Science.gov

Sample records for automated rna extraction

  1. Automated RNA Extraction and Purification for Multiplexed Pathogen Detection

    SciTech Connect

    Bruzek, Amy K.; Bruckner-Lea, Cindy J.

    2005-01-01

    Pathogen detection has become an extremely important part of our nation?s defense in this post 9/11 world where the threat of bioterrorist attacks are a grim reality. When a biological attack takes place, response time is critical. The faster the biothreat is assessed, the faster countermeasures can be put in place to protect the health of the general public. Today some of the most widely used methods for detecting pathogens are either time consuming or not reliable [1]. Therefore, a method that can detect multiple pathogens that is inherently reliable, rapid, automated and field portable is needed. To that end, we are developing automated fluidics systems for the recovery, cleanup, and direct labeling of community RNA from suspect environmental samples. The advantage of using RNA for detection is that there are multiple copies of mRNA in a cell, whereas there are normally only one or two copies of DNA [2]. Because there are multiple copies of mRNA in a cell for highly expressed genes, no amplification of the genetic material may be necessary, and thus rapid and direct detection of only a few cells may be possible [3]. This report outlines the development of both manual and automated methods for the extraction and purification of mRNA. The methods were evaluated using cell lysates from Escherichia coli 25922 (nonpathogenic), Salmonella typhimurium (pathogenic), and Shigella spp (pathogenic). Automated RNA purification was achieved using a custom sequential injection fluidics system consisting of a syringe pump, a multi-port valve and a magnetic capture cell. mRNA was captured using silica coated superparamagnetic beads that were trapped in the tubing by a rare earth magnet. RNA was detected by gel electrophoresis and/or by hybridization of the RNA to microarrays. The versatility of the fluidics systems and the ability to automate these systems allows for quick and easy processing of samples and eliminates the need for an experienced operator.

  2. Automated serial extraction of DNA and RNA from biobanked tissue specimens

    PubMed Central

    2013-01-01

    Background With increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand. The lack of such techniques designed for extraction from tissues results in a bottleneck in downstream genetic analyses, particularly in the field of cancer research. We have developed an automated procedure for tissue homogenization and extraction of DNA and RNA into separate fractions from the same frozen tissue specimen. A purpose developed magnetic bead based technology to serially extract both DNA and RNA from tissues was automated on a Tecan Freedom Evo robotic workstation. Results 864 fresh-frozen human normal and tumor tissue samples from breast and colon were serially extracted in batches of 96 samples. Yields and quality of DNA and RNA were determined. The DNA was evaluated in several downstream analyses, and the stability of RNA was determined after 9 months of storage. The extracted DNA performed consistently well in processes including PCR-based STR analysis, HaloPlex selection and deep sequencing on an Illumina platform, and gene copy number analysis using microarrays. The RNA has performed well in RT-PCR analyses and maintains integrity upon storage. Conclusions The technology described here enables the processing of many tissue samples simultaneously with a high quality product and a time and cost reduction for the user. This reduces the sample preparation bottleneck in cancer research. The open automation format also enables integration with upstream and downstream devices for automated sample quantitation or storage. PMID:23957867

  3. Automated microfluidic DNA/RNA extraction with both disposable and reusable components

    NASA Astrophysics Data System (ADS)

    Kim, Jungkyu; Johnson, Michael; Hill, Parker; Sonkul, Rahul S.; Kim, Jongwon; Gale, Bruce K.

    2012-01-01

    An automated microfluidic nucleic extraction system was fabricated with a multilayer polydimethylsiloxane (PDMS) structure that consists of sample wells, microvalves, a micropump and a disposable microfluidic silica cartridge. Both the microvalves and micropump structures were fabricated in a single layer and are operated pneumatically using a 100 µm PDMS membrane. To fabricate the disposable microfluidic silica cartridge, two-cavity structures were made in a PDMS replica to fit the stacked silica membranes. A handheld controller for the microvalves and pumps was developed to enable system automation. With purified ribonucleic acid (RNA), whole blood and E. coli samples, the automated microfluidic nucleic acid extraction system was validated with a guanidine-based solid phase extraction procedure. An extraction efficiency of ~90% for deoxyribonucleic acid (DNA) and ~54% for RNA was obtained in 12 min from whole blood and E. coli samples, respectively. In addition, the same quantity and quality of extracted DNA was confirmed by polymerase chain reaction (PCR) amplification. The PCR also presented the appropriate amplification and melting profiles. Automated, programmable fluid control and physical separation of the reusable components and the disposable components significantly decrease the assay time and manufacturing cost and increase the flexibility and compatibility of the system with downstream components.

  4. Comparative Evaluation of Commercially Available Manual and Automated Nucleic Acid Extraction Methods for Rotavirus RNA Detection in Stool

    PubMed Central

    Esona, Mathew D.; McDonald, Sharla; Kamili, Shifaq; Kerin, Tara; Gautam, Rashi; Bowen, Michael D.

    2015-01-01

    Rotaviruses are a major cause of viral gastroenteritis in children. For accurate and sensitive detection of rotavirus RNA from stool samples by reverse transcription-polymerase chain reaction (RT-PCR), the extraction process must be robust. However, some extraction methods may not remove the strong RT-PCR inhibitors known to be present in stool samples. The objective of this study was to evaluate and compare the performance of six extraction methods used commonly for extraction of rotavirus RNA from stool, which have never been formally evaluated: the MagNA Pure Compact, KingFisher Flex and NucliSENS® easyMAG® instruments, the NucliSENS® miniMAG® semi-automated system, and two manual purification kits, the QIAamp Viral RNA kit and a modified RNaid® kit. Using each method, total nucleic acid or RNA was extracted from eight rotavirus-positive stool samples with enzyme immunoassay optical density (EIA OD) values ranging from 0.176 to 3.098. Extracts prepared using the MagNA Pure Compact instrument yielded the most consistent results by qRT-PCR and conventional RT-PCR. When extracts prepared from a dilution series were extracted by the 6 methods and tested, rotavirus RNA was detected in all samples by qRT-PCR but by conventional RT-PCR testing, only the MagNA Pure Compact and KingFisher Flex extracts were positive in all cases. RT-PCR inhibitors were detected in extracts produced with the QIAamp Viral RNA Mini kit. The findings of this study should prove useful for selection of extraction methods to be incorporated into future rotavirus detection and genotyping protocols. PMID:24036075

  5. High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

    PubMed Central

    Holmberg, Rebecca C.; Gindlesperger, Alissa; Stokes, Tinsley; Brady, Dane; Thakore, Nitu; Belgrader, Philip; Cooney, Christopher G.; Chandler, Darrell P.

    2013-01-01

    TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively). PMID:23793016

  6. Integrated DNA and RNA extraction and purification on an automated microfluidic cassette from bacterial and viral pathogens causing community-acquired lower respiratory tract infections.

    PubMed

    Van Heirstraeten, Liesbet; Spang, Peter; Schwind, Carmen; Drese, Klaus S; Ritzi-Lehnert, Marion; Nieto, Benjamin; Camps, Marta; Landgraf, Bryan; Guasch, Francesc; Corbera, Antoni Homs; Samitier, Josep; Goossens, Herman; Malhotra-Kumar, Surbhi; Roeser, Tina

    2014-05-01

    In this paper, we describe the development of an automated sample preparation procedure for etiological agents of community-acquired lower respiratory tract infections (CA-LRTI). The consecutive assay steps, including sample re-suspension, pre-treatment, lysis, nucleic acid purification, and concentration, were integrated into a microfluidic lab-on-a-chip (LOC) cassette that is operated hands-free by a demonstrator setup, providing fluidic and valve actuation. The performance of the assay was evaluated on viral and Gram-positive and Gram-negative bacterial broth cultures previously sampled using a nasopharyngeal swab. Sample preparation on the microfluidic cassette resulted in higher or similar concentrations of pure bacterial DNA or viral RNA compared to manual benchtop experiments. The miniaturization and integration of the complete sample preparation procedure, to extract purified nucleic acids from real samples of CA-LRTI pathogens to, and above, lab quality and efficiency, represent important steps towards its application in a point-of-care test (POCT) for rapid diagnosis of CA-LRTI. PMID:24615272

  7. Automated Extraction of Flow Features

    NASA Technical Reports Server (NTRS)

    Dorney, Suzanne (Technical Monitor); Haimes, Robert

    2005-01-01

    Computational Fluid Dynamics (CFD) simulations are routinely performed as part of the design process of most fluid handling devices. In order to efficiently and effectively use the results of a CFD simulation, visualization tools are often used. These tools are used in all stages of the CFD simulation including pre-processing, interim-processing, and post-processing, to interpret the results. Each of these stages requires visualization tools that allow one to examine the geometry of the device, as well as the partial or final results of the simulation. An engineer will typically generate a series of contour and vector plots to better understand the physics of how the fluid is interacting with the physical device. Of particular interest are detecting features such as shocks, re-circulation zones, and vortices (which will highlight areas of stress and loss). As the demand for CFD analyses continues to increase the need for automated feature extraction capabilities has become vital. In the past, feature extraction and identification were interesting concepts, but not required in understanding the physics of a steady flow field. This is because the results of the more traditional tools like; isc-surface, cuts and streamlines, were more interactive and easily abstracted so they could be represented to the investigator. These tools worked and properly conveyed the collected information at the expense of a great deal of interaction. For unsteady flow-fields, the investigator does not have the luxury of spending time scanning only one "snapshot" of the simulation. Automated assistance is required in pointing out areas of potential interest contained within the flow. This must not require a heavy compute burden (the visualization should not significantly slow down the solution procedure for co-processing environments). Methods must be developed to abstract the feature of interest and display it in a manner that physically makes sense.

