytic therapies may necessitate modulation of coagulation and thrombolytic factors to reflect human hemostasis accurately.
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NASA SPoRT JPSS PG Activities in Alaska
NASA Technical Reports Server (NTRS)
Berndt, Emily; Molthan, Andrew; Fuell, Kevin; McGrath, Kevin; Smith, Matt; LaFontaine, Frank; Leroy, Anita; White, Kris
2018-01-01
SPoRT (NASA's Short-term Prediction Research and Transition Center) has collaboratively worked with Alaska WFOs (Weather Forecast Offices) to introduce RGB (Red/Green/Blue false color image) imagery to prepare for NOAA-20 (National Oceanic and Atmospheric Administration, JPSS (Joint Polar Satellite System) series-20 satellite) VIIRS (Visible Infrared Imaging Radiometer Suite) and improve forecasting aviation-related hazards. Last R2O/O2R (Research-to-Operations/Operations-to-Research) steps include incorporating NOAA-20 VIIRS in RGB suite and fully transitioning client-side RGB processing to GINA (Geographic Information Network of Alaska) and Alaska Region. Alaska Region WFOs have been part of the successful R2O/O2R story to assess the use of NESDIS (National Environmental Satellite, Data, and Information Service) Snowfall Rate product in operations. SPoRT introduced passive microwave rain rate and IMERG (Integrated Multi-satellitE Retrievals for GPM (Global Precipitation Measurement)) (IMERG) to Alaska WFOs for use in radar-void areas and assessing flooding potential. SPoRT has been part of the multi-organization collaborative effort to introduce Gridded NUCAPS (NOAA Unique CrIS/ATMS (Crosstrack Infrared Sounder/Advanced Technology Microwave Sounder) Processing System) to the Anchorage CWSU (Center Weather Service Unit) to assess Cold Air Aloft events, [and as part of NOAA's PG (Product Generation) effort].
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Time and Frequency Synchronization on the Virac Radio Telescope RT-32
NASA Astrophysics Data System (ADS)
Bezrukovs, V.
2016-04-01
One of the main research directions of Ventspils International Radio Astronomy Centre (VIRAC) is radio astronomy and astrophysics. The instrumental base for the centre comprised two fully steerable parabolic antennas, RT-16 and RT-32 (i.e. with the mirror diameter of 16 m and 32 m). After long reconstruction, radio telescope RT-32 is currently equipped with the receiving and data acquisition systems that allow observing in a wide frequency range from 327 MHz to 9 GHz. New Antenna Control Unit (ACU) allows stable, fast and precise pointing of antenna. Time and frequency distribution service provide 5, 10 and 100 MHz reference frequency, 1PPS signals and precise time stamps by NTP protocol and in the IRIG-B format by coaxial cable. For the radio astronomical observations, main requirement of spatially Very Long Base Line Interferometric (VLBI) observations for the observatory is precise synchronization of the received and sampled data and linking to the exact time stamps. During October 2015, radio telescope RT-32 performance was tested in several successful VLBI experiments. The obtained results confirm the efficiency of the chosen methods of synchronization and the ability to reproduce them on similar antennas.
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Zhang, Jianhua; Nie, Xianzhou; Boquel, Sébastien; Al-Daoud, Fadi; Pelletier, Yvan
2015-12-01
The sensitivity of reverse transcription-polymerase chain reaction (RT-PCR) for virus detection is influenced by many factors such as specificity of primers and quality of templates. These factors become extremely important for successful detection when virus concentration is low. Total RNA isolated from Potato virus Y (PVY)-infected potato plants using the sodium sulfite RNA isolation method or RNeasy plant mini kit contains a high proportion of host RNA and may also contain trace amount of phenolic and polysaccharide residues, which may inhibit RT-PCR. The goal of this study was to enhance the sensitivity of PVY detection by reducing host RNA in the extract by differential centrifugation followed by extraction using an RNeasy mini kit (DCR method). One-step RT-PCR had relatively low amplification efficiency for PVY RNA when a high proportion of plant RNA was present. SYBR Green-based real time RT-PCR showed that the RNA isolated by the DCR method had a higher cycle threshold value (Ct) for the elongation factor 1-α mRNA (Ef1α) of potato than the Ct value of the RNA extracted using the RNeasy plant mini kit, indicating that the DCR method significantly reduced the proportion of potato RNA in the extract. The detectable amount of RNA extracted using the DCR method was <0.001ng when plant sap from 10 PVY-infected and PVY-free potato leaflets in a 1.5:100 fresh weight ratio was extracted, compared with 0.01 and 0.02ng of RNA using the RNeasy plant mini kit and sodium sulfite RNA isolation methods, respectively. Copyright © 2015. Published by Elsevier B.V.
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Krych, Łukasz; Kot, Witold; Bendtsen, Katja M B; Hansen, Axel K; Vogensen, Finn K; Nielsen, Dennis S
2018-01-01
The Dextran Sulfate Sodium (DSS) induced colitis mouse model is commonly used to investigate human inflammatory bowel disease (IBD). Nucleic acid extracts originating from these animals are often contaminated with DSS, which is a strong inhibitor of many enzymatic based molecular biology reactions including PCR and reverse-transcription (RT). Methods for removing DSS from nucleic acids extracts exist for RNA, but no effective protocol for DNA or cDNA is currently available. However, spermine has previously been shown to be an effective agent for counteracting DSS inhibition of polynucleotide kinase, which led to the hypothesis, that spermine could be used to counteract DSS inhibition of PCR and RT. We investigated the means of adding spermine in an adequate concentration to PCR based protocols (including qPCR, two-step RT-qPCR, and amplicon sequencing library preparation) to remove DSS inhibition. Within the range up to 0.01g/L, spermine can be added to PCR/qPCR or RT prophylactically without a significant reduction of reaction efficiency. Addition of spermine at the concentration of 0.08g/L can be used to recover qualitative PCR signal inhibited by DSS in concentrations up to 0.32g/L. For optimal quantitative analysis, the concentration of spermine requires fine adjustment. Hence, we present here a simple fluorometric based method for adjusting the concentration of spermine ensuring an optimal efficiency of the reaction exposed to an unknown concentration of DSS. In conclusion, we demonstrate a cost effective and easy method to counteract DSS inhibition in PCR and two-step RT-qPCR. Fixed or fine-tuned concentrations of spermine can be administered depending on the qualitative or quantitative character of the analysis. Copyright © 2017 Elsevier B.V. All rights reserved.
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An Overview of NASA SPoRT GOES-R JPSS Proving Ground Testbed Activities
NASA Technical Reports Server (NTRS)
Berndt, Emily; Stano, Geoffrey; Fuell, Kevin; Leroy, Anita; Mcgrath, Kevin; Molthan, Andrew; Schultz, Lori; Smith, Matthew; White, Kris; Schultz, Christopher;
2017-01-01
The Short-term Prediction Research and Transition (SPoRT) Center is funded by NASA's Earth Science Division and NOAA's JPSS and GOES-R Proving Grounds to transition satellite products and capabilities to the NWS to improve short term (0-48 hr) forecasts on a regional and local scale. SPoRT currently collaborates with 30+ NWS WFOs (at least one in each NWS region) and 5 National Centers/Testbeds. SPoRT matches user-identified forecast challenges to specific products, providing access to these data in AWIPS through new plug-in development, and generating applications-based training to use the products for their needs (R20). Upon transition, SPoRT collaborates with the user to assess the product impact in a real-world environment for feedback to product developers (O2R) and to benefit their peers.
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Single-Cell RT-PCR in Microfluidic Droplets with Integrated Chemical Lysis.
Kim, Samuel C; Clark, Iain C; Shahi, Payam; Abate, Adam R
2018-01-16
Droplet microfluidics can identify and sort cells using digital reverse transcription polymerase chain reaction (RT-PCR) signals from individual cells. However, current methods require multiple microfabricated devices for enzymatic cell lysis and PCR reagent addition, making the process complex and prone to failure. Here, we describe a new approach that integrates all components into a single device. The method enables controlled exposure of isolated single cells to a high pH buffer, which lyses cells and inactivates reaction inhibitors but can be instantly neutralized with RT-PCR buffer. Using our chemical lysis approach, we distinguish individual cells' gene expression with data quality equivalent to more complex two-step workflows. Our system accepts cells and produces droplets ready for amplification, making single-cell droplet RT-PCR faster and more reliable.
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Structure of novel rat major histocompatibility complex class II genes RT1.Ha and Hb
DOE Office of Scientific and Technical Information (OSTI.GOV)
Arimura, Yutaka; Tang, Wei Ran; Koda, Toshiaki
1995-03-01
We have cloned the novel rat MHC class II genes, RT1.Ha and Hb, which are homologous to human HLA-DPA and DPB. RT1.Hb is a pseudogene, whereas RT1.Ha is apparently intact and may have transcriptional potential. In addition, with an RT1.Ha probe, we detecteda single Southern hybridization band in the genome of the mouse. This finding may aford an opportunity to analyze the HLA-DPA homologue in the mouse genome. 18 refs., 4 figs., 1 tab.
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The Cost-Effectiveness of Real-Time Continuous Glucose Monitoring (RT-CGM) in Type 2 Diabetes.
Fonda, Stephanie J; Graham, Claudia; Munakata, Julie; Powers, Julia M; Price, David; Vigersky, Robert A
2016-07-01
This analysis models the cost-effectiveness of real-time continuous glucose monitoring (RT-CGM) using evidence from a randomized controlled trial (RCT) that demonstrated RT-CGM reduced A1C, for up to 9 months after using the technology, among patients with type 2 diabetes not on prandial insulin. RT-CGM was offered short-term and intermittently as a self-care tool to inform patients' behavior. The analyses projected lifetime clinical and economic outcomes for RT-CGM versus self-monitoring of blood glucose by fingerstick only. The base-case analysis was consistent with the RCT (RT-CGM for 2 weeks on/1 week off over 3 months). A scenario analysis simulated outcomes of an RT-CGM "refresher" after the active intervention of the RCT. Analyses used the IMS CORE Diabetes Model and were conducted from a US third-party payer perspective, including direct costs obtained from published sources and inflated to 2011 US dollars. Costs and health outcomes were discounted at 3% per annum. Life expectancy (LE) and quality-adjusted life expectancy (QALE) from RT-CGM were 0.10 and 0.07, with a cost of $653/patient over a lifetime. Incremental LE and QALE from a "refresher" were 0.14 and 0.10, with a cost of $1312/patient over a lifetime, and incremental cost-effectiveness ratios were $9319 and $13 030 per LY and QALY gained. RT-CGM, as a self-care tool, is a cost-effective disease management option in the US for people with type 2 diabetes not on prandial insulin. Repeated use of RT-CGM may result in additional cost-effectiveness. © 2016 Diabetes Technology Society.
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NASA Technical Reports Server (NTRS)
Lehmer, R.; Ingram, C.; Jovic, S.; Alderete, J.; Brown, D.; Carpenter, D.; LaForce, S.; Panda, R.; Walker, J.; Chaplin, P.;
2006-01-01
The Virtual Airspace Simulation Technology - Real-Time (VAST-RT) Project, an element cf NASA's Virtual Airspace Modeling and Simulation (VAMS) Project, has been developing a distributed simulation capability that supports an extensible and expandable real-time, human-in-the-loop airspace simulation environment. The VAST-RT system architecture is based on DoD High Level Architecture (HLA) and the VAST-RT HLA Toolbox, a common interface implementation that incorporates a number of novel design features. The scope of the initial VAST-RT integration activity (Capability 1) included the high-fidelity human-in-the-loop simulation facilities located at NASA/Ames Research Center and medium fidelity pseudo-piloted target generators, such as the Airspace Traffic Generator (ATG) being developed as part of VAST-RT, as well as other real-time tools. This capability has been demonstrated in a gate-to-gate simulation. VAST-RT's (Capability 2A) has been recently completed, and this paper will discuss the improved integration of the real-time assets into VAST-RT, including the development of tools to integrate data collected across the simulation environment into a single data set for the researcher. Current plans for the completion of the VAST-RT distributed simulation environment (Capability 2B) and its use to evaluate future airspace capacity enhancing concepts being developed by VAMS will be discussed. Additionally, the simulation environment's application to other airspace and airport research projects is addressed.
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Performance of nested RT-PCR on CSF for tuberculous meningitis diagnosis in HIV-infected patients.
Gualberto, F A S; Gonçalves, M G; Fukasawa, L O; Santos, A M Ramos Dos; Sacchi, C T; Harrison, L H; Boulware, D R; Vidal, J E
2017-10-01
Timely diagnosis of tuberculous meningitis (TBM) in patients with human immunodeficiency virus (HIV) infection remains a challenge. Despite the current scale-up of the Xpert® MTB/RIF assay, other molecular diagnostic tools are necessary, particularly in referral centres in low- and middle-income countries without Xpert testing. To determine the diagnostic performance of nested real-time polymerase chain reaction (nRT-PCR) in HIV-infected TBM patients categorised according to standardised clinical case definitions. Based on clinical, laboratory and imaging data, HIV-infected patients with suspected TBM were prospectively categorised as 'definite TBM', 'probable TBM', 'possible TBM' or 'not TBM'. We evaluated nRT-PCR sensitivity and specificity in diagnosing TBM among definite TBM cases, and among definite + probable TBM cases. Ninety-two participants were enrolled in the study. nRT-PCR sensitivity for definite TBM (n = 8) was 100% (95%CI 67-100) and 86% (95%CI 60-96) for both definite and probable TBM (n = 6). Assuming that 'not TBM' patients (n = 74) were true-negatives, nRT-PCR specificity was 100% (95%CI 95-100). The possible TBM group (n = 4) had no nRT-PCR positives. The nRT-PCR is a useful rule-in test for HIV-infected patients with TBM according to international consensus case definitions. As nRT-PCR cannot exclude TBM, studies comparing and combining nRT-PCR with other assays are necessary for a rule-out test.
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Comparative lytic efficacy of rt-PA and ultrasound in porcine versus human clots
Shekhar, Himanshu; Holland, Christy K.
2017-01-01
Introduction Porcine thrombi are employed routinely in preclinical models of ischemic stroke. In this study, we examined the differential lytic susceptibility of porcine and human whole blood clots with and without the use of microbubbles and ultrasound (US) as an adjuvant. Materials and methods An in vitro system equipped with time-lapse microscopy was used to evaluate recombinant tissue-plasminogen activator (rt-PA) lysis of porcine and human clots in the same species or cross species plasma. Human and porcine whole blood clots were treated with rt-PA and an echo contrast agent, Definity®, and exposed to intermittent 120 kHz US. Results and conclusions The rt-PA lytic efficacy observed for porcine clots in porcine plasma was 22 times lower than for human clots in human plasma reported previously. Further, porcine clots did not exhibit increased lysis with adjuvant Definity® and US exposure. However, the rt-PA lytic susceptibility of the porcine clots in human plasma was similar to that of human clots in human plasma. Human clots perfused with porcine plasma did not respond to rt-PA, but adjuvant use of Definity® and US enhanced lysis. These results reveal considerable differences in lytic susceptibility of porcine clots and human clots to rt-PA. The use of porcine clot models to test new human thrombolytic therapies may necessitate modulation of coagulation and thrombolytic factors to reflect human hemostasis accurately. PMID:28545055
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1977-12-01
related progress p reports concerning the DNA-sponsored effo rt s described herein. - ~~~ Submission of other pertinent informat ion of a related nature...Work Unit 06). 5 5. Atmospheric Chemical Sensitivity and Modeling Invesriga nons—M. Scheibe, MRC (Work Unit 09). 5 6. Low Energy Cross Sections for...Debris Metal Ions—R. Neynaber, D. Vroom . and l.A. Rutherford, IRT, Inc. (Work Unit 12). 5 7. E and F Region Rate Coefficients for Excited Positive
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A novel DANP-coupled hairpin RT-PCR for rapid detection of Chikungunya virus.
Chen, Huixin; Takei, Fumie; Koay, Evelyn Siew-Chuan; Nakatani, Kazuhiko; Chu, Justin Jang Hann
2013-03-01
Chikungunya has re-emerged as an important arboviral infection of global health significance. Because of lack of a vaccine and effective treatment, rapid diagnosis plays an important role in early clinical management of patients. In this study, we have developed a novel molecular diagnostic platform that ensures a rapid and cost-effective one-step RT-PCR assay, with high sensitivity and specificity, for the early detection of the Chikungunya virus (CHIKV). It uses 2,7-diamino-1,8-naphthyridine derivative (DANP)-labeled cytosine-bulge hairpin primers to amplify the nsP2 region of the CHIKV genome, followed by measurement of the fluorescence emitted from DANP-primer complexes after PCRs. The detection limit of our assay was 0.01 plaque-forming units per reaction of CHIKV. Furthermore, the HP-nsP2 primers were highly specific in detecting CHIKV, without any cross-reactivity with the panel of RNA viruses validated in this study. The feasibility of the DANP-coupled hairpin RT-PCR for clinical diagnosis was evaluated using clinical serum samples from CHIKV-infected patients, and the specificity and sensitivity were 100% (95% CI, 80.0% to 100%) and 95.5% (95% CI, 75.1% to 99.8%), respectively. These findings confirmed its potential as a point-of-care clinical molecular diagnostic assay for CHIKV in acute-phase patient serum samples. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.
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Zhou, Jiyun; Lu, Qin; Xu, Ruifeng; He, Yulan; Wang, Hongpeng
2017-08-29
Prediction of DNA-binding residue is important for understanding the protein-DNA recognition mechanism. Many computational methods have been proposed for the prediction, but most of them do not consider the relationships of evolutionary information between residues. In this paper, we first propose a novel residue encoding method, referred to as the Position Specific Score Matrix (PSSM) Relation Transformation (PSSM-RT), to encode residues by utilizing the relationships of evolutionary information between residues. PDNA-62 and PDNA-224 are used to evaluate PSSM-RT and two existing PSSM encoding methods by five-fold cross-validation. Performance evaluations indicate that PSSM-RT is more effective than previous methods. This validates the point that the relationship of evolutionary information between residues is indeed useful in DNA-binding residue prediction. An ensemble learning classifier (EL_PSSM-RT) is also proposed by combining ensemble learning model and PSSM-RT to better handle the imbalance between binding and non-binding residues in datasets. EL_PSSM-RT is evaluated by five-fold cross-validation using PDNA-62 and PDNA-224 as well as two independent datasets TS-72 and TS-61. Performance comparisons with existing predictors on the four datasets demonstrate that EL_PSSM-RT is the best-performing method among all the predicting methods with improvement between 0.02-0.07 for MCC, 4.18-21.47% for ST and 0.013-0.131 for AUC. Furthermore, we analyze the importance of the pair-relationships extracted by PSSM-RT and the results validates the usefulness of PSSM-RT for encoding DNA-binding residues. We propose a novel prediction method for the prediction of DNA-binding residue with the inclusion of relationship of evolutionary information and ensemble learning. Performance evaluation shows that the relationship of evolutionary information between residues is indeed useful in DNA-binding residue prediction and ensemble learning can be used to address the data imbalance
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Metabolic cooperation between co-cultured lung cancer cells and lung fibroblasts.
Koukourakis, Michael I; Kalamida, Dimitra; Mitrakas, Achilleas G; Liousia, Maria; Pouliliou, Stamatia; Sivridis, Efthimios; Giatromanolaki, Alexandra
2017-11-01
Cooperation of cancer cells with stromal cells, such as cancer-associated fibroblasts (CAFs), has been revealed as a mechanism sustaining cancer cell survival and growth. In the current study, we focus on the metabolic interactions of MRC5 lung fibroblasts with lung cancer cells (A549 and H1299) using co-culture experiments and studying changes of the metabolic protein expression profile and of their growth and migration abilities. Using western blotting, confocal microscopy and RT-PCR, we observed that in co-cultures MRC5 respond by upregulating pyruvate dehydrogenase (PDH) and the monocarboxylate transporter MCT1. In contrast, cancer cells increase the expression of glucose transporters (GLUT1), LDH5, PDH kinase and the levels of phosphorylated/inactivated pPDH. H1299 cells growing in the same culture medium with fibroblasts exhibit a 'metastasis-like' phenomenon by forming nests within the fibroblast area. LDH5 and pPDH were drastically upregulated in these nests. The growth rate of both MRC5 and cancer cells increased in co-cultures. Suppression of LDHA or PDK1 in cancer cells abrogates the stimulatory signal from cancer cells to fibroblasts. Incubation of MRC5 fibroblasts with lactate resulted in an increase of LDHB and of PDH expression. Silencing of PDH gene in fibroblasts, or silencing of PDK1 or LDHA gene in tumor cells, impedes cancer cell's migration ability. Overall, a metabolic cooperation between lung cancer cells and fibroblasts has been confirmed in the context of direct Warburg effect, thus the fibroblasts reinforce aerobic metabolism to support the intensified anaerobic glycolytic pathways exploited by cancer cells.
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NASA/SPoRt: GOES-R Activities in Support of Product Development, Management, and Training
NASA Technical Reports Server (NTRS)
Fuell, Kevin; Jedlovec, Gary; Molthan, Andrew; Stano, Geoffrey
2012-01-01
SPoRT is using current capabilities of MODIS and VIIRS, combined with current GOES (i.e. Hybrid Imagery) to demonstrate mesoscale capabilities of future ABI instrument. SPoRT is transitioning RGBs from EUMETSAT standard "recipes" to demonstrate a method to more efficiently handle the increase channels/frequency of ABI. Challenges for RGB production exist. Internal vs. external production, Bit depth needed, Adding quantitative information, etc. SPoRT forming group to address these issues. SPoRT is leading efforts on the application of total lightning in operations and to educate users of this new capability. Training in many forms is used to support testbed activities and is a key part to the transition process.
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Indriati, Dwi Wahyu; Kotaki, Tomohiro; Khairunisa, Siti Qamariyah; Witaningrum, Adiana Mutamsari; Matondang, Muhammad Qushai Yunifiar; Ueda, Shuhei; Nasronudin; Purnama, Asep; Kurniawan, Dwi; Kameoka, Masanori
2018-05-01
Human Immunodeficiency Virus (HIV) is still a major health issue in Indonesia. In recent years, the appearance of drug resistance-associated mutations has reduced the effectiveness of antiretroviral therapy (ART). We conducted genotypic studies, including the detection of drug resistance-associated mutations (from first-line regimen drugs), on HIV-1 genes derived from infected individuals in Maumere, West Nusa Tenggara. Maumere, a transit city in West Nusa Tenggara, which has a high HIV-1 transmission rate. We collected 60 peripheral blood samples from 53 ART-experienced and 7 ART-naive individuals at TC Hillers Hospital, Maumere between 2014 and 2015. The amplification and a sequencing analysis of pol genes encoding protease (the PR gene) and reverse transcriptase (the RT gene) as well as the viral env and gag genes were performed. HIV-1 subtyping and the detection of drug resistance-associated mutations were then conducted. Among 60 samples, 46 PR, 31 RT, 30 env, and 20 gag genes were successfully sequenced. The dominant HIV-1 subtype circulating in Maumere was CRF01_AE. Subtype B and recombinant viruses containing gene fragments of CRF01_AE, subtypes A, B, C, and/or G were also identified as minor populations. The major drug resistance-associated mutations, M184V, K103N, Y188L, and M230I, were found in the RT genes. However, no major drug resistance-associated mutations were detected in the PR genes. CRF01_AE was the major HIV-1 subtype prevalent in Maumere. The appearance of drug resistance-associated mutations found in the present study supports the necessity of monitoring the effectiveness of ART in Maumere. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
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ERIC Educational Resources Information Center
Yoshii, Rika; Milne, Alastair
1998-01-01
Describes DaRT, a computer assisted language-learning system for helping English-as-a-Second-Language students master English articles. DaRT uses a diagrammatic reasoning tool to present communicative contexts for exercises in choosing appropriate articles. This paper describes the development of DaRT and DaRT's system components and concludes…
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Inactivation conditions for human Norovirus measured by an in situ capture-qRT-PCR Method
USDA-ARS?s Scientific Manuscript database
Human noroviruses (HuNoVs) are the major cause of epidemic non-bacterial gastroenteritis. Due to the inability to cultivate HuNoVs, it has been a challenge to determine their infectivity. Quantitative real-time RT-PCR (qRT-PCR) is widely used in detecting HuNoVs. However, qRT-PCR only detects the...
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Investigating the Decision-Making of Response to Intervention (RtI) Teams within the School Setting
ERIC Educational Resources Information Center
Thur, Scott M.
2015-01-01
The purpose of this study was to measure decision-making influences within RtI teams. The study examined the factors that influence school personnel involved in three areas of RtI: determining which RtI measures and tools teams select and implement (i.e. Measures and Tools), evaluating the data-driven decisions that are made based on the…
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WE-H-207B-04: Strategies for Adaptive RT
DOE Office of Scientific and Technical Information (OSTI.GOV)
Green, O.
2016-06-15
In recent years, steady progress has been made towards the implementation of MRI in external beam radiation therapy for processes ranging from treatment simulation to in-room guidance. Novel procedures relying mostly on MR data are currently implemented in the clinic. This session will cover topics such as (a) commissioning and quality control of the MR in-room imagers and simulators specific to RT, (b) treatment planning requirements, constraints and challenges when dealing with various MR data, (c) quantification of organ motion with an emphasis on treatment delivery guidance, and (d) MR-driven strategies for adaptive RT workflows. The content of the sessionmore » was chosen to address both educational and practical key aspects of MR guidance. Learning Objectives: Good understanding of MR testing recommended for in-room MR imaging as well as image data validation for RT chain (e.g. image transfer, filtering for consistency, spatial accuracy, manipulation for task specific); Familiarity with MR-based planning procedures: motivation, core workflow requirements, current status, challenges; Overview of the current methods for the quantification of organ motion; Discussion on approaches for adaptive treatment planning and delivery. T. Stanescu - License agreement with Modus Medical Devices to develop a phantom for the quantification of MR image system-related distortions.; T. Stanescu, N/A.« less
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ULTRASOUND-ENHANCED rt-PA THROMBOLYSIS IN AN EX VIVO PORCINE CAROTID ARTERY MODEL
Hitchcock, Kathryn E.; Ivancevich, Nikolas M.; Haworth, Kevin J.; Caudell Stamper, Danielle N.; Vela, Deborah C.; Sutton, Jonathan T.; Pyne-Geithman, Gail J.; Holland, Christy K.
2014-01-01
Ultrasound is known to enhance recombinant tissue plasminogen activator (rt-PA) thrombolysis. In this study, occlusive porcine whole blood clots were placed in flowing plasma within living porcine carotid arteries. Ultrasonically induced stable cavitation was investigated as an adjuvant to rt-PA thrombolysis. Aged, retracted clots were exposed to plasma alone, plasma containing rt-PA (7.1 ± 3.8 μg/mL) or plasma with rt-PA and Definity® ultrasound contrast agent (0.79 ± 0.47 μL/mL) with and without 120-kHz continuous wave ultrasound at a peak-to-peak pressure amplitude of 0.44 MPa. An insonation scheme was formulated to promote and maximize stable cavitation activity by incorporating ultrasound quiescent periods that allowed for the inflow of Definity®-rich plasma. Cavitation was measured with a passive acoustic detector throughout thrombolytic treatment. Thrombolytic efficacy was measured by comparing clot mass before and after treatment. Average mass loss for clots exposed to rt-PA and Definity® without ultrasound (n = 7) was 34%, and with ultrasound (n = 6) was 83%, which constituted a significant difference (p < 0.0001). Without Definity® there was no thrombolytic enhancement by ultrasound exposure alone at this pressure amplitude (n = 5, p < 0.0001). In the low-oxygen environment of the ischemic artery, significant loss of endothelium occurred but no correlation was observed between arterial tissue damage and treatment type. Acoustic stable cavitation nucleated by an infusion of Definity® enhances rt-PA thrombolysis without apparent treatment-related damage in this ex vivo porcine carotid artery model. PMID:21723448
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Maga, Giovanni; Radi, Marco; Gerard, Marie-Aline; Botta, Maurizio; Ennifar, Eric
2010-01-01
HIV-1 reverse transcriptase (RT) inhibitors currently used in antiretroviral therapy can be divided into two classes: (i) nucleoside analog RT inhibitors (NRTIs), which compete with natural nucleoside substrates and act as terminators of proviral DNA synthesis, and (ii) non-nucleoside RT inhibitors (NNRTIs), which bind to a hydrophobic pocket close to the RT active site. In spite of the efficiency of NRTIs and NNRTIs, the rapid emergence of multidrug-resistant mutations requires the development of new RT inhibitors with an alternative mechanism of action. Recently, several studies reported the discovery of novel non-nucleoside inhibitors with a distinct mechanism of action. Unlike classical NNRTIs, they compete with the nucleotide substrate, thus forming a new class of RT inhibitors: nucleotide-competing RT inhibitors (NcRTIs). In this review, we discuss current progress in the understanding of the peculiar behavior of these compounds. PMID:21994659
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Mann, Steven; Jimenez, Alfonso; Steele, James; Domone, Sarah; Wade, Matthew; Beedie, Chris
2018-03-27
Many sedentary adults have high body fat along with low fitness, strength, and lean body mass (LBM) which are associated with poor health independently of body mass. Physical activity can aid in prevention, management, and treatment of numerous chronic conditions. The potential efficacy of resistance training (RT) in modifying risk factors for cardiovascular and metabolic disease is clear. However, RT is under researched in public health. We report community-based studies of RT in sedentary (Study 1), and overweight and pre-diabetic (Study 2) populations. Study 1 - A semi randomised trial design (48-weeks): Participants choosing either a fitness centre approach, and randomised to structured-exercise (STRUC, n = 107), or free/unstructured gym use (FREE, n = 110), or not, and randomised to physical-activity-counselling (PAC, n = 71) or a measurement only comparator (CONT, n = 76). Study 2 - A randomised wait list controlled trial (12-weeks): Patients were randomly assigned to; traditional-supervised-exercise (STRUC, n = 30), physical-activity-counselling (PAC, n = 23), either combined (COMB, n = 39), or a wait-list comparator (CONT, n = 54). Outcomes for both were BF mass (kg), LBM (kg), BF percentage (%), and strength. Study 1: One-way ANCOVA revealed significant between group effects for BF% and LBM, but not for BF mass or strength. Post hoc paired comparisons revealed significantly greater change in LBM for the STRUC group compared with the CONT group. Within group changes using 95%CIs revealed significant changes only in the STRUC group for both BF% (- 4.1 to - 0.9%) and LBM (0.1 to 4.5 kg), and in FREE (8.2 to 28.5 kg) and STRUC (5.9 to 26.0 kg) for strength. Study 2: One-way ANCOVA did not reveal significant between group effects for strength, BF%, BF mass, or LBM. For strength, 95%CIs revealed significant within group changes for the STRUC (2.4 to 14.1 kg) and COMB (3.7 to 15.0 kg) groups. Strength increased in both
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Li, Wanbo; Dive, Marc; Tamma, Nico; Michaux, Charles; Druet, Tom; Huijbers, Ivo J.; Isacke, Clare M.; Coppieters, Wouter; Georges, Michel; Charlier, Carole
2009-01-01
We herein describe the positional identification of a 2-bp deletion in the open reading frame of the MRC2 receptor causing the recessive Crooked Tail Syndrome in cattle. The resulting frame-shift reveals a premature stop codon that causes nonsense-mediated decay of the mutant messenger RNA, and the virtual absence of functional Endo180 protein in affected animals. Cases exhibit skeletal anomalies thought to result from impaired extracellular matrix remodeling during ossification, and as of yet unexplained muscular symptoms. We demonstrate that carrier status is very significantly associated with desired characteristics in the general population, including enhanced muscular development, and that the resulting heterozygote advantage caused a selective sweep which explains the unexpectedly high frequency (25%) of carriers in the Belgian Blue Cattle Breed. PMID:19779552
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Real-time RT-PCR, a necessary tool to support the diagnosis and surveillance of rotavirus in Mexico.
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.
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SPoRT's Participation in the GOES-R Proving Ground Activity
NASA Astrophysics Data System (ADS)
Jedlovec, G.; Fuell, K.; Smith, M. R.; Stano, G. T.; Molthan, A.
2011-12-01
The next generation geostationary satellite, GOES-R, will carry two new instruments with unique atmospheric and surface observing capabilities, the Advanced Baseline Imager (ABI) and the Geostationary Lightning Mapper (GLM), to study short-term weather processes. The ABI will bring enhanced multispectral observing capabilities with frequent refresh rates for regional and full disk coverage to geostationary orbit to address many existing and new forecast challenges. The GLM will, for the first time, provide the continuous monitoring of total lightning flashes over a hemispherical region from space. NOAA established the GOES-R Proving Ground activity several years ago to demonstrate the new capabilities of these instruments and to prepare forecasters for their day one use. Proving Ground partners work closely with algorithm developers and the end user community to develop and transition proxy data sets representing GOES-R observing capabilities. This close collaboration helps to maximize refine algorithms leading to the delivery of a product that effectively address a forecast challenge. The NASA Short-term Prediction Research and Transition (SPoRT) program has been a participant in the NOAA GOES-R Proving Ground activity by developing and disseminating selected GOES-R proxy products to collaborating WFOs and National Centers. Established in 2002 to demonstrate the weather and forecasting application of real-time EOS measurements, the SPoRT program has grown to be an end-to-end research to operations activity focused on the use of advanced NASA modeling and data assimilation approaches, nowcasting techniques, and unique high-resolution multispectral data from EOS satellites to improve short-term weather forecasts on a regional and local scale. Participation in the Proving Ground activities extends SPoRT's activities and taps its experience and expertise in diagnostic weather analysis, short-term weather forecasting, and the transition of research and experimental
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SPoRT's Participation in the GOES-R Proving Ground Activity
NASA Technical Reports Server (NTRS)
Jedlovec, Gary; Fuell, Kevin; Smith, Matthew; Stano, Geoffrey; Molthan, Andrew
2011-01-01
The next generation geostationary satellite, GOES-R, will carry two new instruments with unique atmospheric and surface observing capabilities, the Advanced Baseline Imager (ABI) and the Geostationary Lightning Mapper (GLM), to study short-term weather processes. The ABI will bring enhanced multispectral observing capabilities with frequent refresh rates for regional and full disk coverage to geostationary orbit to address many existing and new forecast challenges. The GLM will, for the first time, provide the continuous monitoring of total lightning flashes over a hemispherical region from space. NOAA established the GOES-R Proving Ground activity several years ago to demonstrate the new capabilities of these instruments and to prepare forecasters for their day one use. Proving Ground partners work closely with algorithm developers and the end user community to develop and transition proxy data sets representing GOES-R observing capabilities. This close collaboration helps to maximize refine algorithms leading to the delivery of a product that effectively address a forecast challenge. The NASA Short-term Prediction Research and Transition (SPoRT) program has been a participant in the NOAA GOES-R Proving Ground activity by developing and disseminating selected GOES-R proxy products to collaborating WFOs and National Centers. Established in 2002 to demonstrate the weather and forecasting application of real-time EOS measurements, the SPoRT program has grown to be an end-to-end research to operations activity focused on the use of advanced NASA modeling and data assimilation approaches, nowcasting techniques, and unique high-resolution multispectral data from EOS satellites to improve short-term weather forecasts on a regional and local scale. Participation in the Proving Ground activities extends SPoRT s activities and taps its experience and expertise in diagnostic weather analysis, short-term weather forecasting, and the transition of research and experimental
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Development of mRNA-specific RT-PCR for the detection of koi herpesvirus (KHV) replication stage.
Yuasa, Kei; Kurita, Jun; Kawana, Morihiko; Kiryu, Ikunari; Oseko, Norihisa; Sano, Motohiko
2012-08-13
An mRNA-specific reverse transcription (RT)-PCR primer set spanning the exon junction of a spliced putative terminase gene in the koi herpesvirus (KHV) was developed to detect the replicating stage of the virus. The proposed RT-PCR amplified a target gene from the RNA template, but not from a DNA template extracted from common carp brain (CCB) cells infected with KHV. In addition, the RT-PCR did not amplify the target gene of templates extracted from specific cell lines infected with either CyHV-1 or CyHV-2. RT-PCR detected mRNA from the scales of koi experimentally infected with KHV at 24 h post exposure (hpe). However, unlike conventional PCR, RT-PCR could not detect KHV DNA in fish at 0 hpe. The results indicate that the RT-PCR developed in this study is mRNA-specific and that the assay can detect the replicating stage of KHV from both fish and cultured cells infected with the virus.
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Dixit, Swarnita; Dubey, Ramesh Chand; Maheshwari, Dinesh Kumar; Seth, Prahlad Kishore; Bajpai, Vivek K
Strain RT1 was isolated from root nodules of Lens culinaris (a lentil) and characterized as Rhizobium etli (a Gram-negative soil-borne bacterium) by 16S rDNA sequencing and phylogenetic analysis. The signaling molecules produced by R. etli (RT1) were detected and identified by high-performance liquid chromatography coupled with mass spectrometry. The most abundant and biologically active N-acyl homoserine lactone molecules (3-oxo-C 8 -HSL and 3-OH-C 14 -HSL) were detected in the ethyl acetate extract of RT1. The biological role of 3-oxo-C 8 -HSL was evaluated in RT1. Bacterial motility and biofilm formation were affected or modified on increasing concentrations of 3-oxo-C 8 -HSL. Results confirmed the existence of cell communication in RT1 mediated by 3-oxo-C 8 -HSL, and positive correlations were found among quorum sensing, motility and biofilm formation in RT1. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.
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Making the Right Choice: Optimizing rt-PA and eptifibatide lysis, an in vitro study
Shaw, George J.; Meunier, Jason M.; Lindsell, Christopher J.; Pancioli, Arthur M.; Holland, Christy K.
2010-01-01
Introduction Recombinant tissue plasminogen activator (rt-PA) is the only FDA approved lytic therapy for acute ischemic stroke. However, there can be complications such as intra-cerebral hemorrhage. This has led to interest in adjuncts such as GP IIb-IIIa inhibitors. However, there is little data on combined therapies. Here, we measure clot lysis for rt-PA and eptifibatide in an in vitro human clot model, and determine the drug concentrations maximizing lysis. A pharmacokinetic model is used to compare drug concentrations expected in clinical trials with those used here. The hypothesis is that there is a range of rt-PA and eptifibatide concentrations that maximize in vitro clot lysis. Materials and Methods Whole blood clots were made from blood obtained from 28 volunteers, after appropriate institutional approval. Sample clots were exposed to rt-PA and eptifibatide in human fresh-frozen plasma; rt-PA concentrations were 0, 0.5, 1, and 3.15 μg/ml, and eptifibatide concentrations were 0, 0.63, 1.05, 1.26 and 2.31 μg/ml. All exposures were for 30 minutes at 37 C. Clot width was measured using a microscopic imaging technique and mean fractional clot loss (FCL) at 30 minutes was used to determine lytic efficacy. On average, 28 clots (range: 6-148) from 6 subjects (3-24) were used in each group. Results and Conclusions FCL for control clots was 14% (95% Confidence Interval: 13-15%). FCL was 58% (55-61%) for clots exposed to both drugs at all concentrations, except those at an rt-PA concentration of 3.15 μg/ml, and eptifibatide concentrations of 1.26 μg/ml (Epf) or 2.31 μg/ml. Here, FCL was 43% (36-51) and 35% (32-38) respectively. FCL is maximized at moderate rt-PA and eptifibatide concentration; these values may approximate the average concentrations used in some rt-PA and eptifibatide treatments. PMID:20813398
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Detection of canine distemper virus (CDV) through one step RT-PCR combined with nested PCR.
Kim, Y H; Cho, K W; Youn, H Y; Yoo, H S; Han, H R
2001-04-01
A one step reverse transcription PCR (RT-PCR) combined nested PCR was set up to increase efficiency in the diagnosis of canine distemper virus (CDV) infection after developement of nested PCR. Two PCR primer sets were designed based on the sequence of nucleocapsid gene of CDV Onderstepoort strain. One-step RT-PCR with the outer primer pair was revealed to detect 10(2) PFU/ml. The sensitivity was increased hundredfold using the one-step RT-PCR combined with the nested PCR. Specificity of the PCR was also confirmed using other related canine virus and peripheral blood mononuclear cells (PBMC) and body secretes of healthy dogs. Of the 51 blood samples from dogs clinically suspected of CD, 45 samples were revealed as positive by one-step RT-PCR combined with nested PCR. However, only 15 samples were identified as positive with a single one step RT-PCR. Therefore approximately 60% increase in the efficiency of the diagnosis was observed by the combined method. These results suggested that one step RT-PCR combined with nested PCR could be a sensitive, specific, and practical method for diagnosis of CDV infection.
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Conservation of purified protein derivative RT-23 tuberculin.
Fernández-Villar, A; Gorís, A; Otero, M; Chouciño, N; Vázquez, R; Muñoz, M J; Piñeiro, L
2004-07-01
Recommendations that purified protein derivative (PPD) RT-23 tuberculin should not be kept and used more than 24 to 48 hours after opening are rarely complied with. The aim of this study was to determine whether using PPD RT-23 tuberculin from vials that had been open for a longer time could affect results of tuberculin tests and whether the solution could become contaminated. A prospective cross-sectional study was carried out. Two tuberculin tests were simultaneously administered, one in each forearm, to adult contacts of patients with active tuberculosis, one test using a recently opened vial of tuberculin (control) and the other using tuberculin that had been opened a week before (first phase) or a month before (second phase) (study tuberculin). Leftover tuberculin from several of the vials was cultured. For the 127 patients in the first group (tuberculin opened 1 week), the mean (SD) diameter of the induration was 6.2 (6.9) mm for the study tuberculin and 6.3 (6.9) mm for the control (P=.3). For the 83 patients in the second group (tuberculin opened 1 month), the mean diameter of the induration was 5.5 (7.3) mm for the study tuberculin and 5.7 (7.3) mm for the control (P=.5). There were no differences between the number of positive tests found. None of the cultured tuberculins produced bacterial growth. PPD RT-23 tuberculin does not appear to lose potency or sterility when vials have been opened for a week or a month.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Patton, T; Du, K; Bayouth, J
Purpose: Ventilation change caused by radiation therapy (RT) can be predicted using four-dimensional computed tomography (4DCT) and image registration. This study tested the dependency of predicted post-RT ventilation on effort correction and pre-RT lung function. Methods: Pre-RT and 3 month post-RT 4DCT images were obtained for 13 patients. The 4DCT images were used to create ventilation maps using a deformable image registration based Jacobian expansion calculation. The post-RT ventilation maps were predicted in four different ways using the dose delivered, pre-RT ventilation, and effort correction. The pre-RT ventilation and effort correction were toggled to determine dependency. The four different predictedmore » ventilation maps were compared to the post-RT ventilation map calculated from image registration to establish the best prediction method. Gamma pass rates were used to compare the different maps with the criteria of 2mm distance-to-agreement and 6% ventilation difference. Paired t-tests of gamma pass rates were used to determine significant differences between the maps. Additional gamma pass rates were calculated using only voxels receiving over 20 Gy. Results: The predicted post-RT ventilation maps were in agreement with the actual post-RT maps in the following percentage of voxels averaged over all subjects: 71% with pre-RT ventilation and effort correction, 69% with no pre-RT ventilation and effort correction, 60% with pre-RT ventilation and no effort correction, and 58% with no pre-RT ventilation and no effort correction. When analyzing only voxels receiving over 20 Gy, the gamma pass rates were respectively 74%, 69%, 65%, and 55%. The prediction including both pre- RT ventilation and effort correction was the only prediction with significant improvement over using no prediction (p<0.02). Conclusion: Post-RT ventilation is best predicted using both pre-RT ventilation and effort correction. This is the only prediction that provided a significant
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Ultrasound-enhanced rt-PA thrombolysis in an ex vivo porcine carotid artery model.
Hitchcock, Kathryn E; Ivancevich, Nikolas M; Haworth, Kevin J; Caudell Stamper, Danielle N; Vela, Deborah C; Sutton, Jonathan T; Pyne-Geithman, Gail J; Holland, Christy K
2011-08-01
Ultrasound is known to enhance recombinant tissue plasminogen activator (rt-PA) thrombolysis. In this study, occlusive porcine whole blood clots were placed in flowing plasma within living porcine carotid arteries. Ultrasonically induced stable cavitation was investigated as an adjuvant to rt-PA thrombolysis. Aged, retracted clots were exposed to plasma alone, plasma containing rt-PA (7.1 ± 3.8 μg/mL) or plasma with rt-PA and Definity® ultrasound contrast agent (0.79 ± 0.47 μL/mL) with and without 120-kHz continuous wave ultrasound at a peak-to-peak pressure amplitude of 0.44 MPa. An insonation scheme was formulated to promote and maximize stable cavitation activity by incorporating ultrasound quiescent periods that allowed for the inflow of Definity®-rich plasma. Cavitation was measured with a passive acoustic detector throughout thrombolytic treatment. Thrombolytic efficacy was measured by comparing clot mass before and after treatment. Average mass loss for clots exposed to rt-PA and Definity® without ultrasound (n = 7) was 34%, and with ultrasound (n = 6) was 83%, which constituted a significant difference (p < 0.0001). Without Definity® there was no thrombolytic enhancement by ultrasound exposure alone at this pressure amplitude (n = 5, p < 0.0001). In the low-oxygen environment of the ischemic artery, significant loss of endothelium occurred but no correlation was observed between arterial tissue damage and treatment type. Acoustic stable cavitation nucleated by an infusion of Definity® enhances rt-PA thrombolysis without apparent treatment-related damage in this ex vivo porcine carotid artery model. Copyright © 2011. Published by Elsevier Inc.
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Evaluation of sensitivity of TaqMan RT-PCR for rubella virus detection in clinical specimens.
Okamoto, Kiyoko; Mori, Yoshio; Komagome, Rika; Nagano, Hideki; Miyoshi, Masahiro; Okano, Motohiko; Aoki, Yoko; Ogura, Atsushi; Hotta, Chiemi; Ogawa, Tomoko; Saikusa, Miwako; Kodama, Hiroe; Yasui, Yoshihiro; Minagawa, Hiroko; Kurata, Takako; Kanbayashi, Daiki; Kase, Tetsuo; Murata, Sachiko; Shirabe, Komei; Hamasaki, Mitsuhiro; Kato, Takashi; Otsuki, Noriyuki; Sakata, Masafumi; Komase, Katsuhiro; Takeda, Makoto
2016-07-01
An easy and reliable assay for detection of the rubella virus is required to strengthen rubella surveillance. Although a TaqMan RT-PCR assay for detection of the rubella virus has been established in Japan, its utility for diagnostic purposes has not been tested. To allow introduction of the TaqMan RT-PCR into the rubella surveillance system in Japan, the sensitivity of the assay was determined using representative strains for all genotypes and clinical specimens. The detection limits of the method for individual genotypes were examined using viral RNA extracted from 13 representative strains. The assay was also tested at 10 prefectural laboratories in Japan, designated as local reference laboratories for measles and rubella, to allow nationwide application of the assay. The detection limits and amplification efficiencies of the assay were similar among all the representative strains of the 13 genotypes. The TaqMan RT-PCR could detect approximately 90% of throat swab and urine samples taken up to 5days of illness. These samples were determined positive by a highly sensitive nested RT-PCR. The TaqMan RT-PCR could detect at least 10 pfu of rubella virus. Although the sensitivity was somewhat lower than that of the conventional nested RT-PCR, the TaqMan RT-PCR could be more practical to routine tests for rubella laboratory diagnosis and detection in view of the rapid response and reducing risks of contamination. Copyright © 2016 Elsevier B.V. All rights reserved.
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Lau, Katherine A; Wang, Bin; Miranda-Saksena, Monica; Boadle, Ross; Kamarulzaman, Adeeba; Ng, Kee-Peng; Saksena, Nitin K
2010-04-01
In Malaysia, co-circulation of CRF01_AE and subtype B has resulted in the emergence of the second generation derivative; CRF33_01B in approximately 20% of its HIV-1 infected individuals. Our objective was to identify possible biological advantages that CRF33_01B possesses over its progenitors. Biological and molecular comparisons of CRF33_01B against its parental subtypes clearly show that CRF33_01B replicated better in activated whole peripheral blood mononuclear cells (PBMCs) and CD4+ T-lymphocytes, but not monocyte-derived macrophages (MDMs). Also, its acquired fitness was greater than CRF01_AE but not subtype B. Moreover, CRF33_01B has higher rate of apoptotic cell death and syncytia induction compared to subtype B. These adaptive and survival abilities could have been acquired by CRF33_01B due to the incorporation of subtype B fragments into the gag-RT region of its full-length genome. Our studies confirm the previously held belief that HIV-1 strains may harbor enhanced biological fitness upon recombination. We therefore estimate a possible gradual replacement of the current predominance of CRF01_AE, as well as wider dissemination of CRF33_01B, together with the identification of other new CRF01_AE/B inter-subtype recombinants in Malaysia.
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Erectile dysfunction in COPD patients
Ure, Iyimser; Turan, Pakize Ayse
2015-01-01
Sexual dysfunction is a common problem in chronic obstructive pulmonary disease (COPD). We aimed to assess the presence of erectile dysfunction (ED) in COPD patients. Ninety-three outpatients who had been diagnosed as COPD and followed in Bolvadin State Hospital, Afyon, Turkey, were included in the study. All patients underwent pulmonary function tests and arterial blood gas analysis. They completed International Physical Activity Questionnaire (IPAQ), Medical Research Council (MRC) Dyspnea Scale, Short Form 36-item Scale (SF-36), and International Index of Erectile Function (IIEF) Questionnaire. The mean age of 10 (10.8%) mild, 46 (49.5%) moderate, 28 (30.1%) severe, and 9 (9.7%) very severe COPD patients was 61.4 ± 9.8 years. Varying degrees of ED were detected in 67.7% of COPD patients. All patients with hypoxemia had ED. IPAQ score and all SF-36 parameters were low in patients with ED, while MRC score was high. Forced expiratory volume in one second, forced vital capacity, partial pressure of oxygen in blood, oxygen (O2) saturation, IPAQ score, and role-physical parameters were statistically low in ED patients (p = 0.04, 0.02, <0.01, <0.01, 0.02, and 0.04, respectively); MRC score was statistically higher in patients with ED (p = 0.02). Patients with moderate and severe ED had statistically lower score of mental health (p < 0.01 and p = 0.02, respectively). There was a positive correlation between IIEF score and IPAQ scores (p < 0.01), MRC scores (p = 0.01), general health (p < 0.01), role-physical (p < 0.01), role-emotional (p < 0.01), physical functioning (p < 0.01), and mental health (p < 0.01) parameters in SF-36. ED is frequently seen in COPD patients. Hypoxemia, smoking, and limitation of physical activity are thought to be associated with ED in COPD as mechanisms. Quality of life and the functional capacity are negatively affected with the presence of ED. It is important for a physician to question the sexual functions in patients with COPD. The
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Erectile dysfunction in COPD patients.
Turan, Onur; Ure, Iyimser; Turan, Pakize Ayse
2016-02-01
Sexual dysfunction is a common problem in chronic obstructive pulmonary disease (COPD). We aimed to assess the presence of erectile dysfunction (ED) in COPD patients. Ninety-three outpatients who had been diagnosed as COPD and followed in Bolvadin State Hospital, Afyon, Turkey, were included in the study. All patients underwent pulmonary function tests and arterial blood gas analysis. They completed International Physical Activity Questionnaire (IPAQ), Medical Research Council (MRC) Dyspnea Scale, Short Form 36-item Scale (SF-36), and International Index of Erectile Function (IIEF) Questionnaire. The mean age of 10 (10.8%) mild, 46 (49.5%) moderate, 28 (30.1%) severe, and 9 (9.7%) very severe COPD patients was 61.4 ± 9.8 years. Varying degrees of ED were detected in 67.7% of COPD patients. All patients with hypoxemia had ED. IPAQ score and all SF-36 parameters were low in patients with ED, while MRC score was high. Forced expiratory volume in one second, forced vital capacity, partial pressure of oxygen in blood, oxygen (O2) saturation, IPAQ score, and role-physical parameters were statistically low in ED patients (p = 0.04, 0.02, <0.01, <0.01, 0.02, and 0.04, respectively); MRC score was statistically higher in patients with ED (p = 0.02). Patients with moderate and severe ED had statistically lower score of mental health (p < 0.01 and p = 0.02, respectively). There was a positive correlation between IIEF score and IPAQ scores (p < 0.01), MRC scores (p = 0.01), general health (p < 0.01), role-physical (p < 0.01), role-emotional (p < 0.01), physical functioning (p < 0.01), and mental health (p < 0.01) parameters in SF-36. ED is frequently seen in COPD patients. Hypoxemia, smoking, and limitation of physical activity are thought to be associated with ED in COPD as mechanisms. Quality of life and the functional capacity are negatively affected with the presence of ED. It is important for a physician to question the sexual functions in patients with COPD. The
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Linking Action Research to Response to Intervention (RtI): The Strategy Implementation Project
ERIC Educational Resources Information Center
Hoppey, David
2013-01-01
This paper showcases how one teacher preparation program embedded action research within the Response to Intervention (RtI) model. This integration helped preservice teachers gain a deeper knowledge of RtI key concepts and pedagogical decision making for meeting diverse students' needs. Examples from a course assignment are provided to demonstrate…
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Murray, M; Kiryluk, S; Swan, A V
1984-01-01
In the MRC/Derbyshire Smoking Study a cohort of about 6000 adolescents was surveyed annually from 1974 when they entered secondary school aged 11-12 years until 1978 when they reached 15-16 years. In 1981 after the adolescents had left school they were again surveyed by post. Each year from 1974 to 1978 and again in 1981 they answered a questionnaire on their smoking behaviour and other issues. Information on the schools attended by these adolescents was obtained from their teachers and headteachers. This paper examines the relation between the school environment and the adolescents' smoking behaviour both before and after leaving school. The prevalence of smoking was higher among those boys who attended schools that were single sex, non-denominational, or had a parent-teacher association, no health education, no female teachers, or whose headteacher smoked cigarettes. Among girls the prevalence of smoking was higher if they attended a school that had optional school uniform and no health or antismoking education. The importance of these findings for the development of effective preventive measures is discussed. PMID:6747518
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Moving from Batch to Field Using the RT3D Reactive Transport Modeling System
NASA Astrophysics Data System (ADS)
Clement, T. P.; Gautam, T. R.
2002-12-01
The public domain reactive transport code RT3D (Clement, 1997) is a general-purpose numerical code for solving coupled, multi-species reactive transport in saturated groundwater systems. The code uses MODFLOW to simulate flow and several modules of MT3DMS to simulate the advection and dispersion processes. RT3D employs the operator-split strategy which allows the code solve the coupled reactive transport problem in a modular fashion. The coupling between reaction and transport is defined through a separate module where the reaction equations are specified. The code supports a versatile user-defined reaction option that allows users to define their own reaction system through a Fortran-90 subroutine, known as the RT3D-reaction package. Further a utility code, known as BATCHRXN, allows the users to independently test and debug their reaction package. To analyze a new reaction system at a batch scale, users should first run BATCHRXN to test the ability of their reaction package to model the batch data. After testing, the reaction package can simply be ported to the RT3D environment to study the model response under 1-, 2-, or 3-dimensional transport conditions. This paper presents example problems that demonstrate the methods for moving from batch to field-scale simulations using BATCHRXN and RT3D codes. The first example describes a simple first-order reaction system for simulating the sequential degradation of Tetrachloroethene (PCE) and its daughter products. The second example uses a relatively complex reaction system for describing the multiple degradation pathways of Tetrachloroethane (PCA) and its daughter products. References 1) Clement, T.P, RT3D - A modular computer code for simulating reactive multi-species transport in 3-Dimensional groundwater aquifers, Battelle Pacific Northwest National Laboratory Research Report, PNNL-SA-28967, September, 1997. Available at: http://bioprocess.pnl.gov/rt3d.htm.
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Sandstedt, Mikael; Jonsson, Marianne; Asp, Julia; Dellgren, Göran; Lindahl, Anders; Jeppsson, Anders; Sandstedt, Joakim
2015-12-01
Flow cytometry (FCM) has become a well-established method for analysis of both intracellular and cell-surface proteins, while quantitative RT-PCR (RT-qPCR) is used to determine gene expression with high sensitivity and specificity. Combining these two methods would be of great value. The effects of intracellular staining on RNA integrity and RT-qPCR sensitivity and quality have not, however, been fully examined. We, therefore, intended to assess these effects further. Cells from the human lung cancer cell line A549 were fixed, permeabilized and sorted by FCM. Sorted cells were analyzed using RT-qPCR. RNA integrity was determined by RNA quality indicator analysis. A549 cells were then mixed with cells of the mouse cardiomyocyte cell line HL-1. A549 cells were identified by the cell surface marker ABCG2, while HL-1 cells were identified by intracellular cTnT. Cells were sorted and analyzed by RT-qPCR. Finally, cell cultures from human atrial biopsies were used to evaluate the effects of fixation and permeabilization on RT-qPCR analysis of nonimmortalized cells stored prior to analysis by FCM. A large amount of RNA could be extracted even when cells had been fixed and permeabilized. Permeabilization resulted in increased RNA degradation and a moderate decrease in RT-qPCR sensitivity. Gene expression levels were also affected to a moderate extent. Sorted populations from the mixed A549 and HL-1 cell samples showed gene expression patterns that corresponded to FCM data. When samples were stored before FCM sorting, the RT-qPCR analysis could still be performed with high sensitivity and quality. In summary, our results show that intracellular FCM may be performed with only minor impairment of the RT-qPCR sensitivity and quality when analyzing sorted cells; however, these effects should be considered when comparing RT-qPCR data of not fixed samples with those of fixed and permeabilized samples. © 2015 International Society for Advancement of Cytometry.
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Vijayan, R S K; Arnold, Eddy; Das, Kalyan
2014-05-01
HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that is targeted by nucleoside analogs (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). NNRTIs are allosteric inhibitors of RT, and constitute an integral part of several highly active antiretroviral therapy regimens. Under selective pressure, HIV-1 acquires resistance against NNRTIs primarily by selecting mutations around the NNRTI pocket. Complete RT sequencing of clinical isolates revealed that spatially distal mutations arising in connection and the RNase H domain also confer NNRTI resistance and contribute to NRTI resistance. However, the precise structural mechanism by which the connection domain mutations confer NNRTI resistance is poorly understood. We performed 50-ns molecular dynamics (MD) simulations, followed by essential dynamics, free-energy landscape analyses, and network analyses of RT-DNA, RT-DNA-nevirapine (NVP), and N348I/T369I mutant RT-DNA-NVP complexes. MD simulation studies revealed altered global motions and restricted conformational landscape of RT upon NVP binding. Analysis of protein structure network parameters demonstrated a dissortative hub pattern in the RT-DNA complex and an assortative hub pattern in the RT-DNA-NVP complex suggesting enhanced rigidity of RT upon NVP binding. The connection subdomain mutations N348I/T369I did not induce any significant structural change; rather, these mutations modulate the conformational dynamics and alter the long-range allosteric communication network between the connection subdomain and NNRTI pocket. Insights from the present study provide a structural basis for the biochemical and clinical findings on drug resistance caused by the connection and RNase H mutations. Copyright © 2013 Wiley Periodicals, Inc.
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Chow, Wei Zhen; Takebe, Yutaka; Syafina, Nur Ezreen; Prakasa, Malarvelli Soorya; Chan, Kok Gan; Al-Darraji, Haider Abdulrazzaq Abed; Koh, Clayton; Kamarulzaman, Adeeba; Tee, Kok Keng
2014-01-01
The HIV epidemic is primarily characterised by the circulation of HIV-1 group M (main) comprising of 11 subtypes and sub-subtypes (A1, A2, B-D, F1, F2, G, H, J, and K) and to date 55 circulating recombinant forms (CRFs). In Southeast Asia, active inter-subtype recombination involving three main circulating genotypes--subtype B (including subtype B', the Thai variant of subtype B), CRF01_AE, and CRF33_01B--have contributed to the emergence of novel unique recombinant forms. In the present study, we conducted the molecular epidemiological surveillance of HIV-1 gag-RT genes among 258 people who inject drugs (PWIDs) in Kuala Lumpur, Malaysia, between 2009 and 2011 whereby a novel CRF candidate was recently identified. The near full-length genome sequences obtained from six epidemiologically unlinked individuals showed identical mosaic structures consisting of subtype B' and CRF01_AE, with six unique recombination breakpoints in the gag-RT, pol, and env regions. Among the high-risk population of PWIDs in Malaysia, which was predominantly infected by CRF33_01B (>70%), CRF58_01B circulated at a low but significant prevalence (2.3%, 6/258). Interestingly, the CRF58_01B shared two unique recombination breakpoints with other established CRFs in the region: CRF33_01B, CRF48_01B, and CRF53_01B in the gag gene, and CRF15_01B (from Thailand) in the env gene. Extended Bayesian Markov chain Monte Carlo sampling analysis showed that CRF58_01B and other recently discovered CRFs were most likely to have originated in Malaysia, and that the recent spread of recombinant lineages in the country had little influence from neighbouring countries. The isolation, genetic characterization, and evolutionary features of CRF58_01B among PWIDs in Malaysia signify the increasingly complex HIV-1 diversity in Southeast Asia that may hold an implication on disease treatment, control, and prevention.
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Chow, Wei Zhen; Takebe, Yutaka; Syafina, Nur Ezreen; Prakasa, Malarvelli Soorya; Chan, Kok Gan; Al-Darraji, Haider Abdulrazzaq Abed; Koh, Clayton; Kamarulzaman, Adeeba; Tee, Kok Keng
2014-01-01
The HIV epidemic is primarily characterised by the circulation of HIV-1 group M (main) comprising of 11 subtypes and sub-subtypes (A1, A2, B–D, F1, F2, G, H, J, and K) and to date 55 circulating recombinant forms (CRFs). In Southeast Asia, active inter-subtype recombination involving three main circulating genotypes—subtype B (including subtype B′, the Thai variant of subtype B), CRF01_AE, and CRF33_01B—have contributed to the emergence of novel unique recombinant forms. In the present study, we conducted the molecular epidemiological surveillance of HIV-1 gag-RT genes among 258 people who inject drugs (PWIDs) in Kuala Lumpur, Malaysia, between 2009 and 2011 whereby a novel CRF candidate was recently identified. The near full-length genome sequences obtained from six epidemiologically unlinked individuals showed identical mosaic structures consisting of subtype B′ and CRF01_AE, with six unique recombination breakpoints in the gag-RT, pol, and env regions. Among the high-risk population of PWIDs in Malaysia, which was predominantly infected by CRF33_01B (>70%), CRF58_01B circulated at a low but significant prevalence (2.3%, 6/258). Interestingly, the CRF58_01B shared two unique recombination breakpoints with other established CRFs in the region: CRF33_01B, CRF48_01B, and CRF53_01B in the gag gene, and CRF15_01B (from Thailand) in the env gene. Extended Bayesian Markov chain Monte Carlo sampling analysis showed that CRF58_01B and other recently discovered CRFs were most likely to have originated in Malaysia, and that the recent spread of recombinant lineages in the country had little influence from neighbouring countries. The isolation, genetic characterization, and evolutionary features of CRF58_01B among PWIDs in Malaysia signify the increasingly complex HIV-1 diversity in Southeast Asia that may hold an implication on disease treatment, control, and prevention. PMID:24465513
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Nathaniel, Thomas I; Cochran, Thomas; Chaves, Jose; Fulmer, Eric; Sosa, Crystal; Yi, Sara; Fredwall, Megan; Sternberg, Shannon; Blackhurst, Dawn; Nelson, Alfred; Leacock, Rodney
2016-01-01
The objective of this study was to characterize the comorbidities in a population of patients with an acute ischaemic stroke, comparing patients that received recombinant tissue plasminogen activator (rt-PA) to those that did not receive rt-PA. In a retrospective sample of 663 patients admitted for acute ischaemic stroke, this study analysed the effects of co-morbid conditions in the use of rt-PA. It determined non-cerebrovascular risk factors (comorbidities) that differentiate patients who received rt-PA from those who did not receive rt-PA. Patients with a history of carotid stenosis, CHF and previous strokes are significantly (p < 0.05) associated with high risk of not receiving rt-PA. A significant number of patients with a history of hypertension and smoking received rt-PA (p < 0.05). The findings indicate that certain risk factors including carotid stenosis, CHF and previous stroke history impact the treatment of patients with acute ischaemic stroke, specifically the decision to administer rt-PA. Treatment with rt-PA is dependent on stroke severity and onset to treatment time, but the findings suggest that rt-PA use may also depend on patient comorbidities.
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Shen, Xin-Xin; Qiu, Fang-Zhou; Zhao, Huai-Long; Yang, Meng-Jie; Hong, Liu; Xu, Song-Tao; Zhou, Shuai-Feng; Li, Gui-Xia; Feng, Zhi-Shan; Ma, Xue-Jun
2018-03-01
The sensitivity of qRT-PCR assay is not adequate for the detection of the samples with lower viral load, particularly in the cerebrospinal fluid (CSF) of patients. Here, we present the development of a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of human enterovirus (HEV). The clinical performance of both RTN RT-PCR and qRT-PCR was also tested and compared using 140 CSF and fecal specimens. The sensitivities of RTN RT-PCR assay for EV71, Coxsackievirus A (CVA)16, CVA6 and CVA10 achieved 10 -8 dilution with a corresponding Ct value of 38.20, 36.45, 36.75, and 36.45, respectively, which is equal to traditional two-step nested RT-PCR assay and approximately 2-10-fold lower than that of qRT-PCR assay. The specificity of RTN RT-PCR assay was extensively analyzed insilico and subsequently verified using the reference isolates and clinical samples. Sixteen qRT-PCR-negative samples were detected by RTN RT-PCR and a variety of enterovirus serotypes was identified by sequencing of inner PCR products. We conclude RTN RT-PCR is more sensitive than qRT-PCR for the detection of HEV in clinical samples. Copyright © 2017 Elsevier Inc. All rights reserved.
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Vijayan, R.S.K.; Arnold, Eddy; Das, Kalyan
2015-01-01
HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that is targeted by nucleoside analogs (NRTIs) and nonnucleoside inhibitors (NNRTIs). NNRTIs are allosteric inhibitors of RT, and constitute an integral part of the highly active antiretroviral therapy (HAART) regimen. Under selective pressure, HIV-1 acquires resistance against NNRTIs primarily by selecting mutations around the NNRTI pocket. Complete RT sequencing of clinical isolates revealed that spatially distal mutations arising in connection and the RNase H domain also confer NNRTI resistance and contribute to NRTI resistance. However, the precise structural mechanism by which the connection domain mutations confer NNRTI resistance is poorly understood. We performed 50-ns MD simulations, followed by essential dynamics, free-energy landscape analyses and network analyses of RT-DNA, RT-DNA-nevirapine, and N348I/T369I mutant RT-DNA-nevirapine complexes. MD simulation studies revealed altered global motions and restricted conformational landscape of RT upon nevirapine binding. Analysis of protein structure network parameters demonstrated a dissortative hub pattern in the RT-DNA complex and an assortative hub pattern in the RT-DNA-nevirapine complex suggesting enhanced rigidity of RT upon nevirapine binding. The connection subdomain mutations N348I/T369I did not induce any significant structural change; rather, these mutations modulate the conformational dynamics and alter the long-range allosteric communication network between the connection subdomain and NNRTI pocket. Insights from the present study provide a structural basis for the biochemical and clinical findings on drug resistance caused by the connection and RNase H mutations. PMID:24174331
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Nouidui, Thierry; Wetter, Michael
SimulatorToFMU is a software package written in Python which allows users to export a memoryless Python-driven simulation program or script as a Functional Mock-up Unit (FMU) for model exchange or co-simulation.In CyDER (Cyber Physical Co-simulation Platform for Distributed Energy Resources in Smart Grids), SimulatorToFMU will allow exporting OPAL-RT as an FMU. This will enable OPAL-RT to be linked to CYMDIST and GridDyn FMUs through a standardized open source interface.
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Detection and Analysis of Circular RNAs by RT-PCR.
Panda, Amaresh C; Gorospe, Myriam
2018-03-20
Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al ., 2017b; Panda et al ., 2017c). However, the validation of true circRNAs identified by RNA sequencing requires other molecular biology techniques including reverse transcription (RT) followed by conventional or quantitative (q) polymerase chain reaction (PCR), and Northern blot analysis (Jeck and Sharpless, 2014). RT-qPCR analysis of circular RNAs using divergent primers has been widely used for the detection, validation, and sometimes quantification of circRNAs (Abdelmohsen et al ., 2015 and 2017; Panda et al ., 2017b). As detailed here, divergent primers designed to span the circRNA backsplice junction sequence can specifically amplify the circRNAs and not the counterpart linear RNA. In sum, RT-PCR analysis using divergent primers allows direct detection and quantification of circRNAs.
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The value of research : telling the R&T story
DOT National Transportation Integrated Search
2009-07-01
The Federal Highway Administration (FHWA) plays a leadership role in shaping and executing a National Research and Technology (R&T) program. The agency also acts as a convener; collaborations with State, industry, and academic partners provide the fo...
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Liu, Baoming; Yang, Jing-Xian; Yan, Ling; Zhuang, Hui; Li, Tong
2018-01-01
As one of the major global public health concerns, hepatitis B virus (HBV) can be divided into at least eight genotypes, which may be related to disease severity and treatment response. We previously demonstrated that genotypes B and C HBV, with distinct geographical distribution in China, had divergent genotype-dependent amino acid polymorphisms and variations in reverse transcriptase (RT) gene region, a target of antiviral therapy using nucleos(t)ide analogues. Recently recombination between HBV genotypes B and C was reported to occur in the RT region. However, their frequency and clinical significance is poorly understood. Here full-length HBV RT sequences from 201 Chinese chronic hepatitis B (CHB) patients were amplified and sequenced, among which 31.34% (63/201) were genotype B whereas 68.66% (138/201) genotype C. Although no intergenotypic recombination was detected among C-genotype HBV, 38.10% (24/63) of B-genotype HBV had recombination with genotype C in the 3'-terminal RT sequences. The patients with B/C intergenotypic recombinants had significantly (P<0.05) higher serum HBV DNA level than the "pure" B-genotype cohort did. Moreover, the B/C intergenotypic recombinants were prone to more substitutions at several specific residues in the RT region than genotype B or C. Besides, unlike their parental genotypes, the recombinant HBV appeared to display an altered geographic distribution feature in China. Our findings provide novel insight into the virological, clinical and epidemiological features of new HBV B/C intergenotypic recombinants at the 3' end of RT sequences among Chinese CHB patients. The highly complex genetic background of the novel recombinant HBV carrying new mutations affecting RT protein may contribute to an enhanced heterogeneity in treatment response or prognosis among CHB patients. Published by Elsevier B.V.
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Nájera-Martínez, Minerva; García-Latorre, Ethel A; Reyes-Maldonado, Elba; Domínguez-López, M Lilia; Vega-López, Armando
2012-09-01
Halomethanes (HMs) can be formed during the chlorination process to obtain drinking water. In liver cells, HMs had been shown to be mutagenic and carcinogenic; however, their bioactivation by CYP 2E1 and GSTT1 is required. Although inhalation is the most common pathway of exposure, reports on the toxic effects induced by HMs in human lung are contradictory. The aim of this study was therefore to evaluate in vitro cytotoxicity and cell proliferation induced by CH(2)Cl(2), CHCl(3) and BrCHCl(2) in human lung NL20-TA epithelial cells and MRC-5 fibroblasts, and their relationship with CYP 2E1 and GSTT1 activity. High concentrations of these HMs induced cytotoxicity, particularly in cells treated with BrCHCl(2). Low concentrations of BrCHCl(2) stimulated hyperproliferation of fibroblasts, the most probable consequence of which is regenerative proliferation related to collagen induction. Fibroblasts exposed to BrCHCl(2) exhibited low levels of CYP 2E1 activity suggesting that released bromine is able to alter this activity by affecting the active site or auto regulating the activity itself. GSTT1 was up to ten times more active than CYP 2E1 in both cell lines, indicating that potential lung damage is due to formation of pro-carcinogens such as formaldehyde.
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Real-Time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei
2005-10-01
1 Real-time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei Vipin K. Rastogi1, Tu-chen Cheng1, Lisa Collins1 and Jennifer Bagley2 1...A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real-time PCR (RT-PCR) Assays for Burkholderia mallei and B.pseudomallei 5a. CONTRACT NUMBER 5b...pseudomallei and B. mallei , respectively are the causative agents of meliodosis and glanders , primarily in animals (both pathogens), and in humans
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Wagner, Karoline; Springer, Burkard; Imkamp, Frank; Opota, Onya; Greub, Gilbert; Keller, Peter M
2018-04-01
Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix ® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix ® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix ® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was
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A one-step multiplex RT-PCR assay for simultaneous detection of four viruses that infect peach.
Yu, Y; Zhao, Z; Jiang, D; Wu, Z; Li, S
2013-10-01
A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed to enable the simultaneous detection and differentiation of four viruses that infect peach, namely Apple chlorotic leaf spot virus (ACLSV), Cherry green ring mottle virus (CGRMV), Prunus necrotic ringspot virus (PNRSV) and Apricot pseudo-chlorotic leaf spot virus (APCLSV). In this study, four pairs of primers, one specific for each virus, were designed; the corresponding PCR products were 632, 439, 346 and 282 bp in length for ACLSV, CGRMV, PNRSV and APCLSV, respectively, and the fragments could be distinguished clearly by agarose gel electrophoresis. The sensitivity and specificity of the method were tested using individual RT-PCR and enzyme-linked immunosorbent assay (ELISA), and the identity of the RT-PCR amplification products was also confirmed by DNA sequencing. The results of RT-PCR and ELISA, along with batch detection using samples collected from peach orchards, revealed that this rapid and simple technique is an effective way to identify the four viruses simultaneously. The mRT-PCR assay described in this study was developed for the simultaneous detection of four peach viruses from infected peach samples is reliable and sensitive. In contrast to conventional uniplex RT-PCR, mRT-PCR is more efficient, reducing costs, time and handling when testing large numbers of samples. This rapid and simple method is useful for large-scale surveys of viruses that infect peach. © 2013 The Society for Applied Microbiology.
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Masomi-Bornwasser, Julia; Müller-Werkmeister, Hendrik; Kantelhardt, Sven Rainer; König, Jochem; Kempski, Oliver; Giese, Alf
2017-01-01
Hematoma lysis with recombinant tissue plasminogen activator (rtPA) has emerged as an alternative therapy for spontaneous intracerebral hemorrhage (ICH). Optimal dose and schedule are still unclear. The aim of this study was to create a reliable in vitro blood clot model for investigation of optimal drug dose and timing. An in vitro clot model was established, using 25 mL and 50 mL of human blood. Catheters were placed into the clots and three groups, using intraclot application of rtPA, placebo, and catheter alone, were analyzed. Dose-response relationship, repetition, and duration of rtPA treatment and its effectiveness in aged clots were investigated. A significant relative end weight difference was found in rtPA treated clots compared to catheter alone (p = 0.002) and placebo treated clots (p < 0.001). Dose-response analysis revealed 95% effective dose around 1 mg rtPA in 25 and 50 mL clots. Approximately 80% of relative clot lysis could be achieved after 15 min incubation. Lysis of aged clots was less effective. A new clot model for in vitro investigation was established. Our data suggest that current protocols for rtPA based ICH therapy may be optimized by using less rtPA at shorter incubation times. PMID:28459065
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Keric, Naureen; Masomi-Bornwasser, Julia; Müller-Werkmeister, Hendrik; Kantelhardt, Sven Rainer; König, Jochem; Kempski, Oliver; Giese, Alf
2017-01-01
Hematoma lysis with recombinant tissue plasminogen activator (rtPA) has emerged as an alternative therapy for spontaneous intracerebral hemorrhage (ICH). Optimal dose and schedule are still unclear. The aim of this study was to create a reliable in vitro blood clot model for investigation of optimal drug dose and timing. An in vitro clot model was established, using 25 mL and 50 mL of human blood. Catheters were placed into the clots and three groups, using intraclot application of rtPA, placebo, and catheter alone, were analyzed. Dose-response relationship, repetition, and duration of rtPA treatment and its effectiveness in aged clots were investigated. A significant relative end weight difference was found in rtPA treated clots compared to catheter alone ( p = 0.002) and placebo treated clots ( p < 0.001). Dose-response analysis revealed 95% effective dose around 1 mg rtPA in 25 and 50 mL clots. Approximately 80% of relative clot lysis could be achieved after 15 min incubation. Lysis of aged clots was less effective. A new clot model for in vitro investigation was established. Our data suggest that current protocols for rtPA based ICH therapy may be optimized by using less rtPA at shorter incubation times.
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Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay.
Lyu, Xiaodong; Wang, Xianwei; Zhang, Lina; Chen, Zhenzhu; Zhao, Yu; Hu, Jieying; Fan, Ruihua; Song, Yongping
2017-07-25
Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management. We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization. Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL). We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an
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X-ray observations from RT-1 magnetospheric plasmas
NASA Astrophysics Data System (ADS)
Sugata, Tetsuya; Masaki Nishiura Collaboration; Zensho Yoshida Collaboration; Naoki Kenmochi Collaboration; Shotaro Katsura Collaboration; Kaori Nakamura Collaboration
2017-10-01
Planetary magnetospheres like Earth and Jupiter realize stable confinement of high beta plasma. The RT-1 device produces a laboratory magnetosphere by using a levitated superconducting coil for dipole magnetic fields and 8.2 GHz electromagnetic wave for plasma production (ne 1017m-3) and electron heating. In the recent experiments, the RT-1 device has achieved the local beta that exceeds 1. It is considered that the high energy component of electrons contributes to the beta value. Therefore, Si(Li) detectors measured the X-ray spectra from the peripheral plasmas in the range from a few keV to a few ten keV. The density of a few keV component and a few ten keV component are comparable and a few ten keV component dominates the majority of the high beta value that is operated up to 0.8. We found that 150 keV component of electrons exists near the outer of the levitated dipole magnet by using a CdTe detector.
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Oloniniyi, Olamide K; Kurosaki, Yohei; Miyamoto, Hiroko; Takada, Ayato; Yasuda, Jiro
2017-08-01
Ebola virus disease (EVD), a highly virulent infectious disease caused by ebolaviruses, has a fatality rate of 25-90%. Without a licensed chemotherapeutic agent or vaccine for the treatment and prevention of EVD, control of outbreaks requires accurate and rapid diagnosis of cases. In this study, five sets of six oligonucleotide primers targeting the nucleoprotein gene were designed for specific identification of each of the five ebolavirus species using reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay. The detection limits of the ebolavirus species-specific primer sets were evaluated using in vitro transcribed RNAs. The detection limit of species-specific RT-LAMP assays for Zaire ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus, and Bundibugyo ebolavirus was 256 copies/reaction, while the detection limit for Reston ebolavirus was 64 copies/reaction, and the detection time for each of the RT-LAMP assays was 13.3±3.0, 19.8±4.6, 14.3±0.6, 16.1±4.7, and 19.8±2.4min (mean±SD), respectively. The sensitivity of the species-specific RT-LAMP assays were similar to that of the established RT-PCR and quantitative RT-PCR assays for diagnosis of EVD and are suitable for field or point-of-care diagnosis. The RT-LAMP assays were specific for the detection of the respective species of ebolavirus with no cross reaction with other species of ebolavirus and other viral hemorrhagic fever viruses such as Marburg virus, Lassa fever virus, and Dengue virus. The species-specific RT-LAMP assays developed in this study are rapid, sensitive, and specific and could be useful in case of an EVD outbreak. Copyright © 2017 Elsevier B.V. All rights reserved.
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NASA SPoRT Modeling and Data Assimilation Research and Transition Activities Using WRF, LIS and GSI
NASA Technical Reports Server (NTRS)
Case, Jonathan L.; Blankenship, Clay B.; Zavodsky, Bradley T.; Srikishen, Jayanthi; Berndt, Emily B.
2014-01-01
weather research and forecasting ===== The NASA Short-term Prediction Research and Transition (SPoRT) program has numerous modeling and data assimilation (DA) activities in which the WRF model is a key component. SPoRT generates realtime, research satellite products from the MODIS and VIIRS instruments, making the data available to NOAA/NWS partners running the WRF/EMS, including: (1) 2-km northwestern-hemispheric SST composite, (2) daily, MODIS green vegetation fraction (GVF) over CONUS, and (3) NASA Land Information System (LIS) runs of the Noah LSM over the southeastern CONUS. Each of these datasets have been utilized by specific SPoRT partners in local EMS model runs, with select offices evaluating the impacts using a set of automated scripts developed by SPoRT that manage data acquisition and run the NCAR Model Evaluation Tools verification package. SPoRT is engaged in DA research with the Gridpoint Statistical Interpolation (GSI) and Ensemble Kalman Filter in LIS for soil moisture DA. Ongoing DA projects using GSI include comparing the impacts of assimilating Atmospheric Infrared Sounder (AIRS) radiances versus retrieved profiles, and an analysis of extra-tropical cyclones with intense non-convective winds. As part of its Early Adopter activities for the NASA Soil Moisture Active Passive (SMAP) mission, SPoRT is conducting bias correction and soil moisture DA within LIS to improve simulations using the NASA Unified-WRF (NU-WRF) for both the European Space Agency's Soil Moisture Ocean Salinity and upcoming SMAP mission data. SPoRT has also incorporated real-time global GVF data into LIS and WRF from the VIIRS product being developed by NOAA/NESDIS. This poster will highlight the research and transition activities SPoRT conducts using WRF, NU-WRF, EMS, LIS, and GSI.
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RT DDA: A hybrid method for predicting the scattering properties by densely packed media
NASA Astrophysics Data System (ADS)
Ramezan Pour, B.; Mackowski, D.
2017-12-01
The most accurate approaches to predicting the scattering properties of particulate media are based on exact solutions of the Maxwell's equations (MEs), such as the T-matrix and discrete dipole methods. Applying these techniques for optically thick targets is challenging problem due to the large-scale computations and are usually substituted by phenomenological radiative transfer (RT) methods. On the other hand, the RT technique is of questionable validity in media with large particle packing densities. In recent works, we used numerically exact ME solvers to examine the effects of particle concentration on the polarized reflection properties of plane parallel random media. The simulations were performed for plane parallel layers of wavelength-sized spherical particles, and results were compared with RT predictions. We have shown that RTE results monotonically converge to the exact solution as the particle volume fraction becomes smaller and one can observe a nearly perfect fit for packing densities of 2%-5%. This study describes the hybrid technique composed of exact and numerical scalar RT methods. The exact methodology in this work is the plane parallel discrete dipole approximation whereas the numerical method is based on the adding and doubling method. This approach not only decreases the computational time owing to the RT method but also includes the interference and multiple scattering effects, so it may be applicable to large particle density conditions.
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ERIC Educational Resources Information Center
Fan, Chung-Hau; Denner, Peter R.; Bocanegra, Joel O.; Ding, Yi
2016-01-01
After the change in IDEIA, different models of response to intervention (RtI) have been practiced widely in American school systems. School psychologists are in an important position to facilitate RtI practice and provide professional development in order to help their school systems successfully undergo this transformation. However, there is a…
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Molaee, Neda; Abtahi, Hamid; Ghannadzadeh, Mohammad Javad; Karimi, Masoude; Ghaznavi-Rad, Ehsanollah
2015-01-01
Polymerase chain reaction (PCR) is preferred to other methods for detecting Escherichia coli (E. coli) in water in terms of speed, accuracy and efficiency. False positive result is considered as the major disadvantages of PCR. For this reason, reverse transcriptase-polymerase chain reaction (RT-PCR) can be used to solve this problem. The aim of present study was to determine the efficiency of RT-PCR for rapid detection of viable Escherichia coli in drinking water samples and enhance its sensitivity through application of different filter membranes. Specific primers were designed for 16S rRNA and elongation Factor II genes. Different concentrations of bacteria were passed through FHLP and HAWP filters. Then, RT-PCR was performed using 16srRNA and EF -Tu primers. Contamination of 10 wells was determined by RT-PCR in Arak city. To evaluate RT-PCR efficiency, the results were compared with most probable number (MPN) method. RT-PCR is able to detect bacteria in different concentrations. Application of EF II primers reduced false positive results compared to 16S rRNA primers. The FHLP hydrophobic filters have higher ability to absorb bacteria compared with HAWB hydrophilic filters. So the use of hydrophobic filters will increase the sensitivity of RT-PCR. RT-PCR shows a higher sensitivity compared to conventional water contamination detection method. Unlike PCR, RT-PCR does not lead to false positive results. The use of EF-Tu primers can reduce the incidence of false positive results. Furthermore, hydrophobic filters have a higher ability to absorb bacteria compared to hydrophilic filters.
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NASA Astrophysics Data System (ADS)
Bezrukovs, Vl.; Shmeld, I.; Nechaeva, M.; Trokss, J.; Bezrukovs, D.; Klapers, M.; Berzins, A.; Lesins, A.; Dugin, N.
2012-12-01
Radiotelescope RT-32 is a fully steerable 32-m parabolic antenna located at Irbene and belonging to Ventspils International Radio Astronomy Centre (VIRAC). Currently, the work on upgrading and repair of its receiving hardware and data acquisition systems is of high priority for the VIRAC. One of the main scientific objectives for the VIRAC Radioastronomical observatory is VLBI (very long baseline interferometry) observations in centimetre wavelengths in collaboration with world VLBI networks, such as European VLBI network (EVN), Low Frequency VLBI network (LFVN), and others. During the last years the room in the secondary focus of telescope was reconstructed, and several new receivers were installed. Currently, RT-32 observations are carried out in four different bands: 92 cm, 18 cm, 6 cm, and 2.5 cm. First three of them are already successfully employed in diversified VLBI experiments. The receiver on 2.5 cm band has only one linear polarized chain and is used mainly for the methanol maser single dish observations. The apparatus system of RT-32 is equipped with two independent VLBI data acquisition systems: TN-16, and DBBC in combination with MK5b. Both systems are employed in interferometric observations depending on the purpose of experiment and the enabled radiotelescopes. The current status of RT-32, the availability of its receiving and data acquisition units for VLBI observations and the previous VLBI sessions are discussed. Radioteleskops RT-32 ir Ventspils Starptautiskajam Radioastronomijas Centram (VSRC) piederoša pilnas piedziņas 32 m diametra paraboliskā antena. Pašreiz visaktuālākie VSRC veicamie darbi ir saistīti ar RT-32 uztverošās aparatūras un datu reģistrēšanas sistēmas labošanu un modernizāciju. Viens no radioastronomiskās observatorijas galvenajiem zinātniskajiem uzdevumiem ir sevišķi lielas bāzes interferometriskie (VLBI) novērojumi centimetru viļņu garumu diapazonā sadarbībā ar pasaules VLBI tīkla partneriem, t
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BCR-ABL1 expression, RT-qPCR and treatment decisions in chronic myeloid leukaemia.
Latham, Susan; Bartley, Paul A; Budgen, Bradley; Ross, David M; Hughes, Elizabeth; Branford, Susan; White, Deborah; Hughes, Timothy P; Morley, Alexander A
2016-09-01
RT-qPCR is used to quantify minimal residual disease (MRD) in chronic myeloid leukaemia (CML) in order to make decisions on treatment, but its results depend on the level of BCR-ABL1 expression as well as leukaemic cell number. The aims of the study were to quantify inter-individual differences in expression level, to determine the relationship between expression level and response to treatment, and to investigate the effect of expression level on interpretation of the RT-qPCR result. BCR-ABL1 expression was studied in 248 samples from 65 patients with CML by determining the difference between MRD quantified by RT-qPCR and DNA-qPCR. The results were analysed statistically and by simple indicative modelling. Inter-individual levels of expression approximated a normal distribution with an SD of 0.36 log. Expression at diagnosis correlated with expression during treatment. Response to treatment, as measured by the number of leukaemic cells after 3, 6 or 12 months of treatment, was not related to the level of expression. Indicative modelling suggested that interpretation of RT-qPCR results in relation to treatment guidelines could be affected by variation in expression when MRD was around 10% at 3 months and by both expression variation and Poisson variation when MRD was around or below the limit of detection of RT-qPCR. Variation between individuals in expression of BCR-ABL1 can materially affect interpretation of the RT-qPCR when this test is used to make decisions on treatment. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/
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Combination of ultrasound and rtPA enhances fibrinolysis in an In Vitro clot system
Winter, Philipp; Müller-Werkmeister, Hendrik; Strand, Susanne; König, Jochem; Kempski, Oliver; Ringel, Florian; Kantelhardt, Sven R.; Keric, Naureen
2017-01-01
Background Catheter-based lysis with recombinant tissue plasminogen activator (rtPA) is a well-established therapy for spontaneous intracerebral hemorrhage (ICH). The effectiveness of this therapy can be increased with ultrasound, but the optimal conditions are not yet clearly established. Using a novel in vitro system of blood clots previously developed by our group, we investigated various parameters of intralesional sonothrombolysis using an endosonography catheter in combination with rtPA. Methods Standardized human blood clots were equipped with a drainage catheter and weighed before and after 4 treatments: control (drainage only), rtPA only, ultrasound only and the combination of rtPA+ultrasound. The effectiveness of ultrasound was further analysed in terms of optimal frequency, duration and distance to the probe. Temperature and acoustic peak rarefaction pressure (APRP) were assessed to analyse potential adverse effects and quantify lysis. Histo-morphological analysis of the treated clots was performed by H&E staining and confocal laser scanning microscopy using fluorescent fibrinogen. Results The combined treatment rtPA+ultrasound achieved the highest lysis rates with a relative weight of 30.3%±5.5% (p≤0.0001) compared to all other groups. Similar results were observed when treating aged clots. Confocal fluorescent microscopy of the treated clots revealed a rarefied fibrin mesh without cavitations. No relevant temperature increase occurred (0.53±0.75°C). The optimal insonation treatment time was 1 hour. APRP measurements showed a lysis threshold of 515.5±113.4 kPa. Application of 10 MHz achieved optimal lysis and lysis radius, while simultaneously proving to be the best frequency for morphologic imaging of the clot and surrounding tissue. Conclusions These promising data provide the basis for an individualized minimal invasive ICH therapy by rtPA and sonothrombolysis independent of ICH age. PMID:29145482
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USDA-ARS?s Scientific Manuscript database
Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize the viru...
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OR.NET RT: how service-oriented medical device architecture meets real-time communication.
Pfeiffer, Jonas H; Kasparick, Martin; Strathen, Benjamin; Dietz, Christian; Dingler, Max E; Lueth, Tim C; Timmermann, Dirk; Radermacher, Klaus; Golatowski, Frank
2018-02-23
Today's landscape of medical devices is dominated by stand-alone systems and proprietary interfaces lacking cross-vendor interoperability. This complicates or even impedes the innovation of novel, intelligent assistance systems relying on the collaboration of medical devices. Emerging approaches use the service-oriented architecture (SOA) paradigm based on Internet protocol (IP) to enable communication between medical devices. While this works well for scenarios with no or only soft timing constraints, the underlying best-effort communication scheme is insufficient for time critical data. Real-time (RT) networks are able to reliably guarantee fixed latency boundaries, for example, by using time division multiple access (TDMA) communication patterns. However, deterministic RT networks come with their own limitations such as tedious, inflexible configuration and a more restricted bandwidth allocation. In this contribution we overcome the drawbacks of both approaches by describing and implementing mechanisms that allow the two networks to interact. We introduce the first implementation of a medical device network that offers hard RT guarantees for control and sensor data and integrates into SOA networks. Based on two application examples we show how the flexibility of SOA networks and the reliability of RT networks can be combined to achieve an open network infrastructure for medical devices in the operating room (OR).
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TMPA Products 3B42RT & 3B42V6: Evaluation and Application in Qinghai-Tibet Plateau
NASA Astrophysics Data System (ADS)
Hao, Z.; Sun, L.; Wang, J.
2012-04-01
Hydrological researchers in Qinghai-Tibet Plateau tend to be haunted by deficiency of station gauged precipitation data for the sparse and uneven distribution of local meteorological stations. Fortunately, alternative data can be obtained from TRMM (Tropic Rainfall Measurement Mission) satellite. Preliminary evaluation and necessary correction of TRMM satellite rainfall products is required for the sake of reliability and suitability considering that TRMM precipitation is unconventional and natural condition in Qinghai-Tibet Plateau is unusually complicated. 3B42RT and 3B42V6 products from TRMM Multisatellite Precipitation Analysis(TMPA) are evaluated in northeast Qinghai-Tibet Plateau with 50 stations quality-controlled gauged daily precipitation as the benchmark precipitation set. It is found that the RT data overestimates the actual precipitation greatly while V6 only overestimates it slightly. RT data shows different seasonal and inter-annual accuracies. Summer and autumn see better accuracies than winter and spring and wet years see higher accuracies than dry years. Latitude is believed to be an important factor that influences the accuracy of satellite precipitation. Both RT and V6 can reflect the general pattern of the spatial distribution of precipitation even though RT overestimates the quantity greatly. A new parameter, accumulated precipitation weight point (APWP), was introduced to describe the temporal-spatial pattern evolution of precipitation. The APWP of both RT and V6 were moving from south to north in the past decade, but they are all in the west of station gauged precipitation APWP(s).V6 APWP track fit gauged precipitation perfectly while RT APWP track has over-exaggerated legs, indicating that spatial distribution of RT precipitation experienced unreasonable sharp changes. A practical and operational procedure to correct satellite precipitation data is developed. For RT, there are two steps. Step 1, the downscaling, original daily precipitation
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Identification and evaluation of reference genes for qRT-PCR studies in Lentinula edodes
Qin, Peng; He, Maolan; Yu, Xiumei; Zhao, Ke; Zhang, Xiaoping; Ma, Menggen; Chen, Qiang; Chen, Xiaoqiong; Zeng, Xianfu; Gu, Yunfu
2018-01-01
Lentinula edodes (shiitake mushroom) is a common edible mushroom with a number of potential therapeutic and nutritional applications. It contains various medically important molecules, such as polysaccharides, terpenoids, sterols, and lipids, were contained in this mushroom. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to analyze the mechanisms underlying the biosynthetic pathways of these substances. qRT-PCR is used for accurate analyses of transcript levels owing to its rapidity, sensitivity, and reliability. However, its accuracy and reliability for the quantification of transcripts rely on the expression stability of the reference genes used for data normalization. To ensure the reliability of gene expression analyses using qRT-PCR in L. edodes molecular biology research, it is necessary to systematically evaluate reference genes. In the current study, ten potential reference genes were selected from L. edodes genomic data and their expression levels were measured by qRT-PCR using various samples. The expression stability of each candidate gene was analyzed by three commonly used software packages: geNorm, NormFinder, and BestKeeper. Base on the results, Rpl4 was the most stable reference gene across all experimental conditions, and Atu was the most stable gene among strains. 18S was found to be the best reference gene for different development stages, and Rpl4 was the most stably expressed gene under various nutrient conditions. The present work will contribute to qRT-PCR studies in L. edodes. PMID:29293626
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Identification and evaluation of reference genes for qRT-PCR studies in Lentinula edodes.
Xiang, Quanju; Li, Jin; Qin, Peng; He, Maolan; Yu, Xiumei; Zhao, Ke; Zhang, Xiaoping; Ma, Menggen; Chen, Qiang; Chen, Xiaoqiong; Zeng, Xianfu; Gu, Yunfu
2018-01-01
Lentinula edodes (shiitake mushroom) is a common edible mushroom with a number of potential therapeutic and nutritional applications. It contains various medically important molecules, such as polysaccharides, terpenoids, sterols, and lipids, were contained in this mushroom. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to analyze the mechanisms underlying the biosynthetic pathways of these substances. qRT-PCR is used for accurate analyses of transcript levels owing to its rapidity, sensitivity, and reliability. However, its accuracy and reliability for the quantification of transcripts rely on the expression stability of the reference genes used for data normalization. To ensure the reliability of gene expression analyses using qRT-PCR in L. edodes molecular biology research, it is necessary to systematically evaluate reference genes. In the current study, ten potential reference genes were selected from L. edodes genomic data and their expression levels were measured by qRT-PCR using various samples. The expression stability of each candidate gene was analyzed by three commonly used software packages: geNorm, NormFinder, and BestKeeper. Base on the results, Rpl4 was the most stable reference gene across all experimental conditions, and Atu was the most stable gene among strains. 18S was found to be the best reference gene for different development stages, and Rpl4 was the most stably expressed gene under various nutrient conditions. The present work will contribute to qRT-PCR studies in L. edodes.
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ERIC Educational Resources Information Center
Montalvo, Ricardo; Combes, Bertina H.; Kea, Cathy D.
2014-01-01
Response to intervention (RtI) originates from national legislation and critical research of evidence-based practices for low performing students and students at-risk of failing or receiving special education services. RtI proactively facilitates culturally and linguistically responsive pedagogy for culturally and linguistically diverse (CLD)…
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Comparison of monoplex and duplex RT-PCR assays for the detection of measles virus.
Binkhamis, Khalifa; Gillis, Hayley; Lafreniere, Joseph Daniel; Hiebert, Joanne; Mendoza, Lillian; Pettipas, Janice; Severini, Alberto; Hatchette, Todd F; LeBlanc, Jason J
2017-01-01
Rapid and accurate detection of measles virus is important for case diagnosis and public health management. This study compared the performance of two monoplex RT-PCR reactions targeting the H and N genes to a duplex RT-PCR targeting both genes simultaneously. The duplex simplified processing without compromising assay performance characteristic. Copyright © 2016 Elsevier B.V. All rights reserved.
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Goheen, M M; Wegmüller, R; Bah, A; Darboe, B; Danso, E; Affara, M; Gardner, D; Patel, J C; Prentice, A M; Cerami, C
2016-12-01
Iron deficiency causes long-term adverse consequences for children and is the most common nutritional deficiency worldwide. Observational studies suggest that iron deficiency anemia protects against Plasmodium falciparum malaria and several intervention trials have indicated that iron supplementation increases malaria risk through unknown mechanism(s). This poses a major challenge for health policy. We investigated how anemia inhibits blood stage malaria infection and how iron supplementation abrogates this protection. This observational cohort study occurred in a malaria-endemic region where sickle-cell trait is also common. We studied fresh RBCs from anemic children (135 children; age 6-24months; hemoglobin <11g/dl) participating in an iron supplementation trial (ISRCTN registry, number ISRCTN07210906) in which they received iron (12mg/day) as part of a micronutrient powder for 84days. Children donated RBCs at baseline, Day 49, and Day 84 for use in flow cytometry-based in vitro growth and invasion assays with P. falciparum laboratory and field strains. In vitro parasite growth in subject RBCs was the primary endpoint. Anemia substantially reduced the invasion and growth of both laboratory and field strains of P. falciparum in vitro (~10% growth reduction per standard deviation shift in hemoglobin). The population level impact against erythrocytic stage malaria was 15.9% from anemia compared to 3.5% for sickle-cell trait. Parasite growth was 2.4 fold higher after 49days of iron supplementation relative to baseline (p<0.001), paralleling increases in erythropoiesis. These results confirm and quantify a plausible mechanism by which anemia protects African children against falciparum malaria, an effect that is substantially greater than the protection offered by sickle-cell trait. Iron supplementation completely reversed the observed protection and hence should be accompanied by malaria prophylaxis. Lower hemoglobin levels typically seen in populations of African
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Time-extended local rtPA infiltration for acutely thrombosed hemodialysis fistulas.
Regus, Susanne; Lang, Werner; Heinz, Marco; Rother, Ulrich; Meyer, Alexander; Almási-Sperling, Veronika; Uder, Michael; Schmid, Axel
2018-01-01
This study describes results of a modified local thrombolysis technique for acutely thrombosed hemodialysis (HD) arteriovenous fistulas (AVF), which is characterized by prolonged recombinant tissue plasminogen activator (rtPA) local exposure times. Contrary to the standard lyse- and- wait (L&W) technique with local reaction times of 20-40 minutes, the modified protocol allows timing of challenging angioplasty maneuvers to the next regular working day. From February 2009 to April 2014, 84 patients on HD presented with 152 acutely thrombosed AVF. They proceeded to local thrombolysis including a single shot infiltration of rtPA, local reaction time up to 40 hours and finally percutaneous stenosis angioplasty. Success rates, major adverse events and need for temporary catheter placements (TCP) were retrospectively analyzed. The local thrombolysis time after single shot infiltration was 18.6 ± 6.2 (range 2-40) hours. Mean rtPA- dosage was 2.7 mg ± 1.2. The overall success rate was 89.5% and the major complication rate was 3.3%, whereas TCP was necessary in 12.5%. The PP/SP at 1, 3, 6, 12, 18, and 24 month were 86% ± 3%/95% ± 2%, 68% ± 4%/92% ± 2%, 43% ± 4%/90% ± 2%, 28% ± 4%/82% ± 3%, 12% ± 3%/82% ± 3%, 7% ± 2%/63% ± 4%, respectively. The modified L&W technique with prolonged local rtPA reaction times is a safe and effective declotting procedure. The need for TCP was not increased and therefore comparable to the standard technique. © 2017 International Society for Hemodialysis.
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Optical and UV spectroscopy of the peculiar RS CVn system RT Lacertae
NASA Technical Reports Server (NTRS)
Huenemoerder, D. P.; Barden, S. C.
1986-01-01
H-alpha and H-beta spectra of the peculiar double-lined RS CVn binary RT Lacertae have been obtained using the IUE, together with a ground-based coude-feed telescope at KPNO. The ground-based spectra show an asymmetry related to the orbital phase in the H-alpha profile. H-beta profiles showed excess emission in one hemisphere and excess absorption in the other, with a broad Gaussian emission component superposed on the excess H-alpha line. A radial velocity curve was derived to estimate the mass ratio and geometry of the system. It is shown that the component of RT Lac fills 80-90 percent of the equilibrium Roche surface. Low-resolution ultraviolet data show that the supposed cooler component is bluer than its companion, suggesting evidence of a scattering shell or a cloud produced by the splash of a gas stream. The phase behavior of the low resolution ultraviolet data support the conclusion that RT Lac is a mass transfer system and that mass transfer is the primary cause of its activity.
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Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...
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Jia, Peng; Purcell, Maureen; Pan, Guang; Wang, Jinjin; Kan, Shifu; Liu, Yin; Zheng, Xiaocong; SHi, Xiujie; He, Junqiang; Yu, Li; Hua, Qunyi; Lu, Tikang; Lan, Wensheng; Winton, James; Jin, Ningyi; Liu, Hong
2017-01-01
Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/μl while the LOD for the RT-qPCR was 0.2 PFU/μl. Good agreement (69.4–100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.
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In-flight demonstration of a Real-Time Flush Airdata Sensing (RT-FADS) system
NASA Technical Reports Server (NTRS)
Whitmore, Stephen A.; Davis, Roy J.; Fife, John Michael
1995-01-01
A prototype real-time flush airdata sensing (RT-FADS) system has been developed and flight tested at the NASA Dryden Flight Research Center. This system uses a matrix of pressure orifices on the vehicle nose to estimate airdata parameters in real time using nonlinear regression. The algorithm is robust to sensor failures and noise in the measured pressures. The RT-FADS system has been calibrated using inertial trajectory measurements that were bootstrapped for atmospheric conditions using meteorological data. Mach numbers as high as 1.6 and angles of attack greater than 45 deg have been tested. The system performance has been evaluated by comparing the RT-FADS to the ship system airdata computer measurements to give a quantitative evaluation relative to an accepted measurement standard. Nominal agreements of approximately 0.003 in Mach number and 0.20 deg in angle of attack and angle of sideslip have been achieved.
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NASA Astrophysics Data System (ADS)
Documet, Jorge R.; Liu, Brent; Le, Anh; Law, Maria
2008-03-01
During the last 2 years we have been working on developing a DICOM-RT (Radiation Therapy) ePR (Electronic Patient Record) with decision support that will allow physicists and radiation oncologists during their decision-making process. This ePR allows offline treatment dose calculations and plan evaluation, while at the same time it compares and quantifies treatment planning algorithms using DICOM-RT objects. The ePR framework permits the addition of visualization, processing, and analysis tools, which combined with the core functionality of reporting, importing and exporting of medical studies, creates a very powerful application that can improve the efficiency while planning cancer treatments. Usually a Radiation Oncology department will have disparate and complex data generated by the RT modalities as well as data scattered in RT Information/Management systems, Record & Verify systems, and Treatment Planning Systems (TPS) which can compromise the efficiency of the clinical workflow since the data crucial for a clinical decision may be time-consuming to retrieve, temporarily missing, or even lost. To address these shortcomings, the ACR-NEMA Standards Committee extended its DICOM (Digital Imaging & Communications in Medicine) standard from Radiology to RT by ratifying seven DICOM RT objects starting in 1997 [1,2]. However, they are not broadly used yet by the RT community in daily clinical operations. In the past, the research focus of an RT department has primarily been developing new protocols and devices to improve treatment process and outcomes of cancer patients with minimal effort dedicated to integration of imaging and information systems. Our attempt is to show a proof-of-concept that a DICOM-RT ePR system can be developed as a foundation to perform medical imaging informatics research in developing decision-support tools and knowledge base for future data mining applications.
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Lee, Ji Yoon; Pajarillo, Edward Alain B; Kim, Min Jeong; Chae, Jong Pyo; Kang, Dae-Kyung
2013-01-04
Lactobacillus johnsonii PF01 has been reported to be highly resistant to bile, a key property of probiotic microorganisms. Here, we examine the nature of the bile-salt tolerance of L. johnsonii PF01. Growth inhibition and surface morphology and physiology aberrations were observed after overnight exposure to bile stress. Quantitative proteomic profiles using iTRAQ-LC-MS/MS technology identified 8307 peptides from both untreated PF01 cells and those exposed to 0.1%, 0.2%, and 0.3% bile salts. Some 215 proteins exhibited changed levels in response to bile stress; of these, levels of 94 peptides increased while those of 121 decreased. These were classified into the following categories: stress responses, cell division, transcription, translation, nucleotide metabolism, carbohydrate transport and metabolism, cell wall biosynthesis, and amino acid biosynthesis, and 16 of unidentified function. Analysis of the mRNA expression of selected genes by quantitative reverse transcriptase-PCR verified the proteomic data. Both proteomic and mRNA data provided evidence for increased phosphotransferase activity and cell wall biosynthesis. In addition, three bile salt hydrolases were significantly upregulated by bile exposure. These findings provide a basis for future evaluations of the tolerance of potential probiotic strains toward the various gastrointestinal challenges, including bile stress.
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Sun, Xuhong; Berthiller, Julien; Trouillas, Paul; Derex, Laurent; Diallo, Laho; Hanss, Michel
2015-04-15
The purpose of this study was to systematically determine the correlations between the post-thrombolytic changes of hemostasis parameters and the occurrence of early intracerebral hemorrhage (ICH). In 72 consecutive patients with cerebral infarcts treated with rt-PA, plasma levels of fibrinogen, plasminogen, alpha2-antiplasmin, factor XIII, fibrin(ogen) degradation products (FDPs) and d-Dimers were measured at baseline, 2 and 24h after thrombolysis. Correlations were studied between the hemostasis events and early (less than 24h) hemorrhagic infarcts (HIs) or parenchymatous hematomas (PH). Of 72 patients, 6 patients (8.3%) had early PHs, 11 (15.3%) had early HIs, and 55 (76.4%) had no bleeding. Early HIs were not linked to any hemostasis parameter at any time. Univariate comparison of patients having early PHs with non-bleeding patients showed hemostasis abnormalities at 2h: high FDP (p=0.01), high Log FDP (p=0.01), low fibrinogen (p=0.01), and low Log fibrinogen (p=0.01). Logistic regression adjusted for age, NIHSS and diabetes confirmed these 2hour predictors: Log FDP (OR: 7.50; CI: 1.26 to 44.61, p=0.03), and Log fibrinogen (OR: 19.32; CI: 1.81 to 205.98, p=0.01). The decrease in fibrinogen less than 2g/L multiplies the odds of early PH by a factor 12.82. An early fibrinogen degradation coagulopathy involving an increase of FDP and a massive consumption of circulating fibrinogen is predictive of early parenchymal hematomas, indicating the occurrence of a particularly intense lysis of circulating fibrinogen. These results, if confirmed by future studies, suggest that early assays of fibrinogen and FDP may be useful in predicting the risk of post-thrombolytic intracerebral hematoma. Copyright © 2015 Elsevier B.V. All rights reserved.
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Qiao, Yuan; Yang, Ju Xiang; Zhang, Xiao Dong L; Liu, Yu; Zhang, Jian Chao; Zong, Shu Dong; Miao, Shi Ying; Wang, Lin Fang; Koide, Samuel S
2004-06-01
A cDNA, designated as rtSH3p13, was isolated from a rat testis cDNA library. It consists of 1463 bp nuclear acids, which encodes a protein of 312 amino acids and was assigned the GenBank accession number AF227439. The deduced rtSH3p13 protein is a truncated isoform of SH3p13 as a result of mRNA alternative splicing. It is mainly expressed in the rat testis, detected in spermatids at the steps 8-19 of spermiogenesis, and found around the acrosome. During postnatal development, rtSH3p13 appears on day 18 and reaches maximum on day 60. Further experimental results suggested that rtSH3p13 forms a complex with activated epidermal growth factor receptor (EGFR) and interacts with synaptojanin I. Surprisingly, similar to SH3 domain, the V region of rtSH3p13 also inhibits endocytosis in CHO cells. Our results reveal a link between an rtSH3p13-synaptojanin-clathrin complex-mediated formation of pits and the process of spermiogenesis.
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Enteric viruses often contaminate water sources causing frequent outbreaks of gastroenteritis. Reverse transcription-polymerase chain reaction (RT-PCR) assays are commonly used for detection of human enteric viruses in environmental and drinking water samples. RT-PCR provides ...
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Identification and evaluation of reference genes for qRT-PCR normalization in Ganoderma lucidum.
Xu, Jiang; Xu, ZhiChao; Zhu, YingJie; Luo, HongMei; Qian, Jun; Ji, AiJia; Hu, YuanLei; Sun, Wei; Wang, Bo; Song, JingYuan; Sun, Chao; Chen, ShiLin
2014-01-01
Quantitative real-time reverse transcription PCR (qRT-PCR) is a rapid, sensitive, and reliable technique for gene expression studies. The accuracy and reliability of qRT-PCR results depend on the stability of the reference genes used for gene normalization. Therefore, a systematic process of reference gene evaluation is needed. Ganoderma lucidum is a famous medicinal mushroom in East Asia. In the current study, 10 potential reference genes were selected from the G. lucidum genomic data. The sequences of these genes were manually curated, and primers were designed following strict criteria. The experiment was conducted using qRT-PCR, and the stability of each candidate gene was assessed using four commonly used statistical programs-geNorm, NormFinder, BestKeeper, and RefFinder. According to our results, PP2A was expressed at the most stable levels under different fermentation conditions, and RPL4 was the most stably expressed gene in different tissues. RPL4, PP2A, and β-tubulin are the most commonly recommended reference genes for normalizing gene expression in the entire sample set. The current study provides a foundation for the further use of qRT-PCR in G. lucidum gene analysis.
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SPoRT Participation in the GOES-R and JPSS Proving Grounds
NASA Technical Reports Server (NTRS)
Jedlovec, Gary; Fuell, Kevin; Smith, Matthew
2013-01-01
For the last several years, the NASA Short-term Prediction Research and Transition (SPoRT) project at has been working with the various algorithm working groups and science teams to demonstrate the utility of future operational sensors for GOES-R and the suite of instruments for the JPSS observing platforms. For GOES-R, imagery and products have been developed from polar-orbiting sensors such as MODIS and geostationary observations from SEVIRI, simulated imagery, enhanced products derived from existing GOES satellites, and data from ground-based observing systems to generate pseudo or proxy products for the ABI and GLM instruments. The suite of products include GOES-POES basic and RGB hybrid imagery, total lightning flash products, quantitative precipitation estimates, and convective initiation products. SPoRT is using imagery and products from VIIRS, CrIS, ATMS, and OMPS to show the utility of data and products from their operational counterparts on JPSS. The products include VIIRS imagery in swath form, the GOES-POES hybrid, a suite of RGB products including the air mass RGB using water vapor and ozone channels from CrIS, and several DNB products. Over a dozen SPoRT collaborative WFOs and several National Centers are involved in an intensive evaluation of the operational utility of these products.
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NASA Technical Reports Server (NTRS)
Jedlovec, Gary J.
2005-01-01
Over the last three years, NASA/MSFC scientists have embarked on an effort to transition unique NASA EOS data/products and research technology to selected NWSEOs in the southeast U.S. This activity, called the Short-term Prediction and - Research Transition (SPoRT) program, supports the NASA Science Mission Directorate and its Earth-Sun System Mission to develop a scientific understanding of the Earth System and its response to natural or human-induced changes that will enable improved prediction capability for climate, weather, and natural hazards. The overarching question related to weather prediction is "How well can weather forecasting duration and reliability be improved by new space-based observations, data assimilation, and modeling?" The transition activity has included the real-time delivery of MODIS data and products to several NWS Forecast Offices. Local NWS FOs have used the MODIS data to complement the coarse resolution GOES data for a number of applications. Specialized products have also been developed and made available to local and remote offices for their weather applications. Data from &e Lightning Mapping Array (LMA) network has been used in severe storm forecasts at several offices in the region. At the regional scale and forecast horizons from 0-1 day, the next generation of high-resolution mesoscale forecast and data assimilation models have been used to provide local offices with unique weather forecasts not otherwise available. The continued use of near red-time infusion of NASA science products into high-resolution mesoscale forecast and decision-making models can be expected to improve the model initialization as well as short-term forecasts. A current focus of SPoRT is to expand collaborations to include contributions from the assimilation of AMSR-E data in the ADASIARPS forecast system (OU), inclusion of MODIS SSTs and AIRS thermodynamic profiles in the WRF, and to extend the distribution of real-time MODIS and AMSR-E data and products
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A MIV-150/zinc acetate gel inhibits SHIV-RT infection in macaque vaginal explants.
Barnable, Patrick; Calenda, Giulia; Ouattara, Louise; Gettie, Agegnehu; Blanchard, James; Jean-Pierre, Ninochka; Kizima, Larisa; Rodríguez, Aixa; Abraham, Ciby; Menon, Radhika; Seidor, Samantha; Cooney, Michael L; Roberts, Kevin D; Sperling, Rhoda; Piatak, Michael; Lifson, Jeffrey D; Fernandez-Romero, Jose A; Zydowsky, Thomas M; Robbiani, Melissa; Teleshova, Natalia
2014-01-01
To extend our observations that single or repeated application of a gel containing the NNRTI MIV-150 (M) and zinc acetate dihydrate (ZA) in carrageenan (CG) (MZC) inhibits vaginal transmission of simian/human immunodeficiency virus (SHIV)-RT in macaques, we evaluated safety and anti-SHIV-RT activity of MZC and related gel formulations ex vivo in macaque mucosal explants. In addition, safety was further evaluated in human ectocervical explants. The gels did not induce mucosal toxicity. A single ex vivo exposure to diluted MZC (1∶30, 1∶100) and MC (1∶30, the only dilution tested), but not to ZC gel, up to 4 days prior to viral challenge, significantly inhibited SHIV-RT infection in macaque vaginal mucosa. MZC's activity was not affected by seminal plasma. The antiviral activity of unformulated MIV-150 was not enhanced in the presence of ZA, suggesting that the antiviral activity of MZC was mediated predominantly by MIV-150. In vivo administration of MZC and CG significantly inhibited ex vivo SHIV-RT infection (51-62% inhibition relative to baselines) of vaginal (but not cervical) mucosa collected 24 h post last gel exposure, indicating barrier effect of CG. Although the inhibitory effect of MZC (65-74%) did not significantly differ from CG (32-45%), it was within the range of protection (∼75%) against vaginal SHIV-RT challenge 24 h after gel dosing. Overall, the data suggest that evaluation of candidate microbicides in macaque explants can inform macaque efficacy and clinical studies design. The data support advancing MZC gel for clinical evaluation.
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RT-SHIV subpopulation dynamics in infected macaques during anti-HIV therapy
2009-01-01
Background To study the dynamics of wild-type and drug-resistant HIV-1 RT variants, we developed a methodology that follows the fates of individual genomes over time within the viral quasispecies. Single genome sequences were obtained from 3 pigtail macaques infected with a recombinant simian immunodeficiency virus containing the RT coding region from HIV-1 (RT-SHIV) and treated with short-course efavirenz monotherapy 13 weeks post-infection followed by daily combination antiretroviral therapy (ART) beginning at week 17. Bioinformatics tools were constructed to trace individual genomes from the beginning of infection to the end of the treatment. Results A well characterized challenge RT-SHIV inoculum was used to infect three monkeys. The RT-SHIV inoculum had 9 variant subpopulations and the dominant subpopulation accounted for 80% of the total genomes. In two of the three monkeys, the inoculated wild-type virus was rapidly replaced by new wild type variants. By week 13, the original dominant subpopulation in the inoculum was replaced by new dominant subpopulations, followed by emergence of variants carrying known NNRTI resistance mutations. However, during ART, virus subpopulations containing resistance mutations did not outgrow the wide-type subpopulations until a minor subpopulation carrying linked drug resistance mutations (K103N/M184I) emerged. We observed that persistent viremia during ART is primarily made up of wild type subpopulations. We also found that subpopulations carrying the V75L mutation, not known to be associated with NNRTI resistance, emerged initially in week 13 in two macaques. Eventually, all subpopulations from these two macaques carried the V75L mutation. Conclusion This study quantitatively describes virus evolution and population dynamics patterns in an animal model. The fact that wild type subpopulations remained as dominant subpopulations during ART treatment suggests that the presence or absence of at least some known drug resistant
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NASA Technical Reports Server (NTRS)
Molthan, Andrew L.
2010-01-01
The presentation slides include: The SPoRT Center, History and Future of SPoRT, Great Lakes Applications, Great Lakes Forecasting Issues, Applications to the WRF-EMS, Precipitation Science, Lake Effect Precipitation, Sensitivity to Microphysics, Exploring New Schemes, Opportunities for Collaboration, and SPoRT Research and Development.
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NASA Astrophysics Data System (ADS)
Romeo, Stefania; Sannino, Anna; Scarfì, Maria Rosaria; Massa, Rita; D'Angelo, Raffaele; Zeni, Olga
2016-01-01
The last decades have seen increased interest toward possible adverse effects arising from exposure to intense static magnetic fields. This concern is mainly due to the wider and wider applications of such fields in industry and clinical practice; among them, Magnetic Resonance Imaging (MRI) facilities are the main sources of exposure to static magnetic fields for both general public (patients) and workers. In recent investigations, exposures to static magnetic fields have been demonstrated to elicit, in different cell models, both permanent and transient modifications in cellular endpoints critical for the carcinogenesis process. The World Health Organization has therefore recommended in vitro investigations as important research need, to be carried out under strictly controlled exposure conditions. Here we report on the absence of effects on cell viability, reactive oxygen species levels and DNA integrity in MRC-5 human foetal lung fibroblasts exposed to 370 mT magnetic induction level, under different exposure regimens. Exposures have been performed by using an experimental apparatus designed and realized for operating with the static magnetic field generated by permanent magnets, and confined in a magnetic circuit, to allow cell cultures exposure in absence of confounding factors like heating or electric field components.
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A simple competitive RT-PCR assay for quantitation of HIV-1 subtype B and non-B RNA in plasma.
Hamatake, Makiko; Nishizawa, Masako; Yamamoto, Naoki; Kato, Shingo; Sugiura, Wataru
2007-06-01
An easy, inexpensive competitive RT-PCR assay for HIV-1 RNA quantitation was constructed. A 138-bp sequence in the HIV-1 gag p24 region was selected as the target and co-amplified with competitor RNA containing an internal 44-bp deletion. Quantitation of serial dilutions of control RNA samples prepared from the LAI isolate demonstrated a good linearity (R(2)=0.991) within the range between 10 and 250 copies/sample. The detection limit of the assay was determined to be 3.8 copies/sample by Probit analysis and corresponded to 110 copies/ml in plasma. The intra-assay CV value was 9.1%, and the inter-assay value was 25.9%. Both were comparable to those obtained with commercially available HIV-1 RNA quantitation kits. The correlation efficient for the results obtained in 47 plasma samples from HIV-1-infected individuals (subtype A in 1, subtype B in 25, subtype C in 4, subtype F in 1, and CRF01 AE in 16) with the competitive RT-PCR and Cobas Amplicor HIV-1 Monitor test v1.5 was 0.956 for subtype B and 0.947 for subtype non-B. The assay devised is a good alternative for monitoring antiretroviral therapy in resource-poor countries.
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EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. The viral ribonucleic acid (RNA) from water sample concentrates is extracted and tested for enterovirus and norovirus RNA using reverse transcription-quantitative PCR (RT-qPCR). V...