  8. Automated Extraction of Flow Features

    NASA Technical Reports Server (NTRS)

    Dorney, Suzanne (Technical Monitor); Haimes, Robert

    2004-01-01

    Computational Fluid Dynamics (CFD) simulations are routinely performed as part of the design process of most fluid handling devices. In order to efficiently and effectively use the results of a CFD simulation, visualization tools are often used. These tools are used in all stages of the CFD simulation including pre-processing, interim-processing, and post-processing, to interpret the results. Each of these stages requires visualization tools that allow one to examine the geometry of the device, as well as the partial or final results of the simulation. An engineer will typically generate a series of contour and vector plots to better understand the physics of how the fluid is interacting with the physical device. Of particular interest are detecting features such as shocks, recirculation zones, and vortices (which will highlight areas of stress and loss). As the demand for CFD analyses continues to increase the need for automated feature extraction capabilities has become vital. In the past, feature extraction and identification were interesting concepts, but not required in understanding the physics of a steady flow field. This is because the results of the more traditional tools like; iso-surface, cuts and streamlines, were more interactive and easily abstracted so they could be represented to the investigator. These tools worked and properly conveyed the collected information at the expense of a great deal of interaction. For unsteady flow-fields, the investigator does not have the luxury of spending time scanning only one "snapshot" of the simulation. Automated assistance is required in pointing out areas of potential interest contained within the flow. This must not require a heavy compute burden (the visualization should not significantly slow down the solution procedure for (co-processing environments). Methods must be developed to abstract the feature of interest and display it in a manner that physically makes sense.

  9. RCrane: semi-automated RNA model building

    PubMed Central

    Keating, Kevin S.; Pyle, Anna Marie

    2012-01-01

    RNA crystals typically diffract to much lower resolutions than protein crystals. This low-resolution diffraction results in unclear density maps, which cause considerable difficulties during the model-building process. These difficulties are exacerbated by the lack of computational tools for RNA modeling. Here, RCrane, a tool for the partially automated building of RNA into electron-density maps of low or intermediate resolution, is presented. This tool works within Coot, a common program for macromolecular model building. RCrane helps crystallographers to place phosphates and bases into electron density and then automatically predicts and builds the detailed all-atom structure of the traced nucleotides. RCrane then allows the crystallographer to review the newly built structure and select alternative backbone conformations where desired. This tool can also be used to automatically correct the backbone structure of previously built nucleotides. These automated corrections can fix incorrect sugar puckers, steric clashes and other structural problems. PMID:22868764

  10. Automated DNA extraction from pollen in honey.

    PubMed

    Guertler, Patrick; Eicheldinger, Adelina; Muschler, Paul; Goerlich, Ottmar; Busch, Ulrich

    2014-04-15

    In recent years, honey has become subject of DNA analysis due to potential risks evoked by microorganisms, allergens or genetically modified organisms. However, so far, only a few DNA extraction procedures are available, mostly time-consuming and laborious. Therefore, we developed an automated DNA extraction method from pollen in honey based on a CTAB buffer-based DNA extraction using the Maxwell 16 instrument and the Maxwell 16 FFS Nucleic Acid Extraction System, Custom-Kit. We altered several components and extraction parameters and compared the optimised method with a manual CTAB buffer-based DNA isolation method. The automated DNA extraction was faster and resulted in higher DNA yield and sufficient DNA purity. Real-time PCR results obtained after automated DNA extraction are comparable to results after manual DNA extraction. No PCR inhibition was observed. The applicability of this method was further successfully confirmed by analysis of different routine honey samples. PMID:24295710

  11. Avian influenza virus RNA extraction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The efficient extraction and purification of viral RNA is critical for down-stream molecular applications whether it is the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, or quantification of avian influenza (AI) virus by molecular methods from expe...

  12. Automated classification of RNA 3D motifs and the RNA 3D Motif Atlas

    PubMed Central

    Petrov, Anton I.; Zirbel, Craig L.; Leontis, Neocles B.

    2013-01-01

    The analysis of atomic-resolution RNA three-dimensional (3D) structures reveals that many internal and hairpin loops are modular, recurrent, and structured by conserved non-Watson–Crick base pairs. Structurally similar loops define RNA 3D motifs that are conserved in homologous RNA molecules, but can also occur at nonhomologous sites in diverse RNAs, and which often vary in sequence. To further our understanding of RNA motif structure and sequence variability and to provide a useful resource for structure modeling and prediction, we present a new method for automated classification of internal and hairpin loop RNA 3D motifs and a new online database called the RNA 3D Motif Atlas. To classify the motif instances, a representative set of internal and hairpin loops is automatically extracted from a nonredundant list of RNA-containing PDB files. Their structures are compared geometrically, all-against-all, using the FR3D program suite. The loops are clustered into motif groups, taking into account geometric similarity and structural annotations and making allowance for a variable number of bulged bases. The automated procedure that we have implemented identifies all hairpin and internal loop motifs previously described in the literature. All motif instances and motif groups are assigned unique and stable identifiers and are made available in the RNA 3D Motif Atlas (http://rna.bgsu.edu/motifs), which is automatically updated every four weeks. The RNA 3D Motif Atlas provides an interactive user interface for exploring motif diversity and tools for programmatic data access. PMID:23970545

  13. Comparison of automated and manual purification of total RNA for mRNA-based identification of body fluids.

    PubMed

    Akutsu, Tomoko; Kitayama, Tetsushi; Watanabe, Ken; Sakurada, Koichi

    2015-01-01

    Silica column-based RNA purification procedures have widespread use in mRNA profiling for body fluid identification in forensic samples. Also, automated RNA purification systems employing magnetic bead technology have recently become available. In this preliminary study, to ascertain which RNA purification technology is more suitable for the identification of body fluids by real-time reverse transcription polymerase chain reaction (RT-PCR), comparative analyses of the yield and quality of total RNA were performed between automated purification using an EZ1 Advanced Instrument and manual purification using an RNeasy Mini Kit. The yield and size distribution of total RNA were compared by gene expression analysis of two different sized fragments of the β-actin gene. In addition, the relative amounts of several target genes were compared between the purification methods, and the integrity of total RNA was determined by chip-based electrophoresis. The results of this study suggest that RNeasy can purify higher-quality RNA as compared with automated purification using EZ1. The sensitivity of the RT-PCR analysis, however, was higher in the EZ1-purified samples, likely due to the relative efficiency of EZ1 in extracting short-length RNA from degraded samples. We also show that the quantification of relative levels of body fluid-specific genes could be influenced by the purification procedure. Our results indicate that although use of high-quality RNA is generally required for reproducible results in gene expression analysis, the forensic relevance of short RNA fragments in highly degraded samples cannot be ruled out. Furthermore, our results suggest that automated purification procedures as well as silica column-based manual purification procedures can be used for mRNA-based body fluid identification in forensic samples. PMID:25270217

  14. Automated identification of RNA 3D modules with discriminative power in RNA structural alignments.

    PubMed

    Theis, Corinna; Höner Zu Siederdissen, Christian; Hofacker, Ivo L; Gorodkin, Jan

    2013-12-01

    Recent progress in predicting RNA structure is moving towards filling the 'gap' in 2D RNA structure prediction where, for example, predicted internal loops often form non-canonical base pairs. This is increasingly recognized with the steady increase of known RNA 3D modules. There is a general interest in matching structural modules known from one molecule to other molecules for which the 3D structure is not known yet. We have created a pipeline, metaRNAmodules, which completely automates extracting putative modules from the FR3D database and mapping of such modules to Rfam alignments to obtain comparative evidence. Subsequently, the modules, initially represented by a graph, are turned into models for the RMDetect program, which allows to test their discriminative power using real and randomized Rfam alignments. An initial extraction of 22 495 3D modules in all PDB files results in 977 internal loop and 17 hairpin modules with clear discriminatory power. Many of these modules describe only minor variants of each other. Indeed, mapping of the modules onto Rfam families results in 35 unique locations in 11 different families. The metaRNAmodules pipeline source for the internal loop modules is available at http://rth.dk/resources/mrm. PMID:24005040

  15. The RNA 3D Motif Atlas: Computational methods for extraction, organization and evaluation of RNA motifs.

    PubMed

    Parlea, Lorena G; Sweeney, Blake A; Hosseini-Asanjan, Maryam; Zirbel, Craig L; Leontis, Neocles B

    2016-07-01

    RNA 3D motifs occupy places in structured RNA molecules that correspond to the hairpin, internal and multi-helix junction "loops" of their secondary structure representations. As many as 40% of the nucleotides of an RNA molecule can belong to these structural elements, which are distinct from the regular double helical regions formed by contiguous AU, GC, and GU Watson-Crick basepairs. With the large number of atomic- or near atomic-resolution 3D structures appearing in a steady stream in the PDB/NDB structure databases, the automated identification, extraction, comparison, clustering and visualization of these structural elements presents an opportunity to enhance RNA science. Three broad applications are: (1) identification of modular, autonomous structural units for RNA nanotechnology, nanobiology and synthetic biology applications; (2) bioinformatic analysis to improve RNA 3D structure prediction from sequence; and (3) creation of searchable databases for exploring the binding specificities, structural flexibility, and dynamics of these RNA elements. In this contribution, we review methods developed for computational extraction of hairpin and internal loop motifs from a non-redundant set of high-quality RNA 3D structures. We provide a statistical summary of the extracted hairpin and internal loop motifs in the most recent version of the RNA 3D Motif Atlas. We also explore the reliability and accuracy of the extraction process by examining its performance in clustering recurrent motifs from homologous ribosomal RNA (rRNA) structures. We conclude with a summary of remaining challenges, especially with regard to extraction of multi-helix junction motifs. PMID:27125735

  16. Automated building extraction using dense elevation matrices

    NASA Astrophysics Data System (ADS)

    Bendett, A. A.; Rauhala, Urho A.; Pearson, James J.