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The role of pleural fluid MAGE RT-nested PCR in the diagnosis of malignant pleural effusion.
Jeon, Eun Ju; Park, Hye Kyeong; Jeon, Kyeongman; Koh, Won-Jung; Suh, Gee Young; Chung, Man Pyo; Kim, Hojoong; Kwon, O Jung; Ki, Chang-Seok; Kim, Jong-Won; Shim, Young Mog; Um, Sang-Won
2012-11-01
Melanoma antigen (MAGE) genes are expressed in tumor cells, the testis and the placenta. The purpose of this prospective study was to investigate the sensitivity, specificity, and accuracy of the carcinoembryonic antigen (CEA), MAGE reverse transcriptase-nested polymerase chain reaction (RT-nested PCR), and cytology of pleural fluid in the diagnosis of malignant pleural effusion. Patients in whom unilateral pleural effusion was identified on chest radiography from January to December 2009 were included in the study. MAGE genes were analyzed by RT-nested PCR using MAGE A1-6 common primers. Of 81 enrolled patients, 46 were diagnosed as malignant pleural effusion, and 24 were diagnosed as benign pleural effusion. The diagnoses of 11 patients were not confirmed in this study. The diagnostic sensitivity, specificity, and accuracy of MAGE RT-nested PCR were 61.4%, 95.7%, and 73.1%, respectively. The diagnostic sensitivities of cytology and CEA (>5 ng/mL) were 61.4% and 75.0%, respectively. Among 17 patients with negative cytology who had malignant pleural effusion, 12 and 10 patients were positive for CEA (>5.0 ng/mL) and MAGE RT-nested PCR, respectively. However, of five patients with malignant pleural effusion that was not recognized by cytology and CEA, MAGE RT-nested PCR correctly predicted a malignant etiology in only one additional patient (20%). MAGE RT-nested PCR seems to add little on the combination of conventional methods in the diagnosis of malignant effusion. © 2012 Tianjin Lung Cancer Institute and Wiley Publishing Asia Pty. Ltd.
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ERIC Educational Resources Information Center
Tse, Chi-Shing; Hutchison, Keith A.; Li, Yongna
2011-01-01
Participants' reaction time (RT) data in a prime-probe flanker task (e.g., ABA-CAC) were analyzed in terms of the characteristics of RT distribution to examine possible mechanisms that produce negative priming. When the prime and probe were presented in the same context and the proportion of repetition-target trials (TRP) was 0.33, negative…
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Veeranna; Lee, Ju-Hyun; Pareek, Tej K; Jaffee, Howard; Boland, Barry; Vinod, K Yaragudri; Amin, Niranjana; Kulkarni, Ashok B; Pant, Harish C; Nixon, Ralph A
2008-10-01
As axons myelinate, establish a stable neurofilament network, and expand in caliber, neurofilament proteins are extensively phosphorylated along their C-terminal tails, which is recognized by the monoclonal antibody, RT-97. Here, we demonstrate in vivo that RT-97 immunoreactivity (IR) is generated by phosphorylation at KSPXK or KSPXXXK motifs and requires flanking lysines at specific positions. extracellular signal regulated kinase 1,2 (ERK1,2) and pERK1,2 levels increase in parallel with phosphorylation at the RT-97 epitope during early postnatal brain development. Purified ERK1,2 generated RT-97 on both KSP motifs on recombinant NF-H tail domain proteins, while cdk5 phosphorylated only KSPXK motifs. RT-97 epitope generation in primary hippocampal neurons was regulated by extensive cross-talk among ERK1,2, c-Jun N-terminal kinase 1,2 (JNK1,2) and cdk5. Inhibition of both ERK1,2 and JNK1,2 completely blocked RT-97 generation. Cdk5 influenced RT-97 generation indirectly by modulating JNK activation. In mice, cdk5 gene deletion did not significantly alter RT-97 IR or ERK1,2 and JNK activation. In mice lacking the cdk5 activator P35, the partial suppression of cdk5 activity increased RT-97 IR by activating ERK1,2. Thus, cdk5 influences RT-97 epitope generation partly by modulating ERKs and JNKs, which are the two principal kinases regulating neurofilament phosphorylation. The regulation of a single target by multiple protein kinases underscores the importance of monitoring other relevant kinases when the activity of a particular one is blocked.
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NASA Astrophysics Data System (ADS)
Kenmochi, Naoki; Nishiura, Masaki; Yoshida, Zensho; Sugata, Tetsuya; Nakamura, Kaori; Katsura, Shotaro
2017-10-01
The Ring Trap 1 (RT-1) device creates a laboratory magnetosphere that is realized by a levitated superconducting ring magnet in vacuum. The RT-1 experiment has demonstrated the self-organization of a plasma clump with a steep density gradient; a peaked density distribution is spontaneously created through `inward diffusion'. In order to evaluate particle transport characteristics in the RT-1 magnetospheric plasmas which cause these inward diffusion, density modulation experiments were performed in the RT-1. Density modulation is a powerful method for estimating a diffusion coefficient D and a convection velocity V by puffing a periodic neutral gas. The gas puff modulation causes the change in the electron density measured by two chords of microwave interferometer (the radial positions r = 60 and 70 cm, vertical chord). In the case of 2 Hz gas puff modulation, the phase delay and the modulation-amplitude decay at the chord r = 60 cm are obtained with 15 degree and 0.8, respectively, with respect to the phase and the amplitude at r = 70 cm. The particle balance equations are solved on the assumption of profile shapes for D to evaluate D, V and particle source rate. The result suggests the inward convection in high beta magnetospheric plasmas.
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2012-01-01
Background Gametocyte carriage is essential for malaria transmission and endemicity of disease; thereby it is a target for malaria control strategies. Malaria-infected individuals may harbour gametocytes below the microscopic detection threshold that can be detected by reverse transcription polymerase chain reaction (RT-PCR) targeting gametocyte-specific mRNA. To date, RT-PCR has mainly been applied to the diagnosis of Plasmodium falciparum gametocytes but very limited for that of Plasmodium vivax. Methods A multiplex-nested RT-PCR targeting Pfs25 and Pvs25 mRNA specific to mature gametocytes of P. falciparum and P. vivax, respectively, was developed. The assay was evaluated using blood samples collected in rainy and dry seasons from febrile patients,in a malaria-endemic area in Thailand. Malaria diagnosis was performed by Giemsa-stained blood smears and 18S rRNA PCR. Results The multiplex-nested RT-PCR detected Pfs25 mRNA in 75 of 86 (87.2%) P. falciparum-infected individuals and Pvs25 mRNA in 82 of 90 (91.1%) P. vivax malaria patients diagnosed by 18S rRNA PCR. Gametocytes were detected in 38 (eight P. falciparum and 30 P. vivax) of 157 microscopy positive samples, implying that a large number of patients harbour sub-microscopic gametocytaemia. No seasonal differences in gametocyte carriage were observed for both malaria species diagnosed by multiplex-nested RT-PCR. With single-nested RT-PCR targeting Pfs25 or Pvs25 mRNA as standard, the multiplex-nested RT-PCR offered sensitivities of 97.4% and 98.9% and specificities of 100% and 98.8% for diagnosing mature gametocytes of P. falciparum and P. vivax, respectively. The minimum detection limit of the multiplex-nested PCR was 10 copies of templates. Conclusions The multiplex-nested RT-PCR developed herein is useful for simultaneous assessment of both P. falciparum and P. vivax gametocyte carriage that is prevalent and generally sympatric in several malaria-endemic areas outside Africa. PMID:22682065
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Kuamsab, Napaporn; Putaporntip, Chaturong; Pattanawong, Urassaya; Jongwutiwes, Somchai
2012-06-10
Gametocyte carriage is essential for malaria transmission and endemicity of disease; thereby it is a target for malaria control strategies. Malaria-infected individuals may harbour gametocytes below the microscopic detection threshold that can be detected by reverse transcription polymerase chain reaction (RT-PCR) targeting gametocyte-specific mRNA. To date, RT-PCR has mainly been applied to the diagnosis of Plasmodium falciparum gametocytes but very limited for that of Plasmodium vivax. A multiplex-nested RT-PCR targeting Pfs25 and Pvs25 mRNA specific to mature gametocytes of P. falciparum and P. vivax, respectively, was developed. The assay was evaluated using blood samples collected in rainy and dry seasons from febrile patients,in a malaria-endemic area in Thailand. Malaria diagnosis was performed by Giemsa-stained blood smears and 18S rRNA PCR. The multiplex-nested RT-PCR detected Pfs25 mRNA in 75 of 86 (87.2%) P. falciparum-infected individuals and Pvs25 mRNA in 82 of 90 (91.1%) P. vivax malaria patients diagnosed by 18S rRNA PCR. Gametocytes were detected in 38 (eight P. falciparum and 30 P. vivax) of 157 microscopy positive samples, implying that a large number of patients harbour sub-microscopic gametocytaemia. No seasonal differences in gametocyte carriage were observed for both malaria species diagnosed by multiplex-nested RT-PCR. With single-nested RT-PCR targeting Pfs25 or Pvs25 mRNA as standard, the multiplex-nested RT-PCR offered sensitivities of 97.4% and 98.9% and specificities of 100% and 98.8% for diagnosing mature gametocytes of P. falciparum and P. vivax, respectively. The minimum detection limit of the multiplex-nested PCR was 10 copies of templates. The multiplex-nested RT-PCR developed herein is useful for simultaneous assessment of both P. falciparum and P. vivax gametocyte carriage that is prevalent and generally sympatric in several malaria-endemic areas outside Africa.
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Centelleghe, Cinzia; Beffagna, Giorgia; Zanetti, Rossella; Zappulli, Valentina; Di Guardo, Giovanni; Mazzariol, Sandro
2016-09-01
Cetacean Morbillivirus (CeMV) has been identified as the most pathogenic virus for cetaceans. Over the past three decades, this RNA virus has caused several outbreaks of lethal disease in odontocetes and mysticetes worldwide. Isolation and identification of CeMV RNA is very challenging in whales because of the poor preservation status frequently shown by tissues from stranded animals. Nested reverse transcription polymerase chain reaction (nested RT-PCR) is used instead of conventional RT-PCR when it is necessary to increase the sensitivity and the specificity of the reaction. This study describes a new nested RT-PCR technique useful to amplify small amounts of the cDNA copy of Cetacean morbillivirus (CeMV) when it is present in scant quantity in whales' biological specimens. This technique was used to analyze different tissues (lung, brain, spleen and other lymphoid tissues) from one under human care seal and seven cetaceans stranded along the Italian coastline between October 2011 and September 2015. A well-characterized, 200 base pair (bp) fragment of the dolphin Morbillivirus (DMV) haemagglutinin (H) gene, obtained by nested RT-PCR, was sequenced and used to confirm DMV positivity in all the eight marine mammals under study. In conclusion, this nested RT-PCR protocol can represent a sensitive detection method to identify CeMV-positive, poorly preserved tissue samples. Furthermore, this is also a rather inexpensive molecular technique, relatively easy to apply. Copyright © 2016 Elsevier B.V. All rights reserved.
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Ramos-Alemán, Fabiola; González-Jasso, Eva; Pless, Reynaldo C
2018-02-15
Several alkali chlorides were compared for their use in reverse transcription (RT) and PCR of different types of nucleic acid templates. On a test region of biological DNA incapable of forming G quadruplex (G4) structures, Taq DNA polymerase showed similar PCR performance with 50 mM KCl, CsCl, LiCl, and NaCl. In contrast, on a synthetic model polydeoxyribonucleotide prone to G4 formation, good PCR amplification was obtained with 50 mM CsCl, but little or none with LiCl or KCl. Similarly, in RT of a G4-prone model polyribonucleotide, MMLV reverse transcriptase produced a good yield with 50 mM CsCl, mediocre yields with LiCl or without added alkali chloride, and a poor yield with 50 mM KCl. The full RT-PCR assay starting from the G4-prone polyribonucleotide, showed good results with CsCl in both stages, poor results with LiCl, and no product formation with KCl. The model polynucleotides showed fast G quadruplex formation under PCR or RT conditions with 50 mM KCl, but not with CsCl or LiCl. The results argue for the use of CsCl instead of KCl for RT and PCR of G4-prone sequences. No advantage was observed when using the 7-deaza type nucleotide analog c 7 dGTP in PCR amplification of the G4-prone polydeoxyribonucleotide. Copyright © 2017 Elsevier Inc. All rights reserved.
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Remains of abutments for Bridge No. 1575 at MD Rt. ...
Remains of abutments for Bridge No. 1575 at MD Rt. 51 in Spring Gap, Maryland, looking northeast. (Compare with HAER MD-115 photos taken 1988). - Western Maryland Railway, Cumberland Extension, Pearre to North Branch, from WM milepost 125 to 160, Pearre, Washington County, MD
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NASA Astrophysics Data System (ADS)
Oktariyanda, T. A.; Rahaju, T.
2018-01-01
The use of information technology by Surabaya City Government has generated various innovations of public services such as “e-Sapa Warga”, e-Commerce, “e-RT/RW”, e-Budgeting, e-Project, e-Procurement, “e-Pendidikan”. Among the many innovations, e-RT/RW (Rukun Tetangga / Rukun Warga) is an innovation that is quite prominent because there are still many areas that have not implemented. This research can be classified as literature review and critical research. The aims of this research are to describe and analyze e-government strategy in Surabaya City through e-RT / RW to improve public service quality. The result of this research is e-government strategy in Surabaya City through e-RT / RW is already relevant and in accordance with Goldsmith’s theory of e-government. The positive impact of the implementation of e-RT / RW is the increasing quality of public services to bring benefits to all citizens Surabaya City.
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Pandey, Mamta; Singh, Dheer; Onteru, Suneel K
2018-06-01
Transcript analysis is usually performed by costly, time-consuming, and expertise intensive methods, like real time-PCR, microarray, etc. However, they are not much feasible in low-input laboratories. Therefore, we implemented the reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a means of mammalian transcript analysis. Particularly, RT-LAMP was developed for buffalo aromatase cytochrome P450 (CYP19) transcript, to study its expression in 3D-cultured buffalo granulosa cells, which were exposed to lipopolysaccharide (LPS). The CYP19-RT-LAMP assay rapidly identified the LPS-induced downregulation of the CYP19 gene within 30 min at 63°C in a water bath. The assay was visualized via unaided eye by observing the change in turbidity and fluorescence, which were decreased by increasing the LPS exposure time to granulosa cells. Overall, the developed CYP19-RT-LAMP assay provided a hope on the application of RT-LAMP for mammalian transcript analysis in low-input laboratories. © 2017 Wiley Periodicals, Inc.
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Wattjes, M P; Krauter, J; Nagel, S; Heidenreich, O; Ganser, A; Heil, G
2000-02-01
The chromosomal translocation t(8;21)(q22;q22) is one of the most frequent karyotypic aberrations in acute myeloid leukemia (AML) and results in a chimeric fusion transcript AML1/MTG8. Since AML1/MTG8 fusion transcripts remain detectable by RT-PCR in t(8;21) AML patients in long-term hematological remission, quantitative assessment of AML1/MTG8 transcripts is necessary for the monitoring of minimal residual disease (MRD) in these patients. Competitive RT-PCR and recently real-time RT-PCR are increasingly used for detection and quantification of leukemia specific fusion transcripts. For the direct comparison of both methods we cloned a 42 bp DNA fragment into the original AML1/MTG8 sequence. The resulting molecule was used as an internal competitor for our novel competitive nested RT-PCR for AML1/MTG8 and as an external standard for the generation of AML1/MTG8 standard curves in a real-time PCR assay. Using this standard molecule for both PCR techniques, we compared their sensitivity, linearity and reproducibility. Both methods were comparable with regard to all parameters tested irrespective of analyzing serial dilutions of plasmids, cell lines or samples from t(8;21) positive AML patients at different stages of the disease. Therefore, both techniques can be recommended for the monitoring of MRD in these particular AML patients. However, the automatization of the real-time PCR technique offers some technical advantages.
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Challenges in Transitioning Research Data to Operations: The SPoRT Paradigm
NASA Technical Reports Server (NTRS)
Jedloved, Gary J.; Smith, Matt; McGrath, Kevin
2010-01-01
Established in 2002 to demonstrate the weather and forecasting application of real-time EOS measurements, the NASA Short-term Prediction Research and Transition (SPoRT) program has grown to be an end-to-end research to operations activity focused on the use of advanced NASA modeling and data assimilation approaches, nowcasting techniques, and unique high-resolution multispectral data from EOS satellites to improve short-term weather forecasts on a regional and local scale. With the ever-broadening application of real-time high resolution satellite data from current EOS and planned NPP, JPSS, and GOES-R sensors to weather forecast problems, significant challenges arise in the acquisition, delivery, and integration of the new capabilities into the decision making process of the operational weather community. For polar orbiting sensors such as MODIS, AIRS, VIIRS, and CRiS, the use of direct broadcast ground stations is key to the real-time delivery of the data and derived products in a timely fashion. With the ABI on the geostationary GOES-R satellite, the data volume will likely increase by a factor of 5- 10 from current data streams. However, the high data volume and limited bandwidth of end user facilities presents a formidable obstacle to timely access to the data. This challenge can be addressed through the use of subsetting techniques, innovative web services, and the judicious selection of data formats. Many of these approaches have been implemented by SPoRT for the delivery of real-time products to NWS forecast offices and other weather entities. Once available in decision support systems like AWIPS II, these new data and products must be integrated into existing and new displays that allow for the integration of the data with existing operational products in these systems. SPoRT is leading the way in demonstrating this enhanced capability. This paper will highlight the ways SPoRT is overcoming many of the challenges presented by the enormous data volumes of
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Ahamed, Syed Fazil; Vivek, Rosario; Kotabagi, Shalini; Nayak, Kaustuv; Chandele, Anmol; Kaja, Murali-Krishna; Shet, Anita
2017-06-01
Dengue surveillance relies on reverse transcription-polymerase chain reaction (RT-PCR), for confirmation of dengue virus (DENV) serotypes. We compared efficacies of published and modified primer sets targeting envelope (Env) and capsid-premembrane (C-prM) genes for detection of circulating DENV serotypes in southern India. Acute samples from children with clinically-diagnosed dengue were used for RT-PCR testing. All samples were also subjected to dengue serology (NS1 antigen and anti-dengue-IgM/IgG rapid immunochromatographic assay). Nested RT-PCR was performed on viral RNA using three methods targeting 654bp C-prM, 511bp C-prM and 641bp Env regions, respectively. RT-PCR-positive samples were validated by population sequencing. Among 171 children with suspected dengue, 121 were dengue serology-positive and 50 were dengue serology-negative. Among 121 serology-positives, RT-PCR detected 91 (75.2%) by CprM654, 72 (59.5%) by CprM511, and 74 (61.1%) by Env641. Among 50 serology-negatives, 10 (20.0%) were detected by CprM654, 12 (24.0%) by CprM511, and 11 (22.0%) by Env641. Overall detection rate using three methods sequentially was 82.6% (100/121) among serology-positive and 40.0% (20/50) among serology-negative samples; 6.6% (8/120) had co-infection with multiple DENV serotypes. We conclude that detection of acute dengue was enhanced by a modified RT-PCR method targeting the 654bp C-prM region, and further improved by using all three methods sequentially. Copyright © 2017 Elsevier B.V. All rights reserved.
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Request for regular monitoring of the symbiotic variable RT Cru
NASA Astrophysics Data System (ADS)
Waagen, Elizabeth O.
2014-08-01
Dr. Margarita Karovska (Harvard-Smithsonian Center for Astrophysics) and colleagues have requested AAVSO observer assistance in their campaign on the symbiotic variable RT Cru (member of a new class of hard X-ray emitting symbiotic binaries). Weekly or more frequent monitoring (B, V, and visual) beginning now is requested in support of upcoming Chandra observations still to be scheduled. "We plan Chandra observations of RT Cru in the near future that will help us understand the characteristics of the accretion onto the white dwarf in this sub-class of symbiotics. This is an important step for determining the precursor conditions for formation of a fraction of asymmetric Planetary Nebulae, and the potential of symbiotic systems as progenitors of at least a fraction of Type Ia supernovae." Finder charts with sequence may be created using the AAVSO Variable Star Plotter (http://www.aavso.org/vsp). Observations should be submitted to the AAVSO International Database. See full Alert Notice for more details and observations.
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Bluetongue virus RNA detection by real-time rt-PCR in post-vaccination samples from cattle.
De Leeuw, I; Garigliany, M; Bertels, G; Willems, T; Desmecht, D; De Clercq, K
2015-04-01
Bluetongue virus serotype 8 (BTV-8) was responsible for a large outbreak among European ruminant populations in 2006-2009. In spring 2008, a massive vaccination campaign was undertaken, leading to the progressive disappearance of the virus. During surveillance programmes in Western Europe in 2010-2011, a low but significant number of animals were found weakly positive using BTV-specific real-time RT-PCR, raising questions about a possible low level of virus circulation. An interference of the BTV-8 inactivated vaccine on the result of the real-time RT-PCR was also hypothesized. Several studies specifically addressed the potential association between a recent vaccination and BTV-8 RNA detection in the blood of sheep. Results were contradictory and cattles were not investigated. To enlighten this point, a large study was performed to determine the risks of detection of bluetongue vaccine-associated RNA in the blood and spleen of cattle using real-time RT-PCR. Overall, the results presented clearly demonstrate that vaccine viral RNA can reach the blood circulation in sufficient amounts to be detected by real-time RT-PCR in cattle. This BTV-8 vaccine RNA carriage appears as short lasting. © 2013 Blackwell Verlag GmbH.
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[Development of pseudoviral competitive internal controls for RT-PCR detection of dengue virus].
Hang, Xiao-Tong; Li, Jian-Dong; Zhang, Quan-Fu; Li, Chuan; Zhang, Shuo; Liang, Mi-Fang; Li, De-Xin
2010-02-01
Development of pseudoviral competitive internal controls for RT-PCR laboratory detection of dengue virus. The internal controls target gene were obtained by insertion of a 180 bp non-related DNA fragment into RT-PCR detection target of dengue virus between the forward and reverse PCR primer binding regions. A yellow florescence protein reporter gene was induced at downstream of internal controls target gene via internal ribosome entry site gene. HEK 293T cells were transfected with plasmid containing this whole cassette and lentiviral packaging support plasmid. Pseudoviral particle was recovered from the supernatant and analyzed quantitatively and qualitatively in simulated samples at the same tube under different experimental conditions. The established pseudoviral competitive internal controls can be used in the RT-PCR detection of different serotype dengue virus and the whole detection process can be monitored. The obtained fragment is easy to be differentiated in agarose electrophoresis. The pseudoviral competitive internal controls could be used for the quality control of the laboratory diagnosis process, simple to prepare, stable for storage, easy to be transformed into internal controls for other RNA virus.
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A Potent Combination Microbicide that Targets SHIV-RT, HSV-2 and HPV
Kizima, Larisa; Rodríguez, Aixa; Kenney, Jessica; Derby, Nina; Mizenina, Olga; Menon, Radhika; Seidor, Samantha; Zhang, Shimin; Levendosky, Keith; Jean-Pierre, Ninochka; Pugach, Pavel; Villegas, Guillermo; Ford, Brian E.; Gettie, Agegnehu; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D.; Paglini, Gabriela; Teleshova, Natalia; Zydowsky, Thomas M.; Robbiani, Melissa; Fernández-Romero, José A.
2014-01-01
Prevalent infection with human herpes simplex 2 (HSV-2) or human papillomavirus (HPV) is associated with increased human immunodeficiency virus (HIV) acquisition. Microbicides that target HIV as well as these sexually transmitted infections (STIs) may more effectively limit HIV incidence. Previously, we showed that a microbicide gel (MZC) containing MIV-150, zinc acetate (ZA) and carrageenan (CG) protected macaques against simian-human immunodeficiency virus (SHIV-RT) infection and that a ZC gel protected mice against HSV-2 infection. Here we evaluated a modified MZC gel (containing different buffers, co-solvents, and preservatives suitable for clinical testing) against both vaginal and rectal challenge of animals with SHIV-RT, HSV-2 or HPV. MZC was stable and safe in vitro (cell viability and monolayer integrity) and in vivo (histology). MZC protected macaques against vaginal (p<0.0001) SHIV-RT infection when applied up to 8 hours (h) prior to challenge. When used close to the time of challenge, MZC prevented rectal SHIV-RT infection of macaques similar to the CG control. MZC significantly reduced vaginal (p<0.0001) and anorectal (p = 0.0187) infection of mice when 106 pfu HSV-2 were applied immediately after vaginal challenge and also when 5×103 pfu were applied between 8 h before and 4 h after vaginal challenge (p<0.0248). Protection of mice against 8×106 HPV16 pseudovirus particles (HPV16 PsV) was significant for MZC applied up to 24 h before and 2 h after vaginal challenge (p<0.0001) and also if applied 2 h before or after anorectal challenge (p<0.0006). MZC provides a durable window of protection against vaginal infection with these three viruses and, against HSV-2 and HPV making it an excellent candidate microbicide for clinical use. PMID:24740100
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Ji, Jie; Upadhyay, Swapna; Xiong, Xiaomiao; Malmlöf, Maria; Sandström, Thomas; Gerde, Per; Palmberg, Lena
2018-05-02
Diesel exhaust particles (DEP) are a major component of outdoor air pollution. DEP mediated pulmonary effects are plausibly linked to inflammatory and oxidative stress response in which macrophages (MQ), epithelial cells and their cell-cell interaction plays a crucial role. Therefore, in this study we aimed at studying the cellular crosstalk between airway epithelial cells with MQ and MQ polarization following exposure to aerosolized DEP by assessing inflammation, oxidative stress, and MQ polarization response markers. Lung mucosa models including primary bronchial epithelial cells (PBEC) cultured at air-liquid interface (ALI) were co-cultured without (PBEC-ALI) and with MQ (PBEC-ALI/MQ). Cells were exposed to 12.7 μg/cm 2 aerosolized DEP using XposeALI ® . Control (sham) models were exposed to clean air. Cell viability was assessed. CXCL8 and IL-6 were measured in the basal medium by ELISA. The mRNA expression of inflammatory markers (CXCL8, IL6, TNFα), oxidative stress (NFKB, HMOX1, GPx) and MQ polarization markers (IL10, IL4, IL13, MRC1, MRC2 RETNLA, IL12 andIL23) were measured by qRT-PCR. The surface/mRNA expression of TLR2/TLR4 was detected by FACS and qRT-PCR. In PBEC-ALI exposure to DEP significantly increased the secretion of CXCL8, mRNA expression of inflammatory markers (CXCL8, TNFα) and oxidative stress markers (NFKB, HMOX1, GPx). However, mRNA expressions of these markers (CXCL8, IL6, NFKB, and HMOX1) were reduced in PBEC-ALI/MQ models after DEP exposure. TLR2 and TLR4 mRNA expression increased after DEP exposure in PBEC-ALI. The surface expression of TLR2 and TLR4 on PBEC was significantly reduced in sham-exposed PBEC-ALI/MQ compared to PBEC-ALI. After DEP exposure surface expression of TLR2 was increased on PBEC of PBEC-ALI/MQ, while TLR4 was decreased in both models. DEP exposure resulted in similar expression pattern of TLR2/TLR4 on MQ as in PBEC. In PBEC-ALI/MQ, DEP exposure increased the mRNA expression of anti-inflammatory M2 macrophage
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Sarquiz-Martínez, Brenda; González-Bonilla, César R; Santacruz-Tinoco, Clara Esperanza; Muñoz-Medina, José E; Pardavé-Alejandre, Héctor D; Barbosa-Cabrera, Elizabeth; Ramírez-González, José Ernesto; Díaz-Quiñonez, José Alberto
2017-01-01
Enterovirus (EV) and herpes simplex virus 1 and 2 (HSV1 and HSV2) are the main etiologic agents of central nervous system infections. Early laboratory confirmation of these infections is performed by viral culture of the cerebrospinal fluid (CSF), or the detection of specific antibodies in serum (e.g., HSV). The sensitivity of viral culture ranges from 65 to 75%, with a recovery time varying from 3 to 10 days. Serological tests are faster and easy to carry out, but they exhibit cross-reactivity between HSV1 and HSV2. Although molecular techniques are more sensitive (sensitivity >95%), they are more expensive and highly susceptible to cross-contamination. A real-time RT-PCR for the detection of EV, HSV1, and HSV2 was compared with end-point nested PCR. We tested 87 CSF samples of patients with a clinical diagnosis of viral meningitis or encephalitis. Fourteen samples were found to be positive by RT-PCR, but only 8 were positive by end-point PCR. The RT-PCR showed a specificity range of 94-100%, the negative predictive value was 100%, and the positive predictive value was 62, 100, and 28% for HSV1, HSV2, and EV, respectively. Real-time RT-PCR detected EV, HSV1, and HSV2 with a higher sensitivity and specificity than end-point nested RT-PCR. © 2017 S. Karger AG, Basel.
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Bailey, Ryan T.; Morway, Eric D.; Niswonger, Richard G.; Gates, Timothy K.
2013-01-01
A numerical model was developed that is capable of simulating multispecies reactive solute transport in variably saturated porous media. This model consists of a modified version of the reactive transport model RT3D (Reactive Transport in 3 Dimensions) that is linked to the Unsaturated-Zone Flow (UZF1) package and MODFLOW. Referred to as UZF-RT3D, the model is tested against published analytical benchmarks as well as other published contaminant transport models, including HYDRUS-1D, VS2DT, and SUTRA, and the coupled flow and transport modeling system of CATHY and TRAN3D. Comparisons in one-dimensional, two-dimensional, and three-dimensional variably saturated systems are explored. While several test cases are included to verify the correct implementation of variably saturated transport in UZF-RT3D, other cases are included to demonstrate the usefulness of the code in terms of model run-time and handling the reaction kinetics of multiple interacting species in variably saturated subsurface systems. As UZF1 relies on a kinematic-wave approximation for unsaturated flow that neglects the diffusive terms in Richards equation, UZF-RT3D can be used for large-scale aquifer systems for which the UZF1 formulation is reasonable, that is, capillary-pressure gradients can be neglected and soil parameters can be treated as homogeneous. Decreased model run-time and the ability to include site-specific chemical species and chemical reactions make UZF-RT3D an attractive model for efficient simulation of multispecies reactive transport in variably saturated large-scale subsurface systems.
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Walton, Thomas J; Li, Geng; McCulloch, Thomas A; Seth, Rashmi; Powe, Desmond G; Bishop, Michael C; Rees, Robert C
2009-06-01
Real-time quantitative RT-PCR analysis of laser microdissected tissue is considered the most accurate technique for determining tissue gene expression. The discovery of estrogen receptor beta (ERbeta) has focussed renewed interest on the role of estrogen receptors in prostate cancer, yet few studies have utilized the technique to analyze estrogen receptor gene expression in prostate cancer. Fresh tissue was obtained from 11 radical prostatectomy specimens and from 6 patients with benign prostate hyperplasia. Pure populations of benign and malignant prostate epithelium were laser microdissected, followed by RNA isolation and electrophoresis. Quantitative RT-PCR was performed using primers for androgen receptor (AR), estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), progesterone receptor (PGR) and prostate specific antigen (PSA), with normalization to two housekeeping genes. Differences in gene expression were analyzed using the Mann-Whitney U-test. Correlation coefficients were analyzed using Spearman's test. Significant positive correlations were seen when AR and AR-dependent PSA, and ERalpha and ERalpha-dependent PGR were compared, indicating a representative population of RNA transcripts. ERbeta gene expression was significantly over-expressed in the cancer group compared with benign controls (P < 0.01). In contrast, PGR expression was significantly down-regulated in the cancer group (P < 0.05). There were no significant differences in AR, ERalpha or PSA expression between the groups. This study represents the first to show an upregulation of ERbeta gene expression in laser microdissected prostate cancer specimens. In concert with recent studies the findings suggest differential production of ERbeta splice variants, which may play important roles in the genesis of prostate cancer. (c) 2009 Wiley-Liss, Inc.
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Analysis of gene expression in small numbers of purified hemopoietic progenitor cells by RT-PCR.
Ziegler, B L; Lamping, C P; Thoma, S J; Fliedner, T M
1995-05-01
Primitive hemopoietic stem cells represent the most probable targets for genetic alterations due to exposure to ionizing irradiation or chemical carcinogens. We have applied a two-step protocol for the purification of CD34+HLA-DR-/low hemopoietic progenitor cells from cord blood (CB). CD34+ cells were isolated by monoclonal antibody (mAb) against CD34 (My10) and immunomagnetic beads. Beads were cleaved off the CD34+ cells by enzymatic treatment with chymopapain. Due to chymopapain-resistance of epitopes recognized by the used mAbs purity control of CD34+ cells and separation into CD34+HLA-DR-/low and CD34+HLA-DR+ subsets could be performed by using flow cytometry. Two miniaturized procedures were applied to isolate poly(A)+ mRNA for the reverse transcription polymerase chain reaction (RT-PCR) from small numbers of CD34+HLA-DR-/low cells. In five experiments, the mean purity of immunomagnetically isolated CD34+ cells was 93.8% +/- 3.9. Flow cytometry sorting of CD34+ cells resulted in pure CD34+HLA-DR-/low populations (purity > 98.8%; range 98.8% to 99.9%; viability > 96%) with an average yield of 2600 +/- 800 cells/5 x 10(7) low density CB cells. By RT-PCR using both poly(A)+ mRNA isolation procedures, sequences corresponding to CD34 and beta 2-microglobulin were amplified from as few as 20 cells. Furthermore, a sequence-independent RT-PCR (SIP-RT-PCR) was applied to amplify the cDNA derived from five erythroblasts isolated from a burst-forming unit-erythroid (BFU-E). Upon hybridization, full-length c-fos message was detected in the SIP-RT-PCR amplified material. Our data demonstrate that gene expression can be detected at the transcriptional level in small numbers of hemopoietic progenitor cells. In addition, the SIP-RT-PCR may allow the amplification of unique mRNA species when subtractive hybridization procedures are performed. The presented data should be useful to analyze gene expression in rare subsets of radiation-exposed immature hemopoietic stem
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ERIC Educational Resources Information Center
Zahedi, Karynn Jensen
2010-01-01
Response to intervention (RtI) is a multi-tiered process of monitoring student responses to remediation that is designed to help struggling learners succeed within the purview of regular education. Under the RtI model, students are referred to special education only after a series of documented interventions have been attempted. This study…
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Satterwhite, Thomas; Son, Ji; Carey, Joseph; Echo, Anthony; Spurling, Terry; Paro, John; Gurtner, Geoffrey; Chang, James; Lee, Gordon K
2014-05-01
We previously reported results of our on-line microsurgery training program, showing that residents who had access to our website significantly improved their cognitive and technical skills. In this study, we report an objective means for expert evaluators to reliably rate trainees' technical skills under the microscope, with the use of our novel global rating scale. "Microsurgery Essentials" (http://smartmicrosurgery.com) is our on-line training curriculum. Residents were randomly divided into 2 groups: 1 group reviewed this online resource and the other did not. Pre- and post-tests consisted of videotaped microsurgical sessions in which the trainee performed "microsurgery" on 3 different models: latex glove, penrose drain, and the dorsal vessel of a chicken foot. The SMaRT (Stanford Microsurgery and Resident Training) scale, consisting of 9 categories graded on a 5-point Likert scale, was used to assess the trainees. Results were analyzed with ANOVA and Student t test, with P less than 0.05 indicating statistical significance. Seventeen residents participated in the study. The SMaRT scale adequately differentiated the performance of more experienced senior residents (PGY-4 to PGY-6, total average score=3.43) from less experienced junior residents (PGY-1 to PGY-3, total average score=2.10, P<0.0001). Residents who viewed themselves as being confident received a higher score on the SMaRT scale (average score 3.5), compared to residents who were not as confident (average score 2.1) (P<0.001). There were no significant differences in scoring among all 3 evaluators (P>0.05). Additionally, junior residents who had access to our website showed a significant increase in their graded technical performance by 0.7 points when compared to residents who did not have access to the website who showed an improvement of only 0.2 points (P=0.01). Our SMaRT scale is valid and reliable in assessing the microsurgical skills of residents and other trainees. Current trainees are more
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DOE Office of Scientific and Technical Information (OSTI.GOV)
McKenzie, E; Yang, W; Burnison, M
2014-06-15
Purpose: Patients undergoing radiotherapy (RT) for left-sided breast cancer have increased risk of coronary artery disease. Deep Inhalation Breath Hold assisted RT (DIBH-RT) is shown to increase the geometric separation of the target area and heart, reducing cardiac radiation dose. The purposes of this study are to use Cine MV portal images to determine the stability of spirometer-guided DIBH-RT and examine the dosimetric cardiopulmonary impact of this technique. Methods: Twenty consecutive patients with left-sided breast cancer were recruited to the IRB-approved study. Free-breathing (FB) and DIBH-CT's were acquired at simulation. Rigid registration of the FB-CT and DIBH-CT was performed usingmore » primarily breast tissue. Treatment plans were created for each FB-CT and DIBH-CT using identical paired tangent fields with field-in-field or electronic compensation techniques. Dosimetric evaluation included mean and maximum (Dmax) doses for the left anterior descending artery (LAD), mean heart dose, and left lung V20. Cine MV portal images were acquired for medial and lateral fields during treatment. Analysis of Cine images involved chest wall segmentation using an algorithm developed in-house. Intra- and inter-fractional chest wall motion were determined through affine registration to the first frame of each Cine. Results: Dose to each cardiac structure evaluated was significantly (p<0.001) reduced with the DIBH plans. Mean heart dose decreased from 2.9(0.9–6.6) to 1.6(0.6–5.3) Gy; mean LAD dose from 16.6(3–43.6) to 7.4(1.7–32.7) Gy; and LAD Dmax from 35.4 (6.1–53) to 18.4(2.5–51.2) Gy. No statistically significant reduction was found for the left lung V20. Average AP and SI median chest wall motion (intrafractional) was 0.1 (SD=0.9) and 0.5 (SD=1.1) mm, respectively. Average AP inter-fractional chest wall motion was 2.0 (SD=1.4) mm. Conclusion: Spirometer-based DIBH treatments of the left breast are reproducible both inter- and intra-fractionally, and
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Applications of LANCE Data at SPoRT
NASA Technical Reports Server (NTRS)
Molthan, Andrew
2014-01-01
Short term Prediction Research and Transition (SPoRT) Center: Mission: Apply NASA and NOAA measurement systems and unique Earth science research to improve the accuracy of short term weather prediction at the regional/local scale. Goals: Evaluate and assess the utility of NASA and NOAA Earth science data and products and unique research capabilities to address operational weather forecast problems; Provide an environment which enables the development and testing of new capabilities to improve short term weather forecasts on a regional scale; Help ensure successful transition of new capabilities to operational weather entities for the benefit of society
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SMAP Data Assimilation at NASA SPoRT
NASA Technical Reports Server (NTRS)
Blankenship, Clay B.; Case, Jonathan L.; Zavodsky, Bradley T.
2016-01-01
The NASA Short-Term Prediction Research and Transition (SPoRT) Center maintains a near-real- time run of the Noah Land Surface Model within the Land Information System (LIS) at 3-km resolution. Soil moisture products from this model are used by several NOAA/National Weather Service Weather Forecast Offices for flood and drought situational awareness. We have implemented assimilation of soil moisture retrievals from the Soil Moisture Ocean Salinity (SMOS) and Soil Moisture Active/ Passive (SMAP) satellites, and are now evaluating the SMAP assimilation. The SMAP-enhanced LIS product is planned for public release by October 2016.
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1989-04-20
International Business Machines Corporation, IBM Development System. for the Ada Language AIX/RT Ada Compiler, Version 1.1.1, Wright-Patterson APB...Certificate Number: 890420V1.10066 International Business Machines Corporation IBM Development System for the Ada Language AIX/RT Ada Compiler, Version 1.1.1...TEST INFORMATION The compiler was tested using command scripts provided by International Business Machines Corporation and reviewed by the validation
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Superior sulcus non-small cell lung carcinoma: A comparison of IMRT and 3D-RT dosimetry.
Truntzer, Pierre; Antoni, Delphine; Santelmo, Nicola; Schumacher, Catherine; Falcoz, Pierre-Emmanuel; Quoix, Elisabeth; Massard, Gilbert; Noël, Georges
2016-01-01
A dosimetric study comparing intensity modulated radiotherapy (IMRT) by TomoTherapy to conformational 3D radiotherapy (3D-RT) in patients with superior sulcus non-small cell lung cancer (NSCLC). IMRT became the main technique in modern radiotherapy. However it was not currently used for lung cancers. Because of the need to increase the dose to control lung cancers but because of the critical organs surrounding the tumors, the gains obtainable with IMRT is not still demonstrated. A dosimetric comparison of the planned target and organs at risk parameters between IMRT and 3D-RT in eight patients who received preoperative or curative intent irradiation. In the patients who received at least 66 Gy, the mean V95% was significantly better with IMRT than 3D-RT (p = 0.043). IMRT delivered a lower D2% compared to 3D-RT (p = 0.043). The IH was significantly better with IMRT (p = 0.043). The lung V 5 Gy and V 13 Gy were significantly higher in IMRT than 3D-RT (p = 0.043), while the maximal dose (D max) to the spinal cord was significantly lower in IMRT (p = 0.043). The brachial plexus D max was significantly lower in IMRT than 3D-RT (p = 0.048). For patients treated with 46 Gy, no significant differences were found. Our study showed that IMRT is relevant for SS-NSCLC. In patients treated with a curative dose, it led to a reduction of the exposure of critical organs, allowing a better dose distribution in the tumor. For the patients treated with a preoperative schedule, our results provide a basis for future controlled trials to improve the histological complete response by increasing the radiation dose.
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ERIC Educational Resources Information Center
Buckner, Jerry W.
2013-01-01
Conceptually, Response to Intervention (RtI) is a multi-tiered problem solving process rooted in special education yet designed as a framework for early and on-going instructional interventions for students across a continuum of academic need. In recent years, however, RtI has become an increasingly significant part of the discourse on school…
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Parshionikar, Sandhya U; Cashdollar, Jennifer; Fout, G Shay
2004-10-01
Enteric viruses often contaminate water sources causing frequent outbreaks of gastroenteritis. Reverse transcription-polymerase chain reaction (RT-PCR) assays are commonly used for detection of human enteric viruses in environmental and drinking water samples. RT-PCR provides a means to rapidly detect low levels of these viruses, but it is sensitive to inhibitors that are present in water samples. Inhibitors of RT-PCR are concentrated along with viruses during sample processing. While procedures have been developed to remove inhibitors, none of them completely remove all inhibitors from all types of water matrices. This problem requires that adequate controls be used to distinguish true from potentially false-negative results. To address this problem, we have developed homologous viral internal controls for hepatitis A virus (HAV), poliovirus, Norwalk virus and rotavirus. These internal controls can be used in RT-PCR assays for the detection of the above viruses by competitive amplification, thereby allowing the detection of false negatives in processed water samples. The internal controls developed in this study were successfully tested with virus-seeded environmental water sample concentrates.
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Systemic Sustainability in RtI Using Intervention-Based Scheduling Methodologies
ERIC Educational Resources Information Center
Dallas, William P.
2017-01-01
This study evaluated a scheduling methodology referred to as intervention-based scheduling to address the problem of practice regarding the fidelity of implementing Response to Intervention (RtI) in an existing school schedule design. Employing panel data, this study used fixed-effects regressions and first differences ordinary least squares (OLS)…
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How feasible is remote 3D dosimetry for MR guided Radiation Therapy (MRgRT)?
NASA Astrophysics Data System (ADS)
Mein, S.; Rankine, L.; Miles, D.; Juang, T.; Cai, B.; Curcuru, A.; Mutic, S.; Fenoli, J.; Adamovics, J.; Li, H.; Oldham, M.
2017-05-01
To develop and apply a remote dosimetry protocol with PRESAGE® radiochromic plastic and optical-CT readout in the validation of MRI guided radiation therapy (MRgRT) treatments (MRIdian® by ViewRay®). Through multi-institutional collaboration we performed PRESAGE® dosimetry studies in 4ml cuvettes to investigate dose-response linearity, MR-compatibility, and energy-independence. An open calibration field and symmetrical 3-field plans were delivered to 10cm diameter PRESAGE® to examine percent depth dose and response uniformity under a magnetic field. Evidence of non-linear dose response led to a large volume PRESAGE® study where small corrections were developed for temporally- and spatially-dependent behaviors observed between irradiation and delayed readout. TG-119 plans were created in the MRIdian® TPS and then delivered to 14.5cm 2kg PRESAGE® dosimeters. Through the domestic investigation of an off-site MRgRT system, a refined 3D remote dosimetry protocol is presented capable of validation of advanced MRgRT radiation treatments.
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USDA-ARS?s Scientific Manuscript database
Quantitative real-time polymerase chain reaction (qRT-PCR) is the most important tool in measuring levels of gene expression due to its accuracy, specificity, and sensitivity. However, the accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference gene...
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Valle-Maldonado, Marco I; Jácome-Galarza, Irvin E; Gutiérrez-Corona, Félix; Ramírez-Díaz, Martha I; Campos-García, Jesús; Meza-Carmen, Víctor
2015-03-01
Mucor circinelloides is a dimorphic fungal model for studying several biological processes including cell differentiation (yeast-mold transitions) as well as biodiesel and carotene production. The recent release of the first draft sequence of the M. circinelloides genome, combined with the availability of analytical methods to determine patterns of gene expression, such as quantitative Reverse transcription-Polymerase chain reaction (qRT-PCR), and the development of molecular genetic tools for the manipulation of the fungus, may help identify M. circinelloides gene products and analyze their relevance in different biological processes. However, no information is available on M. circinelloides genes of stable expression that could serve as internal references in qRT-PCR analyses. One approach to solve this problem consists in the use of housekeeping genes as internal references. However, validation of the usability of these reference genes is a fundamental step prior to initiating qRT-PCR assays. This work evaluates expression of several constitutive genes by qRT-PCR throughout the morphological differentiation stages of M. circinelloides; our results indicate that tfc-1 and ef-1 are the most stable genes for qRT-PCR assays during differentiation studies and they are proposed as reference genes to carry out gene expression studies in this fungus.
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Development of real-time RT-PCR for the detection of low concentrations of Rift Valley fever virus.
Maquart, Marianne; Temmam, Sarah; Héraud, Jean-Michel; Leparc-Goffart, Isabelle; Cêtre-Sossah, Catherine; Dellagi, Koussay; Cardinale, Eric; Pascalis, Hervé
2014-01-01
In recent years, Madagascar and the Comoros archipelago have been affected by epidemics of Rift Valley fever (RVF), however detection of Rift Valley fever virus (RVFV) in zebu, sheep and goats during the post epidemic periods was frequently unsuccessful. Thus, a highly sensitive real-time RT-PCR assay was developed for the detection of RVFV at low viral loads. A new RVF SYBR Green RT-PCR targeting the M segment was tested on serum from different RVF seronegative ruminant species collected from May 2010 to August 2011 in Madagascar and the Comoros archipelago and compared with a RVF specific quantitative real time RT-PCR technique, which is considered as the reference technique. The specificity was tested on a wide range of arboviruses or other viruses giving RVF similar clinical signs. A total of 38 out of 2756 serum samples tested positive with the new RT-PCR, whereas the reference technique only detected 5 out of the 2756. The described RT-PCR is an efficient diagnostic tool for the investigation of enzootic circulation of the RVF virus. It allows the detection of low viral RNA loads adapted for the investigations of reservoirs or specific epidemiological situations such as inter-epizootic periods. Copyright © 2013 Elsevier B.V. All rights reserved.
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de Moraes, F M; Espósito, D L A; Klein, T M; da Fonseca, B A L
2018-01-01
Clinical manifestations of Zika, dengue, and chikungunya virus infections are very similar, making it difficult to reach a diagnosis based only on clinical grounds. In addition, there is an intense cross-reactivity between antibodies directed to Zika virus and other flaviviruses, and an accurate Zika diagnosis is best achieved by real-time RT-PCR. However, some real-time RT-PCR show better performance than others. To reach the best possible Zika diagnosis, the analytic sensitivity of some probe-based real-time RT-PCR amplifying Zika virus RNA was evaluated in spiked and clinical samples. We evaluated primers and probes to detect Zika virus, which had been published before, and tested sensitivity using serum spiked and patient samples by real-time RT-PCR. When tested against spiked samples, the previously described primers showed different sensitivity, with very similar results when samples from patients (serum and urine) were analyzed. Real-time RT-PCR designed to amplify Zika virus NS1 showed the best analytical sensitivity for all samples.
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Romeo, Stefania; Sannino, Anna; Scarfì, Maria Rosaria; Massa, Rita; d’Angelo, Raffaele; Zeni, Olga
2016-01-01
The last decades have seen increased interest toward possible adverse effects arising from exposure to intense static magnetic fields. This concern is mainly due to the wider and wider applications of such fields in industry and clinical practice; among them, Magnetic Resonance Imaging (MRI) facilities are the main sources of exposure to static magnetic fields for both general public (patients) and workers. In recent investigations, exposures to static magnetic fields have been demonstrated to elicit, in different cell models, both permanent and transient modifications in cellular endpoints critical for the carcinogenesis process. The World Health Organization has therefore recommended in vitro investigations as important research need, to be carried out under strictly controlled exposure conditions. Here we report on the absence of effects on cell viability, reactive oxygen species levels and DNA integrity in MRC-5 human foetal lung fibroblasts exposed to 370 mT magnetic induction level, under different exposure regimens. Exposures have been performed by using an experimental apparatus designed and realized for operating with the static magnetic field generated by permanent magnets, and confined in a magnetic circuit, to allow cell cultures exposure in absence of confounding factors like heating or electric field components. PMID:26762783
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A Novel Pan-Flavivirus Detection and Identification Assay Based on RT-qPCR and Microarray
Sachse, Konrad; Ziegler, Ute; Keller, Markus
2017-01-01
The genus Flavivirus includes arthropod-borne viruses responsible for a large number of infections in humans and economically important animals. While RT-PCR protocols for specific detection of most Flavivirus species are available, there has been also a demand for a broad-range Flavivirus assay covering all members of the genus. It is particularly challenging to balance specificity at genus level with equal sensitivity towards each target species. In the present study, a novel assay combining a SYBR Green-based RT-qPCR with a low-density DNA microarray has been developed. Validation experiments confirmed that the RT-qPCR exhibited roughly equal sensitivity of detection and quantification for all flaviviruses tested. These PCR products are subjected to hybridization on a microarray carrying 84 different oligonucleotide probes that represent all known Flavivirus species. This assay has been used as a screening and confirmation tool for Flavivirus presence in laboratory and field samples, and it performed successfully in international External Quality Assessment of NAT studies. Twenty-six Flavivirus strains were tested with the assay, showing equivalent or superior characteristics compared with the original or even with species-specific RT-PCRs. As an example, test results on West Nile virus detection in a panel of 340 mosquito pool samples from Greece are presented. PMID:28626758
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Shin, Y; Mori, T; Okita, M; Gemma, T; Kai, C; Mikami, T
1995-06-01
For a rapid diagnosis of canine distemper virus (CDV) infection, the reverse transcription-PCR (RT-PCR) was carried out to detect CDV nucleoprotein (NP) gene from canine peripheral blood mononuclear cells (PBMCs). Two sets of primers were targeted to two regions of NP gene of CDV Onderstepoort strain. The NP gene fragments were well amplified by the RT-PCR in each of the RNA extracts from Vero cells infected with 6 laboratory strains of CDV including Onderstepoort strain, and from PBMCs of a dog experimentally infected with CDV. The amplified NP gene was detected in 17 of 32 samples from dogs which were clinically suspected for CDV infection at veterinary hospitals. No RT-PCR product was found in 52 samples from healthy dogs including 40 specific pathogen free beagles vaccinated with an attenuated live virus-vaccine for CDV and 12 stray dogs. The RT-PCR provides a fast, sensitive, and supplementary method for the diagnosis of CDV infection in dogs.
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Using the SPoRT POES/GOES Hybrid Product in OCONUS Forecasting
NASA Technical Reports Server (NTRS)
Smith, Matt; Fuell, Kevin; Nelson, Jim
2014-01-01
The SPoRT (Short-term Prediction and Research Transition) Program at the NASA/Marshall Space Flight Center has been providing unique NASA and NOAA data and techniques to partner Weather Forecast Offices (WFOs) for ten years. Data are provided in the Decision Support System used by WFO forecasters: AWIPS. For the last couple of years, SPoRT has been producing the POES/GOES Hybrid. This suite of products combines the strength ofl5- minute animations of GOES imagery - providing temporal continuity, with the higher resolution, relatively random availability, of polar orbiting (POES) imagery data. The product was first introduced with only MODIS data from NASA's Terra and Aqua satellites, but recently the VIIRS instrument onboard the Suomi-NPP satellite was added, providing better high-resolution coverage. These products represent SPoRT's efforts to prepare for higher resolution, higher frequency GOES-R imagery - as well as helping to move VIIRS (JPSS) data into the mainstream of weather forecasting. SPoRT generates 5 products for this dataset: Visible, Longwave Infrared (11 micrometers), Shortwave IR (3.7 micrometers), Water Vapor (6.7 micrometers), and Fog (Difference of 11 micrometer and 3.7 micrometer channels). The Water Vapor hybrid product has a Red-Blue-Green image from MODIS inlaid, since it provides even more qualitative information than water vapor alone. Animated examples of the products will be shown in this presentation. While the resolution at nadir of GOES imagery is nominally Han (4km for IR channels), the inlaid polar orbiter imagery has a resolution of 250m (lkm for IR channels). This has tremendous application in the continental US. However, in high latitudes, since the usefulness of GOES degrades poleward rapidly, the contrast of GOES and POES data is stark. The consistent temporal nature of GOES, even though at a reduced resolution at high latitudes, provides basic situational awareness, but the introduction of polar data is very helpful in seeing
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Tighe, S.; Holbrook, J.; Nadella, V.; Carmical, R.; Sol-Church, K.; Yueng, A.T.; Chittur, S.
2011-01-01
The Nucleic Acid Research Group (NARG) has previously conducted studies evaluating the impact of RNA integrity and priming strategies on cDNA synthesis and real-time RT-qPCR. The results of last year's field study as it relates to degraded RNA will be presented. In continuation of the RNA integrity theme, this year's study was designed to evaluate the impact of RNA integrity on the analysis of miRNA expression using real-time RT-qPCR. Target section was based on data obtained by the Microarray Research Group (MARG) and other published data from next gen sequencing. These 9 miRNAs represent three groups of miRNA that are expressed at low, medium or high levels in the First Choice human brain reference RNA sample. Two popular RT priming strategies tested in this study include the Megaplex miRNA TaqMan assay (ABI) and the RT2 miRNA qPCR assay (Qiagen/SA Biosciences). The basis for the ABI assay design is a target-specific stem-loop structure and reverse-transcription primer, while the Qiagen design combines poly(A) tailing and a universal reverse transcription in one cDNA synthesis reaction. For this study, the human brain reference RNA was subject to controlled degradation using RNase A to RIN (RNA Integrity Number) values of 7 (good), 4 (moderately degraded), and 2 (severely degraded).These templates were then used to assess both RT methods. In addition to this real-time RT-qPCR data, the same RNA templates were further analyzed using universal poly(A) tailing and hybridization to Affymetrix miRNA GeneChips. This talk will provide insights into RT priming strategies for miRNA and contrast the qPCR results obtained using different technologies.
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Stafford, Mai; Gale, Catharine R; Mishra, Gita; Richards, Marcus; Black, Stephanie; Kuh, Diana L
2015-01-01
Mental wellbeing, conceptualised as positive affect, life satisfaction and realisation of needs that contribute to psychological growth, captures more than the absence of mental ill health. Several nations now aim to monitor and improve mental wellbeing. Whilst many studies document associations between adverse childhood experiences and mental disorders in adulthood, possible links between childhood experiences and adult mental wellbeing have so far received less attention. Using data from 1976 men and women in the MRC National Survey for Health and Development, we investigated prospective associations between childhood socioeconomic and psychosocial environments and the Warwick Edinburgh Mental Wellbeing Scale, designed to capture both hedonic and eudaimonic facets of wellbeing, at age 60-64. Whilst there was no evidence that childhood socioeconomic circumstances were related to later wellbeing independently of other childhood experiences, elements of childrearing and parenting, parental health and adjustment, and childhood illness were related. More advantaged socioeconomic position was associated with greater wellbeing but this did not explain the links between these childhood exposures and adult wellbeing, suggesting alternative explanatory pathways should be considered. Childhood illness and family psychosocial environment are associated with mental wellbeing in early older age, with effects sizes that are larger or comparable to socioeconomic circumstances in adulthood. Initiatives to improve the nation's mental wellbeing that include programmes targeted to supporting families and children may additionally have benefits that continue into older age.
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Detection of 1612 MHz OH emission in the semiregular variable stars RT Vir, R Crt and W Hya
NASA Astrophysics Data System (ADS)
Etoka, S.; Le Squeren, A. M.; Gerard, E.
2003-05-01
We present evidence of 1612 MHz emission in SR variable stars. The two SRb, RT Vir and R Crt, as well as the SRa W Hya have been monitored with the upgraded Nançay radio telescope since February 2001. All three objects have shown a weak 1612 MHz emission occuring in the velocity range of the strongest emission observed in the main-lines. Such a detection is the second observational evidence for emission in the 1612 MHz OH maser satellite line from SRb stars. It also confirms the presence of 1612 MHz emission in the SRa W Hya discovered by Etoka et al. (\\cite{etoka01}). Such a finding strongly suggests that the shell properties of those three objects are quite similar to those of the Mira stars with similar IR characteristics.
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Park, Ilwoo; Tamai, Gregory; Lee, Michael C.; Chuang, Cynthia F.; Chang, Susan M.; Berger, Mitchel S.; Nelson, Sarah J.; Pirzkall, Andrea
2008-01-01
Purpose To determine whether the combined MRI and MR spectroscopy imaging (MRSI) prior to radiation therapy (RT) is valuable for RT target definition, and to evaluate the feasibility of replacing the current definition of uniform margins by custom shaped margins based on the information from MRI and MRSI. Methods and Materials Twenty three GBM patients underwent MRI and MRSI within 4 weeks after surgery but before the initiation of RT and at two month follow-up (FU) intervals thereafter. MRSI data were quantified on the basis of a Choline-to-NAA Index (CNI) as a measure of spectroscopic abnormality. A combined anatomic and metabolic ROI (MRI/S) consisting of T2-weighted hyperintensity, contrast enhancement (CE), resection cavity and CNI2 based on the pre-RT imaging was compared to CNI2 extent and RT dose distribution. The spatial relationship of the pre-RT MRI/S and the RT dose volume was compared to the extent of CE at each FU. Results Nine patients showed new or increased CE during FU, and 14 patients were either stable or had decreased CE. New or increased areas of CE occurred within CNI2 that was covered by 60 Gy in six patients and within the CNI2 that was not entirely covered by 60 Gy in three patients. New or increased CE resided within the pre-RT MRI/S lesion in 89 % (8/9) of the patients with new or increased CE. Conclusion These data indicate that the definition of RT target volumes according to the combined morphologic and metabolic abnormality may be sufficient for RT targeting. PMID:17513061
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NASA Technical Reports Server (NTRS)
Bell, Jordan R.; Case, Jonathan L.; Molthan, Andrew L.
2011-01-01
The NASA Short-term Prediction Research and Transition (SPoRT) Center develops new products and techniques that can be used in operational meteorology. The majority of these products are derived from NASA polar-orbiting satellite imagery from the Earth Observing System (EOS) platforms. One such product is a Greenness Vegetation Fraction (GVF) dataset, which is produced from Moderate Resolution Imaging Spectroradiometer (MODIS) data aboard the NASA EOS Aqua and Terra satellites. NASA SPoRT began generating daily real-time GVF composites at 1-km resolution over the Continental United States (CONUS) on 1 June 2010. The purpose of this study is to compare the National Centers for Environmental Prediction (NCEP) climatology GVF product (currently used in operational weather models) to the SPoRT-MODIS GVF during June to October 2010. The NASA Land Information System (LIS) was employed to study the impacts of the new SPoRT-MODIS GVF dataset on land surface models apart from a full numerical weather prediction (NWP) model. For the 2010 warm season, the SPoRT GVF in the western portion of the CONUS was generally higher than the NCEP climatology. The eastern CONUS GVF had variations both above and below the climatology during the period of study. These variations in GVF led to direct impacts on the rates of heating and evaporation from the land surface. The second phase of the project is to examine the impacts of the SPoRT GVF dataset on NWP using the Weather Research and Forecasting (WRF) model. Two separate WRF model simulations were made for individual severe weather case days using the NCEP GVF (control) and SPoRT GVF (experimental), with all other model parameters remaining the same. Based on the sensitivity results in these case studies, regions with higher GVF in the SPoRT model runs had higher evapotranspiration and lower direct surface heating, which typically resulted in lower (higher) predicted 2-m temperatures (2-m dewpoint temperatures). The opposite was true
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Sequence Optimized Real-Time RT-PCR Assay for Detection of Crimean-Congo Hemorrhagic Fever Virus
2017-03-21
19-23]. Real-56 time reverse-transcription PCR remains the gold standard for quantitative , sensitive, and specific 57 detection of CCHFV; however...five-fold in two different series , and samples were run by real- time RT-PCR 116 in triplicate. The preliminary LOD was the lowest RNA dilution where...1 Sequence optimized real- time RT-PCR assay for detection of Crimean-Congo hemorrhagic fever 1 virus 2 3 JW Koehler1, KL Delp1, AT Hall1, SP
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Fout, G. Shay; Cashdollar, Jennifer L.; Griffin, Shannon M.; Brinkman, Nichole E.; Varughese, Eunice A.; Parshionikar, Sandhya U.
2016-01-01
EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water. PMID:26862985
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NASA Astrophysics Data System (ADS)
Zouari, I.; Sassi, Z.; Seveyrat, L.; Perrin, V.; Zghal, S.; Abdelmoula, N.; Lebrun, L.; Khemakhem, H.
2017-07-01
BaTi0.9Zr0.1O3 (BZT), Ba1- x Ln2 x/3□ x/3Ti0.9Zr0.1O3 (with x = 0.5% mol and Ln = Er3+) (BZT-Er) and Ba1- x Ln2 x/3□ x/3Ti0.9Zr0.1O3 (with x = 0.5% mol and Ln = Pr3+) (BZT-Pr) were prepared via the conventional solid-state reaction method. X-ray diffraction showed that all these ceramics were in the single perovskite phase at room temperature (RT). The temperature dependence of dielectric behavior was investigated in the temperature range 25-225°C and exhibited a classical ferroelectric behavior. A slight decrease of the Curie temperature ( T C) with Pr3+ and Er3+ substitution was observed in addition to an increase in the maximum dielectric permittivity ( \\varepsilon_{r {max} }^' }} ) of about 40% for the BZT-Er. At RT, the ferroelectric and piezoelectric coefficients were decreased for BZT-Pr, but were maintained for BZT-Er with a piezoelectric coefficient ( d 33) of 185 pC/N, a planar electromechanical coupling factor of 30%, and a remanent polarization of 11.6 μC/cm2. The Raman bands as a function of temperature confirmed the paraelectric-ferroelectric phase transition of all those ceramics. The photoluminescence spectra showed that strong red (615 nm and 645 nm) and bright green (523 nm and 545 nm) emission bands were obtained, under excitation by laser at 488 nm at RT, for BZT-Pr and BZT-Er, respectively. These multifunctional materials showed a significant technological promise in coupling device applications.
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The SPoRT concept of bracing for idiopathic scoliosis.
Zaina, Fabio; Fusco, Claudia; Atanasio, Salvatore; Negrini, Stefano
2011-01-01
The SPoRT (acronym: Symmetrical, Patient-oriented, Rigid, Three-dimensional, active) concept of bracing is a new way to build braces based on our 20 years of experience and the biomechanical principles of scoliosis correction, inclusive of the Sibilla and Sforzesco braces. The concept always requires a custom brace, which is made according to the patient's individual requirements. New technologies such as CAD-CAM can be applied, and often for better results, without the customary use of prebuilt forms whose measurements are stored in databases. Once the initial draft brace is completed, a final test must be made on the patient to modify and adapt it, depending on his or her real interaction between the body and the brace. The results that are today available on the SPoRT concept relate to the Sforzesco brace and are necessarily short-term, because the first treated patients are now reaching the fourth-year follow-up examination and haven't yet completed their treatments. On the basis of the initial evaluations, we can state that the Sforzesco brace is more effective than the Lyon brace after 6 months of treatment and that the Sforzesco brace is equally effective as the Risser Plast brace.
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ERIC Educational Resources Information Center
King, Diane; Coughlin, Patricia Kathleen
2016-01-01
There are two approaches for providing Tier 2 interventions within Response to Intervention (RtI): standard treatment protocol (STP) and the problem-solving approach (PSA). This article describes the multi-tiered RtI prevention model being implemented across the United States through an analysis of these two approaches in reading instruction. It…
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Development of an improved RT-LAMP assay for detection of currently circulating rubella viruses.
Abo, H; Okamoto, K; Anraku, M; Otsuki, N; Sakata, M; Icenogle, J; Zheng, Q; Kurata, T; Kase, T; Komase, K; Takeda, M; Mori, Y
2014-10-01
Rubella virus is the causative agent of rubella. The symptoms are usually mild, and characterized by a maculopapular rash and fever. However, rubella infection in pregnant women sometimes can result in the birth of infants with congenital rubella syndrome (CRS). Global efforts have been made to reduce and eliminate CRS. Although a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for detection of rubella virus has been reported, the primers contained several mismatched nucleotides with the genomes of currently circulating rubella virus strains. In the present study, a new RT-LAMP assay was established. The detection limit of this assay was 100-1000PFU/reaction of viruses for all rubella genotypes, except for genotype 2C, which is not commonly found in the current era. Therefore, the new RT-LAMP assay can successfully detect all current rubella virus genotypes, and does not require sophisticated devices like TaqMan real-time PCR systems. This assay should be a useful assay for laboratory diagnosis of rubella and CRS. Copyright © 2014 Elsevier B.V. All rights reserved.
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Villegas, Guillermo; Calenda, Giulia; Ugaonkar, Shweta; Zhang, Shimin; Kizima, Larisa; Mizenina, Olga; Gettie, Agegnehu; Blanchard, James; Cooney, Michael L; Robbiani, Melissa; Fernández-Romero, José A; Zydowsky, Thomas M; Teleshova, Natalia
2016-01-01
Women need multipurpose prevention products (MPTs) that protect against sexually transmitted infections (STIs) and provide contraception. The Population Council has developed a prototype intravaginal ring (IVR) releasing the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 (M), zinc acetate (ZA), carrageenan (CG) and levonorgestrel (LNG) (MZCL IVR) to protect against HIV, HSV-2, HPV and unintended pregnancy. Our objective was to evaluate the anti-SHIV-RT activity of MZCL IVR in genital mucosa. First, macaque vaginal tissues were challenged with SHIV-RT in the presence of (i) MIV-150 ± LNG or (ii) vaginal fluids (VF); available from studies completed earlier) collected at various time points post insertion of MZCL and MZC IVRs. Then, (iii) MZCL IVRs (vs. LNG IVRs) were inserted in non-Depo Provera-treated macaques for 24h and VF, genital biopsies, and blood were collected and tissues were challenged with SHIV-RT. Infection was monitored with one step SIV gag qRT-PCR or p27 ELISA. MIV-150 (LCMS/MS, RIA), LNG (RIA) and CG (ELISA) were measured in different compartments. Log-normal generalized mixed linear models were used for analysis. LNG did not affect the anti-SHIV-RT activity of MIV-150 in vitro. MIV-150 in VF from MZC/MZCL IVR-treated macaques inhibited SHIV-RT in vaginal mucosa in a dose-dependent manner (p<0.05). MIV-150 in vaginal tissue from MZCL IVR-treated animals inhibited ex vivo infection relative to baseline (96%; p<0.0001) and post LNG IVR group (90%, p<0.001). No MIV-150 dose-dependent protection was observed, likely because of high MIV-150 concentrations in all vaginal tissue samples. In cervical tissue, MIV-150 inhibited infection vs. baseline (99%; p<0.05). No cervical tissue was available for MIV-150 measurement. Exposure to LNG IVR did not change tissue infection level. These observations support further development of MZCL IVR as a multipurpose prevention technology to improve women's sexual and reproductive health.
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Hunter, T.C.; Knudtson, K.L.; Nadella, V.; Sol-Church, K.; Taylor, W.L.; Tighe, S.; Yueng, A.T.; Chittur, S.
2010-01-01
r1-1 Real-time reverse transcriptase quantitative PCR (RT-qPCR) is a widely used technique for measuring transcript levels. Priming strategy and reverse transcriptase enzyme are key elements that affect sensitivity and variability of RT-qPCR and microarray results. Previously, the Nucleic Acid Research Group (NARG) had conducted preliminary studies within the group to examine the effects of priming strategy on generating cDNA for use with qPCR. This year's study was an open study in which the qPCR community was invited to participate. Participants received the RT primers and RNA template and were asked to perform the RT reaction using their preferred reaction conditions. Each participating laboratory was provided at least two RNA templates of varying quality. The RT products were returned to the NARG and all RT reactions were used in a qPCR reaction. The qPCR assays looked at three genes of varying abundance, b-actin (high copy), b-glucuronidase (medium copy) and TATA binding protein (low copy) as well as varying distance from the 3? end for each transcript. Results from participating laboratories will be evaluated to determine the impact of priming strategy, assay chemistry and experimental setup on the RT step. Additionally, we will address the impact of RNA integrity on cDNA synthesis.
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Ammour, Y; Faizuloev, E; Borisova, T; Nikonova, A; Dmitriev, G; Lobodanov, S; Zverev, V
2013-01-01
In this study, a rapid quantitative method using TaqMan-based real-time reverse transcription-polymerase chain reaction (qPCR-RT) has been developed for estimating the titers of measles, mumps and rubella (MMR) viruses in infected cell culture supernatants. The qPCR-RT assay was demonstrated to be a specific, sensitive, efficient and reproducible method. For MMR viral samples obtained during MMR viral propagations in Vero cells at a different multiplicity of infection, titers determined by the qPCR-RT assay have been compared with estimates of infectious virus obtained by a traditional commonly used method for MMR viruses - 50% cell culture infective dose (CCID(50)) assay, in paired samples. Pearson analysis evidenced a significant correlation between both methods for a certain period after viral inoculation. Furthermore, the established qPCR-RT assay was faster and less-laborious. The developed method could be used as an alternative method or a supplementary tool for the routine titer estimation during MMR vaccine production. Copyright © 2012 Elsevier B.V. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Hindson, B J; Baker, B R; Bentley Tammero, L F
2007-09-18
A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspectmore » cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRTPCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.« less
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Interactive web-based pulmonary rehabilitation programme: a randomised controlled feasibility trial
Chaplin, Emma; Hewitt, Stacey; Apps, Lindsay; Bankart, John; Pulikottil-Jacob, Ruth; Boyce, Sally; Morgan, Mike; Williams, Johanna; Singh, Sally
2017-01-01
Objectives The aim of this study was to determine if an interactive web-based pulmonary rehabilitation (PR) programme is a feasible alternative to conventional PR. Design Randomised controlled feasibility trial. Setting Participants with a diagnosis of chronic obstructive pulmonary disease were recruited from PR assessments, primary care and community rehabilitation programmes. Patients randomised to conventional rehabilitation started the programme according to the standard care at their referred site on the next available date. Participants 103 patients were recruited to the study and randomised: 52 to conventional rehabilitation (mean (±SD) age 66 (±8) years, Medical Research Council (MRC) 3 (IQR2–4)); 51 to the web arm (mean (±SD) age 66 (±10) years, MRC 3 (IQR2–4)). Participants had to be willing to participate in either arm of the trial, have internet access and be web literate. Interventions Patients randomised to the web-based programme worked through the website, exercising and recording their progress as well as reading educational material. Conventional PR consisted of twice weekly, 2 hourly sessions (an hour for exercise training and an hour for education). Outcome measures Recruitment rates, eligibility, patient preference and dropout and completion rates for both programmes were collected. Standard outcomes for a PR assessment including measures of exercise capacity and quality of life questionnaires were also evaluated. Results A statistically significant improvement (p≤0.01) was observed within each group in the endurance shuttle walk test (WEB: mean change 189±211.1; PR classes: mean change 184.5±247.4 s) and Chronic Respiratory disease Questionnaire-Dyspnoea (CRQ-D; WEB: mean change 0.7±1.2; PR classes: mean change 0.8±1.0). However, there were no significant differences between the groups in any outcome. Dropout rates were higher in the web-based programme (57% vs 23%). Conclusions An interactive web-based PR programme is feasible
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Interactive web-based pulmonary rehabilitation programme: a randomised controlled feasibility trial.
Chaplin, Emma; Hewitt, Stacey; Apps, Lindsay; Bankart, John; Pulikottil-Jacob, Ruth; Boyce, Sally; Morgan, Mike; Williams, Johanna; Singh, Sally
2017-03-31
The aim of this study was to determine if an interactive web-based pulmonary rehabilitation (PR) programme is a feasible alternative to conventional PR. Randomised controlled feasibility trial. Participants with a diagnosis of chronic obstructive pulmonary disease were recruited from PR assessments, primary care and community rehabilitation programmes. Patients randomised to conventional rehabilitation started the programme according to the standard care at their referred site on the next available date. 103 patients were recruited to the study and randomised: 52 to conventional rehabilitation (mean (±SD) age 66 (±8) years, Medical Research Council (MRC) 3 (IQR2-4)); 51 to the web arm (mean (±SD) age 66 (±10) years, MRC 3 (IQR2-4)). Participants had to be willing to participate in either arm of the trial, have internet access and be web literate. Patients randomised to the web-based programme worked through the website, exercising and recording their progress as well as reading educational material. Conventional PR consisted of twice weekly, 2 hourly sessions (an hour for exercise training and an hour for education). Recruitment rates, eligibility, patient preference and dropout and completion rates for both programmes were collected. Standard outcomes for a PR assessment including measures of exercise capacity and quality of life questionnaires were also evaluated. A statistically significant improvement (p≤0.01) was observed within each group in the endurance shuttle walk test (WEB: mean change 189±211.1; PR classes: mean change 184.5±247.4 s) and Chronic Respiratory disease Questionnaire-Dyspnoea (CRQ-D; WEB: mean change 0.7±1.2; PR classes: mean change 0.8±1.0). However, there were no significant differences between the groups in any outcome. Dropout rates were higher in the web-based programme (57% vs 23%). An interactive web-based PR programme is feasible and acceptable when compared with conventional PR. Future trials maybe around choice-based PR
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Nkere, Chukwuemeka K; Oyekanmi, Joshua O; Silva, Gonçalo; Bömer, Moritz; Atiri, Gabriel I; Onyeka, Joseph; Maroya, Norbert G; Seal, Susan E; Kumar, P Lava
2018-04-01
A closed-tube reverse transcription loop-mediated isothermal amplification (CT-RT-LAMP) assay was developed for the detection of yam mosaic virus (YMV, genus Potyvirus) infecting yam (Dioscorea spp.). The assay uses a set of six oligonucleotide primers targeting the YMV coat protein region, and the amplification products in YMV-positive samples are visualized by chromogenic detection with SYBR Green I dye. The CT-RT-LAMP assay detected YMV in leaf and tuber tissues of infected plants. The assay is 100 times more sensitive in detecting YMV than standard RT-PCR, while maintaining the same specificity.
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AbouAssi, Hiba; Slentz, Cris A; Mikus, Catherine R; Tanner, Charles J; Bateman, Lori A; Willis, Leslie H; Shields, A Tamlyn; Piner, Lucy W; Penry, Lorrie E; Kraus, Erik A; Huffman, Kim M; Bales, Connie W; Houmard, Joseph A; Kraus, William E
2015-06-15
Most health organizations recommend a combination of aerobic training (AT) and resistance training (RT), yet few studies have compared their acute (within 24 h of the last exercise bout) and sustained (after 14 days of no exercise training) effects alone and in combination on glucose metabolism. The present study (Studies Targeting Risk Reduction Interventions through Defined Exercise-Aerobic Training and/or Resistance Training) compared the effects of AT, RT, and the combination (AT/RT) on insulin action at both acute and sustained phases. Subjects (N = 196) were 18-70 yr old (mean age = 50 yr), overweight (mean body mass index = 30 kg/m2), sedentary with moderate dyslipidemia, and were randomized into one of three 8-mo exercise groups: 1) RT: 3 days/wk, 8 exercises, 3 sets/exercise, 8-12 repetitions/set; 2) AT: equivalent to ∼19.2 km/wk (12 miles/wk) at 75% peak O2 consumption; 3) AT/RT: the combination of AT and RT. One hundred forty-four subjects completed the intervention. Eighty-eight subjects completed all pre- and postintervention testing visits. Insulin sensitivity, glucose effectiveness, and disposition index were measured via a frequently sampled intravenous glucose tolerance test with subsequent minimal model analyses. AT/RT resulted in greater improvements in insulin sensitivity, β-cell function (disposition index), and glucose effectiveness than either AT or RT alone (all P < 0.05). Approximately 52% of the improvement in insulin sensitivity by AT/RT was retained 14 days after the last exercise training bout. Neither AT or RT led to acute or chronic improvement in sensitivity index. In summary, only AT/RT (which required twice as much time as either alone) led to significant acute and sustained benefits in insulin sensitivity
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Application of RT-PCR in formalin-fixed and paraffin-embedded lung cancer tissues.
Zhang, Fan; Wang, Zhuo-min; Liu, Hong-yu; Bai, Yun; Wei, Sen; Li, Ying; Wang, Min; Chen, Jun; Zhou, Qing-hua
2010-01-01
To analyze gene expression in formalin-fixed, paraffin-embedded lung cancer tissues using modified method. Total RNA from frozen tissues was extracted using TRIZOL reagent. RNA was extracted from formalin-fixed, paraffin-embedded tissues by digestion with proteinase K before the acid-phenol:chloroform extraction and carrier precipitation. We modified this method by using a higher concentration of proteinase K and a longer digestion time, optimized to 16 hours. RT-PCR and real-time RT-PCR were used to check reproducibility and the concordance between frozen and paraffin-embedded samples. The results showed that the RNA extracted from the paraffin-embedded lung tissues had high quality with the most fragment length between 28S and 18S bands (about 1000 to 2000 bases). The housekeeping gene GUSB exhibited low variation of expression in frozen and paraffin-embedded lung tissues, whereas PGK1 had the lowest variation in lymphoma tissues. Furthermore, real-time PCR analysis of the expression of known prognostic genes in non-small cell lung carcinoma (NSCLC) demonstrated an extremely high correlation (r>0.880) between the paired frozen and formalin-fixed, paraffin-embedded specimens. This improved method of RNA extraction is suitable for real-time quantitative RT-PCR, and may be used for global gene expression profiling of paraffin-embedded tissues.
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Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases
Rydbirk, Rasmus; Folke, Jonas; Winge, Kristian; Aznar, Susana; Pakkenberg, Bente; Brudek, Tomasz
2016-01-01
Evaluation of gene expression levels by reverse transcription quantitative real-time PCR (RT-qPCR) has for many years been the favourite approach for discovering disease-associated alterations. Normalization of results to stably expressed reference genes (RGs) is pivotal to obtain reliable results. This is especially important in relation to neurodegenerative diseases where disease-related structural changes may affect the most commonly used RGs. We analysed 15 candidate RGs in 98 brain samples from two brain regions from Alzheimer’s disease (AD), Parkinson’s disease (PD), Multiple System Atrophy, and Progressive Supranuclear Palsy patients. Using RefFinder, a web-based tool for evaluating RG stability, we identified the most stable RGs to be UBE2D2, CYC1, and RPL13 which we recommend for future RT-qPCR studies on human brain tissue from these patients. None of the investigated genes were affected by experimental variables such as RIN, PMI, or age. Findings were further validated by expression analyses of a target gene GSK3B, known to be affected by AD and PD. We obtained high variations in GSK3B levels when contrasting the results using different sets of common RG underlining the importance of a priori validation of RGs for RT-qPCR studies. PMID:27853238
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Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases.
Rydbirk, Rasmus; Folke, Jonas; Winge, Kristian; Aznar, Susana; Pakkenberg, Bente; Brudek, Tomasz
2016-11-17
Evaluation of gene expression levels by reverse transcription quantitative real-time PCR (RT-qPCR) has for many years been the favourite approach for discovering disease-associated alterations. Normalization of results to stably expressed reference genes (RGs) is pivotal to obtain reliable results. This is especially important in relation to neurodegenerative diseases where disease-related structural changes may affect the most commonly used RGs. We analysed 15 candidate RGs in 98 brain samples from two brain regions from Alzheimer's disease (AD), Parkinson's disease (PD), Multiple System Atrophy, and Progressive Supranuclear Palsy patients. Using RefFinder, a web-based tool for evaluating RG stability, we identified the most stable RGs to be UBE2D2, CYC1, and RPL13 which we recommend for future RT-qPCR studies on human brain tissue from these patients. None of the investigated genes were affected by experimental variables such as RIN, PMI, or age. Findings were further validated by expression analyses of a target gene GSK3B, known to be affected by AD and PD. We obtained high variations in GSK3B levels when contrasting the results using different sets of common RG underlining the importance of a priori validation of RGs for RT-qPCR studies.
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Kamau, Everlyn; Agoti, Charles N; Lewa, Clement S; Oketch, John; Owor, Betty E; Otieno, Grieven P; Bett, Anne; Cane, Patricia A; Nokes, D James
2017-03-01
Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples. N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses. An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.
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Rheum emodin inhibits enterovirus 71 viral replication and affects the host cell cycle environment
Zhong, Ting; Zhang, Li-ying; Wang, Zeng-yan; Wang, Yue; Song, Feng-mei; Zhang, Ya-hong; Yu, Jing-hua
2017-01-01
Human enterovirus 71 (EV71) is the primary causative agent of recent large-scale outbreaks of hand, foot, and mouth disease (HFMD) in Asia. Currently, there are no drugs available for the prevention and treatment of HFMD. In this study, we compared the anti-EV71 activities of three natural compounds, rheum emodin, artemisinin and astragaloside extracted from Chinese herbs Chinese rhubarb, Artemisia carvifolia and Astragalus, respectively, which have been traditionally used for the treatment and prevention of epidemic diseases. Human lung fibroblast cell line MRC5 was mock-infected or infected with EV71, and treated with drugs. The cytotoxicity of the drugs was detected with MTT assay. The cytopathic effects such as cell death and condensed nuclei were morphologically observed. The VP1-coding sequence required for EV71 genome replication was assayed with qRT-PCR. Viral protein expression was analyzed with Western blotting. Viral TCID50 was determined to evaluate EV71 virulence. Flow cytometry analysis of propidium iodide staining was performed to analyze the cell cycle distribution of MRC5 cells. Rheum emodin (29.6 μmol/L) effectively protected MRC5 cells from EV71-induced cytopathic effects, which resulted from the inhibiting viral replication: rheum emodin treatment decreased viral genomic levels by 5.34-fold, viral protein expression by less than 30-fold and EV71 virulence by 0.33107-fold. The fact that inhibition of rheum emodin on viral virulence was much stronger than its effects on genomic levels and viral protein expression suggested that rheum emodin inhibited viral maturation. Furthermore, rheum emodin treatment markedly diminished cell cycle arrest at S phase in MRC5 cells, which was induced by EV71 infection and favored the viral replication. In contrast, neither astragaloside (50 μmol/L) nor artemisinin (50 μmol/L) showed similar anti-EV71 activities. Among the three natural compounds tested, rheum emodin effectively suppressed EV71 viral replication
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Rheum emodin inhibits enterovirus 71 viral replication and affects the host cell cycle environment.
Zhong, Ting; Zhang, Li-Ying; Wang, Zeng-Yan; Wang, Yue; Song, Feng-Mei; Zhang, Ya-Hong; Yu, Jing-Hua
2017-03-01
Human enterovirus 71 (EV71) is the primary causative agent of recent large-scale outbreaks of hand, foot, and mouth disease (HFMD) in Asia. Currently, there are no drugs available for the prevention and treatment of HFMD. In this study, we compared the anti-EV71 activities of three natural compounds, rheum emodin, artemisinin and astragaloside extracted from Chinese herbs Chinese rhubarb, Artemisia carvifolia and Astragalus, respectively, which have been traditionally used for the treatment and prevention of epidemic diseases. Human lung fibroblast cell line MRC5 was mock-infected or infected with EV71, and treated with drugs. The cytotoxicity of the drugs was detected with MTT assay. The cytopathic effects such as cell death and condensed nuclei were morphologically observed. The VP1-coding sequence required for EV71 genome replication was assayed with qRT-PCR. Viral protein expression was analyzed with Western blotting. Viral TCID50 was determined to evaluate EV71 virulence. Flow cytometry analysis of propidium iodide staining was performed to analyze the cell cycle distribution of MRC5 cells. Rheum emodin (29.6 μmol/L) effectively protected MRC5 cells from EV71-induced cytopathic effects, which resulted from the inhibiting viral replication: rheum emodin treatment decreased viral genomic levels by 5.34-fold, viral protein expression by less than 30-fold and EV71 virulence by 0.33107-fold. The fact that inhibition of rheum emodin on viral virulence was much stronger than its effects on genomic levels and viral protein expression suggested that rheum emodin inhibited viral maturation. Furthermore, rheum emodin treatment markedly diminished cell cycle arrest at S phase in MRC5 cells, which was induced by EV71 infection and favored the viral replication. In contrast, neither astragaloside (50 μmol/L) nor artemisinin (50 μmol/L) showed similar anti-EV71 activities. Among the three natural compounds tested, rheum emodin effectively suppressed EV71 viral replication
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Evaluation of NASA SPoRT's Pseudo-Geostationary Lightning Mapper Products in the 2011 Spring Program
NASA Technical Reports Server (NTRS)
Stano, Geoffrey T.; Carcione, Brian; Siewert, Christopher; Kuhlman, Kristin M.
2012-01-01
NASA's Short-term Prediction Research and Transition (SPoRT) program is a contributing partner with the GOES-R Proving Ground (PG) preparing forecasters to understand and utilize the unique products that will be available in the GOES-R era. This presentation emphasizes SPoRT s actions to prepare the end user community for the Geostationary Lightning Mapper (GLM). This preparation is a collaborative effort with SPoRT's National Weather Service partners, the National Severe Storms Laboratory (NSSL), and the Hazardous Weather Testbed s Spring Program. SPoRT continues to use its effective paradigm of matching capabilities to forecast problems through collaborations with our end users and working with the developers at NSSL to create effective evaluations and visualizations. Furthermore, SPoRT continues to develop software plug-ins so that these products will be available to forecasters in their own decision support system, AWIPS and eventually AWIPS II. In 2009, the SPoRT program developed the original pseudo geostationary lightning mapper (PGLM) flash extent product to demonstrate what forecasters may see with GLM. The PGLM replaced the previous GLM product and serves as a stepping-stone until the AWG s official GLM proxy is ready. The PGLM algorithm is simple and can be applied to any ground-based total lightning network. For 2011, the PGLM used observations from four ground-based networks (North Alabama, Kennedy Space Center, Oklahoma, and Washington D.C.). While the PGLM is not a true proxy product, it is intended as a tool to train forecasters about total lightning as well as foster discussions on product visualizations and incorporating GLM-resolution data into forecast operations. The PGLM has been used in 2010 and 2011 and is likely to remain the primary lightning training tool for the GOES-R program for the near future. This presentation will emphasize the feedback received during the 2011 Spring Program. This will discuss several topics. Based on feedback
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NASA Technical Reports Server (NTRS)
Bell, Jordan R.; Case, Jonathan L.; LaFontaine, Frank J.; Kumar, Sujay V.
2012-01-01
The NASA Short-term Prediction Research and Transition (SPoRT) Center has developed a Greenness Vegetation Fraction (GVF) dataset, which is updated daily using swaths of Normalized Difference Vegetation Index data from the Moderate Resolution Imaging Spectroradiometer (MODIS) data aboard the NASA EOS Aqua and Terra satellites. NASA SPoRT began generating daily real-time GVF composites at 1-km resolution over the Continental United States (CONUS) on 1 June 2010. The purpose of this study is to compare the National Centers for Environmental Prediction (NCEP) climatology GVF product (currently used in operational weather models) to the SPoRT-MODIS GVF during June to October 2010. The NASA Land Information System (LIS) was employed to study the impacts of the SPoRT-MODIS GVF dataset on a land surface model (LSM) apart from a full numerical weather prediction (NWP) model. For the 2010 warm season, the SPoRT GVF in the western portion of the CONUS was generally higher than the NCEP climatology. The eastern CONUS GVF had variations both above and below the climatology during the period of study. These variations in GVF led to direct impacts on the rates of heating and evaporation from the land surface. In the West, higher latent heat fluxes prevailed, which enhanced the rates of evapotranspiration and soil moisture depletion in the LSM. By late Summer and Autumn, both the average sensible and latent heat fluxes increased in the West as a result of the more rapid soil drying and higher coverage of GVF. The impacts of the SPoRT GVF dataset on NWP was also examined for a single severe weather case study using the Weather Research and Forecasting (WRF) model. Two separate coupled LIS/WRF model simulations were made for the 17 July 2010 severe weather event in the Upper Midwest using the NCEP and SPoRT GVFs, with all other model parameters remaining the same. Based on the sensitivity results, regions with higher GVF in the SPoRT model runs had higher evapotranspiration and
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Sacristán, Carlos; Carballo, Matilde; Muñoz, María Jesús; Bellière, Edwige Nina; Neves, Elena; Nogal, Verónica; Esperón, Fernando
2015-12-15
Cetacean morbillivirus (CeMV) (family Paramyxoviridae, genus Morbillivirus) is considered the most pathogenic virus of cetaceans. It was first implicated in the bottlenose dolphin (Tursiops truncatus) mass stranding episode along the Northwestern Atlantic coast in the late 1980s, and in several more recent worldwide epizootics in different Odontoceti species. This study describes a new one step real-time reverse transcription fast polymerase chain reaction (real-time RT-fast PCR) method based on SYBR(®) Green to detect a fragment of the CeMV fusion protein gene. This primer set also works for conventional RT-PCR diagnosis. This method detected and identified all three well-characterized strains of CeMV: porpoise morbillivirus (PMV), dolphin morbillivirus (DMV) and pilot whale morbillivirus (PWMV). Relative sensitivity was measured by comparing the results obtained from 10-fold dilution series of PMV and DMV positive controls and a PWMV field sample, to those obtained by the previously described conventional phosphoprotein gene based RT-PCR method. Both the conventional and real-time RT-PCR methods involving the fusion protein gene were 100- to 1000-fold more sensitive than the previously described conventional RT-PCR method. Copyright © 2015 Elsevier B.V. All rights reserved.
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Xu, Chun-Wei; Wang, Wen-Xian; Chen, Yan-Ping; Chen, Yu; Liu, Wei; Zhong, Li-Hua; Chen, Fang-Fang; Zhuang, Wu; Song, Zheng-Bo; Chen, Xiao-Hui; Huang, Yun-Jian; Guan, Yan-Fang; Yi, Xin; Lv, Tang-Feng; Zhu, Wei-Feng; Lu, Jian-Ping; Wang, Xiao-Jiang; Shi, Yi; Lin, Xian-Dong; Chen, Gang; Song, Yong
2018-04-11
ALK rearrangement-advanced NSCLC patients respond to crizotinib. ALK rearrangement is currently determined with RT-PCR. VENTANA IHC is a standard method to identify ALK protein overexpression in NSCLC; however, VENTANA IHC has rarely been used to determine the response to crizotinib in Chinese patients with NSCLC and ALK overexpression. To better clarify the clinical implication of VENTANA IHC to detect ALK rearrangements, we conducted this study to analyze VENTANA IHC and RT-PCR in a large cohort of Chinese patients with NSCLC undergoing screening for ALK rearrangements. A total of 1720 patients with NSCLC who had ALK rearrangements detected by VENTANA IHC and/or RT-PCR were included in this analysis. We compared the efficacy and survival of ALK-positive patients detected by VENTANA IHC and RT-PCR. We used NGS to identify patients in whom the two methods were inconsistent. Among 1720 patients, 187 (10.87%) were shown to be ALK-positive by VENTANA IHC and/or RT-PCR, and 66 received crizotinib treatment. We identified 10.27% (172/1674) of patients as ALK-positive by the VENTANA IHC method, and 12.73% (41/322) of patients had ALK rearrangements by the RT-PCR method. Twenty-nine of 276 (10.51%) ALK-positive patients were simultaneously analyzed using VENTANA IHC and RT-PCR. The overall response rates were 65.90% (29/44) by VENTANA IHC and 55.88% (19/34) by RT-PCR. The disease control rates were 86.36% (38/44) by VENTANA IHC and 76.47% (26/34) by RT-PCR. In contrast, the median progression-free survival for VENTANA IHC and RT-PCR was 8.5 and 9.2 months, respectively. The VENTANA IHC and RT-PCR results obtained for 6 of 17 ALK-positive patients were inconsistent based on NGS; specifically, 4 patients had EML4-ALK fusions, 2 patients had non EML4-ALK fusions, 1 patient had a KCL1-ALK fusion, and one patient had a FBXO36-ALK fusion. VENTANA IHC is a reliable and rapid screening tool used in routine pathologic laboratories for the identification of suitable candidates for
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Hindson, B J; Reid, S M; Baker, B R
2007-07-26
A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouthmore » disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.« less
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Nguyen, Duc Quan; Eamens, Andrew L; Grof, Christopher P L
2018-01-01
Quantitative real-time polymerase chain reaction (RT-qPCR) is the key platform for the quantitative analysis of gene expression in a wide range of experimental systems and conditions. However, the accuracy and reproducibility of gene expression quantification via RT-qPCR is entirely dependent on the identification of reliable reference genes for data normalisation. Green foxtail ( Setaria viridis ) has recently been proposed as a potential experimental model for the study of C 4 photosynthesis and is closely related to many economically important crop species of the Panicoideae subfamily of grasses, including Zea mays (maize), Sorghum bicolor (sorghum) and Sacchurum officinarum (sugarcane). Setaria viridis (Accession 10) possesses a number of key traits as an experimental model, namely; (i) a small sized, sequenced and well annotated genome; (ii) short stature and generation time; (iii) prolific seed production, and; (iv) is amendable to Agrobacterium tumefaciens -mediated transformation. There is currently however, a lack of reference gene expression information for Setaria viridis ( S. viridis ). We therefore aimed to identify a cohort of suitable S. viridis reference genes for accurate and reliable normalisation of S. viridis RT-qPCR expression data. Eleven putative candidate reference genes were identified and examined across thirteen different S. viridis tissues. Of these, the geNorm and NormFinder analysis software identified SERINE / THERONINE - PROTEIN PHOSPHATASE 2A ( PP2A ), 5 '- ADENYLYLSULFATE REDUCTASE 6 ( ASPR6 ) and DUAL SPECIFICITY PHOSPHATASE ( DUSP ) as the most suitable combination of reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. To demonstrate the suitability of the three selected reference genes, PP2A , ASPR6 and DUSP , were used to normalise the expression of CINNAMYL ALCOHOL DEHYDROGENASE ( CAD ) genes across the same tissues. This approach readily demonstrated the suitably of the three
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Kaesmacher, Johannes; Kleine, Justus F
2018-03-01
Recent studies suggested that preinterventional intravenous (i. v.) recombinant tissue plasminogen activator (rtPA) as bridging therapy facilitates successful and fast vessel recanalization in endovascular stroke treatment (EST); however, data on this effect and the associated clinical value are discrepant. This study examined if this discrepancy could be related to an effect-modifying variable, specifically to the exact occlusion site. Retrospective analysis of 239 patients with acute occlusion of the middle cerebral artery (MCA) treated with up to date endovascular techniques. Effects of i. v.-rtPA bridging on clinical outcomes and safety/efficacy of EST, defined as the respective rates of successful, first pass and thrombolysis in cerebral infarction (TICI) scale 3 recanalization, were evaluated and stratified according to distal versus proximal occlusion sites. Overall, i. v.-rtPA bridging was associated with a significantly higher rate of successful recanalization (86.9 % vs. 75.7 %, p = 0.028). i. v.-rtPA bridging-related effects, however, were observable only in distal, but not in proximal MCA-occlusions. In distal occlusions, i. v.-rtPA clearly favored successful recanalization (adj. OR 4.6, 95 %-CI 1.5-13.6, p = 0.006) and first-pass successes (adj. OR 2.8, 95 %-CI 1.0-7.6, p = 0.042), but tended to be associated with lower rates of complete (TICI-3) reperfusion (adj. OR 0.4, 95 %-CI 0.2-1.1, p = 0.068). The net effect was a small clinical benefit, reflected in higher rates of strong neurological improvement (adj. OR: 2.8, 95 %-CI: 1.1-6.9, p = 0.03). i. v.-rtPA-bridging-related effects are occlusion site-dependent, paralleling similar effects of systemic i. v.-rtPA when applied without subsequent endovascular therapy. In distal occlusions, i. v.-rtPA facilitates thrombectomy, but may also promote distal embolization, with a small clinical benefit as overall net effect. Randomized trials assessing i.v-rtPA bridging need
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Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis.
Rocha, Danilo J P; Santos, Carolina S; Pacheco, Luis G C
2015-09-01
The appropriate choice of reference genes is essential for accurate normalization of gene expression data obtained by the method of reverse transcription quantitative real-time PCR (RT-qPCR). In 2009, a guideline called the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) highlighted the importance of the selection and validation of more than one suitable reference gene for obtaining reliable RT-qPCR results. Herein, we searched the recent literature in order to identify the bacterial reference genes that have been most commonly validated in gene expression studies by RT-qPCR (in the first 5 years following publication of the MIQE guidelines). Through a combination of different search parameters with the text mining tool MedlineRanker, we identified 145 unique bacterial genes that were recently tested as candidate reference genes. Of these, 45 genes were experimentally validated and, in most of the cases, their expression stabilities were verified using the software tools geNorm and NormFinder. It is noteworthy that only 10 of these reference genes had been validated in two or more of the studies evaluated. An enrichment analysis using Gene Ontology classifications demonstrated that genes belonging to the functional categories of DNA Replication (GO: 0006260) and Transcription (GO: 0006351) rendered a proportionally higher number of validated reference genes. Three genes in the former functional class were also among the top five most stable genes identified through an analysis of gene expression data obtained from the Pathosystems Resource Integration Center. These results may provide a guideline for the initial selection of candidate reference genes for RT-qPCR studies in several different bacterial species.
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A Host-Based RT-PCR Gene Expression Signature to Identify Acute Respiratory Viral Infection
Zaas, Aimee K.; Burke, Thomas; Chen, Minhua; McClain, Micah; Nicholson, Bradly; Veldman, Timothy; Tsalik, Ephraim L.; Fowler, Vance; Rivers, Emanuel P.; Otero, Ronny; Kingsmore, Stephen F.; Voora, Deepak; Lucas, Joseph; Hero, Alfred O.; Carin, Lawrence; Woods, Christopher W.; Ginsburg, Geoffrey S.
2014-01-01
Improved ways to diagnose acute respiratory viral infections could decrease inappropriate antibacterial use and serve as a vital triage mechanism in the event of a potential viral pandemic. Measurement of the host response to infection is an alternative to pathogen-based diagnostic testing and may improve diagnostic accuracy. We have developed a host-based assay with a reverse transcription polymerase chain reaction (RT-PCR) TaqMan low-density array (TLDA) platform for classifying respiratory viral infection. We developed the assay using two cohorts experimentally infected with influenza A H3N2/Wisconsin or influenza A H1N1/Brisbane, and validated the assay in a sample of adults presenting to the emergency department with fever (n = 102) and in healthy volunteers (n = 41). Peripheral blood RNA samples were obtained from individuals who underwent experimental viral challenge or who presented to the emergency department and had microbiologically proven viral respiratory infection or systemic bacterial infection. The selected gene set on the RT-PCR TLDA assay classified participants with experimentally induced influenza H3N2 and H1N1 infection with 100 and 87% accuracy, respectively. We validated this host gene expression signature in a cohort of 102 individuals arriving at the emergency department. The sensitivity of the RT-PCR test was 89% [95% confidence interval (CI), 72 to 98%], and the specificity was 94% (95% CI, 86 to 99%). These results show that RT-PCR–based detection of a host gene expression signature can classify individuals with respiratory viral infection and sets the stage for prospective evaluation of this diagnostic approach in a clinical setting. PMID:24048524
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NASA SPoRT Initialization Datasets for Local Model Runs in the Environmental Modeling System
NASA Technical Reports Server (NTRS)
Case, Jonathan L.; LaFontaine, Frank J.; Molthan, Andrew L.; Carcione, Brian; Wood, Lance; Maloney, Joseph; Estupinan, Jeral; Medlin, Jeffrey M.; Blottman, Peter; Rozumalski, Robert A.
2011-01-01
The NASA Short-term Prediction Research and Transition (SPoRT) Center has developed several products for its National Weather Service (NWS) partners that can be used to initialize local model runs within the Weather Research and Forecasting (WRF) Environmental Modeling System (EMS). These real-time datasets consist of surface-based information updated at least once per day, and produced in a composite or gridded product that is easily incorporated into the WRF EMS. The primary goal for making these NASA datasets available to the WRF EMS community is to provide timely and high-quality information at a spatial resolution comparable to that used in the local model configurations (i.e., convection-allowing scales). The current suite of SPoRT products supported in the WRF EMS include a Sea Surface Temperature (SST) composite, a Great Lakes sea-ice extent, a Greenness Vegetation Fraction (GVF) composite, and Land Information System (LIS) gridded output. The SPoRT SST composite is a blend of primarily the Moderate Resolution Imaging Spectroradiometer (MODIS) infrared and Advanced Microwave Scanning Radiometer for Earth Observing System data for non-precipitation coverage over the oceans at 2-km resolution. The composite includes a special lake surface temperature analysis over the Great Lakes using contributions from the Remote Sensing Systems temperature data. The Great Lakes Environmental Research Laboratory Ice Percentage product is used to create a sea-ice mask in the SPoRT SST composite. The sea-ice mask is produced daily (in-season) at 1.8-km resolution and identifies ice percentage from 0 100% in 10% increments, with values above 90% flagged as ice.
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Pfitzner, Claudia; Schröder, Isabel; Scheungraber, Cornelia; Dogan, Askin; Runnebaum, Ingo Bernhard; Dürst, Matthias; Häfner, Norman
2014-01-01
The detection of circulating tumour cells (CTC) in cancer patients may be useful for therapy monitoring and prediction of relapse. A sensitive assay based on HPV-oncogene transcripts which are highly specific for cervical cancer cells was established. The Digital-Direct-RT-PCR (DD-RT-PCR) combines Ficoll-separation, ThinPrep-fixation and one-step RT-PCR in a low-throughput digital-PCR format enabling the direct analysis and detection of individual CTC without RNA isolation. Experimental samples demonstrated a sensitivity of one HPV-positive cell in 500,000 HPV-negative cells. Spike-in experiments with down to 5 HPV-positive cells per millilitre EDTA-blood resulted in concordant positive results by PCR and immunocytochemistry. Blood samples from 3 of 10 CxCa patients each contained a single HPV-oncogene transcript expressing CTC among 5 to 15*105 MNBC. Only 1 of 7 patients with local but 2 of 3 women with systemic disease had CTC. This highly sensitive DD-RT-PCR for the detection of CTC may also be applied to other tumour entities which express tumour-specific transcripts. Abbreviations: CTC – circulating tumour cells, CxCa – cervical cancer, DD-RT-PCR – Digital-Direct Reverse Transcriptase PCR, HPV – Human Papilloma Virus, MNBC – mononuclear blood cells, ICC – immunocytochemistry. PMID:24496006
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Cai, Yi; Liu, Hao; Chen, Haifeng
2018-03-01
The human immunodeficiency virus (HIV) is a retrovirus which infects T lymphocyte of human body and causes immunodeficiency. Reverse transcriptase inhibitors (RTIs) can inhibit some functions of RT, preventing virus synthesis (double-stranded DNA), so that HIV virus replication can be reduced. Experimental results indicate a series of benzimidazole-based inhibitors which target HIV RT-associated RNase to inhibit the reverse transcription of HIV virus. However, the allosteric mechanism is still unclear. Here, molecular dynamics simulations and dynamics fluctuation network analysis were used to reveal the binding mode between the inhibitors and RT-associated RNase. The most active molecule has more hydrophobic and electrostatic interactions than the less active inhibitor. Dynamics correlation network analysis indicates that the most active inhibitor perturbs the network of RT-associated RNase and decreases the correlation of nodes. 3D-QSAR model suggests that two robust and reliable models were constructed and validated by independent test set. 3D-QSAR model also shows that bulky negatively charged or hydrophilic substituent is favorable to bioactivity. These results reveal the allosteric mechanism of quinoline inhibitors and help to improve the bioactivity. © 2017 John Wiley & Sons A/S.
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Valverde, Estefania J; Borrego, Juan J; Castro, Dolores
2016-12-01
The lymphocystis disease virus (LCDV), a member of the Iridoviridae family, infects a wide range of fish species including gilthead seabream (Sparus aurata L.), the most important species cultured in the Mediterranean. LCDV is difficult to propagate in cell culture and does not produce clear and consistent cytopathic effects (CPE), especially in samples collected from subclinically infected fish. An integrated cell culture reverse transcription-polymerase chain reaction (ICC-RT-PCR) assay, followed by dot-blot hybridization of the RT-PCR products, was developed to improve the detection of infectious LCDV. The sensitivity of the ICC-RT-PCR assay, which can be performed in 7 d, was at least 100-fold higher than viral diagnosis obtained by CPE development. The developed assay thus allows the determination of infectious titres in samples with low viral loads, including those from asymptomatic carrier fish, in which no CPE was recorded after a 14-d incubation period. The ICC-RT-PCR assay enables rapid, specific and sensitive detection and quantification of infectious LCDV, and may be a valuable tool in the study of aspects of LCDV infection including transmission or epizootiology. Copyright © 2016 Elsevier B.V. All rights reserved.
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Lee, Hae-Won; Lee, Hee-Min; Yoon, So-Ra; Kim, Sung Hyun; Ha, Ji-Hyoung
2018-02-01
RT-qPCR allows sensitive detection of viral particles of both infectious and noninfectious viruses in water environments, but cannot discriminate non-infectious from infectious viruses. In this study, we aimed to optimize RT-qPCR-based detection of chlorine-inactivated human norovirus (NoV) and pepper mild mottle virus (PMMoV) in suspension by pretreatment with an optimal combination of a monoazide and a detergent that can efficiently penetrate damaged viral capsids. Four methods were compared to determine the efficacy of chlorine disinfection (at 1, 3, and 5 min mg/L): (A) RT-qPCR alone, (B) RT-qPCR assay preceded by magnetic bead separation for enrichment of viral particles (MBS-RT-qPCR), (C) MBS-RT-qPCR assay with pretreatment with propidium monoazide (PMA-MBS-RT-qPCR), and (D) PMA-MBS-RT-qPCR assay with pretreatment with sodium lauroyl sarcosinate (INCI-PMA-MBS-RT-qPCR). On the basis of a PMA optimization assay, 200 and 300 μM PMA were used in subsequent experiments for NoV GII.4 and PMMoV, respectively. Optimal INCI concentrations, having minimal influence on NoV GII.4 and PMMoV, were found to be 0.5% and 0.2% INCI, respectively. For NoV GII.4, there were significant differences (P < 0.05) in log 10 genome copies between the PMA-treated and the INCI + PMA-treated samples (log 10 genome copies differed by 1.11 and 0.59 log 10 for 3 and 5 min mg/L of chlorine, respectively). For PMMoV, INCI induced differences in log 10 genome copies of 0.92, 1.18, and 1.86, for 1, 3, and 5 min mg/L of chlorine, respectively. Overall, the results of this study indicate that an optimal combination of PMA and INCI could be very useful for evaluating disinfection methods in water treatment strategies. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Stafford, Mai; Gale, Catharine R.; Mishra, Gita; Richards, Marcus; Black, Stephanie; Kuh, Diana L.
2015-01-01
Background Mental wellbeing, conceptualised as positive affect, life satisfaction and realisation of needs that contribute to psychological growth, captures more than the absence of mental ill health. Several nations now aim to monitor and improve mental wellbeing. Whilst many studies document associations between adverse childhood experiences and mental disorders in adulthood, possible links between childhood experiences and adult mental wellbeing have so far received less attention. Methods Using data from 1976 men and women in the MRC National Survey for Health and Development, we investigated prospective associations between childhood socioeconomic and psychosocial environments and the Warwick Edinburgh Mental Wellbeing Scale, designed to capture both hedonic and eudaimonic facets of wellbeing, at age 60-64. Results Whilst there was no evidence that childhood socioeconomic circumstances were related to later wellbeing independently of other childhood experiences, elements of childrearing and parenting, parental health and adjustment, and childhood illness were related. More advantaged socioeconomic position was associated with greater wellbeing but this did not explain the links between these childhood exposures and adult wellbeing, suggesting alternative explanatory pathways should be considered. Conclusions Childhood illness and family psychosocial environment are associated with mental wellbeing in early older age, with effects sizes that are larger or comparable to socioeconomic circumstances in adulthood. Initiatives to improve the nation’s mental wellbeing that include programmes targeted to supporting families and children may additionally have benefits that continue into older age. PMID:26030929
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Rocha-Silva, Fabiana; Gomes, Luciana I; Gracielle-Melo, Cidiane; Goes, Alfredo M; Caligiorne, Rachel B
2017-01-01
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by dimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. It is prevalent in Latin American, mainly in Brazil. Therefore, PCM has fundamental impact on the Brazilian global economy, especially in public health system, since it is affecting economical active population in different country regions. The present study aimed to standardize the Real Time-Polymerase Chain Reaction (rt-PCR) for an efficient and safe PCM diagnosis amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. To standardize a methodology of rt-PCR using species-specific primers and probe designed for annealing in this specific region of the fungi´s genome, amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. Followed by design in silico, experiments were performed in vitro to determine rt-PCR specificity, efficiency and genome detection limit. The primers and probe sequences were deposited in Brazilian Coordination of Technological Innovation and Transfer (CTIT), under patent reference number BR1020160078830. The present study demonstrated the rt-PCR applicability for support on diagnosis of paracoccidioidomycosis, presenting low cost, which makes it affordable for public health services in developing countries as Brazil. It is noteworthy that it is necessary to validate this methodology using clinical samples before to use as a safe method of diagnosis. A review of all patents related to this topic was performed and it was shown that, to date, there are no records of patent on kits for paracoccidioidomycosis´s diagnostic. Indeed, there is still a lot to go to reach this goal. The reaction developed was standardized and patented, opening perspectives to molecular diagnosis development for paracoccidioidomycosis, since rt-PCR can be applied to a broad spectrum of infectious diseases. It would need to be tested in biological
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33 CFR 72.01-15-72.01-20 - [Reserved
Code of Federal Regulations, 2011 CFR
2011-07-01
... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false [Reserved] 72.01-15-72.01-20 Section 72.01-15-72.01-20 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY AIDS TO NAVIGATION MARINE INFORMATION Notices to Mariners §§ 72.01-15—72.01-20 [Reserved] ...
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33 CFR 72.01-15-72.01-20 - [Reserved
Code of Federal Regulations, 2010 CFR
2010-07-01
... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false [Reserved] 72.01-15-72.01-20 Section 72.01-15-72.01-20 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY AIDS TO NAVIGATION MARINE INFORMATION Notices to Mariners §§ 72.01-15—72.01-20 [Reserved] ...
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Kaundal, Nirmal; Sarkate, Purva; Prakash, Charu; Rishi, Narayan
2017-06-01
Environmental surveillance of polioviruses has been used as an important tool in monitoring circulation of wild polioviruses and/or Vaccine derived polioviruses in sewage samples. It is important to distinguish Sabin like isolates from non-Sabin like; ELISA & dual stage real time RT-PCR have been used for the same. Current study was carried out on sewage isolates to compare ELISA & RT-PCR with sequencing to distinguish Sabin like from non-Sabin like. Out of 468 sewage specimens, 91 (19.44%) were non-polio enteroviruses positive and 377 (80.56%) were polio positive by virus isolation method. A total of 488 polio virus isolates were detected by L20B and RD route which were further subjected to ELISA and RT-PCR. The results were compared with sequencing. On comparison, the specificity of ELISA was only 66.67% in spite of very low sensitivity (3.43%). The sensitivity of RT-PCR was 97.71% which makes it a good primary screening test for detection of non-Sabin like viruses. However, the specificity was only 33.33%. RT-PCR appears to be a sensitive tool for detecting non-Sabin like viruses however; the isolates which are non-Sabin like by RT-PCR may not necessarily be mutated viruses. ELISA cannot be used for differentiation of Sabin likes from non-Sabin likes due to low sensitivity.
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Caughey, Byron; Orru, Christina D; Groveman, Bradley R; Hughson, Andrew G; Manca, Matteo; Raymond, Lynne D; Raymond, Gregory J; Race, Brent; Saijo, Eri; Kraus, Allison
2017-01-01
Among the most sensitive, specific and practical of methods for detecting prions are the real-time quaking-induced conversion (RT-QuIC) assays. These assays exploit the fundamental self-propagating activity of prions to amplify the presence of prion seeds by as much as a trillion-fold. The reactions can detect most of the known mammalian prion diseases, often with sensitivities greater than those of animal bioassays. RT-QuIC assays are performed in multiwell plates with fluorescence detection and have now reached the sensitivity and practicality required for routine prion disease diagnostics. Some key strains of prions within particular host species, e.g., humans, cattle, and sheep, can be discriminated by comparison of RT-QuIC responses with different recombinant prion protein substrates. The most thoroughly validated diagnostic application of RT-QuIC is in the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) using cerebrospinal fluid. Diagnostic sensitivities as high as 96% can be achieved in less than 24h with specificities of 98%-100%. The ability, if needed, to also test nasal swab samples can increase the RT-QuIC sensitivity for sCJD to virtually 100%. In addition to diagnostic applications, RT-QuIC has also been used in the testing of prion disinfectants and potential therapeutics. Mechanistically related assays are also now being developed for other protein misfolding diseases. © 2017 Elsevier Inc. All rights reserved.
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Mannose Receptor 2 Attenuates Renal Fibrosis
López-Guisa, Jesús M.; Cai, Xiaohe; Collins, Sarah J.; Yamaguchi, Ikuyo; Okamura, Daryl M.; Bugge, Thomas H.; Isacke, Clare M.; Emson, Claire L.; Turner, Scott M.; Shankland, Stuart J.
2012-01-01
Mannose receptor 2 (Mrc2) expresses an extracellular fibronectin type II domain that binds to and internalizes collagen, suggesting that it may play a role in modulating renal fibrosis. Here, we found that Mrc2 levels were very low in normal kidneys but subsets of interstitial myofibroblasts and macrophages upregulated Mrc2 after unilateral ureteral obstruction (UUO). Renal fibrosis and renal parenchymal damage were significantly worse in Mrc2-deficient mice. Similarly, Mrc2-deficient Col4α3−/− mice with hereditary nephritis had significantly higher levels of total kidney collagen, serum BUN, and urinary protein than Mrc2-sufficient Col4α3−/− mice. The more severe phenotype seemed to be the result of reduced collagen turnover, because procollagen III (α1) mRNA levels and fractional collagen synthesis in the wild-type and Mrc2-deficient kidneys were similar after UUO. Although Mrc2 associates with the urokinase receptor, differences in renal urokinase activity did not account for the increased fibrosis in the Mrc2-deficient mice. Treating wild-type mice with a cathepsin inhibitor, which blocks proteases implicated in Mrc2-mediated collagen degradation, worsened UUO-induced renal fibrosis. Cathepsin mRNA profiles were similar in Mrc2-positive fibroblasts and macrophages, and Mrc2 genotype did not alter relative cathepsin mRNA levels. Taken together, these data establish an important fibrosis-attenuating role for Mrc2-expressing renal interstitial cells and suggest the involvement of a lysosomal collagen turnover pathway. PMID:22095946
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Chalabala, Jan; Uhlig, Frank; Slavíček, Petr
2018-03-29
Ionization in the condensed phase and molecular clusters leads to a complicated chain of processes with coupled electron-nuclear dynamics. It is difficult to describe such dynamics with conventional nonadiabatic molecular dynamics schemes since the number of states swiftly increases as the molecular system grows. It is therefore attractive to use a direct electron and nuclear propagation such as the real-time time-dependent density functional theory (RT-TDDFT). Here we report a RT-TDDFT benchmark study on simulations of singly and doubly ionized states of a water monomer and dimer as a prototype for more complex processes in a condensed phase. We employed the RT-TDDFT based Ehrenfest molecular dynamics with a generalized gradient approximate (GGA) functional and compared it with wave-function-based surface hopping (SH) simulations. We found that the initial dynamics of a singly HOMO ionized water dimer is similar for both the RT-TDDFT/GGA and the SH simulations but leads to completely different reaction channels on a longer time scale. This failure is attributed to the self-interaction error in the GGA functionals and it can be avoided by using hybrid functionals with large fraction of exact exchange (represented here by the BHandHLYP functional). The simulations of doubly ionized states are reasonably described already at the GGA level. This suggests that the RT-TDDFT/GGA method could describe processes following the autoionization processes such as Auger emission, while its applicability to more complex processes such as intermolecular Coulombic decay remains limited.
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Marino-Merlo, Francesca; Frezza, Caterina; Papaianni, Emanuela; Valletta, Elena; Mastino, Antonio; Macchi, Beatrice
2017-11-01
Assessing the actual efficacy of compounds to directly inhibit HIV reverse transcriptase (RT) activity is a main goal in preclinical antiretroviral studies. Our previous studies demonstrated that the effects of inhibitor compounds towards HIV-RT could be efficiently assessed through a simple cell-free assay based on conventional reverse transcription PCR. In the present study, we describe a modified variant of our assay, termed RT real-time quantitative PCR inhibitory assay (RT-qPCR-IA), in which the ability of compounds to restrict the complementary DNA (cDNA) generation by HIV-RT using a specific RNA template is performed by the real-time technique, in order to improve both accuracy and sensitivity of the method. As specific RNA template, RNA extracted from stable transfectants ectopically expressing the herpes simplex virus 1 glycoprotein D gene was utilized. HIV-RT, of both commercial or house-made viral lysate origin, was employed for the assay. To assess the reliability of RT-qPCR-IA, we performed a comparative, quantitative analysis of the dose-dependent effect exerted by known nucleotide and non-nucleotide reverse-transcriptase inhibitors, using the SYBR Green dye chemistry as detection system. The results obtained with RT-qPCR-IA were compared to that obtained using a one-step PicoGreen technology-based commercial kit. The outcome of our study indicates that the development of the novel RT-qPCR-IA will provide rapid and accurate evaluation of the inhibitory efficacy of compounds towards HIV-RT activity. This evaluation could be very useful for large-scale screening of potential new anti-HIV drugs.
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Monteiro, S; Santos, R
2018-04-01
To assess the potential of a viability dye and an enzymatic reverse transcription quantitative PCR (RT-qPCR) pretreatment to discriminate between infectious and noninfectious enteric viruses. Enterovirus (EntV), norovirus (NoV) GII.4 and hepatitis A virus (HAV) were inactivated at 95°C for 10 min, and four methods were used to compare the efficiency of inactivation: (i) cell culture plaque assay for HAV and EntV, (ii) RT-qPCR alone, (iii) RT-qPCR assay preceded by RNase treatment, and (iv) pretreatment with a viability dye (reagent D (RD)) followed by RT-qPCR. In addition, heat-inactivated NoV was treated with RD coupled with surfactants to increase the efficiency of the viability dye. No treatment was able to completely discriminate infectious from noninfectious viruses. RD-RT-qPCR reduced more efficiently the detection of noninfectious viruses with little to no removal observed with RNase. RD-RT-qPCR method was the closest to cell culture assay. The combination of surfactants and RD did not show relevant improvements on the removal of inactivated viruses signal compared with viability RT-qPCR, with the exception of Triton X-100. The use of surfactant/RD-RT-qPCR, although not being able to completely remove the signal from noninfectious viral particles, yielded a better estimation of viral infectivity. Surfactant/RD-RT-qPCR may be an advantageous tool for a better detection of infectious viruses with potential significant impact in the risk assessment of the presence of enteric viruses. © 2017 The Society for Applied Microbiology.