    1997-02-01

    The identification and measurement of buildings in imagery is important to a number of applications including cartography, modeling and simulation, and weapon targeting. Extracting large numbers of buildings manually can be time- consuming and expensive, so the automation of the process is highly desirable. This paper describes and demonstrates such an automated process for extracting rectilinear buildings from stereo imagery. The first step is the generation of a dense elevation matrix registered to the imagery. In the examples shown, this was accomplished using global minimum residual matching (GMRM). GMRM automatically removes y- parallax from the stereo imagery and produces a dense matrix of x-parallax values which are proportional to the local elevation, and, of course, registered to the imagery. The second step is to form a joint probability distribution of the image gray levels and the corresponding height values from the elevation matrix. Based on the peaks of that distribution, the area of interest is segmented into feature and non-feature areas. The feature areas are further refined using length, width and height constraints to yield promising building hypotheses with their corresponding vertices. The gray shade image is used in the third step to verify the hypotheses and to determine precise edge locations corresponding to the approximate vertices and satisfying appropriate orthogonality constraints. Examples of successful application of this process to imagery are presented, and extensions involving the use of dense elevation matrices from other sources are possible.

  17. Automated Fluid Feature Extraction from Transient Simulations

    NASA Technical Reports Server (NTRS)

    Haimes, Robert; Lovely, David

    1999-01-01

    In the past, feature extraction and identification were interesting concepts, but not required to understand the underlying physics of a steady flow field. This is because the results of the more traditional tools like iso-surfaces, cuts and streamlines were more interactive and easily abstracted so they could be represented to the investigator. These tools worked and properly conveyed the collected information at the expense of much interaction. For unsteady flow-fields, the investigator does not have the luxury of spending time scanning only one "snap-shot" of the simulation. Automated assistance is required in pointing out areas of potential interest contained within the flow. This must not require a heavy compute burden (the visualization should not significantly slow down the solution procedure for co-processing environments like pV3). And methods must be developed to abstract the feature and display it in a manner that physically makes sense. The following is a list of the important physical phenomena found in transient (and steady-state) fluid flow: (1) Shocks, (2) Vortex cores, (3) Regions of recirculation, (4) Boundary layers, (5) Wakes. Three papers and an initial specification for the (The Fluid eXtraction tool kit) FX Programmer's guide were included. The papers, submitted to the AIAA Computational Fluid Dynamics Conference, are entitled : (1) Using Residence Time for the Extraction of Recirculation Regions, (2) Shock Detection from Computational Fluid Dynamics results and (3) On the Velocity Gradient Tensor and Fluid Feature Extraction.

  18. Automated Extraction of Secondary Flow Features

    NASA Technical Reports Server (NTRS)

    Dorney, Suzanne M.; Haimes, Robert

    2005-01-01

    The use of Computational Fluid Dynamics (CFD) has become standard practice in the design and development of the major components used for air and space propulsion. To aid in the post-processing and analysis phase of CFD many researchers now use automated feature extraction utilities. These tools can be used to detect the existence of such features as shocks, vortex cores and separation and re-attachment lines. The existence of secondary flow is another feature of significant importance to CFD engineers. Although the concept of secondary flow is relatively understood there is no commonly accepted mathematical definition for secondary flow. This paper will present a definition for secondary flow and one approach for automatically detecting and visualizing secondary flow.

  19. Automated Fluid Feature Extraction from Transient Simulations

    NASA Technical Reports Server (NTRS)

    Haimes, Robert

    2000-01-01

    In the past, feature extraction and identification were interesting concepts, but not required in understanding the physics of a steady flow field. This is because the results of the more traditional tools like iso-surfaces, cuts and streamlines, were more interactive and easily abstracted so they could be represented to the investigator. These tools worked and properly conveyed the collected information at the expense of a great deal of interaction. For unsteady flow-fields, the investigator does not have the luxury of spending time scanning only one 'snap-shot' of the simulation. Automated assistance is required in pointing out areas of potential interest contained within the flow. This must not require a heavy compute burden (the visualization should not significantly slow down the solution procedure for co-processing environments like pV3). And methods must be developed to abstract the feature and display it in a manner that physically makes sense.

  20. Automated Fluid Feature Extraction from Transient Simulations

    NASA Technical Reports Server (NTRS)

    Haimes, Robert

    1998-01-01

    In the past, feature extraction and identification were interesting concepts, but not required to understand the underlying physics of a steady flow field. This is because the results of the more traditional tools like iso-surfaces, cuts and streamlines were more interactive and easily abstracted so they could be represented to the investigator. These tools worked and properly conveyed the collected information at the expense of much interaction. For unsteady flow-fields, the investigator does not have the luxury of spending time scanning only one 'snap-shot' of the simulation. Automated assistance is required in pointing out areas of potential interest contained within the flow. This must not require a heavy compute burden (the visualization should not significantly slow down the solution procedure for co-processing environments like pV3). And methods must be developed to abstract the feature and display it in a manner that physically makes sense. The following is a list of the important physical phenomena found in transient (and steady-state) fluid flow: Shocks; Vortex ores; Regions of Recirculation; Boundary Layers; Wakes.

  1. Automated feature extraction and classification from image sources

    USGS Publications Warehouse

    U.S. Geological Survey

    1995-01-01

    The U.S. Department of the Interior, U.S. Geological Survey (USGS), and Unisys Corporation have completed a cooperative research and development agreement (CRADA) to explore automated feature extraction and classification from image sources. The CRADA helped the USGS define the spectral and spatial resolution characteristics of airborne and satellite imaging sensors necessary to meet base cartographic and land use and land cover feature classification requirements and help develop future automated geographic and cartographic data production capabilities. The USGS is seeking a new commercial partner to continue automated feature extraction and classification research and development.

  2. Efficient oil palm total RNA extraction with a total RNA extraction kit.

    PubMed

    Habib, S H; Saud, H M; Kausar, H

    2014-01-01

    Oil palm tissues are rich in polyphenols, polysaccharides and secondary metabolites; these can co-precipitate with RNA, causing problems for downstream applications. We compared two different methods (one conventional and a kit-based method - Easy-Blue(TM) Total RNA Extraction Kit) to isolate total RNA from leaves, roots and shoot apical meristems of tissue culture derived truncated leaf syndrome somaclonal oil palm seedlings. The quality and quantity of total RNA were compared through spectrophotometry and formaldehyde gel electrophoresis. The specificity and applicability of the protocols were evaluated for downstream applications, including cDNA synthesis and RT-PCR analysis. We found that the conventional method gave higher yields of RNA but took longer, and it was contaminated with genomic DNA. This method required extra genomic DNA removal steps that further reduced the RNA yield. The kit-based method, on the other hand, produced good yields as well as well as good quality RNA, within a very short period of time from a small amount of starting material. Moreover, the RNA from the kit-based method was more suitable for synthesizing cDNA and RT-PCR amplification than the conventional method. Therefore, we conclude that the Easy-BlueTM Total RNA Extraction Kit method is suitable and superior for isolation of total RNA from oil palm leaf, root and shoot apical meristem. PMID:24781991

  3. Automated extraction of free-text from pathology reports.

    PubMed

    Currie, Anne-Marie; Fricke, Travis; Gawne, Agnes; Johnston, Ric; Liu, John; Stein, Barbara

    2006-01-01

    Manually populating a cancer registry from free-text pathology reports is labor intensive and costly. This poster describes a method of automated text extraction to improve the efficiency of this process and reduce cost. FineTooth, a software company, provides an automated service to the Fred Hutchinson Cancer Research Center (FHCRC) to help populate their breast and prostate cancer clinical research database by electronically abstracting over 80 data fields from pathology text reports. PMID:17238518

  4. Automated knowledge extraction from MEDLINE citations.

    PubMed

    Mendonça, E A; Cimino, J J

    2000-01-01

    As part of preliminary studies for the development of a digital library, we have studied the possibility of using the co-occurrence of MeSH terms in MEDLINE citations associated with the search strategies optimal for evidence-based medicine to automate construction of a knowledge base. We use the UMLS semantic types in order to analyze search results to determine which semantic types are most relevant for different types of questions (etiology, diagnosis, therapy, and prognosis). The automated process generated a large amount of information. Seven to eight percent of the semantic pairs generated in each clinical task group co-occur significantly more often than can be accounted for by chance. A pilot study showed good specificity and sensitivity for the intended purposes of this project in all groups. PMID:11079949

  5. Plasmid purification by phenol extraction from guanidinium thiocyanate solution: development of an automated protocol.