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2014-01-01
Background Exercise is consistently recommended for older adults with knee pain related to osteoarthritis. However, the effects from exercise are typically small and short-term, likely linked to insufficient individualisation of the exercise programme and limited attention to supporting exercise adherence over time. The BEEP randomised trial aims to improve patients’ short and long-term outcomes from exercise. It will test the overall effectiveness and cost-effectiveness of two physiotherapy-led exercise interventions (Individually Tailored Exercise and Targeted Exercise Adherence) to improve the individual tailoring of, and adherence to exercise, compared with usual physiotherapy care. Methods/design Based on the learning from a pilot study (ISRCTN 23294263), the BEEP trial is a multi-centre, pragmatic, parallel group, individually randomised controlled trial, with embedded longitudinal qualitative interviews. 500 adults in primary care, aged 45 years and over with knee pain will be randomised to 1 of 3 treatment groups delivered by fully trained physiotherapists in up to 6 NHS services. These are: Usual Physiotherapy Care (control group consisting of up to 4 treatment sessions of advice and exercise), Individually Tailored Exercise (an individualised, supervised and progressed lower-limb exercise programme) or Targeted Exercise Adherence (supporting patients to adhere to exercise and to engage in general physical activity over the longer-term). The primary outcomes are pain and function as measured by the Western Ontario and McMaster Osteoarthritis index. A comprehensive range of secondary outcomes are also included. Outcomes are measured at 3, 6 (primary outcome time-point), 9, 18 and 36 months. Data on adverse events will also be collected. Semi-structured, qualitative interviews with a subsample of 30 participants (10 from each treatment group) will be undertaken at two time-points (end of treatment and 12 to 18 months later) and analysed thematically
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Concordance of IHC, FISH and RT-PCR for EML4-ALK rearrangements.
Teixidó, Cristina; Karachaliou, Niki; Peg, Vicente; Gimenez-Capitan, Ana; Rosell, Rafael
2014-04-01
The echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) has emerged as the second most important driver oncogene in lung cancer and the first targetable fusion oncokinase to be identified in 4-6% of lung adenocarcinomas. Crizotinib, along with a diagnostic test-the Vysis ALK Break Apart fluorescence in situ hybridization (FISH) Probe Kit-is approved for the treatment of ALK positive advanced non-small cell lung cancer (NSCLC). However, the success of a targeted drug is critically dependent on a sensitive and specific screening assay to detect the molecular drug target. In our experience, reverse transcription polymerase chain reaction (RT-PCR)-based detection of EML4-ALK is a more sensitive and reliable approach compared to FISH and immunohistochemistry (IHC). Although ALK FISH is clinically validated, the assay can be technically challenging and other diagnostic modalities, including IHC and RT-PCR should be further explored.
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Santiago, Gilberto A.; Vergne, Edgardo; Quiles, Yashira; Cosme, Joan; Vazquez, Jesus; Medina, Juan F.; Medina, Freddy; Colón, Candimar; Margolis, Harold; Muñoz-Jordán, Jorge L.
2013-01-01
Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV). There are four genetically distinct DENVs (DENV-1–4) that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1–4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA) for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1–4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1–4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases. PMID:23875046
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Pathak, Jyotishman; Murphy, Sean P; Willaert, Brian N; Kremers, Hilal M; Yawn, Barbara P; Rocca, Walter A; Chute, Christopher G
2011-01-01
RxNorm and NDF-RT published by the National Library of Medicine (NLM) and Veterans Affairs (VA), respectively, are two publicly available federal medication terminologies. In this study, we evaluate the applicability of RxNorm and National Drug File-Reference Terminology (NDF-RT) for extraction and classification of medication data retrieved using structured querying and natural language processing techniques from electronic health records at two different medical centers within the Rochester Epidemiology Project (REP). Specifically, we explore how mappings between RxNorm concept codes and NDF-RT drug classes can be leveraged for hierarchical organization and grouping of REP medication data, identify gaps and coverage issues, and analyze the recently released NLM's NDF-RT Web service API. Our study concludes that RxNorm and NDF-RT can be applied together for classification of medication extracted from multiple EHR systems, although several issues and challenges remain to be addressed. We further conclude that the Web service APIs developed by the NLM provide useful functionalities for such activities.
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NASA Technical Reports Server (NTRS)
Case, Jonathan L; White, Kristopher D.
2014-01-01
The NASA Short-term Prediction Research and Transition (SPoRT) Center in Huntsville, AL is running a real-time configuration of the Noah land surface model (LSM) within the NASA Land Information System (LIS) framework (hereafter referred to as the "SPoRT-LIS"). Output from the real-time SPoRT-LIS is used for (1) initializing land surface variables for local modeling applications, and (2) displaying in decision support systems for situational awareness and drought monitoring at select NOAA/National Weather Service (NWS) partner offices. The experimental CONUS run incorporates hourly quantitative precipitation estimation (QPE) from the National Severe Storms Laboratory Multi- Radar Multi-Sensor (MRMS) which will be transitioned into operations at the National Centers for Environmental Prediction (NCEP) in Fall 2014.This paper describes the current and experimental SPoRT-LIS configurations, and documents some of the limitations still remaining through the advent of MRMS precipitation analyses in the SPoRT-LIS land surface model (LSM) simulations.
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ERIC Educational Resources Information Center
Chang, Ming-Mei; Li, Anna; Feissner, Robert; Ahmad, Talal
2016-01-01
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is widely used in diagnosis and research to determine specific mRNA expressions in cells. As RT-qPCR applications increase, it is necessary to provide undergraduates hands-on experience of this modern technique. Here, we report a 3-week laboratory exercise using RT-qPCR to…
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Pfitzner, Claudia; Schröder, Isabel; Scheungraber, Cornelia; Dogan, Askin; Runnebaum, Ingo Bernhard; Dürst, Matthias; Häfner, Norman
2014-02-05
The detection of circulating tumour cells (CTC) in cancer patients may be useful for therapy monitoring and prediction of relapse. A sensitive assay based on HPV-oncogene transcripts which are highly specific for cervical cancer cells was established. The Digital-Direct-RT-PCR (DD-RT-PCR) combines Ficoll-separation, ThinPrep-fixation and one-step RT-PCR in a low-throughput digital-PCR format enabling the direct analysis and detection of individual CTC without RNA isolation. Experimental samples demonstrated a sensitivity of one HPV-positive cell in 500,000 HPV-negative cells. Spike-in experiments with down to 5 HPV-positive cells per millilitre EDTA-blood resulted in concordant positive results by PCR and immunocytochemistry. Blood samples from 3 of 10 CxCa patients each contained a single HPV-oncogene transcript expressing CTC among 5 to 15*10(5) MNBC. Only 1 of 7 patients with local but 2 of 3 women with systemic disease had CTC. This highly sensitive DD-RT-PCR for the detection of CTC may also be applied to other tumour entities which express tumour-specific transcripts.
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Seng, E K; Fang, Q; Lam, T J; Sin, Y M
2004-06-15
A rapid, sensitive and highly specific detection method for Aquareovirus based on reverse-transcription polymerase chain reaction (RT-PCR) was developed. Based on multiple sequence alignment of the cloned sequences of a local isolates, the Threadfin reovirus (TFV) and Guppy reovirus (GPV) with Grass carp reovirus (GCRV), a pair of degenerate primers was selected carefully and synthesized. Using this primer combination, only one specific product, approximately 450 bp in length was obtained when RT-PCR was carried out using the genomic double-stranded RNA (dsRNA) of TFV, GPV and GCRV. Similar results were also obtained when Chum salmon reovirus (CSRV) and Striped bass reovirus (SBRV) dsRNA were used as templates. No products were observed when nucleic acids other than the dsRNA of the aquareoviruses described above were used as RT-PCR templates. This technique could detect not only TFV but also GPV and GCRV in low titer virus-infected cell cultured cells. Furthermore, this method has also been shown to be able to diagnose GPV-infected guppy (Poecilia reticulata) that exhibit clinical symptoms as well as GPV-carrier guppy. Collectively, these results showed that the RT-PCR amplification method using specific degenerate primers described below is very useful for rapid and accurate detection of a variety of aquareovirus strains isolated from different host species and origin.
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Dobhal, Shefali; Olson, Jennifer D; Arif, Mohammad; Garcia Suarez, Johnny A; Ochoa-Corona, Francisco M
2016-06-01
Rose rosette disease is a disorder associated with infection by Rose rosette virus (RRV), a pathogen of roses that causes devastating effects on most garden cultivated varieties, and the wild invasive rose especially Rosa multiflora. Reliable and sensitive detection of this disease in early phases is needed to implement proper control measures. This study assesses a single primer-set based detection method for RRV and demonstrates its application in three different chemistries: Endpoint RT-PCR, TaqMan-quantitative RT-PCR (RT-qPCR) and SYBR Green RT-qPCR with High Resolution Melting analyses. A primer set (RRV2F/2R) was designed from consensus sequences of the nucleocapsid protein gene p3 located in the RNA 3 region of RRV. The specificity of primer set RRV2F/2R was validated in silico against published GenBank sequences and in-vitro against infected plant samples and an exclusivity panel of near-neighbor and other viruses that commonly infect Rosa spp. The developed assay is sensitive with a detection limit of 1fg from infected plant tissue. Thirty rose samples from 8 different states of the United States were tested using the developed methods. The developed methods are sensitive and reliable, and can be used by diagnostic laboratories for routine testing and disease management decisions. Copyright © 2016 Elsevier B.V. All rights reserved.
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Mehta, Komal; Gala, Jayesh; Bhasale, Surendra; Naik, Sattayasheel; Modak, Millind; Thakur, Harshad; Deo, Nivedita; Miller, Mark JS
2007-01-01
Background The efficacy and safety of a dietary supplement derived from South American botanicals was compared to glucosamine sulfate in osteoarthritis subjects in a Mumbai-based multi-center, randomized, double-blind study. Methods Subjects (n = 95) were screened and randomized to receive glucosamine sulfate (n = 47, 1500 mg/day) or reparagen (n = 48, 1800 mg/day), a polyherbal consisting of 300 mg of vincaria (Uncaria guianensis) and 1500 mg of RNI 249 (Lepidium meyenii) administered orally, twice daily. Primary efficacy variable was response rate based on a 20% improvement in WOMAC pain scores. Additional outcomes were WOMAC scores for pain, stiffness and function, visual analog score (VAS) for pain, with assessments at 1, 2, 4, 6 and 8 weeks. Tolerability, investigator and subject global assessments and rescue medication consumption (paracetamol) were measured together with safety assessments including vital signs and laboratory based assays. Results Subject randomization was effective: age, gender and disease status distribution was similar in both groups. The response rates (20% reduction in WOMAC pain) were substantial for both glucosamine (89%) and reparagen (94%) and supported by investigator and subject assessments. Using related criteria response rates to reparagen were favorable when compared to glucosamine. Compared to baseline both treatments showed significant benefits in WOMAC and VAS outcomes within one week (P < 0.05), with a similar, progressive improvement over the course of the 8 week treatment protocol (45–62% reduction in WOMAC or VAS scores). Tolerability was excellent, no serious adverse events were noted and safety parameters were unchanged. Rescue medication use was significantly lower in the reparagen group (p < 0.01) at each assessment period. Serum IGF-1 levels were unaltered by treatments. Conclusion Both reparagen and glucosamine sulfate produced substantial improvements in pain, stiffness and function in subjects with
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Wang, Xiaolong; Zhou, PeiHua; Sun, XueJun; Wei, GuangBing; Zhang, Li; Wang, Hui; Yao, JianFeng; Jia, PengBo; Zheng, JianBao
2016-05-01
One of the current challenges facing cancer gene therapy is the tumour-specific targeting of therapeutic genes. Effective targeting in gene therapy requires accurate spatial and temporal control of gene expression. To develop a sufficient and accurate tumour-targeting method for cancer gene therapy, we have investigated the use of hyperthermia to control the expression of a transgene under the control of the human telomerase reverse transcriptase (hTERT) promoter and eight heat shock elements (8HSEs). Luciferase reporters were constructed by inserting eight HSEs and the hTERT promoter (8HSEs-hTERTp) upstream of the pGL4.20 vector luciferase gene. The luciferase activity of the hTERT promoter and 8HSEs-hTERT promoter were then compared in the presence and absence of heat. The differences in luciferase activity were analysed using dual luciferase assays in SW480 (high hTERT expression), MKN28 and MRC-5 cells (low hTERT expression). The luciferase activity of the Hsp70B promoter was also compared to the 8HSEs-hTERT promoter in the above listed cell lines. Lentiviral vector and heat-induced expression of EGFP expression under the control of the 8HSEs-hTERT promoter in cultured cells and mouse tumour xenografts was measured by reverse transcription polymerase (RT-PCR), Western blot and immunofluorescence assays. hTERT promoter activity was higher in SW480 cells than in MKN28 or MRC-5 cells. At 43 °C, the luciferase activity of the 8HSEs-hTERT promoter was significantly increased in SW480 cells, but not in MKN28 or MRC-5 cells. Importantly, the differences in luciferase activity were much more obvious in both high (SW480) and low (MKN28 and MRC-5) hTERT expressing cells when the activity of the 8HSEs-hTERT promoter was compared to the Hsp70B promoter. Moreover, under the control of 8HSEs-hTERT promoter in vitro and in vivo, EGFP expression was obviously increased by heat treatment in SW480 cells but not in MKN28 or MRC-5 cells, nor was expression increased under
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SPoRT: Transitioning NASA and NOAA Experimental Data to the Operational Weather Community
NASA Technical Reports Server (NTRS)
Jedlovec, Gary J.
2013-01-01
Established in 2002 to demonstrate the weather and forecasting application of real-time EOS measurements, the NASA Short-term Prediction Research and Transition (SPoRT) program has grown to be an end-to-end research to operations activity focused on the use of advanced NASA modeling and data assimilation approaches, nowcasting techniques, and unique high-resolution multispectral data from EOS satellites to improve short-term weather forecasts on a regional and local scale. With the ever-broadening application of real-time high resolution satellite data from current EOS, Suomi NPP, and planned JPSS and GOES-R sensors to weather forecast problems, significant challenges arise in the acquisition, delivery, and integration of the new capabilities into the decision making process of the operational weather community. For polar orbiting sensors such as MODIS, AIRS, VIIRS, and CRiS, the use of direct broadcast ground stations is key to the real-time delivery of the data and derived products in a timely fashion. With the ABI on the geostationary GOES-R satellite, the data volumes will likely increase by a factor of 5-10 from current data streams. However, the high data volume and limited bandwidth of end user facilities presents a formidable obstacle to timely access to the data. This challenge can be addressed through the use of subsetting techniques, innovative web services, and the judicious selection of data formats. Many of these approaches have been implemented by SPoRT for the delivery of real-time products to NWS forecast offices and other weather entities. Once available in decision support systems like AWIPS II, these new data and products must be integrated into existing and new displays that allow for the integration of the data with existing operational products in these systems. SPoRT is leading the way in demonstrating this enhanced capability. This paper will highlight the ways SPoRT is overcoming many of the challenges presented by the enormous data
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Hoferer, Marc; Braun, Anne; Skrypski, Julia; Bock, Sabine; Thalheim, Sabine; Sting, Reinhard
2017-09-01
Infectious pancreatic necrosis virus (IPNV) causes great losses in fish hatcheries world-wide. The detection of IPNV can be challenging in certain circumstances, particularly due to low viral load and the genetic variability of this RNA virus. For the first time, this project created a quantitative triplex real-time reverse transcription PCR (RT-qPCR), including an endogenous control system, for specific, sensitive and rapid detection of IPNV in routine diagnostics. Multiple sequence alignment of 46 nucleotide sequences of the segment A genome obtained from the NCBI database allowed the design of two RT-qPCR systems covering the IPNV genogroup 1 and genogroups 2-5, respectively. The completed triplex RT-qPCR including a salmonid-specific endogenous control showed high specificity and an analytical sensitivity of 20-40 oligonucleotide copies. Testing of dilution series of virus-loaded cell culture suspensions proved equality of the triplex RT-qPCR with virus detection in cell culture and a higher sensitivity than conventional RT-PCR in field samples. In comparative studies of a total of 77 field samples tested, 51 showed identical positive and 19 identical negative results in cell culture and the triplex RT-qPCR. However, seven other samples yielded positive results in the triplex RT-qPCR, but negative results in cell culture. Copyright © 2017 Elsevier B.V. All rights reserved.
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Nicoś, M; Krawczyk, P; Wojas-Krawczyk, K; Bożyk, A; Jarosz, B; Sawicki, M; Trojanowski, T; Milanowski, J
2017-12-01
RT-PCR technique has showed a promising value as pre-screening method for detection of mRNA containing abnormal ALK sequences, but its sensitivity and specificity is still discussable. Previously, we determined the incidence of ALK rearrangement in CNS metastases of NSCLC using IHC and FISH methods. We evaluated ALK gene rearrangement using two-step RT-PCR method with EML4-ALK Fusion Gene Detection Kit (Entrogen, USA). The studied group included 145 patients (45 females, 100 males) with CNS metastases of NSCLC and was heterogeneous in terms of histology and smoking status. 21% of CNS metastases of NSCLC (30/145) showed presence of mRNA containing abnormal ALK sequences. FISH and IHC tests confirmed the presence of ALK gene rearrangement and expression of ALK abnormal protein in seven patients with positive result of RT-PCR analysis (4.8% of all patients, 20% of RT-PCR positive patients). RT-PCR method compared to FISH analysis achieved 100% of sensitivity and only 82.7% of specificity. IHC method compared to FISH method indicated 100% of sensitivity and 97.8% of specificity. In comparison to IHC, RT-PCR showed identical sensitivity with high number of false positive results. Utility of RT-PCR technique in screening of ALK abnormalities and in qualification patients for molecularly targeted therapies needs further validation.
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Kinoshita, Akitoshi; Miyachi, Hayato; Matsushita, Hiromichi; Yabe, Miharu; Taki, Tomohiko; Watanabe, Tomoyuki; Saito, Akiko M; Tomizawa, Daisuke; Taga, Takashi; Takahashi, Hiroyuki; Matsuo, Hidemasa; Kodama, Kumi; Ohki, Kentaro; Hayashi, Yasuhide; Tawa, Akio; Horibe, Keizo; Adachi, Souichi
2014-10-01
The clinical characteristics and prognostic relevance of acute myeloid leukaemia (AML) with myelodysplastic features remains to be clarified in children. We prospectively examined 443 newly diagnosed patients in a multicentre clinical trial for paediatric de novo AML, and found 'AML with myelodysplasia-related changes' (AML-MRC) according to the 2008 World Health Organization classification in 93 (21·0%), in whom 59 were diagnosed from myelodysplasia-related cytogenetics alone, 28 from multilineage dysplasia alone and six from a combination of both. Compared with 111 patients with 'AML, not otherwise specified' (AML-NOS), patients with 'AML-MRC' presented at a younger age, with a lower white blood cell count, higher incidence of 20-30% bone marrow blasts, unfavourable cytogenetics and a lower frequency of Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), NPM1 and CEBPA mutations. Complete remission rate and 3-year probability of event-free survival were significantly worse in 'AML-MRC' patients (67·7 vs. 85·6%, P < 0·01, 37·1% vs. 53·8%, P = 0·02, respectively), but 3-year overall survival and relapse-free survival were comparable with 'AML-NOS' patients. By multivariate analysis, FLT3-ITD was solely associated with worse overall survival. These results support the distinctive features of the category 'AML-MRC' even in children. © 2014 John Wiley & Sons Ltd.
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Recent Upgrades to NASA SPoRT Initialization Datasets for the Environmental Modeling System
NASA Technical Reports Server (NTRS)
Case, Jonathan L.; Lafontaine, Frank J.; Molthan, Andrew L.; Zavodsky, Bradley T.; Rozumalski, Robert A.
2012-01-01
The NASA Short-term Prediction Research and Transition (SPoRT) Center has developed several products for its NOAA/National Weather Service (NWS) partners that can initialize specific fields for local model runs within the NOAA/NWS Science and Training Resource Center Environmental Modeling System (EMS). The suite of SPoRT products for use in the EMS consists of a Sea Surface Temperature (SST) composite that includes a Lake Surface Temperature (LST) analysis over the Great Lakes, a Great Lakes sea-ice extent within the SST composite, a real-time Green Vegetation Fraction (GVF) composite, and NASA Land Information System (LIS) gridded output. This paper and companion poster describe each dataset and provide recent upgrades made to the SST, Great Lakes LST, GVF composites, and the real-time LIS runs.
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Shiroma, Riki; Park, Jeung-yil; Al-Haq, Muhammad Imran; Arakane, Mitsuhiro; Ike, Masakazu; Tokuyasu, Ken
2011-02-01
We improved the CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature (RT). We firstly optimized the RT-lime pretreatment for the lignocellulosic part. When the ratio of lime/dry-biomass was 0.2 (w/w), the RT lime-pretreatment for 7-d resulted in an effect on the enzymatic saccharification of cellulose and xylan equivalent to that of the pretreatment at 120°C for 1h. Sucrose, starch and β-1,3-1,4-glucan, which could be often detected in rice straw, were mostly stable under the RT-lime pretreatment condition. Then, the pretreatment condition in the conventional CaCCO process was modified by the adaptation of the optimized RT lime-pretreatment, resulting in significantly better carbohydrate recoveries via enzymatic saccharification than those of the CaCCO process (120°C for 1 h). Thus, the improved CaCCO process (the RT-CaCCO process) could preserve/pretreat the feedstock at RT in a wet form with minimum loss of carbohydrates. Copyright © 2010 Elsevier Ltd. All rights reserved.
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Sherwood, Matthew S; Kane, Jessica H; Weisend, Michael P; Parker, Jason G
2016-01-01
Real-time functional magnetic resonance imaging (rt-fMRI) neurofeedback can be used to train localized, conscious regulation of blood oxygen level-dependent (BOLD) signals. As a therapeutic technique, rt-fMRI neurofeedback reduces the symptoms of a variety of neurologic disorders. To date, few studies have investigated the use of self-regulation training using rt-fMRI neurofeedback to enhance cognitive performance. This work investigates the utility of rt-fMRI neurofeedback as a tool to enhance human cognition by training healthy individuals to consciously control activity in the left dorsolateral prefrontal cortex (DLPFC). A cohort of 18 healthy participants in the experimental group underwent rt-fMRI neurofeedback from the left DLPFC in five training sessions across two weeks while 7 participants in the control group underwent similar training outside the MRI and without rt-fMRI neurofeedback. Working memory (WM) performance was evaluated on two testing days separated by the five rt-fMRI neurofeedback sessions using two computerized tests. We investigated the ability to control the BOLD signal across training sessions and WM performance across the two testing days. The group with rt-fMRI neurofeedback demonstrated a significant increase in the ability to self-regulate the BOLD signal in the left DLPFC across sessions. WM performance showed differential improvement between testing days one and two across the groups with the highest increases observed in the rt-fMRI neurofeedback group. These results provide evidence that individuals can quickly gain the ability to consciously control the left DLPFC, and this training results in improvements of WM performance beyond that of training alone. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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Okano, Hiroyuki; Baba, Misato; Kawato, Katsuhiro; Hidese, Ryota; Yanagihara, Itaru; Kojima, Kenji; Takita, Teisuke; Fujiwara, Shinsuke; Yasukawa, Kiyoshi
2018-03-01
One-step RT-PCR has not been widely used even though some thermostable DNA polymerases with reverse transcriptase (RT) activity were developed from bacterial and archaeal polymerases, which is owing to low cDNA synthesis activity from RNA. In the present study, we developed highly-sensitive one-step RT-PCR using the single variant of family A DNA polymerase with RT activity, K4pol L329A (L329A), from the hyperthermophilic bacterium Thermotoga petrophila K4 or the 16-tuple variant of family B DNA polymerase with RT activity, RTX, from the hyperthermophilic archaeon Thermococcus kodakarensis. Optimization of reaction condition revealed that the activities for cDNA synthesis and PCR of K4pol L329A and RTX were highly affected by the concentrations of MgCl 2 and Mn(OCOCH 3 ) 2 as well as those of K4pol L329A or RTX. Under the optimized condition, 300 copies/μl of target RNA in 10 μl reaction volumes were successfully detected by the one-step RT-PCR with K4pol L329A or RTX, which was almost equally sensitive enough compared with the current RT-PCR condition using retroviral RT and thermostable DNA polymerase. Considering that K4pol L329A and RTX are stable even at 90-100°C, our results suggest that the one-step RT-PCR with K4pol L329A or RTX is more advantageous than the current one. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
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Singh, R P; Nie, X; Singh, M; Coffin, R; Duplessis, P
2002-01-01
Phenolic compounds from plant tissues inhibit reverse transcription-polymerase chain reaction (RT-PCR). Multiple-step protocols using several additives to inhibit polyphenolic compounds during nucleic acid extraction are common, but time consuming and laborious. The current research highlights that the inclusion of 0.65 to 0.70% of sodium sulphite in the extraction buffer minimizes the pigmentation of nucleic acid extracts and improves the RT-PCR detection of Potato virus Y (PVY) and Potato leafroll virus (PLRV) in potato (Solanum tuberosum) tubers and Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) in leaves and bark in the sweet cherry (Prunus avium) tree. Substituting sodium sulphite in the nucleic acid extraction buffer eliminated the use of proteinase K during extraction. Reagents phosphate buffered saline (PBS)-Tween 20 and polyvinylpyrrolidone (PVP) were also no longer required during RT or PCR phase. The resultant nucleic acid extracts were suitable for both duplex and multiplex RT-PCR. This simple and less expensive nucleic acid extraction protocol has proved very effective for potato cv. Russet Norkotah, which contains a high amount of polyphenolics. Comparing commercially available RNA extraction kits (Catrimox and RNeasy), the sodium sulphite based extraction protocol yielded two to three times higher amounts of RNA, while maintaining comparable virus detection by RT-PCR. The sodium sulphite based extraction protocol was equally effective in potato tubers, and in leaves and bark from the cherry tree.
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Marini, N; Bevilacqua, C B; Büttow, M V; Raseira, M C B; Bonow, S
2017-05-25
Selecting and validating reference genes are the first steps in studying gene expression by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). The present study aimed to evaluate the stability of five reference genes for the purpose of normalization when studying gene expression in various cultivars of Prunus persica with different chilling requirements. Flower bud tissues of nine peach genotypes from Embrapa's peach breeding program with different chilling requirements were used, and five candidate reference genes based on the RT-qPCR that were useful for studying the relative quantitative gene expression and stability were evaluated using geNorm, NormFinder, and bestKeeper software packages. The results indicated that among the genes tested, the most stable genes to be used as reference genes are Act and UBQ10. This study is the first survey of the stability of reference genes in peaches under chilling stress and provides guidelines for more accurate RT-qPCR results.
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Evaluating the Impact of AIRS Observations on Regional Forecasts at the SPoRT Center
NASA Technical Reports Server (NTRS)
Zavodsky, Bradley
2011-01-01
NASA Short-term Prediction Research and Transition (SPoRT) Center collaborates with operational partners of different sizes and operational goals to improve forecasts using targeted projects and data sets. Modeling and DA activities focus on demonstrating utility of NASA data sets and capabilities within operational systems. SPoRT has successfully assimilated the Atmospheric Infrared Sounder (AIRS) radiance and profile data. A collaborative project is underway with the Joint Center for Satellite Data Assimilation (JCSDA) to use AIRS profiles to better understand the impact of AIRS radiances assimilated within Gridpoint Statistical Interpolation (GSI) in hopes of engaging the operational DA community in a reassessment of assimilation methodologies to more effectively assimilate hyperspectral radiances.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Lin, Chao-Feng
Purpose: Vascular smooth muscle cell (VSMC) proliferation plays a critical role in the pathogenesis of atherosclerosis and restenosis. This study investigated piperazinedione derived compound TW-01-mediated inhibitory effects on VSMC proliferation and intimal hyperplasia. Methods: Cell proliferation was determined using [{sup 3}H]-thymidine incorporation and MTT assay; cell cycle distribution was measured using flow cytometry; proteins and mRNA expression were determined using western blotting and RT-PCR analyses; DNA binding activity of nuclear factor-κB (NF-κB), as measured using enzyme-linked immunosorbent assays (ELISA); in vivo effects of TW-01 were determined using balloon angioplasty in the rat. Results: TW-01 significantly inhibited cell proliferation. At themore » concentrations used, no cytotoxic effects were observed. Three predominant signaling pathways were inhibited by TW-01: (a) extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) activation and its downstream effectors of c-fos, c-jun, and c-myc; (b) DNA binding activity of nuclear factor-κB (NF-κB); and, (c) Akt/protein kinase B (PKB) and cell cycle progression. Furthermore, TW-01 also inhibited Ras activation, a shared upstream event of each of these signaling cascades. In vascular injury studies, oral administration of TW-01 significantly suppressed intimal hyperplasia induced by balloon angioplasty. Conclusion: The present study suggests that TW-01 might be a potential candidate for atherosclerosis treatment. - Highlights: • TW-01significantly inhibits vascular smooth muscle cell proliferation. • TW-01 inhibits ERK, Akt and Ras pathway and DNA binding activity of NF-κB. • TW-01 significantly suppresses intimal hyperplasia induced by balloon angioplasty. • TW-01 might be a potential candidate for atherosclerosis treatment.« less
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Jeong, Jae-Kyo; Kang, Min-Hee; Gurunathan, Sangiliyandi; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Kim, Jin-Hoi
2014-09-25
Real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) is the most sensitive, and valuable technique for rare mRNA detection. However, the expression profiles of reference genes under different experimental conditions, such as different mouse strains, developmental stage, and culture conditions have been poorly studied. mRNA stability of the actb, gapdh, sdha, ablim, ywhaz, sptbn, h2afz, tgfb1, 18 s and wrnip genes was analyzed. Using the NormFinder program, the most stable genes are as follows: h2afz for the B6D2F-1 and C57BL/6 strains; sptbn for ICR; h2afz for KOSOM and CZB cultures of B6D2F-1 and C57BL/6 strain-derived embryos; wrnip for M16 culture of B6D2F-1 and C57BL/6 strain-derived embryos; ywhaz, tgfb1, 18 s, 18 s, ywhaz, and h2afz for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst embryonic stages cultured in KSOM medium, respectively; h2afz, wrnip, wrnip, h2afz, ywhaz, and ablim for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in CZB medium, respectively; 18 s, h2afz, h2afz, actb, h2afz, and wrnip for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in M16 medium, respectively. These results demonstrated that candidate reference genes for normalization of target gene expression using RT-qPCR should be selected according to mouse strains, developmental stage, and culture conditions.
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ICG: a wiki-driven knowledgebase of internal control genes for RT-qPCR normalization.
Sang, Jian; Wang, Zhennan; Li, Man; Cao, Jiabao; Niu, Guangyi; Xia, Lin; Zou, Dong; Wang, Fan; Xu, Xingjian; Han, Xiaojiao; Fan, Jinqi; Yang, Ye; Zuo, Wanzhu; Zhang, Yang; Zhao, Wenming; Bao, Yiming; Xiao, Jingfa; Hu, Songnian; Hao, Lili; Zhang, Zhang
2018-01-04
Real-time quantitative PCR (RT-qPCR) has become a widely used method for accurate expression profiling of targeted mRNA and ncRNA. Selection of appropriate internal control genes for RT-qPCR normalization is an elementary prerequisite for reliable expression measurement. Here, we present ICG (http://icg.big.ac.cn), a wiki-driven knowledgebase for community curation of experimentally validated internal control genes as well as their associated experimental conditions. Unlike extant related databases that focus on qPCR primers in model organisms (mainly human and mouse), ICG features harnessing collective intelligence in community integration of internal control genes for a variety of species. Specifically, it integrates a comprehensive collection of more than 750 internal control genes for 73 animals, 115 plants, 12 fungi and 9 bacteria, and incorporates detailed information on recommended application scenarios corresponding to specific experimental conditions, which, collectively, are of great help for researchers to adopt appropriate internal control genes for their own experiments. Taken together, ICG serves as a publicly editable and open-content encyclopaedia of internal control genes and accordingly bears broad utility for reliable RT-qPCR normalization and gene expression characterization in both model and non-model organisms. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Bateman, Lori A; Slentz, Cris A; Willis, Leslie H; Shields, A Tamlyn; Piner, Lucy W; Bales, Connie W; Houmard, Joseph A; Kraus, William E
2011-09-15
Aerobic training (AT) improves the metabolic syndrome (MS) and its component risk factors; however, to our knowledge, no randomized clinical studies have addressed whether resistance training (RT) improves the MS when performed alone or combined with AT. Sedentary, overweight dyslipidemic men and women, aged 18 to 70 years completed a 4-month inactive run-in period and were randomized to 1 of 3 eight-month exercise programs (n = 196). The exercise programs were (1) RT (3 days/week, 3 sets/day of 8 to 12 repetitions of 8 different exercises targeting all major muscle groups); (2) AT (∼120 minutes/week at 75% of the maximum oxygen uptake), and (3) AT and RT combined (AT/RT) (exact combination of AT and RT). Of the 196 randomized patients, 144 completed 1 of the 3 exercise programs. The 86 participants with complete data for all 5 MS criteria were used in the present analysis, and a continuous MS z score was calculated. Eight months of RT did not change the MS score. AT improved the MS score (p <0.07) and showed a trend toward significance compared to RT (p <0.10). AT/RT significantly decreased the MS score and was significantly different from RT alone. In conclusion, RT was not effective at improving the MS score; however, AT was effective. Combined AT and RT was similarly effective but not different from AT alone. When weighing the time commitment versus health benefit, the data suggest that AT alone was the most efficient mode of exercise for improving cardiometabolic health. Published by Elsevier Inc.
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Yuan, Wen; Wang, Jing; Xu, Fengjiao; Huang, Bihong; Lian, Yuexiao; Rao, Dan; Yin, Xueqin; Wu, Miaoli; Zhu, Yujun; Zhang, Yu; Huang, Ren; Guo, Pengju
2016-10-01
Theiler's murine encephalomyelitis virus (TMEV) and rat theilovirus (RTV), the member of the genus Cardiovirus, are widespread in laboratory mice and rats, and are potential contaminants of biological materials. Cardioviruses infection may cause serious complications in biomedical research. To improve the efficiency of routine screening for Cardioviruses infection, a duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection and differentiation of TMEV and RTV. The duplex assay was specific for reference strains of TMEV and RTV, and no cross-reaction was found with seven other rodent viruses. The limits of detection of both TMEV and RTV were 4×10(1) copies RNA/reaction. Reproducibility was estimated using standard dilutions, with coefficients of variation <3.1%. 439 clinical samples were evaluated by both duplex real-time RT-PCR and conventional RT-PCR. For 439 clinical samples，95 samples were positive for TMEV and 72 samples were positive for RTV using duplex real-time RT-PCR approach, whereas only 77 samples were positive for TMEV and 66 samples were positive for RTV when conventional RT-PCR was applied. Mixed infections were found in 20 samples when analyzed by conventional RT-PCR whereas 30 samples were found to be mixed infection when duplex real-time RT-PCR was applied. This duplex assay provides a useful tool for routine health monitoring and screening of contaminated biological materials of these two viruses. Copyright © 2016 Elsevier B.V. All rights reserved.
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Creating Meaning from Collaboration to Implement RtI for At-Risk Students
ERIC Educational Resources Information Center
Diakakis, Julia Ann
2014-01-01
The purpose of this qualitative exploratory case study based on Danielson's (2002) assertion that when teachers learn, student achievement improves was to examine how teachers created a collaborative learning experience through Professional Learning Community (PLC) concepts to implement Response to Intervention (RtI) with at-risk students. The…
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NASA Astrophysics Data System (ADS)
Adlar, F. R.; Bela, B.
2017-08-01
To anticipate the potential use of anti-integrase drugs in Indonesia for treatment of HIV-1 infection, the development of a drug resistance genotyping assay for anti-integrase is crucial in identifying the genetic drug resistance profile of Indonesian HIV-1 strains. This experiment aimed to amplify a target region in the integrase gene of Indonesian HIV-1 subtypes CRF01_AE and B that contain genetic mutations known to confer resistance to anti-integrase drug. Eleven archived plasma samples from individuals living with HIV-1 were obtained from the Virology and Cancer Pathobiology Research Center for Health Service (VCPRC FKUI-RSCM) laboratory. One of the plasma samples contained HIV-1 subtype B, and the remaining plasma samples contained subtype CRF01_AE. The target regions for all samples were amplified through RT-PCR, with an annealing temperature of 55 °C, using the primer pair AE_POL 4086F and AE_POL 5232R that were designed by VCPRC FKUI-RSCM. The results of this experiment show that 18.2% (2/11) of the samples were successfully amplified using the one-step RT-PCR. While the primer pair was effective in amplifying the target region in the integrase gene sequence for subtype B (100%; 1/1), it had a low efficacy (10%, 1/10) for subtype CRF01_AE. In conclusion, the primer pair can be used to amplify the target region in Indonesian HIV-1 strain subtypes CRF01_AE and B. However, optimization of the PCR condition and an increased number of samples would help to determine an accurate representation of the efficacy of the primer pair.
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Eboigbodin, Kevin; Filén, Sanna; Ojalehto, Tuomas; Brummer, Mirko; Elf, Sonja; Pousi, Kirsi; Hoser, Mark
2016-06-01
Rapid and accurate diagnosis of influenza viruses plays an important role in infection control, as well as in preventing the misuse of antibiotics. Isothermal nucleic acid amplification methods offer significant advantages over the polymerase chain reaction (PCR), since they are more rapid and do not require the sophisticated instruments needed for thermal cycling. We previously described a novel isothermal nucleic acid amplification method, 'Strand Invasion Based Amplification' (SIBA®), with high analytical sensitivity and specificity, for the detection of DNA. In this study, we describe the development of a variant of the SIBA method, namely, reverse transcription SIBA (RT-SIBA), for the rapid detection of viral RNA targets. The RT-SIBA method includes a reverse transcriptase enzyme that allows one-step reverse transcription of RNA to complementary DNA (cDNA) and simultaneous amplification and detection of the cDNA by SIBA under isothermal reaction conditions. The RT-SIBA method was found to be more sensitive than PCR for the detection of influenza A and B and could detect 100 copies of influenza RNA within 15 min. The development of RT-SIBA will enable rapid and accurate diagnosis of viral RNA targets within point-of-care or central laboratory settings.
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Quantitative RT-PCR is frequently used to analyze gene expression in different experimental systems. In this assay, housekeeping genes are frequently used to normalize for the variability between samples (relative quantification). We have examined the utility of using qRT-PCR and...
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Detection of enteroviruses and hepatitis a virus in water by consensus primer multiplex RT-PCR
Li, Jun-Wen; Wang, Xin-Wei; Yuan, Chang-Qing; Zheng, Jin-Lai; Jin, Min; Song, Nong; Shi, Xiu-Quan; Chao, Fu-Huan
2002-01-01
AIM: To develop a rapid detection method of enteroviruses and Hepatitis A virus (HAV). METHODS: A one-step, single-tube consensus primers multiplex RT-PCR was developed to simultaneously detect Poliovirus, Coxsackie virus, Echovirus and HAV. A general upstream primer and a HAV primer and four different sets of primers (5 primers) specific for Poliovirus, Coxsacki evirus, Echovirus and HAV cDNA were mixed in the PCR mixture to reverse transcript and amplify the target DNA. Four distinct amplified DNA segments representing Poliovirus, Coxsackie virus, Echovirus and HAV were identified by gel electrophoresis as 589-, 671-, 1084-, and 1128 bp sequences, respectively. Semi-nested PCR was used to confirm the amplified products for each enterovirus and HAV. RESULTS: All four kinds of viral genome RNA were detected, and producing four bands which could be differentiated by the band size on the gel. To confirm the specificity of the multiplex PCR products, semi-nested PCR was performed. For all the four strains tested gave positive results. The detection sensitivity of multiplex PCR was similar to that of monoplex RT-PCR which was 24 PFU for Poliovrus, 21 PFU for Coxsackie virus, 60 PFU for Echovirus and 105 TCID50 for HAV. The minimum amount of enteric viral RNA detected by semi-nested PCR was equivalent to 2.4 PFU for Poliovrus, 2.1 PFU for Coxsackie virus, 6.0 PFU for Echovirus and 10.5 TCID50 for HAV. CONCLUSION: The consensus primers multiplex RT-PCR has more advantages over monoplex RT-PCR for enteric viruses detection, namely, the rapid turnaround time and cost effectiveness. PMID:12174381
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Kanwar, N; Hassan, F; Barclay, L; Langley, C; Vinjé, J; Bryant, P W; George, K St; Mosher, L; Matthews-Greer, J M; Rocha, M A; Beenhouwer, D O; Harrison, C J; Moffatt, M; Shastri, N; Selvarangan, R
2018-04-10
Norovirus is the leading cause of epidemic and sporadic acute gastroenteritis (AGE) in the United States. Widespread prevalence necessitates implementation of accurate norovirus detection assays in clinical diagnostic laboratories. To evaluate RIDA ® GENE norovirus GI/GII real-time RT-PCR assay (RGN RT-PCR) using stool samples from patients with sporadic AGE. Patients between 14 days to 101 years of age with symptoms of AGE were enrolled prospectively at four sites across the United States during 2014-2015. Stool specimens were screened for the presence of norovirus RNA by the RGN RT-PCR assay. Results were compared with a reference method that included conventional RT-PCR and sequencing of a partial region of the 5'end of the norovirus ORF2 gene. A total of 259 (36.0%) of 719 specimens tested positive for norovirus by the reference method. The RGN RT-PCR assay detected norovirus in 244 (94%) of these 259 norovirus positive specimens. The sensitivity and specificity (95% confidence interval) of the RGN RT-PCR assay for detecting norovirus genogroup (G) I was 82.8% (63.5-93.5) and 99.1% (98.0-99.6) and for GII was 94.8% (90.8-97.2) and 98.6% (96.9-99.4), respectively. Seven specimens tested positive by the RGN-RT PCR that were negative by the reference method. The fifteen false negative samples were typed as GII.4 Sydney, GII.13, GI.3, GI.5, GI.2, GII.1, and GII.3 in the reference method. The RGN RT-PCR assay had a high sensitivity and specificity for the detection of norovirus in stool specimens from patients with sporadic AGE. Copyright © 2018. Published by Elsevier B.V.
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Bartsch, Christina; Szabo, Kathrin; Dinh-Thanh, Mai; Schrader, Christina; Trojnar, Eva; Johne, Reimar
2016-12-01
Frozen berries have been repeatedly identified as vehicles for norovirus (NoV) transmission causing large gastroenteritis outbreaks. However, virus detection in berries is often hampered by the presence of RT-PCR-inhibiting substances. Here, several virus extraction methods for subsequent real-time RT-PCR-based NoV-RNA detection in strawberries were compared and optimized. NoV recovery rates (RRs) between 0.21 ± 0.13% and 10.29 ± 6.03% were found when five different artificially contaminated strawberry batches were analyzed by the ISO/TS15216-2 method indicating the presence of different amounts of RT-PCR inhibitors. A comparison of five different virus extraction methods using artificially contaminated strawberries containing high amounts of RT-PCR inhibitors revealed the best NoV RRs for the ISO/TS15216 method. Further improvement of NoV RRs from 2.83 ± 2.92% to 15.28 ± 9.73% was achieved by the additional use of Sephacryl(®)-based columns for RNA purification. Testing of 22 frozen strawberry samples from a batch involved in a gastroenteritis outbreak resulted in 5 vs. 13 NoV GI-positive and in 9 vs. 20 NoV GII-positive samples using the original ISO/TS15216 method vs. the extended protocol, respectively. It can be concluded that the inclusion of an additional RNA purification step can increase NoV detection by the ISO/TS15216-2 method in frozen berries containing high amounts of RT-PCR inhibitors. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.
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Ifejika-Jones, Nneka L.; Harun, Nusrat; Mohammed-Rajput, Nareesa A.; Noser, Elizabeth A.; Grotta, James C.
2011-01-01
Background and Purpose Acute ischemic stroke patients receiving IV tissue plasminogen activator (rt-PA) within 3 hours of symptom onset are 30% more likely to have minimal disability at three months. During hospitalization, short-term disability is subjectively measured by discharge disposition, whether to home, Inpatient Rehabilitation (IR), Skilled Nursing Facility (SNF) or Subacute Care (Sub). There are no studies assessing the role of rt-PA use as a predictor of post-stroke disposition. Methods Retrospective analysis of all ischemic stroke patients admitted to the University of Texas Houston Medical School (UTHMS) Stroke Service between Jan 2004 and Oct 2009. Baseline demographics and National Institute of Health Stroke Scale (NIHSS) score were collected. Cerebrovascular disease risk factors were used for risk stratification. Results Home vs. IR, SNF, Sub Of 2225 acute ischemic stroke patients, 1019 were discharged home, 1206 to another level of care. Patients who received rt-PA therapy were 1.9 times more likely to be discharged home (P = <0.0001; OR 1.945, 95% CI 1.538 to 2.459). IR vs. SNF, Sub / SNF vs. Sub Of 1206 acute ischemic stroke patients, 719 patients were discharged to acute IR, 371 were discharged to SNF, 116 to Sub. There were no differences in disposition between patients who received rt-PA therapy. Conclusions Stroke patients who receive IV rt-PA for acute ischemic stroke are more 1.9 times more likely to be discharged directly home after hospitalization. This study is limited by its retrospective nature and the undetermined role of psychosocial factors related to discharge. PMID:21293014
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NASA Technical Reports Server (NTRS)
Molthan, Andrew L.; Burks, Jason E.; McGrath, Kevin M.; Jedlovec, Gary J.
2012-01-01
NASA s Short-term Prediction Research and Transition (SPoRT) Center supports the transition of unique NASA and NOAA research activities to the operational weather forecasting community. SPoRT emphasizes real-time analysis and prediction out to 48 hours. SPoRT partners with NOAA s National Weather Service (NWS) Weather Forecast Offices (WFOs) and National Centers to improve current products, demonstrate future satellite capabilities and explore new data assimilation techniques. Recently, the SPoRT Center has been involved in several activities related to disaster response, in collaboration with NOAA s National Weather Service, NASA s Applied Sciences Disasters Program, and other partners.
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Cai, Larry; Yeh, Benjamin M; Westphalen, Antonio C; Roberts, John; Wang, Zhen J
2017-03-01
To investigate whether the addition of gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced 3D T1-weighted MR cholangiography (T1w-MRC) to 3D T2-weighted MRC (T2w-MRC) improves the confidence and diagnostic accuracy of biliary anatomy in living liver donors. Two abdominal radiologists retrospectively and independently reviewed pre-operative MR studies in 58 consecutive living liver donors. The second-order bile duct visualization on T1w- and T2w-MRC images was rated on a 4-point scale. The readers also independently recorded the biliary anatomy and their diagnostic confidence using (1) combined T1w- and T2w-MRC, and (2) T2w-MRC. In the 23 right lobe donors, the biliary anatomy at imaging and the imaging-predicted number of duct orifices at surgery were compared to intra-operative findings. T1w-MRC had a higher proportion of excellent visualization than T2w-MRC, 66% vs. 45% for reader 1 and 60% vs. 31% for reader 2. The median confidence score for biliary anatomy diagnosis was significantly higher with combined T1w- and T2w-MRC than T2w-MRC alone for both readers (Reader 1: 3 vs. 2, p < 0.001; Reader 2: 3 vs. 1, p < 0.001). Compared to intra-operative findings, the accuracy of imaging-predicted number of duct orifices using combined T1w-and T2w-MRC was significantly higher than that using T2w-MRC alone (p = 0.034 for reader 1, p = 0.0082 for reader 2). The addition of Gd-EOB-DTPA-enhanced 3D T1w-MRC to 3D T2w-MRC improves second-order bile duct visualization and increases the confidence in biliary anatomy diagnosis and the accuracy in the imaging-predicted number of duct orifices acquired during right lobe harvesting.
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Adams, Richard A; Meade, Angela M; Seymour, Matthew T; Wilson, Richard H; Madi, Ayman; Fisher, David; Kenny, Sarah L; Kay, Edward; Hodgkinson, Elizabeth; Pope, Malcolm; Rogers, Penny; Wasan, Harpreet; Falk, Stephen; Gollins, Simon; Hickish, Tamas; Bessell, Eric M; Propper, David; Kennedy, M John; Kaplan, Richard; Maughan, Timothy S
2011-01-01
Summary Background When cure is impossible, cancer treatment should focus on both length and quality of life. Maximisation of time without toxic effects could be one effective strategy to achieve both of these goals. The COIN trial assessed preplanned treatment holidays in advanced colorectal cancer to achieve this aim. Methods COIN was a randomised controlled trial in patients with previously untreated advanced colorectal cancer. Patients received either continuous oxaliplatin and fluoropyrimidine combination (arm A), continuous chemotherapy plus cetuximab (arm B), or intermittent (arm C) chemotherapy. In arms A and B, treatment continued until development of progressive disease, cumulative toxic effects, or the patient chose to stop. In arm C, patients who had not progressed at their 12-week scan started a chemotherapy-free interval until evidence of disease progression, when the same treatment was restarted. Randomisation was done centrally (via telephone) by the MRC Clinical Trials Unit using minimisation. Treatment allocation was not masked. The comparison of arms A and B is described in a companion paper. Here, we compare arms A and C, with the primary objective of establishing whether overall survival on intermittent therapy was non-inferior to that on continuous therapy, with a predefined non-inferiority boundary of 1·162. Intention-to-treat (ITT) and per-protocol analyses were done. This trial is registered, ISRCTN27286448. Findings 1630 patients were randomly assigned to treatment groups (815 to continuous and 815 to intermittent therapy). Median survival in the ITT population (n=815 in both groups) was 15·8 months (IQR 9·4–26·1) in arm A and 14·4 months (8·0–24·7) in arm C (hazard ratio [HR] 1·084, 80% CI 1·008–1·165). In the per-protocol population (arm A, n=467; arm C, n=511), median survival was 19·6 months (13·0–28·1) in arm A and 18·0 months (12·1–29·3) in arm C (HR 1·087, 0·986–1·198). The upper limits of CIs for HRs
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Kitchener, H; Swart, A M C; Qian, Q; Amos, C; Parmar, M K B
2009-01-10
Hysterectomy and bilateral salpingo-oophorectomy (BSO) is the standard surgery for stage I endometrial cancer. Systematic pelvic lymphadenectomy has been used to establish whether there is extra-uterine disease and as a therapeutic procedure; however, randomised trials need to be done to assess therapeutic efficacy. The ASTEC surgical trial investigated whether pelvic lymphadenectomy could improve survival of women with endometrial cancer. From 85 centres in four countries, 1408 women with histologically proven endometrial carcinoma thought preoperatively to be confined to the corpus were randomly allocated by a minimisation method to standard surgery (hysterectomy and BSO, peritoneal washings, and palpation of para-aortic nodes; n=704) or standard surgery plus lymphadenectomy (n=704). The primary outcome measure was overall survival. To control for postsurgical treatment, women with early-stage disease at intermediate or high risk of recurrence were randomised (independent of lymph-node status) into the ASTEC radiotherapy trial. Analysis was by intention to treat. This study is registered, number ISRCTN 16571884. After a median follow-up of 37 months (IQR 24-58), 191 women (88 standard surgery group, 103 lymphadenectomy group) had died, with a hazard ratio (HR) of 1.16 (95% CI 0.87-1.54; p=0.31) in favour of standard surgery and an absolute difference in 5-year overall survival of 1% (95% CI -4 to 6). 251 women died or had recurrent disease (107 standard surgery group, 144 lymphadenectomy group), with an HR of 1.35 (1.06-1.73; p=0.017) in favour of standard surgery and an absolute difference in 5-year recurrence-free survival of 6% (1-12). With adjustment for baseline characteristics and pathology details, the HR for overall survival was 1.04 (0.74-1.45; p=0.83) and for recurrence-free survival was 1.25 (0.93-1.66; p=0.14). Our results show no evidence of benefit in terms of overall or recurrence-free survival for pelvic lymphadenectomy in women with early
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USDA-ARS?s Scientific Manuscript database
Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring and evaluating changes in gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. In this study, expres...
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Ribeiro, Alex S; Tomeleri, Crisieli M; Souza, Mariana F; Pina, Fábio Luiz C; Schoenfeld, Brad J; Nascimento, Matheus A; Venturini, Danielle; Barbosa, Décio S; Cyrino, Edilson S
2015-12-01
The purpose of this study was to analyze the effects of a progressive resistance training (RT) program on C-reactive protein (CRP), blood glucose (GLU), and lipid profile in older women with differing levels of RT experience. Sixty-five older women (68.9 ± 6.1 years, 67.1 ± 13.1 kg) were separated according to RT experience: an advanced group composed by 35 participants who previously carried out 24 weeks of RT and a novice group composed by 30 participants without previous experience in RT (n = 30). Both groups performed a RT program comprised of eight exercises targeting all the major muscles. Training was carried out 3 days/week for 8 weeks. Serum triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), GLU, and CRP concentrations were determined pre- and post- intervention after 12 h fasting. A significant group by time interaction (P < 0.05) for the TC (novice = -1.9% vs. advanced = 1.0%), and CRP (novice = -22.9% vs. advanced = -54.5%) was observed. A main effect of time (P < 0.05) was identified for the GLU (novice = -2.6% vs. advanced = -6.6%), TG (novice = -12.9% vs. advanced = -5.7%), HDL-C (novice = +6.7% vs. advanced = +2.6%), and LDL-C (novice = -34.0% vs. advanced = -25.4%). These results suggest that RT improves the metabolic profile of older women and that training for a longer period of time seems to produce more pronounced reductions mainly on CRP.
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Hwang, Seoyeon; Kang, Byunghak; Hong, Jiyoung; Kim, Ahyoun; Kim, Hyejin; Kim, Kisang; Cheon, Doo-Sung
2013-07-01
Human enterovirus (EV) 71 is the main etiological agent of hand, foot, and mouth disease (HFMD). It is associated with neurological complications, and caused fatalities during recent outbreaks in the Asia-Pacific region. Infections caused by EV71 could lead to many complications, ranging from brainstem encephalitis to pulmonary oedema, resulting in high mortality. In this study, a duplex real-time RT-PCR assay was developed in order to simultaneously detect pan-EV and EV71. EV71-specific primers and probes were designed based on the highly conserved VP1 region of EV71. Five EV71 strains were detected as positive, and no positive fluorescence signal was observed in the duplex real-time RT-PCR for other viral RNA, which showed 100% specificity for the selected panel, and no cross-reactions were observed in this duplex real-time RT-PCR. The EV71-specific duplex real-time RT-PCR was more sensitive than conventional RT-PCR, and detected viral titers that were 10-fold lower than those measured by the latter. Of the 381 HFMD clinical specimens, 196 (51.4%) cases were pan-EV-positive, of which 170 (86.7%) were EV71-positive when tested by pan-EV and EV71-specific duplex real-time RT-PCR. EV71-specific duplex real-time RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks. Copyright © 2013 Wiley Periodicals, Inc.
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Jothikumar, Narayanan; Cromeans, Theresa L; Robertson, Betty H; Meng, X J; Hill, Vincent R
2006-01-01
Hepatitis E virus (HEV) is transmitted by the fecal-oral route and causes sporadic and epidemic forms of acute hepatitis. Large waterborne HEV epidemics have been documented exclusively in developing countries. At least four major genotypes of HEV have been reported worldwide: genotype 1 (found primarily in Asian countries), genotype 2 (isolated from a single outbreak in Mexico), genotype 3 (identified in swine and humans in the United States and many other countries), and genotype 4 (identified in humans, swine and other animals in Asia). To better detect and quantitate different HEV strains that may be present in clinical and environmental samples, we developed a rapid and sensitive real-time RT-PCR assay for the detection of HEV RNA. Primers and probes for the real-time RT-PCR were selected based on the multiple sequence alignments of 27 sequences of the ORF3 region. Thirteen HEV isolates representing genotypes 1-4 were used to standardize the real-time RT-PCR assay. The TaqMan assay detected as few as four genome equivalent (GE) copies of HEV plasmid DNA and detected as low as 0.12 50% pig infectious dose (PID50) of swine HEV. Different concentrations of swine HEV (120-1.2PID50) spiked into a surface water concentrate were detected in the real-time RT-PCR assay. This is the first reporting of a broadly reactive TaqMan RT-PCR assay for the detection of HEV in clinical and environmental samples.
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Omosa-Manyonyi, Gloria; Mpendo, Juliet; Ruzagira, Eugene; Kilembe, William; Chomba, Elwyn; Roman, François; Bourguignon, Patricia; Koutsoukos, Marguerite; Collard, Alix; Voss, Gerald; Laufer, Dagna; Stevens, Gwynn; Hayes, Peter; Clark, Lorna; Cormier, Emmanuel; Dally, Len; Barin, Burc; Ackland, Jim; Syvertsen, Kristen; Zachariah, Devika; Anas, Kamaal; Sayeed, Eddy; Lombardo, Angela; Gilmour, Jill; Cox, Josephine; Fast, Patricia; Priddy, Frances
2015-01-01
Background Sequential prime-boost or co-administration of HIV vaccine candidates based on an adjuvanted clade B p24, RT, Nef, p17 fusion protein (F4/AS01) plus a non-replicating adenovirus 35 expressing clade A Gag, RT, Int and Nef (Ad35-GRIN) may lead to a unique immune profile, inducing both strong T-cell and antibody responses. Methods In a phase 1, double-blind, placebo-controlled trial, 146 healthy adult volunteers were randomized to one of four regimens: heterologous prime-boost with two doses of F4/AS01E or F4/AS01B followed by Ad35-GRIN; Ad35-GRIN followed by two doses of F4/AS01B; or three co-administrations of Ad35-GRIN and F4/AS01B. T cell and antibody responses were measured. Results The vaccines were generally well-tolerated, and did not cause serious adverse events. The response rate, by IFN-γ ELISPOT, was greater when Ad35-GRIN was the priming vaccine and in the co-administration groups. F4/AS01 induced CD4+ T-cells expressing primarily CD40L and IL2 +/- TNF-α, while Ad35-GRIN induced predominantly CD8+ T-cells expressing IFN-γ +/- IL2 or TNF-α. Viral inhibition was induced after Ad35-GRIN vaccination, regardless of the regimen. Strong F4-specific antibody responses were induced. Immune responses persisted at least a year after the last vaccination. The complementary response profiles, characteristic of each vaccine, were both expressed after co-administration. Conclusion Co-administration of an adjuvanted protein and an adenovirus vector showed an acceptable safety and reactogenicity profile and resulted in strong, multifunctional and complementary HIV-specific immune responses. Trial Registration ClinicalTrials.gov NCT01264445 PMID:25961283
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Ceol, M; Forino, M; Gambaro, G; Sauer, U; Schleicher, E D; D'Angelo, A; Anglani, F
2001-01-01
Gene expression can be examined with different techniques including ribonuclease protection assay (RPA), in situ hybridisation (ISH), and quantitative reverse transcription-polymerase chain reaction (RT/PCR). These methods differ considerably in their sensitivity and precision in detecting and quantifying low abundance mRNA. Although there is evidence that RT/PCR can be performed in a quantitative manner, the quantitative capacity of this method is generally underestimated. To demonstrate that the comparative kinetic RT/PCR strategy-which uses a housekeeping gene as internal standard-is a quantitative method to detect significant differences in mRNA levels between different samples, the inhibitory effect of heparin on phorbol 12-myristate 13-acetate (PMA)-induced-TGF-beta1 mRNA expression was evaluated by RT/PCR and RPA, the standard method of mRNA quantification, and the results were compared. The reproducibility of RT/PCR amplification was calculated by comparing the quantity of G3PDH and TGF-beta1 PCR products, generated during the exponential phases, estimated from two different RT/PCR (G3PDH, r = 0.968, P = 0.0000; TGF-beta1, r = 0.966, P = 0.0000). The quantitative capacity of comparative kinetic RT/PCR was demonstrated by comparing the results obtained from RPA and RT/PCR using linear regression analysis. Starting from the same RNA extraction, but using only 1% of the RNA for the RT/PCR compared to RPA, significant correlation was observed (r = 0.984, P = 0.0004). Moreover the morphometric analysis of ISH signal was applied for the semi-quantitative evaluation of the expression and localisation of TGF-beta1 mRNA in the entire cell population. Our results demonstrate the close similarity of the RT/PCR and RPA methods in giving quantitative information on mRNA expression and indicate the possibility to adopt the comparative kinetic RT/PCR as reliable quantitative method of mRNA analysis. Copyright 2001 Wiley-Liss, Inc.
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Mekuria, Genet; Ramesh, Sunita A; Alberts, Evita; Bertozzi, Terry; Wirthensohn, Michelle; Collins, Graham; Sedgley, Margaret
2003-12-01
A technique based on the reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to detect the presence of Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) simultaneously in almond. This paper presents the results of a 3-year study comparing both enzyme-linked immunosorbent assay (ELISA) and RT-PCR for the detection of PNRSV and PDV using 175 almond leaf samples. Multiplex RT-PCR was found to be more sensitive than ELISA, especially when followed by nested PCR for the detection of PDV. The RT-PCR technique has the added advantage that plant material can be tested at any time throughout the growing season.
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NASA Astrophysics Data System (ADS)
Zhan, Fangfang; Zhou, Xiaoming
2012-12-01
Rotaviruses are double-stranded RNA viruses belonging to the family of enteric pathogens. It is a major cause of diarrhoeal disease in infants and young children worldwide. Consequently, rapid and accurate detection of rotaviruses is of great importance in controlling and preventing food- and waterborne diseases and outbreaks. Reverse transcription-polymerase chain reaction (RT-PCR) is a reliable method that possesses high specificity and sensitivity. It has been widely used to detection of viruses. Electrochemiluminescence (ECL) can be considered as an important and powerful tool in analytical and clinical application with high sensitivity, excellent specificity, and low cost. Here we have developed a method for the detection of rotavirus by combining in situ magnetic beads (MBs) based RT-PCR with ECL. RT of rotavirus RNA was carried out in a traditional way and the resulting cDNA was directly amplified on MBs. Forward primers were covalently bounded to MBs and reverse primers were labeled with tris-(2, 2'-bipyridyl) ruthenium (TBR). During the PCR cycling, the TBR labeled products were directly loaded and enriched on the surface of MBs. Then the MBs-TBR complexes could be analyzed by a magnetic ECL platform without any post-modification or post-incubation which avoid some laborious manual operations and achieve rapid yet sensitive detection. In this study, rotavirus from fecal specimens was successfully detected within 2 h, and the limit of detection was estimated to be 104copies/μL. This novel in situ MBs based RT-PCR with ECL detection method can be used for pathogen detection in food safety field and clinical diagnosis.
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VLA radio upper limit on Type IIn Supernova 2007rt
NASA Astrophysics Data System (ADS)
Chandra, Poonam; Soderberg, Alicia
2008-01-01
Poonam Chandra and Alicia Soderberg report on behalf of a larger collaboration: We observed a Type IIn supernova SN 2007rt (CBET 1148) with the Very Large Array (VLA) in the 8.46 GHz band on 2008, January 12.55 UT. The observations were taken for total duration of one hour in the VLA B-configuration. We do not detect any radio emission at the supernova position (CBET 1148). The flux density at the supernova position is 9 ± 27 uJy.
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Schliep, M.; Pernice, M.; Sinutok, S.; Bryant, C. V.; York, P. H.; Rasheed, M. A.; Ralph, P. J.
2015-01-01
Seagrass meadows are threatened by coastal development and global change. In the face of these pressures, molecular techniques such as reverse transcription quantitative real-time PCR (RT-qPCR) have great potential to improve management of these ecosystems by allowing early detection of chronic stress. In RT-qPCR, the expression levels of target genes are estimated on the basis of reference genes, in order to control for RNA variations. Although determination of suitable reference genes is critical for RT-qPCR studies, reports on the evaluation of reference genes are still absent for the major Australian species Zostera muelleri subsp. capricorni (Z. muelleri). Here, we used three different software (geNorm, NormFinder and Bestkeeper) to evaluate ten widely used reference genes according to their expression stability in Z. muelleri exposed to light limitation. We then combined results from different software and used a consensus rank of four best reference genes to validate regulation in Photosystem I reaction center subunit IV B and Heat Stress Transcription factor A- gene expression in Z. muelleri under light limitation. This study provides the first comprehensive list of reference genes in Z. muelleri and demonstrates RT-qPCR as an effective tool to identify early responses to light limitation in seagrass. PMID:26592440
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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.
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NASA Astrophysics Data System (ADS)
Afzalian, Aryan; Colinge, Jean-Pierre; Flandre, Denis
2011-05-01
A new concept of nanoscale MOSFET, the Gate Modulated Resonant Tunneling Transistor (RT-FET), is presented and modeled using 3D Non-Equilibrium Green's Function simulations enlightening the main physical mechanisms. Owing to the additional tunnel barriers and the related longitudinal confinement present in the device, the density of state is reduced in its off-state, while remaining comparable in its on-state, to that of a MOS transistor without barriers. The RT-FET thus features both a lower RT-limited off-current and a faster increase of the current with V G, i.e. an improved slope characteristic, and hence an improved Ion/ Ioff ratio. Such improvement of the slope can happen in subthreshold regime, and therefore lead to subthreshold slope below the kT/q limit. In addition, faster increase of current and improved slope occur above threshold and lead to high thermionic on-current and significant Ion/ Ioff ratio improvement, even with threshold voltage below 0.2 V and supply voltage V dd of a few hundreds of mV as critically needed for future technology nodes. Finally RT-FETs are intrinsically immune to source-drain tunneling and are therefore promising candidate for extending the roadmap below 10 nm.
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Purcell, Maureen K.; Hart, S. Alexandra; Kurath, Gael; Winton, James R.
2006-01-01
The fish rhabdovirus, Infectious hematopoietic necrosis virus (IHNV), is an important pathogen of salmonids. Cell culture assays have traditionally been used to quantify levels of IHNV in samples; however, real-time or quantitative RT-PCR assays have been proposed as a rapid alternative. For viruses having a single-stranded, negative-sense RNA genome, standard qRT-PCR assays do not distinguish between the negative-sense genome and positive-sense RNA species including mRNA and anti-genome. Thus, these methods do not determine viral genome copy number. This study reports development of strand-specific, qRT-PCR assays that use tagged primers for enhancing strand specificity during cDNA synthesis and quantitative PCR. Protocols were developed for positive-strand specific (pss-qRT-PCR) and negative-strand specific (nss-qRT-PCR) assays for IHNV glycoprotein (G) gene sequences. Validation with synthetic RNA transcripts demonstrated the assays could discriminate the correct strand with greater than 1000-fold fidelity. The number of genome copies in livers of IHNV-infected fish determined by nss-qRT-PCR was, on average, 8000-fold greater than the number of infectious units as determined by plaque assay. We also compared the number of genome copies with the quantity of positive-sense RNA and determined that the ratio of positive-sense molecules to negative-sense genome copies was, on average, 2.7:1. Potential future applications of these IHNV strand-specific qRT-PCR assays are discussed.
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Bisogno, G; Ferrari, A; Bien, E; Brecht, I B; Brennan, B; Cecchetto, G; Godzinski, J; Orbach, D; Reguerre, Y; Stachowicz-Stencel, T; Schneider, D T
2012-10-01
The low incidence and the heterogeneity of very rare tumors (VRTs) demand for international cooperation. In 2008, EXPeRT (European Cooperative Study Group for Pediatric Rare Tumors) was founded by national groups from Italy, France, United Kingdom, Poland and Germany. The first aims of EXPeRT were to agree on a uniform definition of VRTs and to develop the currently most relevant scientific questions. Current initiatives include international data exchange, retrospective and prospective studies of specific entities, and the development of harmonized and internationally recognized guidelines. Moreover, EXPeRT established a network for expert consultation to assist in clinical decision in VRTs. © Georg Thieme Verlag KG Stuttgart · New York.
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Thanarajoo, Sathis Sri; Kong, Lih Ling; Kadir, Jugah; Lau, Wei Hongi; Vadamalai, Ganesan
2014-06-01
A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60 min at 60 °C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd. Copyright © 2014 Elsevier B.V. All rights reserved.
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Lubelchek, Ronald J; Max, Blake; Sandusky, Caroline J; Hota, Bala; Barker, David E
2009-06-23
To explore whether an assay change was responsible for an increasing proportion of patients with undetectable HIV viral loads at our urban HIV clinic, we selected highly stable patients, examining their viral loads before and after changing assays. We compared the proportion with detectable viremia during RT-PCR vs. bDNA periods. We selected patients with > or =1 viral loads assessed during both RT-PCR and bDNA periods. We included patients with stable CD4 counts, excluding patients with viral loads > or =1,000 copies/ml or any significant changes in therapy. Out of 4500 clinic patients, 419 patients (1588 viral loads) were included. 39% of viral loads were reported as detectable by RT-PCR vs. 5% reported as detectable by bDNA. The mean coefficient of variation was higher before vs. after assay change. We found an odds' ratio of 16.7 for having a viral load >75 copies/ml during the RT-PCR vs. bDNA periods. These data support previous reports, suggesting that bDNA may more reliably discriminate between viral suppression and low level viremia in stable patients on therapy. Low-level viremia, noted more with RT-PCR, may promote unneeded testing, while differences in viral load reliability may impact antiretroviral trial and quality assurance endpoints. Commonly used plasma separator tubes may differentially affect RT-PCR and bDNA results.
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Shirato, Kazuya; Semba, Shohei; El-Kafrawy, Sherif A; Hassan, Ahmed M; Tolah, Ahmed M; Takayama, Ikuyo; Kageyama, Tsutomu; Notomi, Tsugunori; Kamitani, Wataru; Matsuyama, Shutoku; Azhar, Esam Ibraheem
2018-05-12
Clinical detection of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in patients is achieved using genetic diagnostic methods, such as real-time RT-PCR assay. Previously, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of MERS-CoV [Virol J. 2014. 11:139]. Generally, amplification of RT-LAMP is monitored by the turbidity induced by precipitation of magnesium pyrophosphate with newly synthesized DNA. However, this mechanism cannot completely exclude the possibility of unexpected reactions. Therefore, in this study, fluorescent RT-LAMP assays using quenching probes (QProbes) were developed specifically to monitor only primer-derived signals. Two primer sets (targeting nucleocapsid and ORF1a sequences) were constructed to confirm MERS cases by RT-LAMP assay only. Our data indicate that both primer sets were capable of detecting MERS-CoV RNA to the same level as existing genetic diagnostic methods, and that both were highly specific with no cross-reactivity observed with other respiratory viruses. These primer sets were highly efficient in amplifying target sequences derived from different MERS-CoV strains, including camel MERS-CoV. In addition, the detection efficacy of QProbe RT-LAMP was comparable to that of real-time RT-PCR assay using clinical specimens from patients in Saudi Arabia. Altogether, these results indicate that QProbe RT-LAMP assays described here can be used as powerful diagnostic tools for rapid detection and surveillance of MERS-CoV infections. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.
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Geologic Controls of Sand Boil Formation at Buck Chute, Mississippi
2017-06-30
12 Figure 6. 1915 MRC hydrographic survey of Buck Chute (Modified from MRC 1975, sheet 48). ............ 13 Figure 7. 1926 MRC...hydrographic survey of Buck Chute (Modified from MRC 1975, sheet 48). ............ 14 Figure 8. Diagram of the evolution of the Mississippi River...library). .................... 17 Figure 11. AGI SuperSting 8 ERT survey equipment
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Ding, Tian; Suo, Yuanjie; Zhang, Zhaohuan; Liu, Donghong; Ye, Xingqian; Chen, Shiguo; Zhao, Yong
2017-01-01
This study firstly developed a multiplex real-time PCR (RT-PCR) technique combined with a pre-enrichment step to simultaneously detect Staphylococcus aureus ( S. aureus ), Listeria monocytogenes ( L. monocytogenes ) and Salmonella spp. in raw milk and the dairy farm environment (feces, soil, feed, water) in one reaction. Brain heart infusion (BHI) broth was selected for the enrichment step to increase the density of the target bacteria by using an incubation of 4 h before multiplex RT-PCR. The results showed that the detection limit of the multiplex real-time assay was approximately 10 2 CFU/mL for pure cultures and artificially contaminated milk without enrichment, while 12, 14, and 10 CFU/25 mL, respectively, for S. aureus, L. monocytogenes , and Salmonella spp. after pre-enrichment. The newly developed multiplex RT-PCR assay was applied to 46 dairy farm environmental samples and raw milk samples covering a wide variety of sample types. The results demonstrated that the multiplex RT-PCR assay coupled with the BHI enrichment broth was suitable for the simultaneous screening of S. aureus, L. monocytogenes , and Salmonella spp. in the pasture environment and in raw milk. The multiplex RT-PCR assay clearly and successfully shortened the total detection time and reduced labor compared to conventional culture-based methods for testing natural samples.
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Orrú, Christina D; Groveman, Bradley R; Raymond, Lynne D; Hughson, Andrew G; Nonno, Romolo; Zou, Wenquan; Ghetti, Bernardino; Gambetti, Pierluigi; Caughey, Byron
2015-06-01
Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested.
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Ding, Tian; Suo, Yuanjie; Zhang, Zhaohuan; Liu, Donghong; Ye, Xingqian; Chen, Shiguo; Zhao, Yong
2017-01-01
This study firstly developed a multiplex real-time PCR (RT-PCR) technique combined with a pre-enrichment step to simultaneously detect Staphylococcus aureus (S. aureus), Listeria monocytogenes (L. monocytogenes) and Salmonella spp. in raw milk and the dairy farm environment (feces, soil, feed, water) in one reaction. Brain heart infusion (BHI) broth was selected for the enrichment step to increase the density of the target bacteria by using an incubation of 4 h before multiplex RT-PCR. The results showed that the detection limit of the multiplex real-time assay was approximately 102 CFU/mL for pure cultures and artificially contaminated milk without enrichment, while 12, 14, and 10 CFU/25 mL, respectively, for S. aureus, L. monocytogenes, and Salmonella spp. after pre-enrichment. The newly developed multiplex RT-PCR assay was applied to 46 dairy farm environmental samples and raw milk samples covering a wide variety of sample types. The results demonstrated that the multiplex RT-PCR assay coupled with the BHI enrichment broth was suitable for the simultaneous screening of S. aureus, L. monocytogenes, and Salmonella spp. in the pasture environment and in raw milk. The multiplex RT-PCR assay clearly and successfully shortened the total detection time and reduced labor compared to conventional culture-based methods for testing natural samples. PMID:28620364
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Williams-Woods, Jacquelina; González-Escalona, Narjol; Burkhardt, William
2011-12-01
Human norovirus (HuNoV) and hepatitis A (HAV) are recognized as leading causes of non-bacterial foodborne associated illnesses in the United States. DNA sequencing is generally considered the standard for accurate viral genotyping in support of epidemiological investigations. Due to the genetic diversity of noroviruses (NoV), degenerate primer sets are often used in conventional reverse transcription (RT) PCR and real-time RT-quantitative PCR (RT-qPCR) for the detection of these viruses and cDNA fragments are generally cloned prior to sequencing. HAV detection methods that are sensitive and specific for real-time RT-qPCR yields small fragments sizes of 89-150bp, which can be difficult to sequence. In order to overcome these obstacles, norovirus and HAV primers were tailed with M13 forward and reverse primers. This modification increases the sequenced product size and allows for direct sequencing of the amplicons utilizing complementary M13 primers. HuNoV and HAV cDNA products from environmentally contaminated oysters were analyzed using this method. Alignments of the sequenced samples revealed ≥95% nucleotide identities. Tailing NoV and HAV primers with M13 sequence increases the cDNA product size, offers an alternative to cloning, and allows for rapid, accurate and direct sequencing of cDNA products produced by conventional or real time RT-qPCR assays. Published by Elsevier B.V.
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Liu, Yun-Xi; Zhao, Zhong-Tang; Cao, Wu-Chun; Xu, Xiao-Qun; Suo, Ji-Jiang; Xing, Yu-Bin; Jia, Ning; Du, Ming-Mei; Liu, Bo-Wei; Yao, Yuan
2013-01-01
The aim of the present study was to evaluate the clinical usefulness of applying RT-nested PCR along with RFLP as a method for diagnosis and genotypic differentiation of Hantavirus in the acute-stage sera of HFRS patients as compared to the ELISA technique. A prospective study of patients with suspected HFRS patients was carried out. Sera were collected for serological evaluation by ELISA and RT-nested PCR testing. Primers were selected from the published sequence of the S segment of HTNV strain 76-118 and SEOV strain SR-11, which made it possible to obtain an amplicon of 403 bp by RT-nested PCR. The genotypic differentiations of the RT-nested PCR amplicons were carried out by RFLP. Sequence analyses of the amplicons were used to confirm the accuracy of the results obtained by RFLP. Of the 48 acute-stage sera from suspected HFRS patients, 35 were ELISA-positive while 41 were positive by RT-nested PCR. With Hind III and Hinf I, RFLP profiles of the RT-nested PCR amplicons of the 41 positive sera exhibited two patterns. 33 had RFLP profiles similar to the reference strain R22, and thus belonged to the SEOV type. The other 8 samples which were collected during October-December had RFLP profiles similar to the reference strain 76-118, and thus belonged to the HTNV type. Sequence phylogenetic analysis of RT-nested PCR amplicons revealed sdp1, sdp2 YXL-2008, and sdp3 as close relatives of HTNV strain 76-118, while sdp22 and sdp37 as close relatives of SEOV strain Z37 and strain R22 located in two separate clusters in the phylogenetic tree. These results were identical to those acquired by RFLP. RT-nested PCR integrated with RFLP was a rapid, simple, accurate method for detecting and differentiating the genotypes of Hantavirus in the acute-stage sera of suspected HFRS patients. In Shandong province, the main genotypes of Hantavirus belonged to the SEOV types, while the HTNV types were observed during the autumn-winter season.
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McMillan, Mary; Pereg, Lily
2014-01-01
Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data. PMID:24841066
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McMillan, Mary; Pereg, Lily
2014-01-01
Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data.
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Ma, Yue-Jiao; Sun, Xiao-Hong; Xu, Xiao-Yan; Zhao, Yong; Pan, Ying-Jie; Hwang, Cheng-An; Wu, Vivian C H
2015-01-01
Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference gene for qRT-PCR. This study evaluated the stability of 6 reference genes (16S rRNA, recA, rpoS, pvsA, pvuA, and gapdh) in 5 V. parahaemolyticus strains (O3:K6-clinical strain-tdh+, ATCC33846-tdh+, ATCC33847-tdh+, ATCC17802-trh+, and F13-environmental strain-tdh+) cultured at 4 different temperatures (15, 25, 37 and 42°C). Stability values were calculated using GeNorm, NormFinder, BestKeeper, and Delta CT algorithms. The results indicated that recA was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that recA can be used as a reference gene for gene expression studies in V. parahaemolyticus.
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Zhang, Jianqiang; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chen, Qi; Zhang, Yan; Chiang, Cheng-Jen; Shen, Yu-Han; Li, Fu-Chun; Chang, Hsiao-Fen Grace; Gauger, Phillip C; Harmon, Karen M; Wang, Hwa-Tang Thomas
2016-08-01
Recent outbreaks of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) in multiple countries have caused significant economic losses and remain a serious challenge to the swine industry. Rapid diagnosis is critical for the implementation of efficient control strategies before and during PEDV and PDCoV outbreaks. Insulated isothermal PCR (iiPCR) on the portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, a singleplex PEDV RT-iiPCR, a singleplex PDCoV RT-iiPCR, and a duplex PEDV/PDCoV real-time RT-PCR (rRT-PCR) commercial reagents targeting the M gene were compared to an N gene-based PEDV rRT-PCR and an M gene-based PDCoV rRT-PCR that were previously published and used as reference PCRs. All PCR assays were highly specific and did not cross react with other porcine enteric pathogens. Analytical sensitivities of the PEDV RT-iiPCR, PDCoV RT-iiPCR and duplex PEDV/PDCoV rRT-PCR were determined using in vitro transcribed RNA as well as viral RNA extracted from ten-fold serial dilutions of PEDV and PDCoV cell culture isolates. Performance of each PCR assay was further evaluated using 170 clinical samples (86 fecal swabs, 24 feces, 19 intestines, and 41 oral fluids). Compared to the reference PEDV rRT-PCR, the sensitivity, specificity and accuracy of the PEDV RT-iiPCR were 97.73%, 98.78%, and 98.24%, respectively, and those of the duplex PEDV/PDCoV rRT-PCR were 98.86%, 96.34%, and 97.65%, respectively. Compared to the reference PDCoV rRT-PCR, the sensitivity, specificity and accuracy of the PDCoV RT-iiPCR were 100%, 100%, and 100%, respectively, and those of the PEDV/PDCoV duplex rRT-PCR were 96.34%, 100%, and 98.24%, respectively. Overall, all three new PCR assays were comparable to the reference rRT-PCRs for detection of PEDV and/or PDCoV. The PEDV and PDCoV RT-iiPCRs are potentially useful tools for on-site detection and the duplex PEDV/PDCoV rRT-PCR provides a convenient method to simultaneously detect
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Williams, Benjamin R; Strauss, Esther H; Hultsch, David F; Hunter, Michael A; Tannock, Rosemary
2007-04-01
Inconsistency across trials of 2-choice reaction time (RT) data was analyzed in 72 adolescents (age 12-17 years) within 4 groups differentiated by the presence or absence of attention deficit/hyperactivity disorder (ADHD) and reading difficulties (RD). ADHD participants were more inconsistent (across all trials, and in the slow portion of the RT distribution) than controls, but only when RD was present. Within the fast portion of the RT distribution, ADHD participants were more inconsistent than controls regardless of RD. The results highlight the importance of fluctuations in cognitive performance in ADHD and suggest that there may be independent sources of variation in inconsistency affecting the fast and slow portions of the RT distribution.
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Sengar, Gyanendra Singh; Deb, Rajib; Raja, T V; Singh, Umesh; Kant, Rajiv; Bhanuprakash, V; Alyethodi, R R; Kumar, Sushil; Verma, Preetam; Chakraborty, Soumendu; Alex, Rani; Singh, Rani
2017-07-01
The purpose of this study is to develop a novel Reverse Transcriptase Loop-mediated isothermal amplification (RT-LAMP) based assay for in vitro profiling of heat shock protein 70 (Hsp70) in bovine peripheral blood mononuclear cell (PBMC) culture model utilizing the absorbance level of magnesium pyrophosphate-a by-product of LAMP reaction. A set of bovine Hsp70 specific RT-LAMP primers were designed to detect the differential absorbance level of magnesium pyrophosphate by-product which signifies the degree of Hsp70 amplification from cDNA of thermally induced cultured cells at different recovery periods. The study revealed significant (P < 0.05) correlation between absorbance level and the fold change of Hsp70 transcripts at different kinetic intervals of heat stress recovery in bovine PBMC cell culture models. RT-LAMP based absorbance assay can be used as an indicator to measure the degree of bovine Hsp70 transcripts produced during thermal stress and can be used as an alternative to the traditional Real time PCR assay. Developed RT-LAMP assay can be used as a cost-effective method for profiling of bovine HSP70 gene.
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A Comprehensive Approach to RtI: Embedding Universal Design for Learning and Technology
ERIC Educational Resources Information Center
Basham, James D.; Israel, Maya; Graden, Janet; Poth, Rita; Winston, Markay
2010-01-01
Response to intervention (RtI) provides tiered levels of supports to all students and allows for increasingly more intensive and individualized instruction. Similarly, universal design for learning (UDL) addresses needs of students by proactively planning for instructional, environmental, and technology supports to allow all students to…
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Extended Maintenance Downtime 01/19 - 01/26
Atmospheric Science Data Center
2018-01-24
Date(s): Friday, January 19, 2018 to Friday, January 26, 2018 Time: 01/19 @ 7 am - 01/26 @ 5 pm EDT Event Impact: The ASDC would like to perform a comprehensive and required maintenance from Friday...
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Vandenbussche, Frank; Lefebvre, David J; De Leeuw, Ilse; Van Borm, Steven; De Clercq, Kris
2017-08-01
The 3D and 5UTR real-time RT-PCR assays (RT-qPCR) from Callahan et al. (2002) and Reid et al. (2002) are commonly used reference methods for the detection of foot-and-mouth disease virus (FMDV). For an optimal detection of FMDV in clinical samples, it is advised to use both assays simultaneously (King et al., 2006). Recently, Vandenbussche et al. (2016) showed that the addition of 5'-tails to the FMDV-specific primers enhances the detection of FMDV in both the 3D and the 5UTR RT-qPCR assay. To validate the 3D and 5UTR RT-qPCR assays with 5'-tailed primers for diagnostic purposes, both assays were run in parallel in a triplex one-step RT-qPCR protocol with beta-actin as an internal control and synthetic RNA as an external control. We obtained low limits of detection and high linearity's, high repeatability and reproducibility, near 100% analytical specificity and >99% diagnostic accuracy for both assays. It was concluded that the 3D and 5UTR RT-qPCR assays with 5'-tailed primers are particularly suited for the detection of FMDV as well as to exclude the presence of FMDV. Copyright © 2017 Elsevier B.V. All rights reserved.
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Osman, Fatima; Dang, Tyler; Bodaghi, Sohrab; Vidalakis, Georgios
2017-07-01
A one-step multiplex reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) based on species-specific minor groove binding (MGB) probes, was developed for the simultaneous detection, identification, and quantification of three citrus viroids belonging to different genera. Citrus exocortis viroid (Pospiviroid), Hop stunt viroid (Hostuviroid), and Citrus bark cracking viroid (Cocadviroid) cause a variety of maladies in agriculturally significant crops. Therefore, reliable assays for their detection are essential tools for various government and industry organizations implementing disease management programs. Singleplex qPCR primers and MGB probes were designed individually for the detection of the three targeted viroids, and subsequently combined in a one-step multiplex RT-qPCR reaction. A wide host range of woody plants, including citrus, grapevines, apricots, plums and herbaceous plants such as tomato, cucumber, eggplant and chrysanthemum different world regions were used to validate the assay. Single, double and triple viroid infections were identified in the tested samples. The developed multiplex RT-qPCR assay was compared with a previously reported SYBR Green I RT-qPCR for the universal detection of citrus viroids. Both assays accurately identified all citrus viroid infected samples. The multiplex assay complemented the SYBR Green I universal detection assay by differentiating among citrus viroid species in the positive samples. The developed multiplex RT-qPCR assay has the potential to simultaneously detect each targeted viroid and could be used in high throughput screenings for citrus viroids in field surveys, germplasm banks, nurseries and other viroid disease management programs. Copyright © 2017. Published by Elsevier B.V.
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Jääskeläinen, Anne J; Kallio-Kokko, Hannimari; Ozkul, Aykut; Bodur, Hurrem; Korukruoglu, Gulay; Mousavi, Mehrdad; Pranav, Patel; Vaheri, Antti; Mirazimi, Ali; Vapalahti, Olli
2014-12-01
Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic disease caused by a nairovirus belonging to family Bunyaviridae. The CCHF virus (CCHFV) can be transmitted to humans by Hyalomma ticks as well as by direct contact with infected body fluids or tissues from viremic livestock or humans. Our aim was to set up a fast RT-qPCR for detection of the different CCHFV genotypes in clinical samples, including an inactivation step to make the sample handling possible in lower biosafety levels (BSL) than BSL-4. This method was evaluated against commercial reference assays and international External Quality Assessment (EQA) samples. The analytical limit of detection for the developed CCHFV-S RT-qPCR was 11 CCHFV genomes per reaction. After exclusion of four dubious samples, we studied 38 CCHFV-positive samples (using reference tests) of which 38 were found positive by CCHFV-S RT-qPCR, suggesting a sensitivity of 100%. CCHFV-S RT q-PCR detected all eight different CCHFV strains representing five different CCHFV genotypes. In conclusion, the CCHFV-S RT-qPCR described in this study was evaluated using various sources of CCHFV samples and shown to be an accurate tool to detect human CCHFV infection caused by different genotypes of the virus.
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Coelho-Lima, Jose; Mohammed, Ashfaq; Cormack, Suzanne; Jones, Samuel; Das, Rajiv; Egred, Mohaned; Panahi, Pedram; Ali, Simi; Spyridopoulos, Ioakim
2018-06-11
Cardiac-enriched micro ribonucleic acids (miRNAs) are released into the circulation following ST-elevation myocardial infarction (STEMI). Lack of standardized approaches for reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) data normalization and presence of RT-qPCR inhibitors (e.g. heparin) in patient blood samples have prevented reproducible miRNA quantification in this cohort and subsequent translation of these biomarkers to clinical practice. Using a RT-qPCR miRNA screening platform, we identified and validated an endogenous circulating miRNA as a normalization control. In addition, we assessed the effects of in vivo and in vitro anticoagulant drugs administration (heparin and bivalirudin) on three RT-qPCR normalization strategies (global miRNA mean, exogenous spike-in control [cel-miR-39] and endogenous miRNA control). Finally, we evaluated the effect of heparin and its in vitro inhibition with heparinase on the quantification of cardiac-enriched miRNAs in STEMI patients. miR-425-5p was validated as an endogenous miRNA control. Heparin administration in vitro and in vivo inhibited all RT-qPCR normalization strategies. In contrast, bivalirudin had no effects on cel-miR-39 or miR-425-5p quantification. In vitro RNA sample treatment with 0.3 U of heparinase overcame heparin-induced over-estimation of cardiac-enriched miRNA levels and improved their correlation with high-sensitivity troponin T. miRNA quantification in STEMI patients receiving heparin is jeopardized by its effect on all RT-qPCR normalization approaches. Use of samples from bivalirudin-treated patients or in vitro treatment of heparin-contaminated samples with heparinase are suitable alternatives for miRNA quantification in this cohort. Finally, we reinforce the evidence that cardiac-enriched miRNAs early after myocardial reperfusion reflect the severity of cardiac injury. Schattauer GmbH Stuttgart.
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Vongsouvath, Manivanh; Phommasone, Koukeo; Sengvilaipaseuth, Onanong; Kosoltanapiwat, Nathamon; Chantratita, Narisara; Blacksell, Stuart D.; Lee, Sue J.; de Lamballerie, Xavier; Mayxay, Mayfong; Keomany, Sommay; Newton, Paul N.; Dubot-Pérès, Audrey
2016-01-01
Background Dengue virus infection causes major public health problems in tropical and subtropical areas. In many endemic areas, including the Lao PDR, inadequate access to laboratory facilities is a major obstacle to surveillance and study of dengue epidemiology. Filter paper is widely used for blood collection for subsequent laboratory testing for antibody and nucleic acid detection. For the first time, we demonstrate that dengue viral RNA can be extracted from dengue rapid diagnostic tests (RDT) and then submitted to real-time RT-PCR for serotyping. Methodology/Principal Findings We evaluated the Standard Diagnostics (SD) Bioline Dengue Duo RDT, a commonly used test in dengue endemic areas. First, using the QIAamp RNA kit, dengue RNA was purified from the sample pad of the NS1 RDT loaded with virus isolates of the four serotypes, then quantified by RT-PCR. We observed greater recovery of virus, with a mean of 27 times more RNA recovered from RDT, than from filter paper. Second, we evaluated dengue NS1 RDTs from patients at Mahosot Hospital, Vientiane, (99 patients) and from rural Salavan Provincial Hospital (362 patients). There was good agreement between dengue RT-PCR from NS1 RDT with RT-PCR performed on RNA extracted from patient sera, either using RDT loaded with blood (82.8% and 91.4%, in Vientiane and Salavan, respectively) or serum (91.9% and 93.9%). There was 100% concordance between RDT and serum RT-PCR of infecting dengue serotype. Conclusions/Significance Therefore, the collection of NS1 positive RDTs, which do not require cold storage, may be a novel approach for dengue serotyping by RT-PCR and offers promising prospects for the collection of epidemiological data from previously inaccessible tropical areas to aid surveillance and public health interventions. PMID:27159058
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Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas)
Chen, I-Hua; Wang, Jiann-Hsiung; Chou, Shih-Jen; Wu, Yeong-Huey; Li, Tsung-Hsien; Leu, Ming-Yih; Chang, Wen-Been
2016-01-01
Reverse transcription quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct results. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from four beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have the potential to serve as an important approach to evaluating cetacean health influenced by environmental insults. PMID:26998411
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Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas).
Chen, I-Hua; Wang, Jiann-Hsiung; Chou, Shih-Jen; Wu, Yeong-Huey; Li, Tsung-Hsien; Leu, Ming-Yih; Chang, Wen-Been; Yang, Wei Cheng
2016-01-01
Reverse transcription quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct results. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from four beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have the potential to serve as an important approach to evaluating cetacean health influenced by environmental insults.
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Cetraz, Maria; Sen, Vasily; Schoch, Sarah; Streule, Karolin; Golubev, Valery; Hartwig, Andrea; Köberle, Beate
2017-02-01
The therapeutic efficacy of the anticancer drug cisplatin is limited by the development of resistance. We therefore investigated newly synthesized platinum-nitroxyl complexes (PNCs) for their potential to circumvent cisplatin resistance. The complexes used were PNCs with bivalent cis-Pt II (R · NH 2 )(NH 3 )Cl 2 and cis-Pt II (DAPO)Ox and four-valent platinum cis,trans,cis-Pt IV (R · NH 2 )(NH 3 )(OR) 2 Cl 2 and cis,trans,cis-Pt IV (DAPO)(OR) 2 Ox, where R · are TEMPO or proxyl nitroxyl radicals, DAPO is trans-3,4-diamino-2,2,6,6-tetramethylpiperidine-1-oxyl, and OR and Ox are carboxylato and oxalato ligands, respectively. The complexes were characterized by spectroscopic methods, HPLC, log P ow data and elemental analysis. We studied intracellular platinum accumulation, DNA platination and cytotoxicity upon treatment with the PNCs in a model system of the bladder cancer cell line RT112 and its cisplatin-resistant subline RT112-CP. Platinum accumulation and DNA platination were similar in RT112 and RT112-CP cells for both bivalent and four-valent PNCs, in contrast to cisplatin for which a reduction in intracellular accumulation and DNA platination was observed in the resistant subline. The PNCs were found to platinate DNA in relation to the length of their axial RO-ligands. Furthermore, the PNCs were increasingly toxic in relation to the elongation of their axial RO-ligands, with similar toxicities in RT112 and its cisplatin-resistant subline. Using a cell-free assay, we observed induction of oxidative DNA damage by cisplatin but not PNCs suggesting that cisplatin exerts its toxic action by platination and oxidative DNA damage, while cells treated with PNCs are protected against oxidatively induced lesions. Altogether, our study suggests that PNCs may provide a more effective treatment for tumors which have developed resistance toward cisplatin.
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NASA Astrophysics Data System (ADS)
Altieri, Bruno; Dannerbauer, Helmut
We present Herschel and APEX LABOCA 870 μm imaging of the field of the high-redshift radio galaxy MRC1138 at z = 2.16. We detect 16 submillimeter galaxies in this ˜140 arcmin2 large bolometer map, with flux densities in the range 3-11 mJy. The pure number counts indicate an overdensity of SMGs by a factor of five compared to blank field surveys. Based on an exquisite multi-wavelength database including VLA 1.4 GHz radio and infrared observations, we verifiy whether these sources are members of the proto-cluster structure at z = 2.2 or not. Based on Herschel PACS+ SPIRE and Spitzer MIPS photometry, we derived reliable far-infrared photometric redshifts for all of our sources. VLT-ISAAC near-infrared spectroscopic observations confirmed redshifts of z ≈ 2.2 for four of these SMGs. We conclude that in total at least seven sources are part of this proto-cluster at z = 2.16. We measure a star formation rate density S FRD ˜ 1500 M⊙ yr-1 Mpc-3, four magntiudes higher compared to the global SFRD at this redshift. Striklingly, these seven sources are concentrated within a region of 2 Mpc (the typical size of clusters in the local universe) and are not distributed in the filaments as predicted by theories and traced by the Hα emitters at z ≈ 2.2. This concentration of massive, dusty starbursts is not centered on the radio galaxy which is submm bright. A significant fraction, six out of 11 SMGs with z ≈ 2.2 Hα imaging coverage are associated with Hα emitters, demonstrating the potential of tracing SMG counterparts with this source population. Our results demonstrate that indeed submm observations enable us to reveal clusters of massive, dusty starbursts and will pave the road for systematic and detailed investigations with this technique in the future.
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Crosbie, Emma J; Roberts, Chris; Qian, Wendi; Swart, Ann Marie; Kitchener, Henry C; Renehan, Andrew G
2012-04-01
Body mass index (BMI) is a major risk factor for endometrial cancer incidence but its impact on post-treatment survival is unclear. We investigated the relationships of BMI (categorised using the WHO definitions) with clinico-pathological characteristics and outcome in women treated within the MRC ASTEC randomised trial, which provides data from patients who received standardised allocated treatments and therefore reduces biases. The impact of BMI on both recurrence-free survival (RFS) and overall survival (OS) was analysed using the Cox regression models. An apriori framework of evaluating potential biases was explored. From 1408 participants, there were 1070 women with determinable BMI (median=29.1 kg/m(2)). Histological types were endometrioid (type 1) in 893 and non-endometrioid (type 2) in 146 women; the proportion of the latter decreasing with increasing BMI (8% versus 19% for obese III WHO category versus normal weight, p(trend)=0.003). For type 1 carcinomas, increasing BMI was associated with less aggressive histopathological features (depth of invasion, p=0.006; tumour grade, p=0.015). With a median follow-up of 34.3 months, there was no influence of BMI on RFS - adjusted HRs per 5 kg/m(2) were 0.98 (95% CI 0.86, 1.13) and 0.95 (0.74, 1.24), for type 1 and 2 carcinomas; and no influence on OS - adjusted HRs per 5 kg/m(2) were 0.96 (0.81, 1.14) and 0.92 (0.70, 1.23), respectively. These findings demonstrate an important principle: that an established link between an exposure (here, obesity) and increased incident cancer risk, does not necessarily translate into an inferior outcome following treatment for that cancer. Copyright © 2011 Elsevier Ltd. All rights reserved.
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Lubelchek, Ronald J.; Max, Blake; Sandusky, Caroline J.; Hota, Bala; Barker, David E.
2009-01-01
Introduction To explore whether an assay change was responsible for an increasing proportion of patients with undetectable HIV viral loads at our urban HIV clinic, we selected highly stable patients, examining their viral loads before and after changing assays. We compared the proportion with detectable viremia during RT-PCR vs. bDNA periods. Methodology/Principal Findings We selected patients with ≥1 viral loads assessed during both RT-PCR and bDNA periods. We included patients with stable CD4 counts, excluding patients with viral loads ≥1,000 copies/ml or any significant changes in therapy. Out of 4500 clinic patients, 419 patients (1588 viral loads) were included. 39% of viral loads were reported as detectable by RT-PCR vs. 5% reported as detectable by bDNA. The mean coefficient of variation was higher before vs. after assay change. We found an odds' ratio of 16.7 for having a viral load >75 copies/ml during the RT-PCR vs. bDNA periods. Discussion These data support previous reports, suggesting that bDNA may more reliably discriminate between viral suppression and low level viremia in stable patients on therapy. Low-level viremia, noted more with RT-PCR, may promote unneeded testing, while differences in viral load reliability may impact antiretroviral trial and quality assurance endpoints. Commonly used plasma separator tubes may differentially affect RT-PCR and bDNA results. PMID:19547711
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Daniele, Lorenzo; Annaratone, Laura; Allia, Elena; Mariani, Sara; Armando, Enrico; Bosco, Martino; Macrì, Luigia; Cassoni, Paola; D'Armento, Giuseppe; Bussolati, Gianni; Cserni, Gabor; Sapino, Anna
2009-09-01
The optimal pathological assessment of sentinel nodes (SLNs) in breast cancer is a matter of debate. Currently, multilevel histological evaluation and immunohistochemistry (IHC) are recommended, but alternative RT-PCR procedures have been developed. To assess the reliability of these different procedures, we devised a step-sectioning protocol at 100 micron-intervals of 74 SLNs using methacarn fixation. mRNA was extracted from sections collected from levels 4 to 5. Mammaglobin, CEA and CK19 were used for RT-PCR. mRNA extraction was successful in 69 SLNs. Of these, 7 showed macrometastases (>2mm), 2 showed micrometastases (<2 mm) and 7 showed isolated tumour cells (ITC) by IHC. RT-PCR was positive for the three markers in 6 of 7 macrometastases and in 1 of 2 micrometastases. In the 2 RT-PCR negative cases, metastases were detected only on sections distant from those analysed by RT-PCR. CEA and/or CK19 were positive by RT-PCR in 3 of 7 ITC and in 23 morphologically negative SLNs. In conclusion, the main goal of our study was to show that the use of alternate sections of the same sample for different procedures is the key reason for the discrepancies between molecular and morphological analyses of SLN. We believe that only prospective studies with quantitative mRNA analysis of specific metastatic markers on the whole lymph node can elucidate the utility of molecular assessments of SLN.
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Yaka, Reyhan; Birand, Ayşegül; Yılmaz, Yasemin; Caner, Ceren; Açan, Sinan Can; Gündüzalp, Sidar; Parvizi, Poorya; Erim Özdoğan, Aslı; Togan, İnci; Somel, Mehmet
2018-05-01
North Mesopotamia has witnessed dramatic social change during the Holocene, but the impact of these events on its demographic history is poorly understood. Here, we study this question by analysing genetic data from the recently excavated Late Iron Age settlement of Çemialo Sırtı in Batman, southeast Turkey. Archaeological and radiocarbon evidence indicate that the site was inhabited during the second and first millennia BCE. Çemialo Sırtı reveals nomadic items of the Early Iron Age, as well as items associated with the Late Achaemenid and subsequent Hellenistic Periods. We compare Çemialo Sırtı mitochondrial DNA profiles with earlier and later populations from west Eurasia to describe genetic continuity patterns in the region. A total of 16 Çemialo Sırtı individuals' remains were studied. PCR and Sanger sequencing were used to obtain mitochondrial DNA HVRI-HVRII sequences. We studied haplotype diversity and pairwise genetic distances using F ST , comparing the Çemialo Sırtı population with ancient and modern-day populations from west Eurasia. Coalescent simulations were carried out to test continuity for specific population comparisons. Mitochondrial DNA (mtDNA) haplotypes from 12 Çemialo Sırtı individuals reveal high haplotype diversity in this population, conspicuously higher than early Holocene west Eurasian populations, which supports the notion of increasing population admixture in west Eurasia through the Holocene. In its mtDNA composition, Çemialo Sırtı shows highest affinity to Neolithic north Syria and Neolithic Anatolia among ancient populations studied, and to modern-day southwest Asian populations. Based on population genetic simulations we cannot reject continuity between Neolithic and Iron Age, or between Iron Age and present-day populations of the region. Despite the region's complex sociopolitical history and indication for increased genetic diversity over time, we find no evidence for sharp shifts in north Mesopotamian
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MIV-150-containing intravaginal rings protect macaque vaginal explants against SHIV-RT infection.
Ouattara, Louise A; Barnable, Patrick; Mawson, Paul; Seidor, Samantha; Zydowsky, Thomas M; Kizima, Larisa; Rodriguez, Aixa; Fernández-Romero, José A; Cooney, Michael L; Roberts, Kevin D; Gettie, Agegnehu; Blanchard, James; Robbiani, Melissa; Teleshova, Natalia
2014-05-01
Recent studies demonstrated that intravaginal rings (IVRs) containing 100 mg of the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 significantly protect macaques against a chimeric simian-human immunodeficiency virus that expresses the HIV-1 HxB2 reverse transcriptase (SHIV-RT) when present before and after vaginal challenge. The objectives of this study were to (i) evaluate the pharmacodynamics (PD) of MIV-150 in vaginal fluids (VF) and in ectocervical and vaginal tissues following 100-mg MIV-150 IVR exposure and to (ii) gain more insight whether pharmacokinetics (PK) of MIV-150 can predict PD. MIV-150 in VF collected at 1 day and 14 days post-MIV-150 IVR insertion inhibited ex vivo SHIV-RT infection in vaginal biopsy specimens from untreated animals (not carrying IVRs) in a dose-dependent manner. Previous PK studies demonstrated a significant increase of ectocervical and vaginal tissue MIV-150 concentrations 14 days versus 1 day post-IVR insertion, with the highest increase in vaginal tissue. Therefore, we tested PD of MIV-150 in tissues 14 days post-MIV-150 IVR insertion. Ex vivo SHIV-RT infection of vaginal, but not ectocervical, tissues collected 14 days post-MIV-150 IVR insertion was significantly inhibited compared to infection at the baseline (prior to MIV-150 IVR exposure). No changes in vaginal and ectocervical tissue infection were observed after placebo IVR exposure. Overall, these data underscore the use of the ex vivo macaque explant challenge models to evaluate tissue and VF PK/PD of candidate microbicides before in vivo animal efficacy studies. The data support further development of MIV-150-containing IVRs.
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Liu, Zhiying; Li, Wei; Xu, Meng; Sheng, Bo; Yang, Zixuan; Jiao, Yanmei; Zhang, Tong; Mou, Danlei; Chen, Dexi; Wu, Hao
2015-01-01
The detection of acute HIV infection (AHI) among high risk populations can help reduce secondary transmission of HIV. The nucleic acid testing (NAT) can shorten the test window period by up to 7-12 days. In this study, we describe an in-house NAT based on the multiplex nested RT-PCR method to detect the HIV RNA. We also evaluated it in a high risk cohort in Beijing. Four primer pairs were designed and evaluated for the detection of different HIV-1 subtypes in group M. Multiplex RT-PCR and nested PCR were performed. The sensitivity, specialty, primers compatibility among HIV subtypes were evaluated simultaneously. In an MSM cohort in Beijing during a 3-year period, a total of 11,808 blood samples that were negative by ELISA or indeterminate by Western blot were analyzed by this multiplex nested RT-PCR with pooling strategy. The multiplex nested RT-PCR was successfully applied for the detection of at least six HIV-1 subtypes. The sensitivity was 40 copies/ml and the specificity was 100%. A total of 29 people were tested HIV-1 positive with acute infection in a MSM cohort of Beijing during a 3 years period. This multiplex nested RT-PCR provides a useful tool for the rapid detection of acute HIV-1 infection. When used in combination with the 3(rd) generation ELISA, it can improve the detection rate of HIV infection, especially in the source limited regions.
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1989-11-29
nvmbe’j International Business Machines Corporation Wright-Patterson AFB, The IBM Development System for the Ada Language AIX/RT follow-on, Version 1.1...Certificate Number: 891129W1.10198 International Business Machines Corporation The IBM Development System for the Ada Language AIX/RT Follow-on, Version 1.1 IBM...scripts provided by International Business Machines Corporation and reviewed by the validation team. The compiler was tested using all the following
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The Role of RT Carry-Over for Congruence Sequence Effects in Masked Priming
ERIC Educational Resources Information Center
Huber-Huber, Christoph; Ansorge, Ulrich
2017-01-01
The present study disentangles 2 sources of the congruence sequence effect with masked primes: congruence and response time of the previous trial (reaction time [RT] carry-over). Using arrows as primes and targets and a metacontrast masking procedure we found congruence as well as congruence sequence effects. In addition, congruence sequence…
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Hout, David R; Schweitzer, Brock L; Lawrence, Kasey; Morris, Stephan W; Tucker, Tracy; Mazzola, Rosetta; Skelton, Rachel; McMahon, Frank; Handshoe, John; Lesperance, Mary; Karsan, Aly; Saltman, David L
2017-08-01
Patients with lung cancers harboring an activating anaplastic lymphoma kinase ( ALK ) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off Δ C t of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK.
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Hout, David R.; Lawrence, Kasey; Morris, Stephan W.; Tucker, Tracy; Mazzola, Rosetta; Skelton, Rachel; McMahon, Frank; Handshoe, John; Lesperance, Mary; Karsan, Aly
2017-01-01
Patients with lung cancers harboring an activating anaplastic lymphoma kinase (ALK) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off ΔCt of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK. PMID:28763012
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Cheng, Keding; Sloan, Angela; Avery, Kristen M; Coulthart, Michael; Carpenter, Michael; Knox, J David
2014-01-01
Real-time quaking-induced conversion (RT-QuIC), a highly specific and sensitive assay able to detect low levels of the disease-inducing isoform of the prion protein (PrP(d)) in brain tissue biopsies and cerebral spinal fluid, has great potential to become a method for diagnosing prion disease ante mortem. In order to standardize the assay method for routine analysis, an understanding of how physical and chemical factors affect the stability of the recombinant prion protein (rPrP) substrate and the RT-QuIC assay's sensitivity, specificity, and reproducibility is required. In this study, using sporadic Creutzfeldt-Jakob Disease brain homogenate to seed the reactions and an in vitro-expressed recombinant prion protein, hamster rPrP, as the substrate, the following factors affecting the RT-QuIC assay were examined: salt and substrate concentrations, substrate storage, and pH. Results demonstrated that both the generation of the quality and quantities of rPrP substrate critical to the reaction, as well as the RT-QuIC reaction itself required strict adherence to specific physical and chemical conditions. Once optimized, the RT-QuIC assay was confirmed to be a very specific and sensitive assay method for sCJD detection. Findings in this study indicate that further optimization and standardization of RT-QuIC assay is required before it can be adopted as a routine diagnostic test.
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Recent Upgrades to NASA SPoRT Initialization Datasets for the Environmental Modeling System
NASA Technical Reports Server (NTRS)
Case, Jonathan L.; LaFontaine, Frank J.; Molthan, Andrew L.; Zavodsky, Bradley T.; Rozumalski, Robert A.
2012-01-01
The NASA Short-term Prediction Research and Transition (SPoRT) Center has developed several products for its National Weather Service (NWS) partners that can initialize specific fields for local model runs within the NOAA/NWS Science and Training Resource Center (STRC) Environmental Modeling System (EMS). In last year's NWA abstract on this topic, the suite of SPoRT products supported in the STRC EMS was presented, which includes a Sea Surface Temperature (SST) composite, a Great Lakes sea-ice extent, a Green Vegetation Fraction (GVF) composite, and NASA Land Information System (LIS) gridded output. This abstract and companion presentation describes recent upgrades made to the SST and GVF composites, as well as the real-time LIS runs. The Great Lakes sea-ice product is unchanged from 2011. The SPoRT SST composite product has been expanded geographically and as a result, the resolution has been coarsened from 1 km to 2 km to accommodate the larger domain. The expanded domain covers much of the northern hemisphere from eastern Asia to western Europe (0 N to 80 N latitude and 150 E to 10 E longitude). In addition, the NESDIS POES-GOES product was added to fill in gaps caused by the Moderate Resolution Imaging Spectroradiometer (MODIS) being unable to sense in cloudy regions, replacing the recently-lost Advanced Microwave Scanning Radiometer for EOS with negligible change to product fidelity. The SST product now runs twice per day for Terra and Aqua combined data collections from 0000 to 1200 UTC and from 1200 to 0000 UTC, with valid analysis times at 0600 and 1800 UTC. The twice-daily compositing technique reduces the overall latency of the previous version while still representing the diurnal cycle characteristics. The SST composites are available at approximately four hours after the end of each collection period (i.e. 1600 UTC for the nighttime analysis and 0400 UTC for the daytime analysis). The real-time MODIS GVF composite has only received minor updates in the
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Beaulieu, Robert; López-Mondéjar, Rubén; Tittarelli, Fabio; Ros, Margarita; Pascual, José Antonio
2011-02-01
To ensure proper use of Trichoderma harzianum in agriculture, accurate data must be obtained in population monitoring. The effectiveness of qRT-PCR to quantify T. harzianum in different growing media was compared to the commonly used techniques of colony counting and qPCR. Results showed that plate counting and qPCR offered similar T. harzianum quantification patterns of an initial rapid increase in fungal population that decreased over time. However, data from qRT-PCR showed a population curve of active T. harzianum with a delayed onset of initial growth which then increased throughout the experiment. Results demonstrated that T. harzianum can successfully grow in these media and that qRT-PCR can offer a more distinct representation of active T. harzianum populations. Additionally, compost amended with T. harzianum exhibited a lower Fusarium oxysporum infection rate (67%) and lower percentage of fresh weight loss (11%) in comparison to amended peat (90% infection rate, 23% fresh weight loss). Copyright © 2010 Elsevier Ltd. All rights reserved.
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Saba Shirvan, Aylar; Mardani, Karim
2014-01-01
Infectious bronchitis (IB) and Newcastle disease (ND) are highly contagious and the most economically important diseases of the poultry affecting respiratory tract and causing economic losses in poultry industry throughout the world. In the present study, the simultaneous detection and differentiation of causative agents of these diseases were investigated using duplex-RT-PCR. RNA was extracted from vaccinal and reference strains of infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) and then cDNA was synthesized. Using two universal primer sets for detection of IBV and NDV, the duplex-RT-PCR was developed. In order to assess the efficiency of the developed duplex RT-PCR, a number of 12 broiler farms with the symptoms of respiratory tract infection was sampled (trachea, lung and kidney were sampled from affected birds suspicious for IBV and NDV infections). After RNA extraction from tissues and cDNA synthesis, the presence of IBV and NDV genome were investigated using duplex-PCR. The results showed that three of twelve examined broiler farms were positive for IBV and two farms were positive for NDV and IBV. The results revealed that the duplex-RT-PCR is a quick and sensitive procedure for simultaneously detecting IBV and NDV in birds with respiratory infections.
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Developing strong response capacity: training volunteers in the medical reserve corps.
Ye, Jiali; Stanford, Stacy; Gousse, Tahlia; Tosatto, Robert J
2014-12-01
The success of the Medical Reserve Corps (MRC) is dependent on the ability of volunteers to respond in a timely and effective manner. This study aimed to assess the current status of MRC volunteer training and to examine the association between MRC characteristics and provision of training. The data for this study were drawn from the 2013 Network Profile Survey of the MRC, which was administered to active MRC unit leaders or designated alternates of 962 units across the country in April to May of 2013. Over 80% of MRCs had a training plan. Ninety-one percent of MRCs offered one or more training courses to volunteers, and 73% indicated requirements for mandatory training. Approximately 84% of MRC units collaborated with other organizations to conduct trainings. Units with more volunteers (>150) were 3 times as likely to have a plan for volunteer training as were those with fewer volunteers (≤50). Compared to units with a full-time leader, those with leaders who were volunteers were only 0.57 times as likely to have a training plan. An overwhelming majority of MRC units provide critical training to their volunteers prior to an emergency deployment. To further strengthen the overall MRC capacities, it is important for MRC units to have a training plan tailored to their community needs and features, make full use of available training resources, and collaborate with partner organizations.
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Echovirus 30 meningitis epidemic followed by an outbreak-specific RT-qPCR.
Österback, Riikka; Kalliokoski, Teemu; Lähdesmäki, Tuire; Peltola, Ville; Ruuskanen, Olli; Waris, Matti
2015-08-01
An outbreak of enteroviral aseptic meningitis emerged in Southwestern Finland in August 2009. The same enterovirus reappeared with increasing incidence of meningitis in other parts of Finland in 2010. To identify the incidence and molecular epidemiology of enteroviral meningitis outbreak. The causative agent was identified as echovirus 30 (E-30) by sequencing partial viral protein 1 capsid genome, and a virus type-specific RT-qPCR was set up for sensitive detection of the virus in cerebrospinal fluid specimens. Enterovirus positive CSF specimens were subjected to the E-30-specific assay to investigate this unusual occurrence of aseptic meningitis and facilitate case confirmation during the outbreaks between August 2009 and September 2010. E-30 was detected in 106 (72%) enterovirus positive cerebrospinal fluid specimens. All the meningitis cases in 2009 and most of them in 2010 were among adolescents and several were members of sport teams. Between August 2009 and September 2010, E-30 caused an extensive outbreak with two peaks in Finland. Type-specific RT-PCR allowed rapid diagnostic follow-up of the epidemic. Copyright © 2015 Elsevier B.V. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Pyakuryal, A; Lee, C; Lee, C
Purpose: Prior to 3D conformal radiation therapy planning, patient anatomy information was mostly limited to 2D beams-eye-view from the conventional simulator. To analyze the outcomes of such treatments for radiation late effects, 3D computational human models are often used in commercial treatment planning systems (TPSs). However, several underlying difficulties such as time-consuming manual delineation procedures of a large number of structures in the model have always limited its applications. Primary objective of this work was to develop a human model library for the epidemiological study by converting 3D-surface model organs to DICOM-RT format (DICOM-RT structure) using an in-house built software.more » We converted the ICRP reference human models to DICOM-RT models, which can be readily adopted for various dose calculations. Methods: MATLAB based code were utilized to convert the contour drawings extracted in text-format from the 3D graphic-tool, Rhinoceros into DICOM-RT structure format for 50 different organs of each model using a 16GB dual-core processor. The conversion periods were measured for each DICOM-RT models, and the reconstructed structure volumes were validated against the original 3D-surface models in the TPS. Ten reference hybrid whole-body models (8-pediatric and 2-adults) were automatically processed to create DICOM-RT computational human model library. Results: Mean contour conversion period was found to be 580 (N=2) and 394.5 (N=8) seconds for 50 organs in the adult and pediatric models respectively. A good agreement for large organs (NRMSD <1.0%) and small organs (NRMSD <7.7%) was also observed between the original volumes and corresponding DICOM-RT structure volumes of the organs. Conclusion: The ICRP reference human models were converted into DICOM-RT format to support the epidemiological study using a large cohort of conventional radiotherapy patients. Due to its DICOM-compatibility, the library may be implemented to many other
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NASA/SPoRT's GOES-R Activities in Support of Product Development, Management, and Training
NASA Astrophysics Data System (ADS)
Fuell, K. K.; Jedlovec, G.; Molthan, A.; Stano, G. T.
2012-12-01
The NASA Short-term Prediction Research and Transition (SPoRT) Center supports many activities within the GOES-R Proving Grounds (PG). These include the development of imagery from existing instrumentation as a proxy to future Advanced Baseline Imager (ABI) capabilities on GOES-R. The Moderate Resolution Imaging Spectroradiometer (MODIS) and the Visible/Infrared Imager/Radiometer Suite (VIIRS) instruments are used to provide a glimpse of the multi-spectral capabilities that will become the norm as the number of channels and data rate dramatically increase with GOES-R. The NOAA/NWS has plans to provide operational users with all ABI channels at the highest resolution. Data fusion of individual channels into composite red, green, and blue imagery products will assist the end user with this future wave of information. While increasing the efficiency in the operational use of ABI channels, these composites provide only qualitative information. Within the GOES-R PG, SPoRT and other partners are exploring ways to include quantitative information as part of the composite imagery. However, limitations in local hardware processing and/or data bandwidth for users of the GOES-R data stream are challenges to overcome. This presentation will discuss the creation of these composite images as well as possible solutions to address these processing challenges. In a similar manner the Geostationary Lightning Mapper (GLM) to be launched on GOES-R presents several data management challenges. The GLM is a pioneering instrument to quantify total lightning from a geostationary platform. The expected data frequency from the GLM is to be at a sub-minute interval. Users of such a data set may have little experience in handling such a rapid update of information. To assist users, SPoRT is working with the NWS to develop tools within the user's decision support system to allow tracking and analysis of total lightning from a storm-based perspective. This presentation will discuss the
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NASA/SPoRT's GOES-R Activities in Support of Product Development, Management, and Training
NASA Technical Reports Server (NTRS)
Fuell, Kevin K.; Jedlovec, Gary; Molthan, Andrew L.; Stano, Geoffrey T.