    PubMed

    Fisher, J A; Favreau, M B

    1991-05-01

    We have developed a novel plasmid isolation procedure and have adapted it for use on an automated nucleic acid extraction instrument. The protocol is based on the finding that phenol extraction of a 1 M guanidinium thiocyanate solution at pH 4.5 efficiently removes genomic DNA from the aqueous phase, while supercoiled plasmid DNA is retained in the aqueous phase. S1 nuclease digestion of the removed genomic DNA shows that it has been denatured, which presumably confers solubility in the organic phase. The complete automated protocol for plasmid isolation involves pretreatment of bacterial cells successively with lysozyme, RNase A, and proteinase K. Following these digestions, the solution is extracted twice with a phenol/chloroform/water mixture and once with chloroform. Purified plasmid is then collected by isopropanol precipitation. The purified plasmid is essentially free of genomic DNA, RNA, and protein and is a suitable substrate for DNA sequencing and other applications requiring highly pure supercoiled plasmid. PMID:1713749

  6. DNA, RNA, and Protein Extraction: The Past and The Present

    PubMed Central

    Tan, Siun Chee; Yiap, Beow Chin

    2009-01-01

    Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples; the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination. PMID:20011662

  7. Automated vasculature extraction from placenta images

    NASA Astrophysics Data System (ADS)

    Almoussa, Nizar; Dutra, Brittany; Lampe, Bryce; Getreuer, Pascal; Wittman, Todd; Salafia, Carolyn; Vese, Luminita

    2011-03-01

    Recent research in perinatal pathology argues that analyzing properties of the placenta may reveal important information on how certain diseases progress. One important property is the structure of the placental blood vessels, which supply a fetus with all of its oxygen and nutrition. An essential step in the analysis of the vascular network pattern is the extraction of the blood vessels, which has only been done manually through a costly and time-consuming process. There is no existing method to automatically detect placental blood vessels; in addition, the large variation in the shape, color, and texture of the placenta makes it difficult to apply standard edge-detection algorithms. We describe a method to automatically detect and extract blood vessels from a given image by using image processing techniques and neural networks. We evaluate several local features for every pixel, in addition to a novel modification to an existing road detector. Pixels belonging to blood vessel regions have recognizable responses; hence, we use an artificial neural network to identify the pattern of blood vessels. A set of images where blood vessels are manually highlighted is used to train the network. We then apply the neural network to recognize blood vessels in new images. The network is effective in capturing the most prominent vascular structures of the placenta.

  8. AN RNA EXTRACTION PROTOCOL FOR SHELLFISH-BORNE VIRUSES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The GPTT virus RNA extraction method, originally developed for extraction of human norovirus and hepatitis A virus RNAs from contaminated shellfish, was evaluated for extraction of RNA from Aichi virus strain A846/88 (AiV), coxsackievirus strains A9 (CAV9) and B5 (CBV5), murine norovirus (strain MNV...

  9. Automated Image Registration Using Morphological Region of Interest Feature Extraction

    NASA Technical Reports Server (NTRS)

    Plaza, Antonio; LeMoigne, Jacqueline; Netanyahu, Nathan S.

    2005-01-01

    With the recent explosion in the amount of remotely sensed imagery and the corresponding interest in temporal change detection and modeling, image registration has become increasingly important as a necessary first step in the integration of multi-temporal and multi-sensor data for applications such as the analysis of seasonal and annual global climate changes, as well as land use/cover changes. The task of image registration can be divided into two major components: (1) the extraction of control points or features from images; and (2) the search among the extracted features for the matching pairs that represent the same feature in the images to be matched. Manual control feature extraction can be subjective and extremely time consuming, and often results in few usable points. Automated feature extraction is a solution to this problem, where desired target features are invariant, and represent evenly distributed landmarks such as edges, corners and line intersections. In this paper, we develop a novel automated registration approach based on the following steps. First, a mathematical morphology (MM)-based method is used to obtain a scale-orientation morphological profile at each image pixel. Next, a spectral dissimilarity metric such as the spectral information divergence is applied for automated extraction of landmark chips, followed by an initial approximate matching. This initial condition is then refined using a hierarchical robust feature matching (RFM) procedure. Experimental results reveal that the proposed registration technique offers a robust solution in the presence of seasonal changes and other interfering factors. Keywords-Automated image registration, multi-temporal imagery, mathematical morphology, robust feature matching.

  10. Automated sea floor extraction from underwater video

    NASA Astrophysics Data System (ADS)

    Kelly, Lauren; Rahmes, Mark; Stiver, James; McCluskey, Mike

    2016-05-01

    Ocean floor mapping using video is a method to simply and cost-effectively record large areas of the seafloor. Obtaining visual and elevation models has noteworthy applications in search and recovery missions. Hazards to navigation are abundant and pose a significant threat to the safety, effectiveness, and speed of naval operations and commercial vessels. This project's objective was to develop a workflow to automatically extract metadata from marine video and create image optical and elevation surface mosaics. Three developments made this possible. First, optical character recognition (OCR) by means of two-dimensional correlation, using a known character set, allowed for the capture of metadata from image files. Second, exploiting the image metadata (i.e., latitude, longitude, heading, camera angle, and depth readings) allowed for the determination of location and orientation of the image frame in mosaic. Image registration improved the accuracy of mosaicking. Finally, overlapping data allowed us to determine height information. A disparity map was created using the parallax from overlapping viewpoints of a given area and the relative height data was utilized to create a three-dimensional, textured elevation map.

  11. An automated approach for global identification of sRNA-encoding regions in RNA-Seq data from Mycobacterium tuberculosis.

    PubMed

    Wang, Ming; Fleming, Joy; Li, Zihui; Li, Chuanyou; Zhang, Hongtai; Xue, Yunxin; Chen, Maoshan; Zhang, Zongde; Zhang, Xian-En; Bi, Lijun

    2016-06-01

    Deep-sequencing of bacterial transcriptomes using RNA-Seq technology has made it possible to identify small non-coding RNAs, RNA molecules which regulate gene expression in response to changing environments, on a genome-wide scale in an ever-increasing range of prokaryotes. However, a simple and reliable automated method for identifying sRNA candidates in these large datasets is lacking. Here, after generating a transcriptome from an exponential phase culture of Mycobacterium tuberculosis H37Rv, we developed and validated an automated method for the genome-wide identification of sRNA candidate-containing regions within RNA-Seq datasets based on the analysis of the characteristics of reads coverage maps. We identified 192 novel candidate sRNA-encoding regions in intergenic regions and 664 RNA transcripts transcribed from regions antisense (as) to open reading frames (ORF), which bear the characteristics of asRNAs, and validated 28 of these novel sRNA-encoding regions by northern blotting. Our work has not only provided a simple automated method for genome-wide identification of candidate sRNA-encoding regions in RNA-Seq data, but has also uncovered many novel candidate sRNA-encoding regions in M. tuberculosis, reinforcing the view that the control of gene expression in bacteria is more complex than previously anticipated. PMID:27174874

  12. Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities.

    PubMed

    Oldham, Athenia L; Drilling, Heather S; Stamps, Blake W; Stevenson, Bradley S; Duncan, Kathleen E

    2012-01-01

    The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources. PMID:23168231

  13. Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities

    PubMed Central

    2012-01-01

    The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources. PMID:23168231

  14. Automated extraction of radiation dose information for CT examinations.

    PubMed

    Cook, Tessa S; Zimmerman, Stefan; Maidment, Andrew D A; Kim, Woojin; Boonn, William W

    2010-11-01

    Exposure to radiation as a result of medical imaging is currently in the spotlight, receiving attention from Congress as well as the lay press. Although scanner manufacturers are moving toward including effective dose information in the Digital Imaging and Communications in Medicine headers of imaging studies, there is a vast repository of retrospective CT data at every imaging center that stores dose information in an image-based dose sheet. As such, it is difficult for imaging centers to participate in the ACR's Dose Index Registry. The authors have designed an automated extraction system to query their PACS archive and parse CT examinations to extract the dose information stored in each dose sheet. First, an open-source optical character recognition program processes each dose sheet and converts the information to American Standard Code for Information Interchange (ASCII) text. Each text file is parsed, and radiation dose information is extracted and stored in a database which can be queried using an existing pathology and radiology enterprise search tool. Using this automated extraction pipeline, it is possible to perform dose analysis on the >800,000 CT examinations in the PACS archive and generate dose reports for all of these patients. It is also possible to more effectively educate technologists, radiologists, and referring physicians about exposure to radiation from CT by generating report cards for interpreted and performed studies. The automated extraction pipeline enables compliance with the ACR's reporting guidelines and greater awareness of radiation dose to patients, thus resulting in improved patient care and management. PMID:21040869

  15. Improved Automated Seismic Event Extraction Using Machine Learning

    NASA Astrophysics Data System (ADS)

    Mackey, L.; Kleiner, A.; Jordan, M. I.

    2009-12-01

    Like many organizations engaged in seismic monitoring, the Preparatory Commission for the Comprehensive Test Ban Treaty Organization collects and processes seismic data from a large network of sensors. This data is continuously transmitted to a central data center, and bulletins of seismic events are automatically extracted. However, as for many such automated systems at present, the inaccuracy of this extraction necessitates substantial human analyst review effort. A significant opportunity for improvement thus lies in the fact that these systems currently fail to fully utilize the valuable repository of historical data provided by prior analyst reviews. In this work, we present the results of the application of machine learning approaches to several fundamental sub-tasks in seismic event extraction. These methods share as a common theme the use of historical analyst-reviewed bulletins as ground truth from which they extract relevant patterns to accomplish the desired goals. For instance, we demonstrate the effectiveness of classification and ranking methods for the identification of false events -- that is, those which will be invalidated and discarded by analysts -- in automated bulletins. We also show gains in the accuracy of seismic phase identification via the use of classification techniques to automatically assign seismic phase labels to station detections. Furthermore, we examine the potential of historical association data to inform the direct association of new signal detections with their corresponding seismic events. Empirical results are based upon parametric historical seismic detection and event data received from the Preparatory Commission for the Comprehensive Test Ban Treaty Organization.