2012-01-01
The NASA Short-term Prediction Research and Transition (SPoRT) Center supports many activities within the GOES-R Proving Grounds (PG). These include the development of imagery from existing instrumentation as a proxy to future Advanced Baseline Imager (ABI) capabilities on GOES-R. The Moderate Resolution Imaging Spectroradiometer (MODIS) and the Visible/Infrared Imager/Radiometer Suite (VIIRS) instruments are used to provide a glimpse of the multi-spectral capabilities that will become the norm as the number of channels and data rate dramatically increase with GOES-R. The NOAA/NWS has plans to provide operational users with all ABI channels at the highest resolution. Data fusion of individual channels into composite red, green, and blue imagery products will assist the end user with this future wave of information. While increasing the efficiency in the operational use of ABI channels, these composites provide only qualitative information. Within the GOES-R PG, SPoRT and other partners are exploring ways to include quantitative information as part of the composite imagery. However, limitations in local hardware processing and/or data bandwidth for users of the GOES-R data stream are challenges to overcome. This presentation will discuss the creation of these composite images as well as possible solutions to address these processing challenges. In a similar manner the Geostationary Lightning Mapper (GLM) to be launched on GOES-R presents several data management challenges. The GLM is a pioneering instrument to quantify total lightning from a geostationary platform. The expected data frequency from the GLM is to be at a sub-minute interval. Users of such a data set may have little experience in handling such a rapid update of information. To assist users, SPoRT is working with the NWS to develop tools within the user fs decision support system to allow tracking and analysis of total lightning from a storm-based perspective. This presentation will discuss the
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Redondo, Javier Jareño; Keller, Peter M; Zbinden, Reinhard; Wagner, Karoline
2018-01-01
In this study we evaluated the commercially available LightMix® RT-PCR assay for Helicobacter pylori detection and identification of clarithromycin (CLR) resistance in culture and clinical specimens (gastric biopsies and stool). The H. pylori LightMix® RT-PCR detects a 97bp long fragment of the 23S rRNA gene and allows the identification of 3 distinct point mutations conferring CLR resistance via melting curve analysis. The performance of the H. pylori LightMix® RT-PCR was evaluated using a set of 60 H. pylori strains showing phenotypical CLR susceptibility or CLR resistance (Minimum inhibitory concentrations from 0.016 to 256mg/L). We found high concordance (95%) between phenotypical CLR resistance screening by E-Test® and the Lightmix® RT-PCR. Discrepant results were verified by sequencing of the 23S rRNA gene that always confirmed the results obtained by Lightmix® RT-PCR. Furthermore, H. pylori was detected in clinical biopsy and stool specimens by Lightmix® RT-PCR that identified the correct H. pylori genotype. The LightMix® RT-PCR is an accurate, sensitive and easy to use test for H. pylori and CLR resistance detection and can therefore be readily implemented in any diagnostic laboratory. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
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Kumar, Sandeep; Baranwal, V K; Joshi, Subodh; Arya, Meenakshi; Majumder, S
2010-06-01
Reduced seed production in onion is associated with Onion yellow dwarf virus (OYDV), a filamentous Potyvirus. Onion is also infected with other filamentous virus particles suspected to be Allexivirus. RT-PCR was used to detect mixed infection of both the viruses in leaves and bulbs. A duplex RT-PCR was developed, which simultaneously detected the presence of these two viruses in winter (Rabi) onion bulb. In summer (Kharif) onion bulbs only Allexivirus was detected. The absence of OYDV in summer crop is discussed. The sequencing of RT-PCR amplified products confirmed the identity of OYDV and Allexivirus, the latter showing closer identity to Garlic virus C (GVC)/Garlic mite-borne mosaic virus. This makes the first detection of an Allexivirus in onion crop in India. The duplex RT-PCR to detect these viruses (OYDV and Allexivirus) would be an improvement for indexing of viruses in onion bulbs for seed production.
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2009-01-01
Summary Background Hysterectomy and bilateral salpingo-oophorectomy (BSO) is the standard surgery for stage I endometrial cancer. Systematic pelvic lymphadenectomy has been used to establish whether there is extra-uterine disease and as a therapeutic procedure; however, randomised trials need to be done to assess therapeutic efficacy. The ASTEC surgical trial investigated whether pelvic lymphadenectomy could improve survival of women with endometrial cancer. Methods From 85 centres in four countries, 1408 women with histologically proven endometrial carcinoma thought preoperatively to be confined to the corpus were randomly allocated by a minimisation method to standard surgery (hysterectomy and BSO, peritoneal washings, and palpation of para-aortic nodes; n=704) or standard surgery plus lymphadenectomy (n=704). The primary outcome measure was overall survival. To control for postsurgical treatment, women with early-stage disease at intermediate or high risk of recurrence were randomised (independent of lymph-node status) into the ASTEC radiotherapy trial. Analysis was by intention to treat. This study is registered, number ISRCTN 16571884. Findings After a median follow-up of 37 months (IQR 24–58), 191 women (88 standard surgery group, 103 lymphadenectomy group) had died, with a hazard ratio (HR) of 1·16 (95% CI 0·87–1·54; p=0·31) in favour of standard surgery and an absolute difference in 5-year overall survival of 1% (95% CI −4 to 6). 251 women died or had recurrent disease (107 standard surgery group, 144 lymphadenectomy group), with an HR of 1·35 (1·06–1·73; p=0·017) in favour of standard surgery and an absolute difference in 5-year recurrence-free survival of 6% (1–12). With adjustment for baseline characteristics and pathology details, the HR for overall survival was 1·04 (0·74–1·45; p=0·83) and for recurrence-free survival was 1·25 (0·93–1·66; p=0·14). Interpretation Our results show no evidence of benefit in terms of overall
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Detecting the Presence of Nora Virus in "Drosophila" Utilizing Single Fly RT-PCR
ERIC Educational Resources Information Center
Munn, Bethany; Ericson, Brad; Carlson, Darby J.; Carlson, Kimberly A.
2015-01-01
A single fly RT-PCR protocol has recently been developed to detect the presence of the persistent, horizontally transmitted Nora virus in "Drosophila." Wild-caught flies from Ohio were tested for the presence of the virus, with nearly one-fifth testing positive. The investigation presented can serve as an ideal project for biology…
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Graeber, Kai; Linkies, Ada; Wood, Andrew T.A.; Leubner-Metzger, Gerhard
2011-01-01
Comparative biology includes the comparison of transcriptome and quantitative real-time RT-PCR (qRT-PCR) data sets in a range of species to detect evolutionarily conserved and divergent processes. Transcript abundance analysis of target genes by qRT-PCR requires a highly accurate and robust workflow. This includes reference genes with high expression stability (i.e., low intersample transcript abundance variation) for correct target gene normalization. Cross-species qRT-PCR for proper comparative transcript quantification requires reference genes suitable for different species. We addressed this issue using tissue-specific transcriptome data sets of germinating Lepidium sativum seeds to identify new candidate reference genes. We investigated their expression stability in germinating seeds of L. sativum and Arabidopsis thaliana by qRT-PCR, combined with in silico analysis of Arabidopsis and Brassica napus microarray data sets. This revealed that reference gene expression stability is higher for a given developmental process between distinct species than for distinct developmental processes within a given single species. The identified superior cross-species reference genes may be used for family-wide comparative qRT-PCR analysis of Brassicaceae seed germination. Furthermore, using germinating seeds, we exemplify optimization of the qRT-PCR workflow for challenging tissues regarding RNA quality, transcript stability, and tissue abundance. Our work therefore can serve as a guideline for moving beyond Arabidopsis by establishing high-quality cross-species qRT-PCR. PMID:21666000
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Ye, Yingwang; Wu, Qingping; Zhang, Jumei; Jiang, He; Hu, Wang
2012-11-01
Cronobacter are opportunistic food-borne pathogens associated with meningitis, sepsis, and necrotizing enterocolitis. Little attempt has focused on detection of viable cell of Cronobacter spp. in dry aquatic products, which were frequently used for raw materials of infant foods due to high nutrition. In this paper, one-step reverse transcription polymerase chain reaction (RT-PCR) was developed for detection of viable Cronobacter spp. in dry aquatic products. Specificity test indicated that clearly expected amplicon in size 469 bp was amplified from RNA of Cronobacter, but not from RNA of negative controls and DNA of Cronobacter strains. The sensitivity was 10(4) CFU/mL of Cronobacter strain in artificially fish meal samples and 10(1) CFU/mL of Cronobacter after 10-h enrichment. In a total of 81 dry aquatic products, 9.8%, 8.6%, and 9.8% of samples were found to be positive for Cronobacter by one-step RT-PCR, U.S. Food and Drug Administration method, and Druggan-Forsythe-Iversen medium, respectively. The results clearly indicated that one-step RT-PCR could avoid the interference of residual DNA of Cronobacter in food samples and be used to specifically detect viable Cronobacter spp. for large-scale monitoring of food samples. The use of rapid and specific detection of food borne pathogens in food samples was most of importance for control and precaution of food borne diseases. In this study, one-step RT-PCR was developed for detection of Cronobacter spp. in aquatic products. A comparison of different methods for detection of Cronobacter indicated that the newly developed method could be widely used to specifically detect Cronobacter spp. in food samples. © 2012 Institute of Food Technologists®
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Validation of reference genes for RT-qPCR analysis in Herbaspirillum seropedicae.
Pessoa, Daniella Duarte Villarinho; Vidal, Marcia Soares; Baldani, José Ivo; Simoes-Araujo, Jean Luiz
2016-08-01
The RT-qPCR technique needs a validated set of reference genes for ensuring the consistency of the results from the gene expression. Expression stabilities for 9 genes from Herbaspirillum seropedicae, strain HRC54, grown with different carbon sources were calculated using geNorm and NormFinder, and the gene rpoA showed the best stability values. Copyright © 2016 Elsevier B.V. All rights reserved.
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Regional Data Assimilation of AIRS Profiles and Radiances at the SPoRT Center
NASA Technical Reports Server (NTRS)
Zavodsky, Brad; Chou, Shih-hung; Jedlovec, Gary
2009-01-01
This slide presentation reviews the Short Term Prediction Research and Transition (SPoRT) Center's mission to improve short-term weather prediction at the regional and local scale. It includes information on the cold bias in Weather Research and Forcasting (WRF), troposphere recordings from the Atmospheric Infrared Sounder (AIRS), and vertical resolution of analysis grid.
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DETECTION OF HUMAN ENTERIC VIRUSES IN STREAM WATER WITH RT-PCR AND CELL CULTURE
A multiplex RT-PCR method was used to measure virus occurrence at five stream water sites that span a range of hydroclimatic, water-quality, and land-use characteristics. The performance of the molecular method was evaluated in comparison to traditional cell culture and Escherich...
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Zhou, Xiaodie; Song, Yong; Zhou, Xiaojun; Yu, Like; Wang, Jiandong
2015-01-01
Anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) gene rearrangements occur in approximately 5% of non-small-cell lung cancers (NSCLC), leading to the overexpression of anaplastic lymphoma kinase and predicting a response to the targeted inhibitor, crizotinib. Malignant pleural effusion occurs in most patients with advanced lung cancer, especially adenocarcinoma, and tissue samples are not always available from these patients. We attempted to clarify the feasibility of detecting the EML4-ALK fusion gene in pleural effusion cells using different methods. We obtained 66 samples of pleural effusion from NSCLC patients. The pleural effusion fluid was centrifuged, and the cellular components obtained were formalin fixed and paraffin embedded. The EML4-ALK fusion gene status was determined with fluorescent in situ hybridization (FISH), reverse transcription—polymerase chain reaction (RT-PCR), and immunohistochemistry (IHC). EML4-ALK was detected in three of 66 patient samples (4.5%) with RT-PCR. When the RT-PCR data were used as the standard, one false positive and one false negative samples were identified with IHC; and one false negative sample was identified with FISH. These results suggest that a block of pleural effusion cells can be used to detect the EML4-ALK fusion gene. IHC had good sensitivity, but low specificity. FISH had low sensitivity, but high specificity. RT-PCR is a good candidate method for detecting EML4-ALK in blocks of pleural effusion cells from lung cancer patients. PMID:25785456
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Liu, Leilei; Zhan, Ping; Zhou, Xiaodie; Song, Yong; Zhou, Xiaojun; Yu, Like; Wang, Jiandong
2015-01-01
Anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) gene rearrangements occur in approximately 5% of non-small-cell lung cancers (NSCLC), leading to the overexpression of anaplastic lymphoma kinase and predicting a response to the targeted inhibitor, crizotinib. Malignant pleural effusion occurs in most patients with advanced lung cancer, especially adenocarcinoma, and tissue samples are not always available from these patients. We attempted to clarify the feasibility of detecting the EML4-ALK fusion gene in pleural effusion cells using different methods. We obtained 66 samples of pleural effusion from NSCLC patients. The pleural effusion fluid was centrifuged, and the cellular components obtained were formalin fixed and paraffin embedded. The EML4-ALK fusion gene status was determined with fluorescent in situ hybridization (FISH), reverse transcription-polymerase chain reaction (RT-PCR), and immunohistochemistry (IHC). EML4-ALK was detected in three of 66 patient samples (4.5%) with RT-PCR. When the RT-PCR data were used as the standard, one false positive and one false negative samples were identified with IHC; and one false negative sample was identified with FISH. These results suggest that a block of pleural effusion cells can be used to detect the EML4-ALK fusion gene. IHC had good sensitivity, but low specificity. FISH had low sensitivity, but high specificity. RT-PCR is a good candidate method for detecting EML4-ALK in blocks of pleural effusion cells from lung cancer patients.
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Damhorst, Gregory L.; Duarte-Guevara, Carlos; Chen, Weili; Ghonge, Tanmay; Cunningham, Brian T.; Bashir, Rashid
2015-01-01
Viral load measurements are an essential tool for the long-term clinical care of hum an 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. PMID:26705482
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Li, Tao; Wang, Jing; Lu, Miao; Zhang, Tianyi; Qu, Xinyun; Wang, Zhezhi
2017-01-01
Due to its sensitivity and specificity, real-time quantitative PCR (qRT-PCR) is a popular technique for investigating gene expression levels in plants. Based on the Minimum Information for Publication of Real-Time Quantitative PCR Experiments (MIQE) guidelines, it is necessary to select and validate putative appropriate reference genes for qRT-PCR normalization. In the current study, three algorithms, geNorm, NormFinder, and BestKeeper, were applied to assess the expression stability of 10 candidate reference genes across five different tissues and three different abiotic stresses in Isatis indigotica Fort. Additionally, the IiYUC6 gene associated with IAA biosynthesis was applied to validate the candidate reference genes. The analysis results of the geNorm, NormFinder, and BestKeeper algorithms indicated certain differences for the different sample sets and different experiment conditions. Considering all of the algorithms, PP2A-4 and TUB4 were recommended as the most stable reference genes for total and different tissue samples, respectively. Moreover, RPL15 and PP2A-4 were considered to be the most suitable reference genes for abiotic stress treatments. The obtained experimental results might contribute to improved accuracy and credibility for the expression levels of target genes by qRT-PCR normalization in I. indigotica. PMID:28702046
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Li, Bai; Liu, Wei Jia; Adab, Peymane; Pallan, Miranda; Hemming, Karla; Frew, Emma; Lin, Rong; Martin, James; Liu, Wei; Cheng, Kar Keung
2017-01-01
Introduction Childhood obesity in China has increased more rapidly and over a shorter time period than in other countries. However, there is a paucity of rigorously developed and evaluated prevention interventions. We aim to evaluate the clinical and cost-effectiveness as well as the implementation process of a complex multicomponent intervention developed using the UK Medical Research Council (MRC) framework. This study provides one of the first examples of rigorous development and evaluation of a childhood obesity prevention programme in a non-western population using the MRC methods. Methods and analysis A cluster-randomised controlled trial in 40 primary schools in Guangzhou, China, including children aged 6–7 years at baseline. Schools will be randomly allocated to either the usual practice (n=20) or intervention arm (n=20). The 12-month intervention consists of four components targeting diet and physical activity behaviours in and outside school, with family involvement. The primary objective is to compare the difference in mean body mass index (BMI) z-score between the intervention and control arms at the end of the intervention (starting March/April 2017). A sample size of 1640 pupils recruited from 40 schools is sufficient to detect a difference of 0.17 units in the mean BMI z-score with a power of 80% (ICC=0.01. ICC, intraclass correlation coefficient) and a significance level of 5%. Treatment effects will be tested using a mixed linear model in STATA adjusting for the child baseline BMI z-score and clustering by school. All analyses will be by intention to treat. Secondary analyses will additionally adjust for prespecified school-level and child-level covariates. The incremental cost-effectiveness ratio for the intervention versus usual practice will be ‘cost per quality-adjusted life year (QALY)’. Cost per change in BMI z-score will also be assessed. A range of methods will be used to evaluate intervention implementation, mechanisms of impact and
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Bekaert, Michaël; Bakheit, Mohammed; Frischmann, Sieghard; Patel, Pranav; Simon-Loriere, Etienne; Lambrechts, Louis; Duong, Veasna; Dussart, Philippe; Harold, Graham; Fall, Cheikh; Faye, Oumar; Sall, Amadou Alpha; Weidmann, Manfred
2018-01-01
Background 4 one-step, real-time, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed for the detection of dengue virus (DENV) serotypes by considering 2,056 full genome DENV sequences. DENV1 and DENV2 RT-LAMP assays were validated with 31 blood and 11 serum samples from Tanzania, Senegal, Sudan and Mauritania. DENV3 and DENV4 RT-LAMP assays were validated with 25 serum samples from Cambodia Methodology/Principal findings 4 final reaction primer mixes were obtained by using a combination of Principal Component Analysis of the full DENV genome sequences, and LAMP primer design based on sequence alignments using the LAVA software. These mixes contained 14 (DENV1), 12 (DENV2), 8 (DENV3) and 3 (DENV4) LAMP primer sets. The assays were evaluated with an External Quality Assessment panel from Quality Control for Molecular Diagnostics. The assays were serotype-specific and did not cross-detect with other flaviviruses. The limits of detection, with 95% probability, were 22 (DENV1), 542 (DENV2), 197 (DENV3) and 641 (DENV4) RNA molecules, and 100% reproducibility in the assays was obtained with up to 102 (DENV1) and 103 RNA molecules (DENV2, DENV3 and DENV4). Validation of the DENV2 assay with blood samples from Tanzania resulted in 23 samples detected by RT-LAMP, demonstrating that the assay is 100% specific and 95.8% sensitive (positive predictive value of 100% and a negative predictive value of 85.7%). All serum samples from Senegal, Sudan and Mauritania were detected and 3 untyped as DENV1. The sensitivity of RT-LAMP for DENV4 samples from Cambodia did not quite match qRT-PCR. Conclusions/Significance We have shown a novel approach to design LAMP primers that makes use of fast growing sequence databases. The DENV1 and DENV2 assays were validated with viral RNA extracted clinical samples, showing very good performance parameters. PMID:29813062
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Godoy, M G; Kibenge, F S; Kibenge, M J; Olmos, P; Ovalle, L; Yañez, A J; Avendaño-Herrera, R
2010-05-18
The objective of this study was to evaluate the application of a TaqMan real-time reverse transcriptase PCR (RT-PCR) assay for the detection of infectious salmon anaemia virus (ISAV) in formalin-fixed paraffin-embedded (FFPE) fish tissues from Atlantic salmon Salmo salar with and without clinical signs of infection, and to compare it with histological and immunohistochemical (IHC) techniques. Sixteen fish samples obtained in 2007 and 2008 from 4 different farms in Chile were examined. The real-time RT-PCR allowed the detection of ISAV in FFPE samples from 9 of 16 fish, regardless of the organs analyzed, whereas 4 of the real-time RT-PCR negative fish were positive as indicated by histological examination and 3 of the real-time RT-PCR positive fish were negative as indicated by immunohistochemistry evaluation. The presence of ISAV in RT-PCR positive samples was confirmed by amplicon sequencing. This work constitutes the first report on the use of real-time RT-PCR for the detection of ISAV in FFPE sections. The assay is very useful for the examination of archival wax-embedded tissues, and allows for both prospective and retrospective evaluation of tissue samples for the presence of ISAV. However, the method only confirms the presence of the pathogen and should be used in combination with histopathology, which is a more precise tool. The combination of both techniques would be invaluable for confirmatory diagnosis of infectious salmon anaemia (ISA), which is essential for solving salmon farm problems.
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Transition and Evaluation of RGB Imagery to WFOs and National Centers by NASA SPoRT
NASA Technical Reports Server (NTRS)
Fuell, Kevin K.; Molthan, Andrew L.
2012-01-01
MODIS Snow/Cloud and True Color RGB imagery has been used by SPoRT partners since 2004 to examine changes in surface features such as snow cover, vegetation, ocean color, fires, smoke plumes, and oil spills.
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Kang, Xiao-ping; Jiang, Tao; Li, Yong-qiang; Lin, Fang; Liu, Hong; Chang, Guo-hui; Zhu, Qing-yu; Qin, E-de; Qin, Cheng-feng; Yang, Yin-hui
2010-06-02
A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009) influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose) for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same as that of each single-target RT-PCR for pandemic H1N1 and even more sensitive for H5N1 with the same primers and probes. No cross reactivity of detecting other subtype influenza viruses or respiratory tract viruses was observed. Two hundred and thirty-six clinical specimens were tested by comparing with single real-time RT-PCR and result from the duplex assay was 100% consistent with the results of single real-time RT-PCR and sequence analysis.
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Pichler, Martin; Zatloukal, Kurt
2013-01-01
Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application
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Wilhelm, B J; Leblanc, D; Avery, B; Pearl, D L; Houde, A; Rajić, A; McEwen, S A
2016-03-01
We collected 599 Canadian retail pork chops and 283 pork livers routinely (usually weekly) from April 2011 to March 2012 using the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) retail sampling platform. Samples were assayed using validated real-time (q) reverse transcriptase polymerase chain reaction (RT-PCR) and nested classical RT-PCR for the detection of hepatitis E virus (HEV), porcine enteric calicivirus (PEC) and rotavirus (RV). The presence of Escherichia coli, Salmonella spp. and Campylobacter spp. was measured on a subset of our samples. Exact logistic regression models were fitted for predictors for HEV detection, for each assay. For both assays, sample type (pork chop versus liver) was a significant predictor for HEV RNA detection. For nested classical RT-PCR but not qRT-PCR, region of sample collection was a significant predictor (P = 0.008) of HEV detection. Odds of HEV detection were greatest in spring relative to other seasons. E. coli was a significant predictor for HEV RNA detection using the qRT-PCR (P = 0.03). Overall, the prevalence of E. coli, Salmonella spp. and Campylobacter spp. was significantly greater than HEV, PEC or RV on our retail pork samples. Our sparse data set for the detection of PEC and RV precluded modelling of risk factors for the detection of these viruses. © 2015 Zoonoses and Public Health © 2015 Her Majesty the Queen in Right of Canada Reproduced with the permission of the Minister of the Public Health Agency of Canada.
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Mahakapuge, T A N; Scheerlinck, J-P Y; Rojas, C A Alvarez; Every, A L; Hagen, J
2016-03-01
With the availability of genetic sequencing data, quantitative reverse transcription PCR (RT-qPCR) is increasingly being used for the quantification of gene transcription across species. Too often there is little regard to the selection of reference genes and the impact that a poor choice has on data interpretation. Indeed, RT-qPCR provides a snapshot of relative gene transcription at a given time-point, and hence is highly dependent on the stability of the transcription of the reference gene(s). Using ovine efferent lymph cells and peripheral blood mono-nuclear cells (PBMCs), the two most frequently used leukocytes in immunological studies, we have compared the stability of transcription of the most commonly used ovine reference genes: YWHAZ, RPL-13A, PGK1, B2M, GAPDH, HPRT, SDHA and ACTB. Using established algorithms for reference gene normalization "geNorm" and "Norm Finder", PGK1, GAPDH and YWHAZ were deemed the most stably transcribed genes for efferent leukocytes and PGK1, YWHAZ and SDHA were optimal in PBMCs. These genes should therefore be considered for accurate and reproducible RT-qPCR data analysis of gene transcription in sheep. Copyright © 2016. Published by Elsevier B.V.
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Use of armored RNA as a standard to construct a calibration curve for real-time RT-PCR.
Donia, D; Divizia, M; Pana', A
2005-06-01
Armored Enterovirus RNA was used to standardize a real-time reverse transcription (RT)-PCR for environmental testing. Armored technology is a system to produce a robust and stable RNA standard, trapped into phage proteins, to be used as internal control. The Armored Enterovirus RNA protected sequence includes 263 bp of highly conserved sequences in 5' UTR region. During these tests, Armored RNA has been used to produce a calibration curve, comparing three different fluorogenic chemistry: TaqMan system, Syber Green I and Lux-primers. The effective evaluation of three amplifying commercial reagent kits, in use to carry out real-time RT-PCR, and several extraction procedures of protected viral RNA have been carried out. The highest Armored RNA recovery was obtained by heat treatment while chemical extraction may decrease the quantity of RNA. The best sensitivity and specificity was obtained using the Syber Green I technique since it is a reproducible test, easy to use and the cheapest one. TaqMan and Lux-primer assays provide good RT-PCR efficiency in relationship to the several extraction methods used, since labelled probe or primer request in these chemistry strategies, increases the cost of testing.
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Cuccia, C; Franzoni, P; Volpini, M; Scalvini, S; Volterrani, M; Musmeci, G; Metra, M
1990-09-01
In 4 consecutive patients admitted for multiple pulmonary embolism 2-dimensional echocardiography showed large right atrial migrant thromboemboli in transit, floating and prolapsing into the right ventricle in diastole. This pattern was always associated with the echocardiographic signs of pulmonary hypertension. All the patients were treated with intravenous infusion of 100 mg of rt-PA in 3 hours. rt-PA determined the dissolution and disappearance of the right atrial thromboemboli (it took 4 hours in 2 patients and 5 hours in the remaining 2), and the concomitant disappearance of the echocardiographic signs of pulmonary hypertension. During and after the rt-PA therapy there was no evidence of further pulmonary embolism. The fibrinolytic treatment for right atrial thromboemboli during multiple pulmonary embolism is a promising alternative to right atrial thrombectomy: our results indicate that rt-PA acts rapidly and is effective and safe; if these results will be confirmed in a larger group of patients, rt-PA could become the first-choice therapy of right atrial thromboembolus.
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Ellis, Chelsey; Misir, Amita; Hui, Charles; Jabbour, Mona; Barrowman, Nicholas; Langill, Jonathan; Bowes, Jennifer; Slinger, Robert
2016-05-01
Rapid detection of the wide range of viruses and bacteria that cause respiratory infection in children is important for patient care and antibiotic stewardship. We therefore designed and evaluated a ready-to-use 22 target respiratory infection reverse-transcription real-time polymerase chain reaction (RT-qPCR) panel to determine if this would improve detection of these agents at our pediatric hospital. RT-qPCR assays for twenty-two target organisms were dried-down in individual wells of 96 well plates and saved at room temperature. Targets included 18 respiratory viruses and 4 bacteria. After automated nucleic acid extraction of nasopharyngeal aspirate (NPA) samples, rapid qPCR was performed. RT-qPCR results were compared with those obtained by the testing methods used at our hospital laboratories. One hundred fifty-nine pediatric NPA samples were tested with the RT-qPCR panel. One or more respiratory pathogens were detected in 132/159 (83%) samples. This was significantly higher than the detection rate of standard methods (94/159, 59%) (P<0.001). This difference was mainly due to improved RT-qPCR detection of rhinoviruses, parainfluenza viruses, bocavirus, and coronaviruses. The panel internal control assay performance remained stable at room temperature storage over a two-month testing period. The RT-qPCR panel was able to identify pathogens in a high proportion of respiratory samples. The panel detected more positive specimens than the methods in use at our hospital. The pre-made panel format was easy to use and rapid, with results available in approximately 90 minutes. We now plan to determine if use of this panel improves patient care and antibiotic stewardship.
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Chávez-Valencia, V; Espinosa-Ortega, H F; Espinoza-Peralta, D; Arce-Salinas, C A
2009-01-01
Obstructive jaundice in patients with previous cholecystectomy requires a precise diagnosis. In the diagnostic algorithm, biliary ultrasound (BUS) and magnetic resonance cholangiogram (MRC) are used, although the accuracy of each method is unknown in our setting. No previous comparison of US and MRC in subjects with cholecystectomy has been made. To determine diagnostic accuracy of BUS and MRC in patients with recurrent biliary obstruction. Patients with endoscopic retrograde cholangiopacreatography (ERCP) demonstrating recurrent biliary obstruction by stones were included. All patients underwent BUS and MRC. We determined the diagnostic performance of each image study compared with ERCP. Twenty-seven patients with a mean age of 62.9 +/- 17.3 years-old were included. Sensitivity and specificity of BUS were 0.12 and 0.58, respectively. Figures for MRC were 0.88 and 0.82. Diagnostic agreement between ERCP and MRC was k= 0.66 whereas BUS had a k of only 0.26. MRC had good diagnostic performance for recurrent choledocolithiasis. BUS demonstrated lower accuracy compared with previous reports, so should not be considered in the initial approach of recurrent choledocus obstruction.
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Real-Time-Simulation of IEEE-5-Bus Network on OPAL-RT-OP4510 Simulator
NASA Astrophysics Data System (ADS)
Atul Bhandakkar, Anjali; Mathew, Lini, Dr.
2018-03-01
The Real-Time Simulator tools have high computing technologies, improved performance. They are widely used for design and improvement of electrical systems. The advancement of the software tools like MATLAB/SIMULINK with its Real-Time Workshop (RTW) and Real-Time Windows Target (RTWT), real-time simulators are used extensively in many engineering fields, such as industry, education, and research institutions. OPAL-RT-OP4510 is a Real-Time Simulator which is used in both industry and academia. In this paper, the real-time simulation of IEEE-5-Bus network is carried out by means of OPAL-RT-OP4510 with CRO and other hardware. The performance of the network is observed with the introduction of fault at various locations. The waveforms of voltage, current, active and reactive power are observed in the MATLAB simulation environment and on the CRO. Also, Load Flow Analysis (LFA) of IEEE-5-Bus network is computed using MATLAB/Simulink power-gui load flow tool.
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Bandla, Aishwarya; Liao, Lun-De; Chan, Su Jing; Ling, Ji Min; Liu, Yu-Hang; Shih, Yen-Yu Ian; Pan, Han-Chi; Wong, Peter Tsun-Hon; Lai, Hsin-Yi; King, Nicolas Kon Kam; Chen, You-Yin; Ng, Wai Hoe; Thakor, Nitish V
2018-06-01
The advance of thrombolytic therapy has been hampered by the lack of optimization of the therapy during the hyperacute phase of focal ischemia. Here, we investigate neurovascular dynamics using a custom-designed hybrid electrocorticography (ECoG)-functional photoacoustic microscopy (fPAM) imaging system during the hyperacute phase (first 6 h) of photothrombotic ischemia (PTI) in male Wistar rats following recombinant tissue plasminogen activator (rtPA)-mediated thrombolysis. We reported, for the first time, the changes in neural activity and cerebral hemodynamic responses following rtPA infusion at different time points post PTI. Interestingly, very early administration of rtPA (< 1 h post PTI) resulted in only partial recovery of neurovascular dynamics (specifically , neural activity recovered to 71 ± 3.5% of baseline and hemodynamics to only 52 ± 2.6% of baseline) and late administration of rtPA (> 4 h post PTI) resulted in the deterioration of neurovascular function. A therapeutic window between 1 and 3 h post PTI was found to improve recovery of neurovascular function (i.e. significant restoration of neural activity to 93 ± 4.2% of baseline and hemodynamics to 81 ± 2.1% of baseline, respectively). The novel combination of fPAM and ECoG enables direct mapping of neurovascular dynamics and serves as a platform to evaluate potential interventions for stroke.
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Henritzi, Dinah; Zhao, Na; Starick, Elke; Simon, Gaelle; Krog, Jesper S; Larsen, Lars Erik; Reid, Scott M; Brown, Ian H; Chiapponi, Chiara; Foni, Emanuela; Wacheck, Silke; Schmid, Peter; Beer, Martin; Hoffmann, Bernd; Harder, Timm C
2016-11-01
A diversifying pool of mammalian-adapted influenza A viruses (IAV) with largely unknown zoonotic potential is maintained in domestic swine populations worldwide. The most recent human influenza pandemic in 2009 was caused by a virus with genes originating from IAV isolated from swine. Swine influenza viruses (SIV) are widespread in European domestic pig populations and evolve dynamically. Knowledge regarding occurrence, spread and evolution of potentially zoonotic SIV in Europe is poorly understood. Efficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost-effective large-scale analysis. New SIV haemagglutinin (HA) and neuraminidase (NA) subtype- and lineage-specific multiplex real-time RT-PCRs (RT-qPCR) have been developed and validated with reference virus isolates and clinical samples. A diagnostic algorithm is proposed for the combined detection in clinical samples and subtyping of SIV strains currently circulating in Europe that is based on a generic, M-gene-specific influenza A virus RT-qPCR. In a second step, positive samples are examined by tetraplex HA- and triplex NA-specific RT-qPCRs to differentiate the porcine subtypes H1, H3, N1 and N2. Within the HA subtype H1, lineages "av" (European avian-derived), "hu" (European human-derived) and "pdm" (human pandemic A/H1N1, 2009) are distinguished by RT-qPCRs, and within the NA subtype N1, lineage "pdm" is differentiated. An RT-PCR amplicon Sanger sequencing method of small fragments of the HA and NA genes is also proposed to safeguard against failure of multiplex RT-qPCR subtyping. These new multiplex RT-qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe. © 2016 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.
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Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples
2010-01-01
Background Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. Results Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. Conclusions We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system. PMID:20492695
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Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples.
Mehta, Rohini; Birerdinc, Aybike; Hossain, Noreen; Afendy, Arian; Chandhoke, Vikas; Younossi, Zobair; Baranova, Ancha
2010-05-21
Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.
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Effect and mechanism of calpains on pediatric lobar pneumonia.
Yin, Genquan; Zeng, Qiang; Zhao, Haijin; Wu, Peiqiong; Cai, Shaoxi; Deng, Li; Jiang, Wenhui
2017-07-04
Lobar pneumonia, one of the community-acquired pneumonia (CAP), is a common pediatric low respiratory tract infection. Calpains are Ca 2+ -activated cysteine proteases whose activation mechanism is elusive. The present study was undertaken to detect the role and mechanism of calpains in pediatric lobar pneumonia. The human acute lung infection model (ALIM) was constructed and infected by Streptococcus. Enzyme-linked immunosorbent assay (ELISA) was used to measure interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α. We observed the lactate dehydrogenase (LDH) release, calpains activity and calpain inhibitor effects in ALIM. The expression of proliferating cell nuclear antigen (PCNA) protein was quantified by western blotting. Then the effects of calpain 1 and 2 knockdown on expressions of inflammation factors and PCNA protein, LDH release and apoptosis were evaluated in lung MRC-5 cells. In constructed ALIM, expressions of IL-6 (P < 0.01), IL-8 (P < 0.01), TNF-α (P < 0.05) and PCNA protein (P < 0.05) were significantly reduced by the calpain inhibitor. Expressions of IL-6, IL-8, TNF-α, PCNA protein and relative LDH release were statistically reduced by the small interfering (si) RNA-calpain 1 and 2 in MRC-5 cells (P < 0.05). Calpains silence increased apoptotic cells from 5% (negative control) to more than 20% in MRC-5 cells. The present study suggests that calpains possess a significant effect on inflammations, cell proliferation and apoptosis. Suppression of calpains may provide a potential therapeutic target of lobar pneumonia.
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Arita, Minetaro; Ling, Hua; Yan, Dongmei; Nishimura, Yorihiro; Yoshida, Hiromu; Wakita, Takaji; Shimizu, Hiroyuki
2009-12-16
In the global eradication program for poliomyelitis, the laboratory diagnosis plays a critical role by isolating poliovirus (PV) from the stool samples of acute flaccid paralysis (AFP) cases. In this study, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) system for a rapid and highly sensitive detection of enterovirus including PV to identify stool samples positive for enterovirus including PV. A primer set was designed for RT-LAMP to detect enterovirus preferably those with PV-like 5'NTRs of the viral genome. The sensitivity of RT-LAMP system was evaluated with prototype strains of enterovirus. Detection of enterovirus from stool extracts was examined by using RT-LAMP system. We detected at least 400 copies of the viral genomes of PV(Sabin) strains within 90 min by RT-LAMP with the primer set. This RT-LAMP system showed a preference for Human enterovirus species C (HEV-C) strains including PV, but exhibited less sensitivity to the prototype strains of HEV-A and HEV-B (detection limits of 7,400 to 28,000 copies). Stool extracts, from which PV, HEV-C, or HEV-A was isolated in the cell culture system, were mostly positive by RT-LAMP method (positive rates of 15/16 (= 94%), 13/14 (= 93%), and 4/4 (= 100%), respectively). The positive rate of this RT-LAMP system for stool extracts from which HEV-B was isolated was lower than that of HEV-C (positive rate of 11/21 (= 52%)). In the stool samples, which were negative for enterovirus isolation by the cell culture system, we found that two samples were positive for RT-LAMP (positive rates of 2/38 (= 5.3%)). In these samples, enterovirus 96 was identified by sequence analysis utilizing a seminested PCR system. RT-LAMP system developed in this study showed a high sensitivity comparable to that of the cell culture system for the detection of PV, HEV-A, and HEV-C, but less sensitivity to HEV-B. This RT-LAMP system would be useful for the direct detection of enterovirus from the stool
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Muthukrishnan, Madhanmohan; Singanallur, Nagendrakumar B; Ralla, Kumar; Villuppanoor, Srinivasan A
2008-08-01
Foot-and-mouth disease virus (FMDV) samples transported to the laboratory from far and inaccessible areas for serodiagnosis pose a major problem in a tropical country like India, where there is maximum temperature fluctuation. Inadequate storage methods lead to spoilage of FMDV samples collected from clinically positive animals in the field. Such samples are declared as non-typeable by the typing laboratories with the consequent loss of valuable epidemiological data. The present study evaluated the usefulness of FTA Classic Cards for the collection, shipment, storage and identification of the FMDV genome by RT-PCR and real-time RT-PCR. The stability of the viral RNA, the absence of infectivity and ease of processing the sample for molecular methods make the FTA cards a useful option for transport of FMDV genome for identification and serotyping. The method can be used routinely for FMDV research as it is economical and the cards can be transported easily in envelopes by regular document transport methods. Live virus cannot be isolated from samples collected in FTA cards, which is a limitation. This property can be viewed as an advantage as it limits the risk of transmission of live virus.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yao, R; Chisela, W; Dorbu, G
2016-06-15
Purpose: To evaluate clinical usefulness of AlignRT (Vision RT Ltd., London, UK) in reducing patient positioning errors in breast irradiation. Methods: 60 patients undergoing whole breast irradiation were selected for this study. Patients were treated to the left or right breast lying on Qfix Access breast board (Qfix, Avondale, PA) in supine position for 28 fractions using tangential fields. 30 patients were aligned using AlignRT by aligning a breast surface region of interest (ROI) to the same area from a reference surface image extracted from planning CT. When the patient’s surface image deviated from the reference by more than 3mmmore » on one or more translational and rotational directions, a new reference was acquired using AlignRT in-room cameras. The other 30 patients were aligned to the skin marks with room lasers. On-Board MV portal images of medial field were taken daily and matched to the DRRs. The magnitude and frequency of positioning errors were determined from measured translational shifts. Kolmogorov-Smirnov test was used to evaluate statistical differences of positional accuracy and precision between AlignRT and non-AlignRT patients. Results: The percentage of port images with no shift required was 46.5% and 27.0% in vertical, 49.8% and 25.8% in longitudinal, 47.6% and 28.5% in lateral for AlignRT and non-AlignRT patients, respectively. The percentage of port images requiring more than 3mm shifts was 18.1% and 35.1% in vertical, 28.6% and 50.8% in longitudinal, 11.3% and 24.2% in lateral for AlignRT and non-AlignRT patients, respectively. Kolmogorov-Smirnov test showed that there were significant differences between the frequency distributions of AlignRT and non-AlignRT in vertical, longitudinal, and lateral shifts. Conclusion: As confirmed by port images, AlignRT-assisted patient positioning can significantly reduce the frequency and magnitude of patient setup errors in breast irradiation compared to the use of lasers and skin marks.« less
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USDA-ARS?s Scientific Manuscript database
Optical detection of bacteria has been approached in recent years as a bacteria detection method that can counter time restraints of traditional plating or the high reoccurring cost of real-time polymerase chain reaction (RT-PCR). The goal of optical detection is to identify bacteria with spectral s...
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Application of a Master Equation for Quantitative mRNA Analysis Using qRT-PCR
USDA-ARS?s Scientific Manuscript database
The qRT-PCR has been widely accepted as the assay of choice for mRNA quantification. Gene expression as measured by mRNA dynamics varies in response to different conditions and environmental stimuli. For conventional practice, housekeeping genes have been applied as internal reference for data nor...
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Zhu, Yu; Wang, Gui-Hua; Cui, Yu-Dong; Cui, Shang-Jin
2016-09-01
Porcine epidemic diarrhea virus (PEDV) can cause serious disease and even death in neonatal piglets, resulting in serious damage to the swine industry worldwide. Open reading frame 3 (ORF3) is the only accessory gene in the PEDV genome. Previous studies have indicated that PEDV vaccine strains have a partial deletion in ORF3. In this study, a nanoparticle-assisted polymerase chain reaction (nanoparticle-assisted RT-PCR) assay targeting the ORF3 of PEDV was developed to distinguish PEDV field strains from attenuated strains by using a specific pair of primers. The PCR products of field strains and attenuated strains were 264 bp and 215 bp in length, respectively. The sensitivity and specificity of this assay were also assessed. The nanoparticle-assisted RT-PCR assay was 10-100 times more sensitive than the conventional RT-PCR assay, with no cross-reactions when amplifying porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (RV), and porcine transmissible gastroenteritis virus (TGEV). The nanoparticle-assisted RT-PCR assay we describe here can be used to distinguish field strains from vaccine strains of PEDV, and it shows promise for reducing economic loss due to PEDV infection.
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A tool for design of primers for microRNA-specific quantitative RT-qPCR.
Busk, Peter K
2014-01-28
MicroRNAs are small but biologically important RNA molecules. Although different methods can be used for quantification of microRNAs, quantitative PCR is regarded as the reference that is used to validate other methods. Several commercial qPCR assays are available but they often come at a high price and the sequences of the primers are not disclosed. An alternative to commercial assays is to manually design primers but this work is tedious and, hence, not practical for the design of primers for a larger number of targets. I have developed the software miRprimer for automatic design of primers for the method miR-specific RT-qPCR, which is one of the best performing microRNA qPCR methods available. The algorithm is based on an implementation of the previously published rules for manual design of miR-specific primers with the additional feature of evaluating the propensity of formation of secondary structures and primer dimers. Testing of the primers showed that 76 out of 79 primers (96%) worked for quantification of microRNAs by miR-specific RT-qPCR of mammalian RNA samples. This success rate corresponds to the success rate of manual primer design. Furthermore, primers designed by this method have been distributed to several labs and used successfully in published studies. The software miRprimer is an automatic and easy method for design of functional primers for miR-specific RT-qPCR. The application is available as stand-alone software that will work on the MS Windows platform and in a developer version written in the Ruby programming language.
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[Detection of gene mutation in glucose-6-phosphate dehydrogenase deficiency by RT-PCR sequencing].
Lyu, Rong-Yu; Chen, Xiao-Wen; Zhang, Min; Chen, Yun-Sheng; Yu, Jie; Wen, Fei-Qiu
2016-07-01
Since glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common hereditary hemolytic erythrocyte enzyme deficiency, most cases have single nucleotide mutations in the coding region, and current test methods for gene mutation have some missed detections, this study aimed to investigate the feasibility of RT-PCR sequencing in the detection of gene mutation in G6PD deficiency. According to the G6PD/6GPD ratio, 195 children with anemia of unknown cause or who underwent physical examination between August 2013 and July 2014 were classified into G6PD-deficiency group with 130 children (G6PD/6GPD ratio <1.00) and control group with 65 children (G6PD/6GPD ratio≥1.00). The primer design and PCR amplification conditions were optimized, and RT-PCR sequencing was used to analyze the complete coding sequence and verify the genomic DNA sequence in the two groups. In the G6PD-deficiency group, the detection rate of gene mutation was 100% and 13 missense mutations were detected, including one new mutation. In the control group, no missense mutation was detected in 28 boys; 13 heterozygous missense mutations, 1 homozygous same-sense mutation (C1191T) which had not been reported in China and abroad, and 14 single nucleotide polymorphisms of C1311T were detected in 37 girls. The control group showed a high rate of missed detection of G6PD deficiency (carriers) in the specimens from girls (35%, 13/37). RT-PCR sequencing has a high detection rate of G6PD gene mutation and a certain value in clinical diagnosis of G6PD deficiency.
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Hao, Lu; Xie, Jipeng; Chen, Shanyi; Wang, Shaojie; Gong, Zhuoqun; Ling, Kai-Shu; Guo, Liyun; Fan, Zaifeng; Zhou, Tao
2016-08-01
Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV) are three latent viruses frequently occurring in apple trees worldwide. In field orchards, these viruses are frequently found in a mixed infection with viroids in the genus Apscarviroid, including Apple scar skin viroid, and Apple dimple fruit viroid. Together these viruses and viroids could cause serious damage to apple fruit production worldwide. Rapid and efficient detection methods are pivotal to identify and select the virus-free propagation material for healthy apple orchard management. In this study a multiplex Reverse Transcription-PCR (RT-PCR) was developed and optimized for simultaneous detection and differentiation of the three latent viruses and apscarviroids. With newly designed specific primers for ACLSV, ASGV, APSV, and EF-1α (as an internal control), and a pair of degenerate primers for apscarviroids, optimized parameters for multiplex RT-PCR were determined. The resulting PCR products from each target virus and viroid could be easily identified because their product sizes differ by at least a 100bp. The multiplex RT-PCR method is expected to detect different variants of the viruses as the test results showed that a variety of isolates from different regions in China gave positive results. To the best of our knowledge, this multiplex RT-PCR assay is the first to simultaneously detect multiple viruses and viroids infecting apple trees in a single reaction tube. This assay, therefore, offers a useful tool for routine certification and quarantine programs. Copyright © 2016 Elsevier B.V. All rights reserved.
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Development and evaluation of the RT-PCR kit for the rabies virus diagnosis.
Dedkov, Vladimir G; Deviatkin, A A; Poleschuk, E M; Safonova, M V; Markelov, M L; Shipulin, G A
To improve the diagnosis, surveillance, and control for the rabies virus, a kit for hybridization-triggered fluorescence detection of rabies virus DNA by the RT-PCR technique was developed and evaluated. The analytical sensitivity of the kit was 4*10 GE per ml. High specificity of the kit was shown using representative sampling of viral, bacterial, and human nucleic acids.
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Rhee, Chin Kook; Kim, Jin Woo; Hwang, Yong Il; Lee, Jin Hwa; Jung, Ki-Suck; Lee, Myung Goo; Yoo, Kwang Ha; Lee, Sang Haak; Shin, Kyeong-Cheol; Yoon, Hyoung Kyu
2015-01-01
Background and objective According to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines, either a modified Medical Research Council (mMRC) dyspnea score of ≥2 or a chronic obstructive pulmonary disease (COPD) assessment test (CAT) score of ≥10 is considered to represent COPD patients who are more symptomatic. We aimed to identify the ideal CAT score that exhibits minimal discrepancy with the mMRC score. Methods A receiver operating characteristic curve of the CAT score was generated for an mMRC scores of 1 and 2. A concordance analysis was applied to quantify the association between the frequencies of patients categorized into GOLD groups A–D using symptom cutoff points. A κ-coefficient was calculated. Results For an mMRC score of 2, a CAT score of 15 showed the maximum value of Youden’s index with a sensitivity and specificity of 0.70 and 0.66, respectively (area under the receiver operating characteristic curve [AUC] 0.74; 95% confidence interval [CI], 0.70–0.77). For an mMRC score of 1, a CAT score of 10 showed the maximum value of Youden’s index with a sensitivity and specificity of 0.77 and 0.65, respectively (AUC 0.77; 95% CI, 0.72–0.83). The κ value for concordance was highest between an mMRC score of 1 and a CAT score of 10 (0.66), followed by an mMRC score of 2 and a CAT score of 15 (0.56), an mMRC score of 2 and a CAT score of 10 (0.47), and an mMRC score of 1 and a CAT score of 15 (0.43). Conclusion A CAT score of 10 was most concordant with an mMRC score of 1 when classifying patients with COPD into GOLD groups A–D. However, a discrepancy remains between the CAT and mMRC scoring systems. PMID:26316736
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Gong, Zu-Kang; Wang, Shuang-Jie; Huang, Yong-Qi; Zhao, Rui-Qiang; Zhu, Qi-Fang; Lin, Wen-Zhen
2014-12-01
RT-qPCR is a commonly used method for evaluating gene expression; however, its accuracy and reliability are dependent upon the choice of appropriate reference gene(s), and there is limited information available on suitable reference gene(s) that can be used in mouse testis at different stages. In this study, using the RT-qPCR method, we investigated the expression variations of six reference genes representing different functional classes (Actb, Gapdh, Ppia, Tbp, Rps29, Hprt1) in mice testis during embryonic and postnatal development. The expression stabilities of putative reference genes were evaluated using five algorithms: geNorm, NormFinder, Bestkeeper, the comparative delta C(t) method and integrated tool RefFinder. Analysis of the results showed that Ppia, Gapdh and Actb were identified as the most stable genes and the geometric mean of Ppia, Gapdh and Actb constitutes an appropriate normalization factor for gene expression studies. The mRNA expression of AT1 as a test gene of interest varied depending upon which of the reference gene(s) was used as an internal control(s). This study suggested that Ppia, Gapdh and Actb are suitable reference genes among the six genes used for RT-qPCR normalization and provide crucial information for transcriptional analyses in future studies of gene expression in the developing mouse testis.
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A universal TaqMan-based RT-PCR protocol for cost-efficient detection of small noncoding RNA.
Jung, Ulrike; Jiang, Xiaoou; Kaufmann, Stefan H E; Patzel, Volker
2013-12-01
Several methods for the detection of RNA have been developed over time. For small RNA detection, a stem-loop reverse primer-based protocol relying on TaqMan RT-PCR has been described. This protocol requires an individual specific TaqMan probe for each target RNA and, hence, is highly cost-intensive for experiments with small sample sizes or large numbers of different samples. We describe a universal TaqMan-based probe protocol which can be used to detect any target sequence and demonstrate its applicability for the detection of endogenous as well as artificial eukaryotic and bacterial small RNAs. While the specific and the universal probe-based protocol showed the same sensitivity, the absolute sensitivity of detection was found to be more than 100-fold lower for both than previously reported. In subsequent experiments, we found previously unknown limitations intrinsic to the method affecting its feasibility in determination of mature template RISC incorporation as well as in multiplexing. Both protocols were equally specific in discriminating between correct and incorrect small RNA targets or between mature miRNA and its unprocessed RNA precursor, indicating the stem-loop RT-primer, but not the TaqMan probe, triggers target specificity. The presented universal TaqMan-based RT-PCR protocol represents a cost-efficient method for the detection of small RNAs.
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Sandercock, Peter; Lindley, Richard; Wardlaw, Joanna; Dennis, Martin; Innes, Karen; Cohen, Geoff; Whiteley, Will; Perry, David; Soosay, Vera; Buchanan, David; Venables, Graham; Czlonkowska, Anna; Kobayashi, Adam; Berge, Eivind; Slot, Karsten Bruins; Murray, Veronica; Peeters, Andre; Hankey, Graeme J; Matz, Karl; Brainin, Michael; Ricci, Stefano; Cantisani, Teresa A; Gubitz, Gordon; Phillips, Stephen J; Antonio, Arauz; Correia, Manuel; Lyrer, Phillippe; Kane, Ingrid; Lundstrom, Erik
2011-11-30
Intravenous recombinant tissue plasminogen activator (rtPA) is approved in Europe for use in patients with acute ischaemic stroke who meet strictly defined criteria. IST-3 sought to improve the external validity and precision of the estimates of the overall treatment effects (efficacy and safety) of rtPA in acute ischaemic stroke, and to determine whether a wider range of patients might benefit. International, multi-centre, prospective, randomized, open, blinded endpoint (PROBE) trial of intravenous rtPA in acute ischaemic stroke. Suitable patients had to be assessed and able to start treatment within 6 hours of developing symptoms, and brain imaging must have excluded intracranial haemorrhage and stroke mimics. The initial pilot phase was double blind and then, on 01/08/2003, changed to an open design. Recruitment began on 05/05/2000 and closed on 31/07/2011, by which time 3035 patients had been included, only 61 (2%) of whom met the criteria for the 2003 European approval for thrombolysis. 1617 patients were aged over 80 years at trial entry. The analysis plan will be finalised, without reference to the unblinded data, and published before the trial data are unblinded in early 2012. The main trial results will be presented at the European Stroke Conference in Lisbon in May 2012 with the aim to publish simultaneously in a peer-reviewed journal. The trial result will be presented in the context of an updated Cochrane systematic review. We also intend to include the trial data in an individual patient data meta-analysis of all the relevant randomised trials. The data from the trial will: improve the external validity and precision of the estimates of the overall treatment effects (efficacy and safety) of iv rtPA in acute ischaemic stroke; provide: new evidence on the balance of risk and benefit of intravenous rtPA among types of patients who do not clearly meet the terms of the current EU approval; and, provide the first large-scale randomised evidence on effects in
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Löf, Marie; Henriksson, Hanna; Forsum, Elisabet
2013-01-01
Activity energy expenditure (AEE) during free-living conditions can be assessed using devices based on different principles. To make proper comparisons of different devices' capacities to assess AEE, they should be evaluated in the same population. Thus, in the present study we evaluated, in the same group of subjects, the ability of three devices to assess AEE in groups and individuals during free-living conditions. In twenty women, AEE was assessed using RT3 (three-axial accelerometry) (AEERT3), Actiheart (a combination of heart rate and accelerometry) (AEEActi) and IDEEA (a multi-accelerometer system) (AEEIDEEA). Reference AEE (AEEref) was assessed using the doubly labelled water method and indirect calorimetry. Average AEEActi was 5760 kJ per 24 h and not significantly different from AEEref (5020 kJ per 24 h). On average, AEERT3 and AEEIDEEA were 2010 and 1750 kJ per 24 h lower than AEEref, respectively (P < 0·001). The limits of agreement (± 2 sd) were 2940 (Actiheart), 1820 (RT3) and 2650 (IDEEA) kJ per 24 h. The variance for AEERT3 was lower than for AEEActi (P = 0·006). The RT3 classified 60 % of the women in the correct activity category while the corresponding value for IDEEA and Actiheart was 30 %. In conclusion, the Actiheart may be useful for groups and the RT3 for individuals while the IDEEA requires further development. The results are likely to be relevant for a large proportion of Western women of reproductive age and demonstrate that the procedure selected to assess physical activity can greatly influence the possibilities to uncover important aspects regarding interactions between physical activity, diet and health.
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Mulick, Amy; Walker, Jane; Puntis, Stephen; Burke, Katy; Symeonides, Stefan; Gourley, Charlie; Wanat, Marta; Frost, Chris; Sharpe, Michael
2018-04-01
Comorbid major depression has been associated with worse survival in patients with cancer. However, we do not know if treating depression improves survival. In the SMaRT Oncology-2 (good prognosis cancers) and SMaRT Oncology-3 (lung cancer, a poor prognosis cancer) trials, we found that a depression treatment programme, Depression Care for People with Cancer (DCPC), was effective in reducing comorbid major depression. In this analysis, we aimed to identify whether DCPC also had an effect on survival. The trials were conducted in three cancer centres and their associated clinics in Scotland, UK. In SMaRT Oncology-2, outpatients with good prognosis cancers and major depression were randomly assigned in a 1:1 ratio to DCPC or usual care, with stratification (by trial centre) and minimisation (by age, primary cancer, and sex) with allocation concealment. In SMaRT Oncology-3, outpatients with lung cancer and major depression were randomly assigned (1:1 ratio) to DCPC or usual care with stratification (by trial centre) and minimisation (by age, sex, and cancer type) with allocation concealment. For this analysis, we obtained long-term data on deaths (all causes) in the SMaRT Oncology-2 and 3 trial participants, censored at July 31, 2015, and analysed survival as a trial outcome. We estimated unadjusted hazard ratios (HRs) for each trial using Cox regression, and pooled the log HRs in a fixed-effects meta-analysis. We recruited 642 participants; between May 12, 2008, and May 13, 2011, 500 participants were recruited to the SMaRT Oncology-2 trial and between Jan 5, 2009, and Sept 9, 2011, 142 participants were recruited to the SMaRT Oncology-3 trial. We followed up SMaRT Oncology-2 and SMaRT Oncology-3 participants for a median of 5 years and 1 year, respectively. 135 (27%) of 500 SMaRT Oncology-2 participants and 114 (80%) of 142 SMaRT Oncology-3 participants died within this period. We found no significant effect of DCPC on survival in the total follow-up period for
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Ram, Chet; Koramutla, Murali Krishna; Bhattacharya, Ramcharan
2017-07-01
Brassica juncea is a chief oil yielding crop in many parts of the world including India. With advancement of molecular techniques, RT-qPCR based study of gene-expression has become an integral part of experimentations in crop breeding. In RT-qPCR, use of appropriate reference gene(s) is pivotal. The virtue of the reference genes, being constant in expression throughout the experimental treatments, needs to be validated case by case. Appropriate reference gene(s) for normalization of gene-expression data in B. juncea during the biotic stress of aphid infestation is not known. In the present investigation, 11 reference genes identified from microarray database of Arabidopsis-aphid interaction at a cut off FDR ≤0.1, along with two known reference genes of B. juncea, were analyzed for their expression stability upon aphid infestation. These included 6 frequently used and 5 newly identified reference genes. Ranking orders of the reference genes in terms of expression stability were calculated using advanced statistical approaches such as geNorm, NormFinder, delta Ct and BestKeeper. The analysis suggested CAC, TUA and DUF179 as the most suitable reference genes. Further, normalization of the gene-expression data of STP4 and PR1 by the most and the least stable reference gene, respectively has demonstrated importance and applicability of the recommended reference genes in aphid infested samples of B. juncea. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
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Expósito-Rodríguez, Marino; Borges, Andrés A; Borges-Pérez, Andrés; Pérez, José A
2008-01-01
Background The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR. Results In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (CAC), SGN-U321250 (TIP41), SGN-U346908 ("Expressed") and SGN-U316474 (SAND) genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process. Conclusion This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time RT-PCR studies of gene
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Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M.; Zhao, Hui; Ma, Xiaoyue; Ellison, James A.; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian
2017-01-01
Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high
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Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M; Zhao, Hui; Ma, Xiaoyue; Ellison, James A; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian; Li, Yu
2017-01-01
Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high
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NASA Astrophysics Data System (ADS)
Deshpande, Ruchi; Thuptimdang, Wanwara; DeMarco, John; Liu, Brent J.
2014-03-01
We have built a decision support system that provides recommendations for customizing radiation therapy treatment plans, based on patient models generated from a database of retrospective planning data. This database consists of relevant metadata and information derived from the following DICOM objects - CT images, RT Structure Set, RT Dose and RT Plan. The usefulness and accuracy of such patient models partly depends on the sample size of the learning data set. Our current goal is to increase this sample size by expanding our decision support system into a collaborative framework to include contributions from multiple collaborators. Potential collaborators are often reluctant to upload even anonymized patient files to repositories outside their local organizational network in order to avoid any conflicts with HIPAA Privacy and Security Rules. We have circumvented this problem by developing a tool that can parse DICOM files on the client's side and extract de-identified numeric and text data from DICOM RT headers for uploading to a centralized system. As a result, the DICOM files containing PHI remain local to the client side. This is a novel workflow that results in adding only relevant yet valuable data from DICOM files to the centralized decision support knowledge base in such a way that the DICOM files never leave the contributor's local workstation in a cloud-based environment. Such a workflow serves to encourage clinicians to contribute data for research endeavors by ensuring protection of electronic patient data.
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Sprigg, Nikola; O’Connor, Rebecca; Woodhouse, Lisa; Krishnan, Kailash; England, Timothy J.; Connell, Louise A.; Walker, Marion F.; Bath, Philip M.
2016-01-01
is not supported. Future work should occur earlier after stroke, alongside on-going clinical rehabilitation. Trial Registration ISRCTN.com ISRCTN16714730 PMID:27610616
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de Oliveira, Anapolino Macedo; Fonseca, Antônio Augusto; Camargos, Marcelo Fernandes; Orzil, Lívia Maria; Laguardia-Nascimento, Mateus; Oliveira, Anna Gabriella Guimarães; Rodrigues, Jacqueline Gomes; Sales, Mariana Lázaro; de Oliveira, Tatiana Flávia Pinheiro; de Melo, Cristiano Barros
2018-07-01
Vesicular stomatitis is an infectious disease that occurs mainly in countries of the Western Hemisphere and affects cattle, swine and horses. The clinical symptoms in cattle and swine are similar to foot-and-mouth disease and include vesicular ulceration of the tongue and mouth. The disease requires a rapid and accurate differential diagnosis, aiming for immediate implementation of control measures. The objective of the present study was to develop and perform validation tests of multiplex RT-qPCR(s) for the detection of RNA from Alagoas vesiculovirus, considering the parameters of sensitivity and analytical specificity, analytical performance (repeatability and reproducibility criteria) and the uncertainty of the measurement. The threshold cycle values obtained in triplicate from each sample were evaluated by considering the variations between days, analysts and equipment in an analysis of variance aimed at determining the variances of repeatability and reproducibility. The results showed that RT-qPCRs had excellent sensitivity and specificity in the detection of RNA of the Alagoas vesiculovirus. The validation parameters showed low coefficients of variation and were equivalent to those found in other validation studies, indicating that the tests presented excellent repeatability and reproducibility. Copyright © 2018 Elsevier B.V. All rights reserved.
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An improved RT-IPCR for detection of pyrene and related polycyclic aromatic hydrocarbons.
Meng, X Y; Li, Y S; Zhou, Y; Sun, Y; Qiao, B; Si, C C; Hu, P; Lu, S Y; Ren, H L; Liu, Z S; Qiu, H J; Liu, J Q
2016-04-15
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous homogeneous chemicals which are well known by carcinogens, mutagens and endocrine disorder. Here, an improved real-time immuno-PCR (RT-IPCR) was developed for detection of pyrene and its homologs in water samples. The PAHs in sample compete with pyrene-modified DNA to bind with monoclonal antibody (McAb) coated on PCR plate. The reporter DNA was exponentially amplified by real-time PCR instrument using Fast Start universal SYBR Green Master (ROX) kit. Only two reaction steps were needed to accomplish the detection. The assay had a good linear range from 5 pmol L(-1) to 5 nmol L(-1) with a detection limit of 3.5 pmol L(-1). For application assay, the average recoveries from tap water, lake water and mineral water were 98.4%, 98.2% and 99.7%, respectively which showed a good correlation (R(2)=0.9906) with those from GC-MS. The results indicated that the improved RT-IPCR seems to be a potential method for simple and ultrasensitive detection of pyrene and some homologues in environment water samples. Copyright © 2015 Elsevier B.V. All rights reserved.
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Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress
USDA-ARS?s Scientific Manuscript database
Lolium temulentum is a valuable model grass species for the study of stress in forage and turf grasses. Gene expression analysis by quantitative real time RT-PCR relies on the use of proper internal standards. The aim of this study was to identify and evaluate reference genes for use in real-time q...
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NASA Technical Reports Server (NTRS)
Nebenfuhr, A.; Lomax, T. L.
1998-01-01
We have developed an improved method for determination of gene expression levels with RT-PCR. The procedure is rapid and does not require extensive optimization or densitometric analysis. Since the detection of individual transcripts is PCR-based, small amounts of tissue samples are sufficient for the analysis of expression patterns in large gene families. Using this method, we were able to rapidly screen nine members of the Aux/IAA family of auxin-responsive genes and identify those genes which vary in message abundance in a tissue- and light-specific manner. While not offering the accuracy of conventional semi-quantitative or competitive RT-PCR, our method allows quick screening of large numbers of genes in a wide range of RNA samples with just a thermal cycler and standard gel analysis equipment.
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Lee, Se Hee; Baek, Yun Hee; Kim, Yang-Hoon; Choi, Young-Ki; Song, Min-Suk; Ahn, Ji-Young
2017-01-01
Due to the limitation of rapid development of specific antiviral drug or vaccine for novel emerging viruses, an accurate and rapid diagnosis is a key to manage the virus spread. We developed an efficient and rapid method with high specificity for the Middle East Respiratory Syndrome coronavirus (MERS-CoV), based on one-pot reverse transcription loop-mediated isothermal amplification (one-pot RT-LAMP). A set of six LAMP primers [F3, B3, FIP, BIP, LF (Loop-F), and LB (Loop-B)] were designed using the sequence of nucleocapsid (N) gene with optimized RT-LAMP enzyme conditions: 100 U M-MLV RTase and 4 U Bst polymerase, implying that the reaction was able to detect four infectious viral genome copies of MERS-CoV within a 60 min reaction time period. Significantly, EvaGreen dye has better signal read-out properties in one-pot RT-LAMP reaction and is more compatible with DNA polymerase than SYBR green I. Isothermally amplified specific N genes were further evaluated using field-deployable microchamber devices, leading to the specific identification of as few as 0.4 infectious viral genome copies, with no cross-reaction to the other acute respiratory disease viruses, including influenza type A (H1N1 and H3N2), type B, human coronavirus 229E, and human metapneumovirus. This sensitive, specific and feasible method provides a large-scale technical support in emergencies, and is also applied as a sample-to-detection module in Point of Care Testing devices. PMID:28119682
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2009-01-01
Background In the global eradication program for poliomyelitis, the laboratory diagnosis plays a critical role by isolating poliovirus (PV) from the stool samples of acute flaccid paralysis (AFP) cases. In this study, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) system for a rapid and highly sensitive detection of enterovirus including PV to identify stool samples positive for enterovirus including PV. Methods A primer set was designed for RT-LAMP to detect enterovirus preferably those with PV-like 5'NTRs of the viral genome. The sensitivity of RT-LAMP system was evaluated with prototype strains of enterovirus. Detection of enterovirus from stool extracts was examined by using RT-LAMP system. Results We detected at least 400 copies of the viral genomes of PV(Sabin) strains within 90 min by RT-LAMP with the primer set. This RT-LAMP system showed a preference for Human enterovirus species C (HEV-C) strains including PV, but exhibited less sensitivity to the prototype strains of HEV-A and HEV-B (detection limits of 7,400 to 28,000 copies). Stool extracts, from which PV, HEV-C, or HEV-A was isolated in the cell culture system, were mostly positive by RT-LAMP method (positive rates of 15/16 (= 94%), 13/14 (= 93%), and 4/4 (= 100%), respectively). The positive rate of this RT-LAMP system for stool extracts from which HEV-B was isolated was lower than that of HEV-C (positive rate of 11/21 (= 52%)). In the stool samples, which were negative for enterovirus isolation by the cell culture system, we found that two samples were positive for RT-LAMP (positive rates of 2/38 (= 5.3%)). In these samples, enterovirus 96 was identified by sequence analysis utilizing a seminested PCR system. Conclusions RT-LAMP system developed in this study showed a high sensitivity comparable to that of the cell culture system for the detection of PV, HEV-A, and HEV-C, but less sensitivity to HEV-B. This RT-LAMP system would be useful for the direct
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2004-01-01
of RNA From Peripheral Blood Cells: A Validation Study for Molecular Diagnostics by Microarray and Kinetic RT-PCR Assays Application in...VALIDATION STUDY FOR MOLECULAR DIAGNOSTICS BY MICROARRAY AND KINETIC RT-PCR ASSAYS APPLICATION IN AEROSPACE MEDICINE INTRODUCTION Extraction of cellular
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Llerena, Juan P P; Araújo, Pedro; Mazzafera, Paulo
2018-01-01
Saccharum spontaneum has been used for the development of energy cane a crop aimed to be used for the production of second-generation ethanol, or lignocellulosic ethanol. Lignin is a main challenge in the conversion of cell wall sugars into ethanol. In our studies to isolate the genes the lignin biosynthesis in S. spontaneum we have had great difficulty in RT-PCR reactions. Thus, we evaluated the effectiveness of different additives in the amplification of these genes. While COMT and CCoAOMT genes did not need any additives for other genes there was no amplification (HCT, F5H, 4CL and CCR) or the yield was very low (CAD and C4H). The application of supplementary cDNA was enough to overcome the non-specificity and low yield for C4H and C3H, while the addition of 0.04% BSA + 2% formamide was effective to amplify 4CL, CCR, F5H and CCR. HCT was amplified only by addition of 0.04% BSA + 2% formamide + 0.1 M trehalose and amplification of PAL was possible with addition of 2% of DMSO. Besides optimization of expression assays, the results show that additives can act independently or synergistically.
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Policy Implications at the State and District Level with RtI for Gifted students
ERIC Educational Resources Information Center
Brown, Elissa F.; Abernethy, Sherry H.
2009-01-01
As a field, gifted education does not endorse any one approach to serving students because of the range of student abilities and resulting concomitant diverse needs. Therefore, service delivery in gifted education is still heavily teacher dependent. Yet, many of the components of Response to Intervention (RtI) are employed in gifted education,…
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Implementing psychological first-aid training for medical reserve corps volunteers.
Chandra, Anita; Kim, Jee; Pieters, Huibrie C; Tang, Jennifer; McCreary, Michael; Schreiber, Merritt; Wells, Kenneth
2014-02-01
We assessed the feasibility and impact on knowledge, attitudes, and reported practices of psychological first-aid (PFA) training in a sample of Medical Reserve Corps (MRC) members. Data have been limited on the uptake of PFA training in surge responders (eg, MRC) who are critical to community response. Our mixed-methods approach involved self-administered pre- and post-training surveys and within-training focus group discussions of 76 MRC members attending a PFA training and train-the-trainer workshop. Listen, protect, connect (a PFA model for lay persons) focuses on listening and understanding both verbal and nonverbal cues; protecting the individual by determining realistic ways to help while providing reassurance; and connecting the individual with resources in the community. From pre- to post-training, perceived confidence and capability in using PFA after an emergency or disaster increased from 71% to 90% (P < .01), but no significant increase was found in PFA-related knowledge. Qualitative analyses suggest that knowledge and intentions to use PFA increased with training. Brief training was feasible, and while results were modest, the PFA training resulted in greater reported confidence and perceived capability in addressing psychological distress of persons affected by public health threats. PFA training is a promising approach to improve surge responder confidence and competency in addressing postdisaster needs.
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Detection of Apoptosis and Necrosis in Normal Human Lung Cells Using 1H NMR Spectroscopy
NASA Astrophysics Data System (ADS)
Shih, Chwen-Ming; Ko, Wun-Chang; Yang, Liang-Yo; Lin, Chien-Ju; Wu, Jui-Sheng; Lo, Tsui-Yun; Wang, Shwu-Huey; Chen, Chien-Tsu
2005-05-01
This study aimed to detect apoptosis and necrosis in MRC-5, a normal human lung cell line, by using noninvasive proton nuclear magnetic resonance (1H NMR). Live MRC-5 cells were processed first for 1H NMR spectroscopy; subsequently their types and the percentage of cell death were assessed on a flow cytometer. Cadmium (Cd) and mercury (Hg) induced apoptosis and necrosis in MRC-5 cells, respectively, as revealed by phosphatidylserine externalization on a flow cytometer. The spectral intensity ratio of methylene (CH2) resonance (at 1.3 ppm) to methyl (CH3) resonance (at 0.9 ppm) was directly proportional to the percentage of apoptosis and strongly and positively correlated with PI staining after Cd treatment (r2 = 0.9868, P < 0.01). In contrast, this ratio only increased slightly within 2-h Hg treatment, and longer Hg exposure failed to produce further increase. Following 2-h Hg exposure, the spectral intensity of choline resonance (at 3.2 ppm) was abolished, but this phenomenon was absent in Cd-induced apoptosis. These findings together demonstrate that 1H NMR is a novel tool with a quantitative potential to distinguish apoptosis from necrosis as early as the onset of cell death in normal human lung cells.
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Takekawa, John Y.; Iverson, Samuel A.; Schultz, Annie K.; Hill, Nichola J.; Cardona, Carol J.; Boyce, Walter M.; Dudley, Joseph P.
2010-01-01
Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAIV) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds is often conducted in remote regions, but results are often delayed because of limited local analytical capabilities, difficulties with sample transportation and permitting, or problems keeping samples cold in the field. In response to these challenges, the performance of a portable real-time, reverse transcriptase-polymerase chain reaction (rRT-PCR) unit (RAPID(Registered), Idaho Technologies, Salt Lake City, UT) that employed lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies) was compared to virus isolation combined with real-time RT-PCR conducted in a laboratory. This study included both field and experimental-based sampling. Field samples were collected from migratory shorebirds captured in northern California, while experimental samples were prepared by spiking fecal material with an H6N2 AIV isolate. Results indicated that the portable rRT-PCR unit had equivalent specificity to virus isolation with no false positives, but sensitivity was compromised at low viral titers. Use of portable rRT-PCR with lyophilized reagents may expedite surveillance results, paving the way to a better understanding of wild bird involvement in HPAIV H5N1 transmission.
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Minguzzi, Stefano; Terlizzi, Federica; Lanzoni, Chiara; Poggi Pollini, Carlo; Ratti, Claudio
2016-01-01
Many efforts have been made to develop a rapid and sensitive method for phytoplasma and virus detection. Taking our cue from previous works, different rapid sample preparation methods have been tested and applied to Candidatus Phytoplasma prunorum (‘Ca. P. prunorum’) detection by RT-qPCR. A duplex RT-qPCR has been optimized using the crude sap as a template to simultaneously amplify a fragment of 16S rRNA of the pathogen and 18S rRNA of the host plant. The specific plant 18S rRNA internal control allows comparison and relative quantification of samples. A comparison between DNA and RNA contribution to qPCR detection is provided, showing higher contribution of the latter. The method presented here has been validated on more than a hundred samples of apricot, plum and peach trees. Since 2013, this method has been successfully applied to monitor ‘Ca. P. prunorum’ infections in field and nursery. A triplex RT-qPCR assay has also been optimized to simultaneously detect ‘Ca. P. prunorum’ and Plum pox virus (PPV) in Prunus. PMID:26742106
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Use of Medical Reserve Corps Volunteers in a Hospital-based Disaster Exercise.
Gist, Ramon; Daniel, Pia; Grock, Andrew; Lin, Chou-Jui; Bryant, Clarence; Kohlhoff, Stephan; Roblin, Patricia; Arquilla, Bonnie
2016-06-01
Introduction The Medical Reserve Corps (MRC) is a national network of community-based volunteer groups created in 2002 by the Office of the United States Surgeon General (Rockville, Maryland USA) to augment the nation's ability to respond to medical and public health emergencies. However, there is little evidence-based literature available to guide hospitals on the optimal use of medical volunteers and hesitancy on the part of hospitals to use them. Hypothesis/Problem This study sought to determine how MRC volunteers can be used in hospital-based disasters through their participation in a full-scale exercise. A full-scale exercise was designed as a "Disaster Olympics," in which the Emergency Medicine residents were divided into teams tasked with completing one of the following five challenges: victim decontamination, mass casualty/decontamination tent assembly, patient triage and registration during a disaster, point of distribution (POD) site set-up and operation, and infection control management. A surge of patients potentially exposed to avian influenza was the scenario created for the latter three challenges. Some MRC volunteers were assigned clinical roles. These roles included serving as members of the suit support team for victim decontamination, distributing medications at the POD, and managing infection control. Other MRC volunteers functioned as "victim evaluators," who portrayed the potential avian influenza victims while simultaneously evaluating various aspects of the disaster response. The MRC volunteers provided feedback on their experience and evaluators provided feedback on the performance of the MRC volunteers using evaluation tools. Twenty-eight (90%) MRC volunteers reported that they worked well with the residents and hospital staff, felt the exercise was useful, and were assigned clearly defined roles. However, only 21 (67%) reported that their qualifications were assessed prior to role assignment. For those MRC members who functioned as
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U.S. EPA, Pesticide Product Label, , 01/30/1985
2011-04-14
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de Wit, Bianca; Kinoshita, Sachiko
2015-01-01
The magnitude of the semantic priming effect is known to increase as the proportion of related prime-target pairs in an experiment increases. This relatedness proportion (RP) effect was studied in a lexical decision task at a short prime-target stimulus onset asynchrony (240 ms), which is widely assumed to preclude strategic prospective usage of the prime. The analysis of the reaction time (RT) distribution suggested that the observed RP effect reflected a modulation of a retrospective semantic matching process. The pattern of the RP effect on the RT distribution found here is contrasted to that reported in De Wit and Kinoshita's (2014) semantic categorization study, and it is concluded that the RP effect is driven by different underlying mechanisms in lexical decision and semantic categorization.
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Hoffmann, Bernd; Hoffmann, Donata; Henritzi, Dinah; Beer, Martin; Harder, Timm C
2016-06-03
Rapid and sensitive diagnostic approaches are of the utmost importance for the detection of humans and animals infected by specific influenza virus subtype(s). Cascade-like diagnostics starting with the use of pan-influenza assays and subsequent subtyping devices are normally used. Here, we demonstrated a novel low density array combining 32 TaqMan(®) real-time RT-PCR systems in parallel for the specific detection of the haemagglutinin (HA) and neuraminidase (NA) subtypes of avian and porcine hosts. The sensitivity of the newly developed system was compared with that of the pan-influenza assay, and the specificity of all RT-qPCRs was examined using a broad panel of 404 different influenza A virus isolates representing 45 different subtypes. Furthermore, we analysed the performance of the RT-qPCR assays with diagnostic samples obtained from wild birds and swine. Due to the open format of the array, adaptations to detect newly emerging influenza A virus strains can easily be integrated. The RITA array represents a competitive, fast and sensitive subtyping tool that requires neither new machinery nor additional training of staff in a lab where RT-qPCR is already established.
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Daou, Badih; Deprince, Maureen; D'Ambrosio, Robin; Tjoumakaris, Stavropoula; Rosenwasser, Robert H; Ackerman, Daniel J; Bell, Rodney; Tzeng, Diana L; Ghobrial, Michelle; Fernandez, Andres; Shah, Qaisar; Gzesh, Dan J; Murphy, Deborah; Castaldo, John E; Mathiesen, Claranne; Pineda, Maria Carissa; Jabbour, Pascal
2015-12-01
Recently, the FDA guidelines regarding the eligibility of patients with acute ischemic stroke to receive IV rt-PA have been modified and are not in complete accord with the latest AHA/ASA guidelines. The resultant differences may result in discrepancies in patient selection for intravenous thrombolysis. Several comprehensive stroke centers in the state of Pennsylvania have undertaken a collaborative effort to clarify and unify our own recommendations regarding how to reconcile these different guidelines. Seizure at onset of stroke, small previous strokes that are subacute or chronic, multilobar infarct involving more than one third of the middle cerebral artery territory on CT scan, hypoglycemia, minor or rapidly improving symptoms should not be considered as contraindications for intravenous thrombolysis. It is recommended to follow the AHA/ASA guidelines regarding blood pressure management and bleeding diathesis. Patients receiving factor Xa inhibitors and direct thrombin inhibitors within the preceding 48 h should be excluded from receiving IV rt-PA. CT angiography is effective in identifying candidates for endovascular therapy. Consultation with and/or transfer to a comprehensive stroke center should be an option where indicated. Patients should receive IV rt-PA up to 4.5h after the onset of stroke. The process of identifying patients who will benefit the most from IV rt-PA is still evolving. Considering the rapidity with which patients need to be evaluated and treated, it remains imperative that systems of care adopt protocols to quickly gather the necessary data and have access to expert consultation as necessary to facilitate best practices. Copyright © 2015 Elsevier B.V. All rights reserved.
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Using 4+ to grade near-normal muscle strength does not improve agreement.
O'Neill, Søren; Jaszczak, Sofie Louise Thomsen; Steffensen, Anne Katrine Søndergaard; Debrabant, Birgit
2017-01-01
Manual assessment of muscle strength is often graded using the ordinal Medical Research Council (MRC) scale. The scale has a number of inherent weaknesses, including poorly defined limits between grades '4' and '5' and very large differences in the span of muscle strength encompassed by each of the six grades. It is not necessarily obvious how to convert a manual muscle test finding into an MRC grade. Several modifications which include intermediate grades have been suggested to improve the MRC scale and the current study examines whether agreement improves and variation in ratings decrease, with an intermediate grade between '4' and '5', in circumstances where such a grade would seem appropriate. The present study examined the hypothesis, that a modified MRC-scale which included the commonly used '4+' option, resulted in greater agreement between clinicians compared to the standard MRC-scale. A questionnaire containing five simple clinical cases were distributed to a large convenience sample of chiropractors in Northern Europe, with instructions to grade the described muscle strength findings using the MRC scale. The scale was adapted (with/without an intermediate '4+' grade) depending on the preference of the individual respondent. The cases were designed in such a way as to suggest a muscle weakness in the grey area between '4' and '5', i.e. grade '4+' on the modified MRC scale. A total of 225 questionnaires were returned (7% response rate). The average percentage agreement (across cases) in the standard MRC group was 64% [range 51%: 73%] (grade '4' in all cases). In the modified MRC group, the corresponding findings was 48% [38%: 74%] (grade '4' or '4+' in all cases). The mean average deviation analogue in the standard MRC group was 0.34 (range 0.34: 0.40), compared to 0.51 (range 0.39: 0.73) in the modified MRC group, indicating greater dispersion of scores in the modified MRC group. The Fleiss kappa was 0.02 ( p < 0.001) and 0.13 ( p < 0
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2009-01-01
Quantization of gene expression requires that an accurate measurement of a specific transcript is made. In this paper, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) by competition for tilapia growth hormone receptor type I is designed and validated. This experimental procedure was used to determine the abundance of growth hormone receptor type I transcript in different tilapia tissues. The results obtained with this developed competitive RT-PCR were similar to real-time PCR results reported recently. This protocol provides a reliable alternative, but less expensive than real-time PCR to quantify specific genes. PMID:19495916
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Hu, Hui; Jung, Kwonil; Wang, Qiuhong; Saif, Linda J; Vlasova, Anastasia N
2018-06-01
Coronaviruses (CoVs) are critical human and animal pathogens because of their potential to cause severe epidemics of respiratory or enteric diseases. In pigs, the newly emerged porcine deltacoronavirus (PDCoV) and re-emerged porcine epidemic diarrhea virus (PEDV) reported in the US and Asia, as well as the discovery of novel CoVs in wild bats or birds, has necessitated development of improved detection and control measures for these CoVs. Because the previous pancoronavirus (panCoV) RT-PCR established in our laboratory in 2007-2011 did not detect deltacoronaviruses (δ-CoVs) in swine fecal and serum samples, our goal was to develop a new panCoV RT-PCR assay to detect known human and animal CoVs, including δ-CoVs. In this study, we designed a new primer set to amplify a 668 bp-region within the RNA-dependent RNA polymerase (RdRP) gene that encodes the most conserved protein domain of α-, β-, γ-, and δ-CoVs. We established a one-step panCoV RT-PCR assay and standardized the assay conditions. The newly established panCoV RT-PCR assay was demonstrated to have a high sensitivity and specificity. Using a panel of 60 swine biological samples (feces, intestinal contents, and sera) characterized by PEDV, PDCoV and transmissible gastroenteritis virus-specific RT-PCR assays, we demonstrated that sensitivity and specificity of the newly established panCoV RT-PCR assay were 100%. 400 avian fecal (RNA) samples were further tested simultaneously for CoV by the new panCoV RT-PCR and a one-step RT-PCR assay with the δ-CoV nucleocapsid-specific universal primers. Four of 400 avian samples were positive for CoV, three of which were positive for δ-CoV by the conventional RT-PCR. PanCoV RT-PCR fragments for 3 of the 4 CoVs were sequenced. Phylogenetic analysis revealed the presence of one γ-CoV and two δ-CoV in the sequenced samples. The newly designed panCoV RT-PCR assay should be useful for the detection of currently known CoVs in animal biological samples. Copyright © 2018
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Untersuchung der Mira-Sterne RT Boo, TV Peg und VX Aur
NASA Astrophysics Data System (ADS)
Raetz, Kerstin; Berthold, Thomas
2015-02-01
The light-change of the Mira stars RT Boo, TV Peg and VX Aur was analyzed on three ways for a long time. In the first period I estimated the brightness of the variables on sky monitoring photo plates (red spectral range) from Sonneberg Observatory with Argelander¥s Method, in the last years I measured scanned photo plates with a photometry program and in addition I used visual observations from A.A.V.S.O. for the analysis. The behavior of the periods of the tree stars from 1965 to 2013 is described here.
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Letovanec, Igor; Finn, Stephen; Zygoura, Panagiota; Smyth, Paul; Soltermann, Alex; Bubendorf, Lukas; Speel, Ernst-Jan; Marchetti, Antonio; Nonaka, Daisuke; Monkhorst, Kim; Hager, Henrik; Martorell, Miguel; Sejda, Aleksandra; Cheney, Richard; Hernandez-Losa, Javier; Verbeken, Eric; Weder, Walter; Savic, Spasenija; Di Lorito, Alessia; Navarro, Atilio; Felip, Enriqueta; Warth, Arne; Baas, Paul; Meldgaard, Peter; Blackhall, Fiona; Dingemans, Anne-Marie; Dienemann, Hendrik; Dziadziuszko, Rafal; Vansteenkiste, Johan; O'Brien, Cathal; Geiger, Thomas; Sherlock, Jon; Schageman, Jeoffrey; Dafni, Urania; Kammler, Roswitha; Kerr, Keith; Thunnissen, Erik; Stahel, Rolf; Peters, Solange
2018-03-01
The reported prevalence of ALK receptor tyrosine kinase gene (ALK) rearrangement in NSCLC ranges from 2% to 7%. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proved to be a reproducible and sensitive technique. Reverse-transcriptase polymerase chain reaction (RT-PCR) has also been advocated, and most recently, the advent of targeted next-generation sequencing (NGS) for ALK and other fusions has become possible. This study compares anaplastic lymphoma kinase (ALK) evaluation with all four techniques in resected NSCLC from the large European Thoracic Oncology Platform Lungscape cohort. A total of 96 cases from the European Thoracic Oncology Platform Lungscape iBiobank, with any ALK immunoreactivity were examined by FISH, central RT-PCR, and NGS. An H-score higher than 120 defines IHC positivity. RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers covered the most frequent ALK translocations. For NGS, the Oncomine Solid Tumour Fusion Transcript Kit (Thermo Fisher Scientific, Waltham, MA) was used. The concordance was assessed using the Cohen κ coefficient (two-sided α ≤ 5%). NGS provided results for 77 of the 95 cases tested (81.1%), whereas RT-PCR provided results for 77 of 96 (80.2%). Concordance occurred in 55 cases of the 60 cases tested with all four methods (43 ALK negative and 12 ALK positive). Using ALK copositivity for IHC and FISH as the criterion standard, we derived a sensitivity for RT-PCR/NGS of 70.0%/85.0%, with a specificity of 87.1%/79.0%. When either RT-PCR or NGS was combined with IHC, the sensitivity remained the same, whereas the specificity increased to 88.7% and 83.9% respectively. NGS evaluation with the Oncomine Solid Tumour Fusion transcript kit and RT-PCR proved to have high sensitivity and specificity, advocating their use in routine practice. For maximal sensitivity and specificity, ALK status should be
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Warghane, Ashish; Misra, Pragati; Bhose, Sumit; Biswas, Kajal Kumar; Sharma, Ashwani Kumar; Reddy, M Krishna; Ghosh, Dilip Kumar
2017-12-01
Tristeza is a devastating disease of citrus and reported to be present in almost all countries where it is cultivated as a commercial crop. The etiological agent of this disease is Citrus tristeza virus (CTV), a member of the genus Closterovirus with in the family Closteroviridae. The pathogen is restricted to the phloem tissue of the infected citrus plant and has a monopartite ss (+) RNA genome of ∼20kb size. Till date, there is no effective control measure available for this virus. Management of tristeza depends on destruction of CTV infected field plants, production of virus-free planting material for new orchard establishment and controlling viruliferous aphid vectors responsible for field spread of the pathogen. Availability of rapid diagnostic assay is essential for rapid and efficient detection of the pathogen. In the present investigation, RT-LAMP (reverse transcription-loop mediated isothermal amplification), a highly sensitive, robust and low cost assay has been developed for rapid detection of CTV in infected citrus plant samples. Based on conserved nucleotide sequences available in GenBank and specific to p25 gene (major coat protein gene) of predominant CTV isolates of India, four primer sets (CTV-F3, CTV-B3, CTV-FIP and CTV-BIP) ware designed and custom synthesized. The amplified LAMP products obtained after maintaining isothermal condition of 65°C for 60min duration could be visible easily with necked eyes in presence of SYBR Green I (100X). Subsequently, LAMP products were verified by electrophoresis run in 1.5% agarose gel. The RT-LAMP results obtained with known CTV isolates maintained in screen house of CCRI, Nagpur were validated using field samples and thereafter it was further confirmed by conventional RT-PCR (reveres transcription-polymerase chain reaction) assay. The sensitivity of CTV-RT-LAMP protocol standardized in the present study was 100 times more than conventional one step RT-PCR assay. It also has maximum detection limit up to 0
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Monazah, A; Zeinoddini, M; Saeeidinia, A R
2017-08-01
Coxsackievirus B3 (CVB3) is a member of the genus Enterovirus within the family Picornaviridae and is an important pathogen of viral myocarditis, which accounts for more than 50% viral myocarditis cases. VP1 is major capsid protein that this region has a low homology in both amino acid and nucleotide sequences among Enteroviruses. Therefore we have chosen this region for designed a set of RT-LAMP primers for CVB3 detection. For this the total RNA was extracted from 24-h post infected-HeLa cells with complete cytopathic effect (CPE), and applied to a one-step reverse transcription loop-mediated isothermal amplification reaction (RT-LAMP) using CVB3-specific primers. The optimization of RT-LAMP reaction was carried out with three variables factors including MgSO 4 concentration, temperature and time of incubation. Amplification was analyzed by using 2% agarose gel electrophoresis and ethidium bromide and SYBR Green staining. Our results were shown the ladder-like pattern of the VP1 gene amplification. The LAMP reaction mix was optimized and the best result observed at 4mM MgSO 4 and 60°C for 90min incubation. RT-LAMP had high sensitivity and specificity for detection of CVB3 infection. This method can be used as a rapid and easy diagnostic test for detection of CVB3 in clinical laboratories. Copyright © 2017 Elsevier B.V. All rights reserved.
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Rawat, B; Loewy, J
1996-08-01
To evaluate the role of magnetic resonance cholangiography (MRC) in patients with suspected choledocholithiasis. Twenty-six consecutive patients with suspected choledocholithiasis (11 men and 15 women ranging in age from 25 to 81 years) underwent three-dimensional gradient-echo MRC; each patient also underwent endoscopic retrograde cholangiography or operative cholangiography. Each set of images for each patient was reviewed independently by a radiologist who was unaware of the results of the other type of imaging. Diagnostic-quality MRC images were obtained for 17 of the patients. Of these, 13 had stones in the common bile duct, as confirmed by another imaging method, and MRC indicated the presence of these stones in all 13 patients. In the other four patients bile duct obstruction was due to either acute pancreatitis (in three) or cholangiocarcinoma (in one). For seven of the nine nondiagnostic-quality MRC studies, the bile duct was not obstructed, so there was no bile stasis and the MRC images could not be obtained. Motion artifacts due to inability to hold the breath were the limiting factors in the other two patients. Although MRC has some limitations, this new noninvasive technique may be used as a screening test in selected patients with suspected choledocholithiasis.
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77 FR 57088 - Agency Information Collection Activities; Proposed Collection; Public Comment Request
Federal Register 2010, 2011, 2012, 2013, 2014
2012-09-17
... States, and to continue planning for future emergencies that are national in scope, detailed information... the Surgeon General. MRC Unit Leaders are asked to update this information on the MRC Web site at... Total burden respondents respondent per response hours MRC Unit Leader 1,000 6 1.0 6,000 Keith A. Tucker...
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Functional Consequences of Mannose and Asialoglycoprotein Receptor Ablation*
Mi, Yiling; Coonce, Marcy; Fiete, Dorothy; Steirer, Lindsay; Dveksler, Gabriela; Townsend, R. Reid; Baenziger, Jacques U.
2016-01-01
The mannose receptor (ManR, Mrc1) and asialoglycoprotein receptor (ASGR, Asgr1 and Asgr2) are highly abundant endocytic receptors expressed by sinusoidal endothelial cells and parenchymal cells in the liver, respectively. We genetically manipulated either receptor individually or in combination, revealing phenotypic changes in female and male mice associated with changes in circulating levels of many glycoproteins. Both receptors rise and fall in response to progesterone during pregnancy. Thirty percent of Asgr2−/− and 65% of Mrc1−/−Asgr2−/− mice are unable to initiate parturition at the end of pregnancy, whereas Mrc1−/− mice initiate normally. Twenty five percent of Mrc1−/−Asgr2−/− male mice develop priapism when mating due to thrombosis of the penile vein, but neither Mrc1−/− nor Asgr2−/− mice do so. The half-life for luteinizing hormone (LH) clearance increases in Mrc1−/− and Mrc1−/−Asgr2−/− mice but not in Asgr2−/− mice; however, LH and testosterone are elevated in all three knockouts. The ManR clears LH thus regulating testosterone production, whereas the ASGR appears to mediate clearance of an unidentified glycoprotein that increases LH levels. More than 40 circulating glycoproteins are elevated >3.0-fold in pregnant Mrc1−/−Asgr2−/− mice. Pregnancy-specific glycoprotein 23, undetectable in WT mice (<50 ng/ml plasma), reaches levels of 1–10 mg/ml in the plasma of Mrc1−/−Asgr2−/− and Asgr2−/− mice, indicating it is cleared by the ASGR. Elevation of multiple coagulation factors in Mrc1−/−Asgr2−/− mice may account for priapism seen in males. These male and female phenotypic changes underscore the key roles of the ManR and ASGR in controlling circulating levels of numerous glycoproteins critical for regulating reproductive hormones and blood coagulation. PMID:27405760
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DOE Office of Scientific and Technical Information (OSTI.GOV)
McGurk, R; Green, R; Lawrence, M
2015-06-15
Purpose: The dose-dependent nature of radiation therapy (RT)-induced lung injury following hypo-fractionated stereotactic RT is unclear. We herein report preliminary results of a prospective study assessing the magnitude of RT-induced reductions in regional lung perfusion following hypo-fractionated stereotactic RT. Methods: Four patients undergoing hypo-fractionated stereotactic lung RT (SBRT: 12 Gy x 4 fractions or 10 Gy x 5 fractions) had a pre-treatment SPECT (single-photon emission computed tomography) perfusion scan providing a 3D map of regional lung perfusion. Scans were repeated 3–6 months post-treatment. Pre- and post SPECT scans were registered to the planning CT scan (and hence the 3D dosemore » data). Changes in regional perfusion (counts per cc on the pre-post scans) were computed in regions of the lung exposed to different doses of radiation (in 5 Gy intervals), thus defining a dose-response function. SPECT scans were internally normalized to the regions receiving <5 Gy. Results: At 3 months post-RT, the changes in perfusion are highly variable. At 6 months, there is a consistent dose-dependent reduction in regional perfusion. The average percent decline in regional perfusion was 10% at 15–20 Gy, 20% at 20–25 Gy, and 30% at 25–30 Gy representing a relatively linear dose response with an approximate 2% reduction per Gray for doses in excess of 10 Gy. There was a subtle increase in perfusion in the lung receiving <10 Gy. Conclusion: Hypo-fractionated stereotactic RT appears to cause a dose-dependent reduction in regional lung perfusion. There appears to be a threshold effect with no apparent perfusion loss at doses <10 Gy, though this might be in part due to the normalization technique used. Additional data is needed from a larger number of patients to better assess this issue. This sort of data can be used to assist optimizing RT treatment plans that minimize the risk of lung injury. Partly supported by the NIH (CA69579) and the Lance
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Zubakov, Dmitry; Boersma, Anton W. M.; Choi, Ying; van Kuijk, Patricia F.; Wiemer, Erik A. C.
2010-01-01
MicroRNAs (miRNAs) are non-protein coding molecules with important regulatory functions; many have tissue-specific expression patterns. Their very small size in principle makes them less prone to degradation processes, unlike messenger RNAs (mRNAs), which were previously proposed as molecular tools for forensic body fluid identification. To identify suitable miRNA markers for forensic body fluid identification, we first screened total RNA samples derived from saliva, semen, vaginal secretion, and venous and menstrual blood for the expression of 718 human miRNAs using a microarray platform. All body fluids could be easily distinguished from each other on the basis of complete array-based miRNA expression profiles. Results from quantitative reverse transcription PCR (RT-PCR; TaqMan) assays for microarray candidate markers confirmed strong over-expression in the targeting body fluid of several miRNAs for venous blood and several others for semen. However, no candidate markers from array experiments for other body fluids such as saliva, vaginal secretion, or menstrual blood could be confirmed by RT-PCR. Time-wise degradation of venous blood and semen stains for at least 1 year under lab conditions did not significantly affect the detection sensitivity of the identified miRNA markers. The detection limit of the TaqMan assays tested for selected venous blood and semen miRNA markers required only subpicogram amounts of total RNA per single RT-PCR test, which is considerably less than usually needed for reliable mRNA RT-PCR detection. We therefore propose the application of several stable miRNA markers for the forensic identification of blood stains and several others for semen stain identification, using commercially available TaqMan assays. Additional work remains necessary in search for suitable miRNA markers for other forensically relevant body fluids. Electronic supplementary material The online version of this article (doi:10.1007/s00414-009-0402-3) contains
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Gautam, Rashi; Mijatovic-Rustempasic, Slavica; Esona, Mathew D; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D
2016-01-01
Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8-100% sensitivity, 99.7-100% specificity, 85-95% efficiency and a limit of detection of 4-60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81-92% efficiency and limit of detection of 150-600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98
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Mijatovic-Rustempasic, Slavica; Esona, Mathew D.; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D.
2016-01-01
Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8
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Li, B; Matter, E K; Hoppert, H T; Grayson, B E; Seeley, R J; Sandoval, D A
2014-02-01
Obesity has a complicated metabolic pathology, and defining the underlying mechanisms of obesity requires integrative studies with molecular end points. Real-time quantitative PCR (RT-qPCR) is a powerful tool that has been widely utilized. However, the importance of using carefully validated reference genes in RT-qPCR seems to have been overlooked in obesity-related research. The objective of this study was to select a set of reference genes with stable expressions to be used for RT-qPCR normalization in rats under fasted vs re-fed and chow vs high-fat diet (HFD) conditions. Male long-Evans rats were treated under four conditions: chow/fasted, chow/re-fed, HFD/fasted and HFD/re-fed. Expression stabilities of 13 candidate reference genes were evaluated in the rat hypothalamus, duodenum, jejunum and ileum using the ReFinder software program. The optimal number of reference genes needed for RT-qPCR analyses was determined using geNorm. Using geNorm analysis, we found that it was sufficient to use the two most stably expressed genes as references in RT-qPCR analyses for each tissue under specific experimental conditions. B2M and RPLP0 in the hypothalamus, RPS18 and HMBS in the duodenum, RPLP2 and RPLP0 in the jejunum and RPS18 and YWHAZ in the ileum were the most suitable pairs for a normalization study when the four aforementioned experimental conditions were considered. Our study demonstrates that gene expression levels of reference genes commonly used in obesity-related studies, such as ACTB or RPS18, are altered by changes in acute or chronic energy status. These findings underline the importance of using reference genes that are stable in expression across experimental conditions when studying the rat hypothalamus and intestine, because these tissues have an integral role in the regulation of energy homeostasis. It is our hope that this study will raise awareness among obesity researchers on the essential need for reference gene validation in gene expression
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Chen, Jingfang; Zhang, Rusheng; Ou, Xinhua; Yao, Dong; Huang, Zheng; Li, Linzhi; Sun, Biancheng
2017-06-01
A TaqMan based duplex one-step real time RT-PCR (rRT-PCR) assay was developed for the rapid detection of Coxsackievirus A10 (CV-A10) and other enterovirus (EVs) in clinical samples. The assay was fully evaluated and found to be specific and sensitive. When applied in 115 clinical samples, a 100% diagnostic sensitivity in CV-A10 detection and 97.4% diagnostic sensitivity in other EVs were found. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Tang, Yi; Chen, Hao; Diao, Youxiang
2016-01-01
Tembusu virus (TMUV) is a mosquito-borne flavivirus which threatens both poultry production and public health. In this study we developed a complete open reading frame alignment-based rRT-LAMP method for the universal detection of TUMV. To prevent false-positive results, the reaction was supplemented with uracil DNA glycosylase (UDG) to eliminate carryover contamination. The detection limit of the newly developed UDG-rRT-LAMP for TMUV was as low as 100 copies/reaction of viral RNA and 1 × 100.89 − 1 × 101.55 tissue culture infectious dose/100 μL of viruses. There were no cross-reactions with other viruses, and the reproducibility of the assay was confirmed by intra- and inter-assay tests with variability ranging from 0.22–3.33%. The new UDG-rRT-LAMP method for TMUV produced the same results as viral isolation combined with RT-PCR as the “gold standard” in 96.88% of cases for 81 clinical samples from subjects with suspected TMUV infection. The addition of UDG can eliminate as much as 1 × 10−16 g/reaction of contaminants, which can significantly reduce the likelihood of false-positive results during the rRT-LAMP reaction. Our result indicated that our UDG-rRT-LAMP is a rapid, sensitive, specific, and reliable method that can effectively prevent carryover contamination in the detection of TMUV. PMID:27270462
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Single reaction, real time RT-PCR detection of all known avian and human metapneumoviruses.
Lemaitre, E; Allée, C; Vabret, A; Eterradossi, N; Brown, P A
2018-01-01
Current molecular methods for the detection of avian and human metapneumovirus (AMPV, HMPV) are specifically targeted towards each virus species or individual subgroups of these. Here a broad range SYBR Green I real time RT-PCR was developed which amplified a highly conserved fragment of sequence in the N open reading frame. This method was sufficiently efficient and specific in detecting all MPVs. Its validation according to the NF U47-600 norm for the four AMPV subgroups estimated low limits of detection between 1000 and 10copies/μL, similar with detection levels described previously for real time RT-PCRs targeting specific subgroups. RNA viruses present a challenge for the design of durable molecular diagnostic test due to the rate of change in their genome sequences which can vary substantially in different areas and over time. The fact that the regions of sequence for primer hybridization in the described method have remained sufficiently conserved since the AMPV and HMPV diverged, should give the best chance of continued detection of current subgroups and of potential unknown or future emerging MPV strains. Copyright © 2017 Elsevier B.V. All rights reserved.
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Stable confinement of electron plasma and initial results on positron injection in RT-1
NASA Astrophysics Data System (ADS)
Saitoh, H.; Yoshida, Z.; Morikawa, J.; Yano, Y.; Kasaoka, N.; Sakamoto, W.; Nogami, T.
2013-03-01
The Ring Trap 1 (RT-1) device is a dipole field configuration generated by a levitated superconducting magnet. It offers very interesting opportunities for research on the fundamental properties on non-neutral plasmas, such as self-organization of charged particles in the strongly positive and negative charged particles on magnetic surfaces. When strong positron sources will be available in the future, the dipole field configuration will be potentially applicable to the formation of an electron-positron plasma. We have realized stable, long trap of toroidal pure electron plasma in RT-1; Magnetic levitation of the superconducting magnet resulted in more than 300s of confinement for electron plasma of ˜ 1011 m-3. Aiming for the confinement of positrons as a next step, we started a positron injection experiment. For the formation of positron plasma in the closed magnetic surfaces, one of the key issues to be solved is the efficient injection method of positron across closed magnetic surfaces. In contrast to linear configurations, toroidal configurations have the advantage that they are capable of trapping high energy positrons in the dipole field configuration and consider the possibility of direct trapping of positrons emitted from a 22Na source.
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Impact Evaluation of the Minnesota Reading Corps K-3 Program
ERIC Educational Resources Information Center
Markovitz, Carrie E.; Hernandez, Marc W.; Hedberg, Eric C.; Silberglitt, Benjamin
2014-01-01
Minnesota Reading Corps (MRC) is the largest AmeriCorps State program in the country. The goal of MRC is to ensure that students become successful readers and meet reading proficiency targets by the end of the third grade. To meet this goal, the MRC program, and its host organization, ServeMinnesota Action Network, recruit, train, place and…
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[Evolution in muscle strength in critical patients with invasive mechanical ventilation].
Via Clavero, G; Sanjuán Naváis, M; Menéndez Albuixech, M; Corral Ansa, L; Martínez Estalella, G; Díaz-Prieto-Huidobro, A
2013-01-01
To assess the evolution of muscle strength in critically ill patients with mechanical ventilation (MV) from withdrawal of sedatives to hospital discharge. A cohort study was conducted in two intensive care units in the Hospital Universitari de Bellvitge from November 2011 to March 2012. Consecutive patients with MV > 72h. Dependent outcome: Muscle strength measured with the Medical Research Council (MRC) scale beginning on the first day the patient was able to answer 3 out of 5 simple orders (day 1), every week, at ICU discharge and at hospital discharge or at day 60 Independent outcomes: factors associated with muscle strength loss, ventilator-free days, ICU length of stay and hospital length of stay. The patients were distributed into two groups (MRC< 48, MRC ≥ 48) after the first measurement. Thirty-four patients were assessed. Independent outcomes associated with muscle strength weakness were: days with cardiovascular SOFA >2 (P<.001) and days with costicosteroids (P<.001). Initial MRC in MRC<48 group was 38 (27-43), and 52 (50-54) in MRC ≥ 48. The largest muscle strength gain was obtained the first week (31% versus 52%). A MRC < 48 value was associated with more MV days (P<.007) and a longer ICU stay. (P<.003). The greatest muscle strength gain after withdrawing of the sedatives was achieved in the first week. Muscle strength loss was associated with a cardiovascular SOFA > 2 and costicosteroids. Patients with a MRC < 48 required more days with MV and a longer ICU stay. Copyright © 2013 Elsevier España, S.L. y SEEIUC. All rights reserved.
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Seymour, Matthew T; Thompson, Lindsay C; Wasan, Harpreet S; Middleton, Gary; Brewster, Alison E; Shepherd, Stephen F; O'Mahony, M Sinead; Maughan, Timothy S; Parmar, Mahesh; Langley, Ruth E
2011-05-21
Elderly and frail patients with cancer, although often treated with chemotherapy, are under-represented in clinical trials. We designed FOCUS2 to investigate reduced-dose chemotherapy options and to seek objective predictors of outcome in frail patients with advanced colorectal cancer. We undertook an open, 2 × 2 factorial trial in 61 UK centres for patients with previously untreated advanced colorectal cancer who were considered unfit for full-dose chemotherapy. After comprehensive health assessment (CHA), patients were randomly assigned by minimisation to: 48-h intravenous fluorouracil with levofolinate (group A); oxaliplatin and fluorouracil (group B); capecitabine (group C); or oxaliplatin and capecitabine (group D). Treatment allocation was not masked. Starting doses were 80% of standard doses, with discretionary escalation to full dose after 6 weeks. The two primary outcome measures were: addition of oxaliplatin ([A vs B] + [C vs D]), assessed with progression-free survival (PFS); and substitution of fluorouracil with capecitabine ([A vs C] + [B vs D]), assessed by change from baseline to 12 weeks in global quality of life (QoL). Analysis was by intention to treat. Baseline clinical and CHA data were modelled against outcomes with a novel composite measure, overall treatment utility (OTU). This study is registered, number ISRCTN21221452. 459 patients were randomly assigned (115 to each of groups A-C, 114 to group D). Factorial comparison of addition of oxaliplatin versus no addition suggested some improvement in PFS, but the finding was not significant (median 5·8 months [IQR 3·3-7·5] vs 4·5 months [2·8-6·4]; hazard ratio 0·84, 95% CI 0·69-1·01, p=0·07). Replacement of fluorouracil with capecitabine did not improve global QoL: 69 of 124 (56%) patients receiving fluorouracil reported improvement in global QoL compared with 69 of 123 (56%) receiving capecitabine. The risk of having any grade 3 or worse toxic effect was not significantly increased
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Kumar, Jyoti S; Saxena, Divyasha; Parida, Manmohan
2014-01-01
The recent outbreaks of West Nile Virus (WNV) in the Northeastern American continents and other regions of the world have made it essential to develop an efficient protocol for surveillance of WN virus. Nucleic acid based techniques like, RT-PCR have the advantage of sensitivity, specificity and rapidity. A one step single tube Env gene specific real-time RT-PCR was developed for early and reliable clinical diagnosis of WNV infection in clinical samples. The applicability of this assay for clinical diagnosis was validated with 105 suspected acute-phase serum and plasma samples from the recent epidemic of mysterious fever in Tamil Nadu, India in 2009-10. The comparative evaluation revealed the higher sensitivity of real-time RT-PCR assay by picking up 4 additional samples with low copy number of template in comparison to conventional RT-PCR. All the real-time positive samples further confirmed by CDC reported TaqMan real-time RT-PCR and quantitative real-time RT-PCR assays for the simultaneous detection of WNV lineage 1 and 2 strains. The quantitation of the viral load samples was done using a standard curve. These findings demonstrated that the assay has the potential usefulness for clinical diagnosis due to detection and quantification of WNV in acute-phase patient serum samples. Copyright © 2014 Elsevier Ltd. All rights reserved.
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NASA Technical Reports Server (NTRS)
Jedlovec, Gary J.; Molthan, Andrew; Zavodsky, Bradley T.; Case, Jonathan L.; LaFontaine, Frank J.; Srikishen, Jayanthi
2010-01-01
The NASA Short-term Prediction Research and Transition Center (SPoRT)'s new "Weather in a Box" resources will provide weather research and forecast modeling capabilities for real-time application. Model output will provide additional forecast guidance and research into the impacts of new NASA satellite data sets and software capabilities. By combining several research tools and satellite products, SPoRT can generate model guidance that is strongly influenced by unique NASA contributions.
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Transitioning NPOESS Data to Weather Offices: The SPoRT Paradigm with EOS Data
NASA Technical Reports Server (NTRS)
Jedlovec, Gary
2009-01-01
Real-time satellite information provides one of many data sources used by NWS weather forecast offices (WFOs) to diagnose current weather conditions and to assist in short-term forecast preparation. While GOES satellite data provides relatively coarse spatial resolution coverage of the continental U.S. on a 10-15 minute repeat cycle, polar orbiting imagery has the potential to provide snapshots of weather conditions at high-resolution in many spectral channels. Additionally, polar orbiting sounding data can provide additional information on the thermodynamic structure of the atmosphere in data sparse regions of at asynoptic observation times. The NASA Short-term Prediction Research and Transition (SPoRT) project has demonstrated the utility of polar orbiting MODIS and AIRS data on the Terra and Aqua satellites to improve weather diagnostics and short-term forecasting on the regional and local scales. SPoRT scientists work directly forecasters at selected WFOS in the Southern Region (SR) to help them ingest these unique data streams into their AWIPS system, understand how to use the data (through on-site and distance learn techniques), and demonstrate the utility of these products to address significant forecast problems. This process also prepares forecasters for the use of similar observational capabilities from NPOESS operational sensors. NPOESS environmental data records (EDRs) from the Visible 1 Infrared Imager I Radiometer Suite (VIIRS), the Cross-track Infrared Sounder (CrlS) and Advanced Technology Microwave Sounder (ATMS) instruments and additional value-added products produced by NESDIS will be available in near real-time and made available to WFOs to extend their use of NASA EOS data into the NPOESS era. These new data streams will be integrated into the NWs's new AWIPS II decision support tools. The AWIPS I1 system to be unveiled in WFOs in 2009 will be a JAVA-based decision support system which preserves the functionality of the existing systems and
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2011-01-01
. Trial registration ISRCTN: ISRCTN02694183 PMID:21952034
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Prolongation of ERP latency and reaction time (RT) in simultaneous EEG/fMRI data acquisition.
Chun, Jinsoo; Peltier, Scott J; Yoon, Daehyun; Manschreck, Theo C; Deldin, Patricia J
2016-08-01
Recording EEG and fMRI data simultaneously inside a fully-operating scanner has been recognized as a novel approach in human brain research. Studies have demonstrated high concordance between the EEG signals and hemodynamic response. However, a few studies reported altered cognitive process inside the fMRI scanner such as delayed reaction time (RT) and reduced and/or delayed N100 and P300 event-related brain potential (ERP) components. The present study investigated the influence of electromagnetic field (static magnetic field, radio frequency (RF) pulse, and gradient switching) and experimental environment on posterior N100 and P300 ERP components in four different settings with six healthy subjects using a visual oddball task: (1) classic fMRI acquisition inside the scanner (e.g., supine position, mirror glasses for stimulus presentation), (2) standard behavioral experiment outside the scanner (e.g., seated position, keyboard response), (3) controlled fMRI acquisition inside the scanner (e.g., organic light-emitting diode (OLED) goggles for stimulus presentation) inside; and (4) modified behavioral experiment outside the scanner (e.g., supine position, OLED goggles). The study findings indicated that the experimental environment in simultaneous EEG/fMRI acquisition could substantially delay N1P, P300 latency, and RT inside the scanner, and was associated with a reduced N1P amplitude. There was no effect of electromagnetic field in the prolongation of RT, N1P and P300 latency inside the scanner. N1P, but not P300, latency was sensitive to stimulus presentation method inside the scanner. Future simultaneous EEG/fMRI data collection should consider experimental environment in both design and analysis. Copyright © 2016 Elsevier B.V. All rights reserved.
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Green, Clayton B; Cheng, Georgina; Chandra, Jyotsna; Mukherjee, Pranab; Ghannoum, Mahmoud A; Hoyer, Lois L
2004-02-01
An RT-PCR assay was developed to analyse expression patterns of genes in the Candida albicans ALS (agglutinin-like sequence) family. Inoculation of a reconstituted human buccal epithelium (RHE) model of mucocutaneous candidiasis with strain SC5314 showed destruction of the epithelial layer by C. albicans and also formation of an upper fungal layer that had characteristics similar to a biofilm. RT-PCR analysis of total RNA samples extracted from C. albicans-inoculated buccal RHE showed that ALS1, ALS2, ALS3, ALS4, ALS5 and ALS9 were consistently detected over time as destruction of the RHE progressed. Detection of transcripts from ALS7, and particularly from ALS6, was more sporadic, but not associated with a strictly temporal pattern. The expression pattern of ALS genes in C. albicans cultures used to inoculate the RHE was similar to that observed in the RHE model, suggesting that contact of C. albicans with buccal RHE does little to alter ALS gene expression. RT-PCR analysis of RNA samples extracted from model denture and catheter biofilms showed similar gene expression patterns to the buccal RHE specimens. Results from the RT-PCR analysis of biofilm RNA specimens were consistent between various C. albicans strains during biofilm development and were comparable to gene expression patterns in planktonic cells. The RT-PCR assay described here will be useful for analysis of human clinical specimens and samples from other disease models. The method will provide further insight into the role of ALS genes and their encoded proteins in the diverse interactions between C. albicans and its host.
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Biggerstaff, Brad J.; Tanner, Nathan A.; Lauterbach, Molly; Lanciotti, Robert S.
2017-01-01
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. PMID:28945787
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Calvert, Amanda E; Biggerstaff, Brad J; Tanner, Nathan A; Lauterbach, Molly; Lanciotti, Robert S
2017-01-01
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.
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Wu, Yi-Cheng; Chang, Il-Chi; Wang, Chi-Liang; Chen, Tai-Di; Chen, Ya-Ting; Liu, Hui-Ping; Chu, Yen; Chiu, Yu-Ting; Wu, Tzu-Hua; Chou, Li-Hui; Chen, Yi-Rong; Huang, Shiu-Feng
2013-01-01
Recently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK) fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib) treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain. Reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in Situ hybridization (FISH) and Immunohistochemical (IHC) stain were performed on tumor tissues of 312 NSCLC patients for detection of ALK rearrangements. Mutation analyses for EGFR and KRAS genes were also performed. Thirteen of the 312 patients (4.17%) had ALK rearrangements detected by RT-PCR. If RT-PCR data was used as the gold standard, FISH tests had a low sensitivity (58.33%), but very good specificity (99.32%). IHC stain had better sensitivity (91.67%) than FISH, but lower specificity (79.52%), when the cut off was IHC2+. All of the 8 patients with high abundance of EML4-ALK positive cells in tumor tissues (assessed by the signal intensities of the RT-PCR product), were also have high expression of ALK protein (IHC3+), and positive for FISH, except one failed in FISH. Variants 3a+3b (4/5, 80%) of EML4-ALK fusion gene were more common to have high abundance of EML4-ALK positive cells in tumor tissues than variant 1 (1/3, 33.3%). Meta-analysis of the published data of 2273 NSCLC patients revealed that variant 3 (23/44, 52.3%) was the most common type in Chinese population, while variant 1 (28/37, 75.7%) was most common in Caucasian. Among the three detection methods, RT-PCR could detect not only the presence of EML4-ALK fusion gene and their variant types, but also the abundance of EML4-ALK positive cells in NSCLC tumor tissues. The latter two factors might affect the treatment response to anti-ALK inhibitor. Including RT-PCR as a diagnostic test for ALK inhibitor treatment in the prospective clinical trials is recommended.
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Wang, Chi-Liang; Chen, Tai-Di; Chen, Ya-Ting; Liu, Hui-Ping; Chu, Yen; Chiu, Yu-Ting; Wu, Tzu-Hua; Chou, Li-Hui; Chen, Yi-Rong; Huang, Shiu-Feng
2013-01-01
Background Recently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK) fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib) treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain. Methods Reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in Situ hybridization (FISH) and Immunohistochemical (IHC) stain were performed on tumor tissues of 312 NSCLC patients for detection of ALK rearrangements. Mutation analyses for EGFR and KRAS genes were also performed. Results Thirteen of the 312 patients (4.17%) had ALK rearrangements detected by RT-PCR. If RT-PCR data was used as the gold standard, FISH tests had a low sensitivity (58.33%), but very good specificity (99.32%). IHC stain had better sensitivity (91.67%) than FISH, but lower specificity (79.52%), when the cut off was IHC2+. All of the 8 patients with high abundance of EML4-ALK positive cells in tumor tissues (assessed by the signal intensities of the RT-PCR product), were also have high expression of ALK protein (IHC3+), and positive for FISH, except one failed in FISH. Variants 3a+3b (4/5, 80%) of EML4-ALK fusion gene were more common to have high abundance of EML4-ALK positive cells in tumor tissues than variant 1 (1/3, 33.3%). Meta-analysis of the published data of 2273 NSCLC patients revealed that variant 3 (23/44, 52.3%) was the most common type in Chinese population, while variant 1 (28/37, 75.7%) was most common in Caucasian. Conclusions Among the three detection methods, RT-PCR could detect not only the presence of EML4-ALK fusion gene and their variant types, but also the abundance of EML4-ALK positive cells in NSCLC tumor tissues. The latter two factors might affect the treatment response to anti-ALK inhibitor. Including RT-PCR as a diagnostic test for ALK inhibitor treatment in the prospective clinical trials is recommended. PMID:23951022
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Ensor, Hannah M; Schwab, Claire; Russell, Lisa J; Richards, Sue M; Morrison, Heather; Masic, Dino; Jones, Lisa; Kinsey, Sally E; Vora, Ajay J; Mitchell, Christopher D; Harrison, Christine J; Moorman, Anthony V
2011-02-17
Deregulated expression of CRLF2 (CRLF2-d) arises via its juxtaposition to the IGH@ enhancer or P2RY8 promoter. Among 865 BCP-ALL children treated on MRC ALL97, 52 (6%) had CRLF2-d, but it was more prevalent among Down syndrome patients (54%). P2RY8-CRLF2 (n = 43) was more frequent than IGH@-CRLF2 (n = 9). CRLF2-d was not associated with age, sex, or white cell count, but IGH@-CRLF2 patients were older than P2RY8-CRLF2 patients (median 8 vs 4 years, P = .0017). Patients with CRLF2-d were more likely to present with enlarged livers and spleens (38% vs 18%, P < .001). CRLF2-d was not seen in conjunction with established chromosomal translocations but 6 (12%) cases had high hyperdiploidy, and 5 (10%) had iAMP21. Univariate analysis suggested that CRLF2-d was associated with an inferior outcome: (event-free survival [EFS] hazard ratio 2.27 [95% confidence interval 1.48-3.47], P < .001; OS 3.69 [2.34-5.84], P < .001). However, multivariate analysis indicated that its effect was mediated by other risk factors such as cytogenetics and DS status (EFS 1.45 [0.88-2.39], P = .140; OS 1.90 [1.08-3.36], P = .027). Although the outcome of IGH@-CRLF2 patients appeared inferior compared with P2RY8-CRLF2 patients, the result was not significant (EFS 2.69 [1.15-6.31], P = .023; OS 2.86 [1.15-6.79], P = .021). Therefore, we concluded that patients with CRLF2-d should be classified into the intermediate cytogenetic risk group.
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Szabó-Fodor, J; Bors, I; Szabó, A; Kovács, M
2016-08-01
In this study the occurrence of hidden fumonisin B1 (FB1) and fumonisin B2 (FB2) was analysed, on two cereal substrates (maize and rice), inoculated with Fusarium verticillioides (MRC 826), in order to determine the ratio of hidden FB1 and FB2. Two parallel methods were applied: an in vitro human digestion sample pre-treatment and the routine extraction procedure, in both cases with subsequent LC-MS analysis. It was found that all samples showed higher concentration of total fumonisin B1 after digestion, as compared to that of free fumonisin analysed only after extraction. The percentage of the hidden form by maize was 18.8 % (±2.4) for FB1 and 36.8 % (±3.8) for FB2, while for rice it was 32.3 % (±11.3) and 58.0 (±6.8), respectively, expressed as the proportion to total fumonisin B1, for the total dataset. Significant differences were found in the FB1 and FB2 concentration measured after the different digestion phases (saliva, gastric and duodenal) in case of both matrixes. The results are useful for human risk assessment, since both humans and animals may be exposed to markedly higher toxin load, as determined merely by conventional analytical methods.