  16. Arduino-based automation of a DNA extraction system.

    PubMed

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile. PMID:26409535

  17. Automated tools for phenotype extraction from medical records.

    PubMed

    Yetisgen-Yildiz, Meliha; Bejan, Cosmin A; Vanderwende, Lucy; Xia, Fei; Evans, Heather L; Wurfel, Mark M

    2013-01-01

    Clinical research studying critical illness phenotypes relies on the identification of clinical syndromes defined by consensus definitions. Historically, identifying phenotypes has required manual chart review, a time and resource intensive process. The overall research goal of C ritical I llness PH enotype E xt R action (deCIPHER) project is to develop automated approaches based on natural language processing and machine learning that accurately identify phenotypes from EMR. We chose pneumonia as our first critical illness phenotype and conducted preliminary experiments to explore the problem space. In this abstract, we outline the tools we built for processing clinical records, present our preliminary findings for pneumonia extraction, and describe future steps. PMID:24303281

  18. Automated labeling of bibliographic data extracted from biomedical online journals

    NASA Astrophysics Data System (ADS)

    Kim, Jongwoo; Le, Daniel X.; Thoma, George R.

    2003-01-01

    A prototype system has been designed to automate the extraction of bibliographic data (e.g., article title, authors, abstract, affiliation and others) from online biomedical journals to populate the National Library of Medicine"s MEDLINE database. This paper describes a key module in this system: the labeling module that employs statistics and fuzzy rule-based algorithms to identify segmented zones in an article"s HTML pages as specific bibliographic data. Results from experiments conducted with 1,149 medical articles from forty-seven journal issues are presented.

  19. Feature extraction from Doppler ultrasound signals for automated diagnostic systems.

    PubMed

    Ubeyli, Elif Derya; Güler, Inan

    2005-11-01

    This paper presented the assessment of feature extraction methods used in automated diagnosis of arterial diseases. Since classification is more accurate when the pattern is simplified through representation by important features, feature extraction and selection play an important role in classifying systems such as neural networks. Different feature extraction methods were used to obtain feature vectors from ophthalmic and internal carotid arterial Doppler signals. In addition to this, the problem of selecting relevant features among the features available for the purpose of classification of Doppler signals was dealt with. Multilayer perceptron neural networks (MLPNNs) with different inputs (feature vectors) were used for diagnosis of ophthalmic and internal carotid arterial diseases. The assessment of feature extraction methods was performed by taking into consideration of performances of the MLPNNs. The performances of the MLPNNs were evaluated by the convergence rates (number of training epochs) and the total classification accuracies. Finally, some conclusions were drawn concerning the efficiency of discrete wavelet transform as a feature extraction method used for the diagnosis of ophthalmic and internal carotid arterial diseases. PMID:16278106

  20. Automated Feature Extraction of Foredune Morphology from Terrestrial Lidar Data

    NASA Astrophysics Data System (ADS)

    Spore, N.; Brodie, K. L.; Swann, C.

    2014-12-01

    Foredune morphology is often described in storm impact prediction models using the elevation of the dune crest and dune toe and compared with maximum runup elevations to categorize the storm impact and predicted responses. However, these parameters do not account for other foredune features that may make them more or less erodible, such as alongshore variations in morphology, vegetation coverage, or compaction. The goal of this work is to identify other descriptive features that can be extracted from terrestrial lidar data that may affect the rate of dune erosion under wave attack. Daily, mobile-terrestrial lidar surveys were conducted during a 6-day nor'easter (Hs = 4 m in 6 m water depth) along 20km of coastline near Duck, North Carolina which encompassed a variety of foredune forms in close proximity to each other. This abstract will focus on the tools developed for the automated extraction of the morphological features from terrestrial lidar data, while the response of the dune will be presented by Brodie and Spore as an accompanying abstract. Raw point cloud data can be dense and is often under-utilized due to time and personnel constraints required for analysis, since many algorithms are not fully automated. In our approach, the point cloud is first projected into a local coordinate system aligned with the coastline, and then bare earth points are interpolated onto a rectilinear 0.5 m grid creating a high resolution digital elevation model. The surface is analyzed by identifying features along each cross-shore transect. Surface curvature is used to identify the position of the dune toe, and then beach and berm morphology is extracted shoreward of the dune toe, and foredune morphology is extracted landward of the dune toe. Changes in, and magnitudes of, cross-shore slope, curvature, and surface roughness are used to describe the foredune face and each cross-shore transect is then classified using its pre-storm morphology for storm-response analysis.

  1. Extraction of Bacterial RNA from Soil: Challenges and Solutions

    PubMed Central

    Wang, Yong; Hayatsu, Masahito; Fujii, Takeshi

    2012-01-01

    Detection of bacterial gene expression in soil emerged in the early 1990s and provided information on bacterial responses in their original soil environments. As a key procedure in the detection, extraction of bacterial RNA from soil has attracted much interest, and many methods of soil RNA extraction have been reported in the past 20 years. In addition to various RT-PCR-based technologies, new technologies for gene expression analysis, such as microarrays and high-throughput sequencing technologies, have recently been applied to examine bacterial gene expression in soil. These technologies are driving improvements in RNA extraction protocols. In this mini-review, progress in the extraction of bacterial RNA from soil is summarized with emphasis on the major difficulties in the development of methodologies and corresponding strategies to overcome them. PMID:22791042

  2. Parallel RNA extraction using magnetic beads and a droplet array

    PubMed Central

    Shi, Xu; Chen, Chun-Hong; Gao, Weimin; Meldrum, Deirdre R.

    2015-01-01

    Nucleic acid extraction is a necessary step for most genomic/transcriptomic analyses, but it often requires complicated mechanisms to be integrated into a lab-on-a-chip device. Here, we present a simple, effective configuration for rapidly obtaining purified RNA from low concentration cell medium. This Total RNA Extraction Droplet Array (TREDA) utilizes an array of surface-adhering droplets to facilitate the transportation of magnetic purification beads seamlessly through individual buffer solutions without solid structures. The fabrication of TREDA chips is rapid and does not require a microfabrication facility or expertise. The process takes less than 5 minutes. When purifying mRNA from bulk marine diatom samples, its repeatability and extraction efficiency are comparable to conventional tube-based operations. We demonstrate that TREDA can extract the total mRNA of about 10 marine diatom cells, indicating that the sensitivity of TREDA approaches single-digit cell numbers. PMID:25519439

  3. An automated procedure for covariation-based detection of RNA structure

    SciTech Connect

    Winker, S.; Overbeek, R.; Woese, C.R.; Olsen, G.J.; Pfluger, N.

    1989-12-01

    This paper summarizes our investigations into the computational detection of secondary and tertiary structure of ribosomal RNA. We have developed a new automated procedure that not only identifies potential bondings of secondary and tertiary structure, but also provides the covariation evidence that supports the proposed bondings, and any counter-evidence that can be detected in the known sequences. A small number of previously unknown bondings have been detected in individual RNA molecules (16S rRNA and 7S RNA) through the use of our automated procedure. Currently, we are systematically studying mitochondrial rRNA. Our goal is to detect tertiary structure within 16S rRNA and quaternary structure between 16S and 23S rRNA. Our ultimate hope is that automated covariation analysis will contribute significantly to a refined picture of ribosome structure. Our colleagues in biology have begun experiments to test certain hypotheses suggested by an examination of our program's output. These experiments involve sequencing key portions of the 23S ribosomal RNA for species in which the known 16S ribosomal RNA exhibits variation (from the dominant pattern) at the site of a proposed bonding. The hope is that the 23S ribosomal RNA of these species will exhibit corresponding complementary variation or generalized covariation. 24 refs.

  4. Automated extraction of chemical structure information from digital raster images

    PubMed Central

    Park, Jungkap; Rosania, Gus R; Shedden, Kerby A; Nguyen, Mandee; Lyu, Naesung; Saitou, Kazuhiro

    2009-01-01

    Background To search for chemical structures in research articles, diagrams or text representing molecules need to be translated to a standard chemical file format compatible with cheminformatic search engines. Nevertheless, chemical information contained in research articles is often referenced as analog diagrams of chemical structures embedded in digital raster images. To automate analog-to-digital conversion of chemical structure diagrams in scientific research articles, several software systems have been developed. But their algorithmic performance and utility in cheminformatic research have not been investigated. Results This paper aims to provide critical reviews for these systems and also report our recent development of ChemReader – a fully automated tool for extracting chemical structure diagrams in research articles and converting them into standard, searchable chemical file formats. Basic algorithms for recognizing lines and letters representing bonds and atoms in chemical structure diagrams can be independently run in sequence from a graphical user interface-and the algorithm parameters can be readily changed-to facilitate additional development specifically tailored to a chemical database annotation scheme. Compared with existing software programs such as OSRA, Kekule, and CLiDE, our results indicate that ChemReader outperforms other software systems on several sets of sample images from diverse sources in terms of the rate of correct outputs and the accuracy on extracting molecular substructure patterns. Conclusion The availability of ChemReader as a cheminformatic tool for extracting chemical structure information from digital raster images allows research and development groups to enrich their chemical structure databases by annotating the entries with published research articles. Based on its stable performance and high accuracy, ChemReader may be sufficiently accurate for annotating the chemical database with links to scientific research

  5. Automated Extraction of Family History Information from Clinical Notes

    PubMed Central

    Bill, Robert; Pakhomov, Serguei; Chen, Elizabeth S.; Winden, Tamara J.; Carter, Elizabeth W.; Melton, Genevieve B.

    2014-01-01

    Despite increased functionality for obtaining family history in a structured format within electronic health record systems, clinical notes often still contain this information. We developed and evaluated an Unstructured Information Management Application (UIMA)-based natural language processing (NLP) module for automated extraction of family history information with functionality for identifying statements, observations (e.g., disease or procedure), relative or side of family with attributes (i.e., vital status, age of diagnosis, certainty, and negation), and predication (“indicator phrases”), the latter of which was used to establish relationships between observations and family member. The family history NLP system demonstrated F-scores of 66.9, 92.4, 82.9, 57.3, 97.7, and 61.9 for detection of family history statements, family member identification, observation identification, negation identification, vital status, and overall extraction of the predications between family members and observations, respectively. While the system performed well for detection of family history statements and predication constituents, further work is needed to improve extraction of certainty and temporal modifications. PMID:25954443

  6. Text Mining approaches for automated literature knowledge extraction and representation.

    PubMed

    Nuzzo, Angelo; Mulas, Francesca; Gabetta, Matteo; Arbustini, Eloisa; Zupan, Blaz; Larizza, Cristiana; Bellazzi, Riccardo

    2010-01-01

    Due to the overwhelming volume of published scientific papers, information tools for automated literature analysis are essential to support current biomedical research. We have developed a knowledge extraction tool to help researcher in discovering useful information which can support their reasoning process. The tool is composed of a search engine based on Text Mining and Natural Language Processing techniques, and an analysis module which process the search results in order to build annotation similarity networks. We tested our approach on the available knowledge about the genetic mechanism of cardiac diseases, where the target is to find both known and possible hypothetical relations between specific candidate genes and the trait of interest. We show that the system i) is able to effectively retrieve medical concepts and genes and ii) plays a relevant role assisting researchers in the formulation and evaluation of novel literature-based hypotheses. PMID:20841825

  7. Automated extraction of knowledge for model-based diagnostics

    NASA Technical Reports Server (NTRS)

    Gonzalez, Avelino J.; Myler, Harley R.; Towhidnejad, Massood; Mckenzie, Frederic D.; Kladke, Robin R.

    1990-01-01

    The concept of accessing computer aided design (CAD) design databases and extracting a process model automatically is investigated as a possible source for the generation of knowledge bases for model-based reasoning systems. The resulting system, referred to as automated knowledge generation (AKG), uses an object-oriented programming structure and constraint techniques as well as internal database of component descriptions to generate a frame-based structure that describes the model. The procedure has been designed to be general enough to be easily coupled to CAD systems that feature a database capable of providing label and connectivity data from the drawn system. The AKG system is capable of defining knowledge bases in formats required by various model-based reasoning tools.

  8. Automated Extraction of Substance Use Information from Clinical Texts

    PubMed Central

    Wang, Yan; Chen, Elizabeth S.; Pakhomov, Serguei; Arsoniadis, Elliot; Carter, Elizabeth W.; Lindemann, Elizabeth; Sarkar, Indra Neil; Melton, Genevieve B.

    2015-01-01

    Within clinical discourse, social history (SH) includes important information about substance use (alcohol, drug, and nicotine use) as key risk factors for disease, disability, and mortality. In this study, we developed and evaluated a natural language processing (NLP) system for automated detection of substance use statements and extraction of substance use attributes (e.g., temporal and status) based on Stanford Typed Dependencies. The developed NLP system leveraged linguistic resources and domain knowledge from a multi-site social history study, Propbank and the MiPACQ corpus. The system attained F-scores of 89.8, 84.6 and 89.4 respectively for alcohol, drug, and nicotine use statement detection, as well as average F-scores of 82.1, 90.3, 80.8, 88.7, 96.6, and 74.5 respectively for extraction of attributes. Our results suggest that NLP systems can achieve good performance when augmented with linguistic resources and domain knowledge when applied to a wide breadth of substance use free text clinical notes. PMID:26958312

  9. Automated Dsm Extraction from Uav Images and Performance Analysis

    NASA Astrophysics Data System (ADS)

    Rhee, S.; Kim, T.

    2015-08-01

    As technology evolves, unmanned aerial vehicles (UAVs) imagery is being used from simple applications such as image acquisition to complicated applications such as 3D spatial information extraction. Spatial information is usually provided in the form of a DSM or point cloud. It is important to generate very dense tie points automatically from stereo images. In this paper, we tried to apply stereo image-based matching technique developed for satellite/aerial images to UAV images, propose processing steps for automated DSM generation and to analyse the possibility of DSM generation. For DSM generation from UAV images, firstly, exterior orientation parameters (EOPs) for each dataset were adjusted. Secondly, optimum matching pairs were determined. Thirdly, stereo image matching was performed with each pair. Developed matching algorithm is based on grey-level correlation on pixels applied along epipolar lines. Finally, the extracted match results were united with one result and the final DSM was made. Generated DSM was compared with a reference DSM from Lidar. Overall accuracy was 1.5 m in NMAD. However, several problems have to be solved in future, including obtaining precise EOPs, handling occlusion and image blurring problems. More effective interpolation technique needs to be developed in the future.

  10. Automated Tract Extraction via Atlas Based Adaptive Clustering

    PubMed Central

    Tunç, Birkan; Parker, William A.; Ingalhalikar, Madhura; Verma, Ragini

    2014-01-01

    Advancements in imaging protocols such as the high angular resolution diffusion-weighted imaging (HARDI) and in tractography techniques are expected to cause an increase in the tract-based analyses. Statistical analyses over white matter tracts can contribute greatly towards understanding structural mechanisms of the brain since tracts are representative of the connectivity pathways. The main challenge with tract-based studies is the extraction of the tracts of interest in a consistent and comparable manner over a large group of individuals without drawing the inclusion and exclusion regions of interest. In this work, we design a framework for automated extraction of white matter tracts. The framework introduces three main components, namely a connectivity based fiber representation, a fiber clustering atlas, and a clustering approach called Adaptive Clustering. The fiber representation relies on the connectivity signatures of fibers to establish an easy correspondence between different subjects. A group-wise clustering of these fibers that are represented by the connectivity signatures is then used to generate a fiber bundle atlas. Finally, Adaptive Clustering incorporates the previously generated clustering atlas as a prior, to cluster the fibers of a new subject automatically. Experiments on the HARDI scans of healthy individuals acquired repeatedly, demonstrate the applicability, the reliability and the repeatability of our approach in extracting white matter tracts. By alleviating the seed region selection or the inclusion/exclusion ROI drawing requirements that are usually handled by trained radiologists, the proposed framework expands the range of possible clinical applications and establishes the ability to perform tract-based analyses with large samples. PMID:25134977

  11. Evaluation of commercial RNA extraction kits for the isolation of viral MS2 RNA from soil.

    PubMed

    Dineen, Shauna M; Aranda, Roman; Dietz, Marianne E; Anders, Douglas L; Robertson, James M

    2010-09-01

    Nucleic acid extraction is a critical step in the detection of an unknown biological agent. However, success can vary depending on the isolation and identification methods chosen and the difficulty of extraction from environmental matrices. In this work, bacteriophage MS2 RNA was extracted from three soil matrices, sand, clay, and loam, using five commercially available kits: the PowerSoil Total RNA Isolation, E.Z.N.A. Soil RNA, FastRNA Pro Soil-Direct, FastRNA Pro Soil-Indirect, and IT 1-2-3 Platinum Path kits. Success of the isolation was determined using an MS2-specific RT-PCR assay. The reproducibility and sensitivity of each method in the hands of both experienced and novice users were assessed and compared. Cost, operator time, and storage conditions were also considered in the evaluation. The RNA isolation method that yielded the best results, as defined by reproducibility and sensitivity, was the E.Z.N.A. Soil RNA kit for sand, the IT 1-2-3 Platinum Path Sample Purification kit for clay, and the FastRNA Pro Soil-Indirect kit for loam. However, if time and storage conditions are important considerations, the IT 1-2-3 Platinum Path kit may be appropriate for use with all soils since the kit has the shortest processing time and fewest temperature requirements. PMID:20417664

  12. Evaluation of microbial RNA extractions from Streptococcus pneumoniae.

    PubMed

    Li-Korotky, Ha-Sheng; Kelly, Lori A; Piltcher, Otavio; Hebda, Patricia A; Doyle, William J

    2007-02-01

    The mechanisms that control Streptococcus pneumoniae's ability to colonize the nasopharynx or to invade the middle ear and cause acute otitis media are not understood. Focused study of these mechanisms requires efficient methods for the extraction of microbial RNA from minute clinical samples. Several lysis/extraction methods were tested and compared to determine the optimal conditions for isolating intact total RNA from pneumococcal cells. The sensitivity and efficiency of the extractions were evaluated by reverse transcription polymerase chain reaction (RT-PCR). Compared to other methods, mechanical homogenization in TRIZOL was the most efficient for releasing microbial RNA, and addition of polyinosinic acid (Poly I) as an RNA carrier increased the assay sensitivity to 10(2) colony forming units when detected by RT-PCR amplification of 16S ribosomal RNA or messenger RNA for penicillin binding protein 2b. Quantitative results were confirmed using a ribonuclease protection assay. Penicillin binding protein 2b was also detected in rat middle ear mucosa recovered 5 weeks after middle ear challenge with S. pneumoniae. This study describes a useful core methodology for use in identifying pneumococcal virulence genes from small titer samples and has promising applications in clinical studies of pneumococcal nasopharyngeal colonization and otitis media pathogenesis. PMID:17095113

  13. Development and verification of an automated sample processing protocol for quantitation of human immunodeficiency virus type 1 RNA in plasma.

    PubMed

    Lee, Brenda G; Fiebelkorn, Kristin R; Caliendo, Angela M; Nolte, Frederick S

    2003-05-01

    We developed and verified an automated sample processing protocol for use with the AMPLICOR HIV-1 MONITOR test, version 1.5 (Roche Diagnostics, Indianapolis, Ind.). The automated method uses the MagNA Pure LC instrument and total nucleic acid reagents (Roche Applied Science, Indianapolis, Ind.) to extract human immunodeficiency virus type 1 (HIV-1) RNA from plasma specimens. We compared the HIV-1 load results for a dilution series (1 to 5 nominal log(10) copies/ml) and 175 clinical specimens processed by the automated method to those for the same samples processed by the manual methods specified by the manufacturer. The sensitivity, dynamic range, and precision of the viral load assay obtained by automated processing of specimens were similar to those obtained by an ultrasensitive manual processing method. The results were highly correlated (R(2), 0.95), and were in close agreement, with a mean difference of 0.09 log(10) (standard deviation, 0.292). The limits of agreement were +/-0.58 log(10) for results for samples processed by both the manual and the automated methods. These performance characteristics were achieved with a smaller sample volume (200 versus 500 microl) and without a high-speed centrifugation step and required only 15 min of labor for a batch of 32 samples. In conclusion, the automated sample preparation protocol can replace both the standard and the ultrasensitive manual methods used with the AMPLICOR HIV-1 MONITOR test and can substantially reduce the labor associated with this test. PMID:12734249

  14. SMiRK: an Automated Pipeline for miRNA Analysis

    PubMed Central

    Milholland, Brandon; Gombar, Saurabh; Suh, Yousin

    2015-01-01

    Background Micro RNAs (miRNAs), important regulators of cell function, can be interrogated by high-throughput sequencing in a rapid and cost-effective manner. However, the tremendous amount of data generated by such methods is not easily analyzed. In order to extract meaningful information and draw biological conclusions from miRNA data, many challenges in quality control, alignment, normalization, and analysis must be overcome. Typically, these would only be possible with the dedicated efforts of a specialized computational biologist for a sustained period of time. Results Here, we present SMiRK, an automated pipeline that allows such tasks to be completed with minimal time and without dedicated bioinformatics personnel. SMiRK’s flexibility also allows experienced users to exert more control, if they wish. We describe how SMiRK automatically normalizes the data, removes low-information miRNAs, and produces heatmaps of the processed data. We give details on SMiRK’s implementation and use cases for novice and advanced users. As a demonstration of its capabilities, SMiRK was used to rapidly and automatically analyze a dataset taken from the literature. Conclusion SMiRK is a useful and efficient tool that can be used by investigators at multiple skill levels. Those who lack bioinformatics training can use it to easily and automatically analyze their data, while those with experience will find it beneficial to not need to write tools from scratch. PMID:26613105

  15. Avian influenza virus RNA extraction from tissue and swab material

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Viral RNA extraction is a critical step for any molecular downstream application and correct sample collection and handling is critical to obtaining reliable results for virus recovery or detection. The choice of Specimen type depends on numerous factors and must be compatible with the downstream ap...

  16. ACIS Extract: A Chandra/ACIS Tool for Automated Point Source Extraction and Spectral Fitting

    NASA Astrophysics Data System (ADS)

    Townsley, L.; Broos, P.; Bauer, F.; Getman, K.

    2003-03-01

    ACIS Extract (AE) is an IDL program that assists the observer in performing the many tasks involved in analyzing the spectra of large numbers of point sources observed with the ACIS instrument on Chandra. Notably, all tasks are performed in a context that may include multiple observations of the field. Features of AE and its several accessory tools include refining the accuracy of source positions, defining extraction regions based on the PSF of each source in each observation, generating single-observation and composite ARFs and RMFs, applying energy-dependent aperture corrections to the ARFs, computing light curves and K-S tests for source variability, automated broad-band photometry, automated spectral fitting and review of fitting results, and compilation of results into LaTeX tables. A variety of interactive plots are produced showing various source properties across the catalog. This poster details the capabilities of the package and shows example output. The code and a detailed users' manual are available to the community at http://www.astro.psu.edu/xray/docs/TARA/ae_users_guide.html. Support for this effort was provided by NASA contract NAS8-38252 to Gordon Garmire, the ACIS Principal Investigator.

  17. Assessing an improved protocol for plasma microRNA extraction.

    PubMed

    Moret, Inés; Sánchez-Izquierdo, Dolors; Iborra, Marisa; Tortosa, Luis; Navarro-Puche, Ana; Nos, Pilar; Cervera, José; Beltrán, Belén

    2013-01-01

    The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. This issue is critical when considering peripheral blood samples (plasma and serum) that are clinically interesting but meet several methodological problems, mainly complexity and low biomarker concentration. Analysis of small molecules, such as circulating microRNAs, should overcome these disadvantages. The present study describes an optimal RNA extraction method of microRNAs from human plasma samples. Different reagents and commercially available kits have been analyzed, identifying also the best pre-analytical conditions for plasma isolation. Between all of them, the column-based approaches were shown to be the most effective. In this context, miRNeasy Serum/Plasma Kit (from Qiagen) rendered more concentrated RNA, that was better suited for microarrays studies and did not require extra purification steps for sample concentration and purification than phenol based extraction methods. We also present evidences that the addition of low doses of an RNA carrier before starting the extraction process improves microRNA purification while an already published carrier dose can result in significant bias over microRNA profiles. Quality controls for best protocol selection were developed by spectrophotometry measurement of contaminants and microfluidics electrophoresis (Agilent 2100 Bioanalyzer) for RNA integrity. Selected donor and patient plasma samples and matched biopsies were tested by Affymetrix microarray technology to compare differentially expressed microRNAs. In summary, this study defines an optimized protocol for microRNA purification from human blood samples, increasing the performance of assays and shedding light over the best way to discover and use these biomarkers in clinical practice. PMID:24376572

  18. A COMPARISON OF AUTOMATED AND TRADITIONAL METHODS FOR THE EXTRACTION OF ARSENICALS FROM FISH

    EPA Science Inventory

    An automated extractor employing accelerated solvent extraction (ASE) has been compared with a traditional sonication method of extraction for the extraction of arsenicals from fish tissue. Four different species of fish and a standard reference material, DORM-2, were subjected t...

  19. AUTOMATED SOLID PHASE EXTRACTION GC/MS FOR ANALYSIS OF SEMIVOLATILES IN WATER AND SEDIMENTS

    EPA Science Inventory

    Data is presented on the development of a new automated system combining solid phase extraction (SPE) with GC/MS spectrometry for the single-run analysis of water samples containing a broad range of organic compounds. The system uses commercially available automated in-line sampl...

  20. RNASwift: A rapid, versatile RNA extraction method free from phenol and chloroform.

    PubMed

    Nwokeoji, Alison O; Kilby, Peter M; Portwood, David E; Dickman, Mark J

    2016-11-01

    RNASwift is an inexpensive, versatile method for the rapid extraction of RNA. Existing RNA extraction methods typically use hazardous chemicals including phenol, chloroform and formamide which are often difficult to completely remove from the extracted RNA. RNASwift uses sodium chloride and sodium dodecyl sulphate to lyse the cells and isolate the RNA from the abundant cellular components in conjunction with solid phase extraction or isopropanol precipitation to rapidly purify the RNA. Moreover, the purified RNA is directly compatible with downstream analysis. Using spectrophotometry in conjunction with ion pair reverse phase chromatography to analyse the extracted RNA, we show that RNASwift extracts and purifies RNA of higher quality and purity in comparison to alternative RNA extraction methods. The RNASwift method yields approximately 25 μg of RNA from only 10(8)Escherichia coli cells. Furthermore, RNASwift is versatile; the same simple reagents can be used to rapidly extract RNA from a variety of different cells including bacterial, yeast and mammalian cells. In addition to the extraction of total RNA, the RNASwift method can also be used to extract double stranded RNA from genetically modified E. coli in higher yields compared to alternative methods. PMID:27495141

  1. Disposable and removable nucleic acid extraction and purification cartridges for automated flow-through systems

    DOEpatents

    Regan, John Frederick

    2014-09-09

    Removable cartridges are used on automated flow-through systems for the purpose of extracting and purifying genetic material from complex matrices. Different types of cartridges are paired with specific automated protocols to concentrate, extract, and purifying pathogenic or human genetic material. Their flow-through nature allows large quantities sample to be processed. Matrices may be filtered using size exclusion and/or affinity filters to concentrate the pathogen of interest. Lysed material is ultimately passed through a filter to remove the insoluble material before the soluble genetic material is delivered past a silica-like membrane that binds the genetic material, where it is washed, dried, and eluted. Cartridges are inserted into the housing areas of flow-through automated instruments, which are equipped with sensors to ensure proper placement and usage of the cartridges. Properly inserted cartridges create fluid- and air-tight seals with the flow lines of an automated instrument.

  2. Plant RNA processing: soybean pre-mRNA in a pea cell-free extract

    SciTech Connect

    Schuler, M.A.; Hanley, B.A.

    1987-05-01

    Using a pea cell-free extract they have demonstrated the splicing of an SP6 fusion transcript containing an intron derived from the soybean seed storage protein ..beta..-subunit gene. Intron 115 from the conglycinin gene was cloned into a SP6 vector and transcribed using standard recombinant DNA techniques. Incubation of radioactively labeled fusion transcripts in the cell-free system produced a number of products which were identified by primer extension and S1 nuclease analysis. All the products are linear RNA molecules. Lariat intermediates, similar to those found in the yeast and HeLa cell RNA processing systems, have not been detected. The linear RNA products detected in their plant in vitro processing system have various portions of the intron removed which suggests that alternative splice sites are used in processing of this plant intron due to activation of cryptic splice sites or creation of splice sites in the fusion construction. The kinetics of the reactions and parameters of the extract are similar to those determined for the HeLa cell system. Sucrose gradient analysis has demonstrated that the plant RNA products sedimented in a 30S particle, similar in size to that found for the spliceosome of the HeLa cell system.

  3. Computational strategies for the automated design of RNA nanoscale structures from building blocks using NanoTiler☆

    PubMed Central

    Bindewald, Eckart; Grunewald, Calvin; Boyle, Brett; O’Connor, Mary; Shapiro, Bruce A.

    2013-01-01

    One approach to designing RNA nanoscale structures is to use known RNA structural motifs such as junctions, kissing loops or bulges and to construct a molecular model by connecting these building blocks with helical struts. We previously developed an algorithm for detecting internal loops, junctions and kissing loops in RNA structures. Here we present algorithms for automating or assisting many of the steps that are involved in creating RNA structures from building blocks: (1) assembling building blocks into nanostructures using either a combinatorial search or constraint satisfaction; (2) optimizing RNA 3D ring structures to improve ring closure; (3) sequence optimisation; (4) creating a unique non-degenerate RNA topology descriptor. This effectively creates a computational pipeline for generating molecular models of RNA nanostructures and more specifically RNA ring structures with optimized sequences from RNA building blocks. We show several examples of how the algorithms can be utilized to generate RNA tecto-shapes. PMID:18838281

  4. Spatial resolution requirements for automated cartographic road extraction

    USGS Publications Warehouse

    Benjamin, S.; Gaydos, L.

    1990-01-01

    Ground resolution requirements for detection and extraction of road locations in a digitized large-scale photographic database were investigated. A color infrared photograph of Sunnyvale, California was scanned, registered to a map grid, and spatially degraded to 1- to 5-metre resolution pixels. Road locations in each data set were extracted using a combination of image processing and CAD programs. These locations were compared to a photointerpretation of road locations to determine a preferred pixel size for the extraction method. Based on road pixel omission error computations, a 3-metre pixel resolution appears to be the best choice for this extraction method. -Authors

  5. Automated Algorithm for Extraction of Wetlands from IRS Resourcesat Liss III Data

    NASA Astrophysics Data System (ADS)

    Subramaniam, S.; Saxena, M.

    2011-09-01

    Wetlands play significant role in maintaining the ecological balance of both biotic and abiotic life in coastal and inland environments. Hence, understanding of their occurrence, spatial extent of change in wetland environment is very important and can be monitored using satellite remote sensing technique. The extraction of wetland features using remote sensing has so far been carried out using visual/ hybrid digital analysis techniques, which is time consuming. To monitor the wetland and their features at National/ State level, there is a need for the development of automated technique for the extraction of wetland features. A knowledge based algorithm has been developed using hierarchical decision tree approach for automated extraction of wetland features such as surface water spread, wet area, turbidity and wet vegetation including aquatic for pre and post monsoon period. The results obtained for Chhattisgarh, India using the automated technique has been found to be satisfactory, when compared with hybrid digital/visual analysis technique.

  6. Automated insertion of sequences into a ribosomal RNA alignment: An application of computational linguistics in molecular biology

    SciTech Connect

    Taylor, R.C.

    1991-11-01

    This thesis involved the construction of (1) a grammar that incorporates knowledge on base invariancy and secondary structure in a molecule and (2) a parser engine that uses the grammar to position bases into the structural subunits of the molecule. These concepts were combined with a novel pinning technique to form a tool that semi-automates insertion of a new species into the alignment for the 16S rRNA molecule (a component of the ribosome) maintained by Dr. Carl Woese's group at the University of Illinois at Urbana. The tool was tested on species extracted from the alignment and on a group of entirely new species. The results were very encouraging, and the tool should be substantial aid to the curators of the 16S alignment. The construction of the grammar was itself automated, allowing application of the tool to alignments for other molecules. The logic programming language Prolog was used to construct all programs involved. The computational linguistics approach used here was found to be a useful way to attach the problem of insertion into an alignment.

  7. Automated insertion of sequences into a ribosomal RNA alignment: An application of computational linguistics in molecular biology

    SciTech Connect

    Taylor, R.C.

    1991-11-01

    This thesis involved the construction of (1) a grammar that incorporates knowledge on base invariancy and secondary structure in a molecule and (2) a parser engine that uses the grammar to position bases into the structural subunits of the molecule. These concepts were combined with a novel pinning technique to form a tool that semi-automates insertion of a new species into the alignment for the 16S rRNA molecule (a component of the ribosome) maintained by Dr. Carl Woese`s group at the University of Illinois at Urbana. The tool was tested on species extracted from the alignment and on a group of entirely new species. The results were very encouraging, and the tool should be substantial aid to the curators of the 16S alignment. The construction of the grammar was itself automated, allowing application of the tool to alignments for other molecules. The logic programming language Prolog was used to construct all programs involved. The computational linguistics approach used here was found to be a useful way to attach the problem of insertion into an alignment.

  8. The evaluation of a concept for a Canadian-made automated multipurpose materials extraction facility

    NASA Astrophysics Data System (ADS)

    Kleinberg, H.

    Long-term habitation of space will eventually require use of off-Earth resources to reduce long-term program costs and risks to personnel and equipment due to launch from Earth. Extraction of oxygen from lunar soil is a prime example. Processes currently under study for such activities focus on the extraction of only one element / chemical from one type of soil on one world, and they produce large amounts of waste material. This paper presents the results of an examination by Spar Aerospace of a plasma separation concept as part of a materials extraction facility that might be used in space. Such a process has the far-reaching potential for extracting any or all of the elements available in soil samples, extraction of oxygen from lunar soil being the near-term application. Plasma separation has the potential for a 100 percent yield of extracted elements from input samples, and the versatility to be used on many non-terrestrial sites for the extraction of available elemental resources. The development of new materials extraction processes for each world would thus be eliminated. Such a facility could also reduce the generation of waste products by decomposing soil samples into pure, stable elements. Robotics, automation, and a plasma separation facility could be used to gather, prepare, process, separate, collect and ship the available chemical elements. The following topics are discussed: automated soil-gathering using robotics; automated soil pre-processing; plasma dissociation and separation of soil, and collection of sorted elements in an automated process; containment of gases, storage of pure elements, metals; and automated shipment of materials to a manned base, or pick-up site.

  9. Comparison of an automated nucleic acid extraction system with the column-based procedure

    PubMed Central

    Hinz, Rebecca; Hagen, Ralf Matthias

    2015-01-01

    Here, we assessed the extraction efficiency of a deployable bench-top nucleic acid extractor EZ1 in comparison to the column-based approach with complex sample matrices. A total of 48 EDTA blood samples and 81 stool samples were extracted by EZ1 automated extraction and the column-based QIAamp DNA Mini Kit. Blood sample extractions were assessed by two real-time malaria PCRs, while stool samples were analyzed by six multiplex real-time PCR assays targeting bacterial, viral, and parasitic stool pathogens. Inhibition control PCR testing was performed as well. In total, 147 concordant and 13 discordant pathogen-specific PCR results were obtained. The latter comprised 11 positive results after column-based extraction only and two positive results after EZ1 extraction only. EZ1 extraction showed a higher frequency of inhibition. This phenomenon was, however, inconsistent for the different PCR schemes. In case of concordant PCR results, relevant differences of cycle threshold numbers for the compared extraction schemes were not observed. Switches from well-established column-based extraction to extraction with the automated EZ1 system do not lead to a relevantly reduced yield of target DNA when complex sample matrices are used. If sample inhibition is observed, column-based extraction from another sample aliquot may be considered. PMID:25883797

  10. Feature Extraction and Selection Strategies for Automated Target Recognition

    NASA Technical Reports Server (NTRS)

    Greene, W. Nicholas; Zhang, Yuhan; Lu, Thomas T.; Chao, Tien-Hsin

    2010-01-01

    Several feature extraction and selection methods for an existing automatic target recognition (ATR) system using JPLs Grayscale Optical Correlator (GOC) and Optimal Trade-Off Maximum Average Correlation Height (OT-MACH) filter were tested using MATLAB. The ATR system is composed of three stages: a cursory region of-interest (ROI) search using the GOC and OT-MACH filter, a feature extraction and selection stage, and a final classification stage. Feature extraction and selection concerns transforming potential target data into more useful forms as well as selecting important subsets of that data which may aide in detection and classification. The strategies tested were built around two popular extraction methods: Principal Component Analysis (PCA) and Independent Component Analysis (ICA). Performance was measured based on the classification accuracy and free-response receiver operating characteristic (FROC) output of a support vector machine(SVM) and a neural net (NN) classifier.

  11. Chemical documents: machine understanding and automated information extraction.

    PubMed

    Townsend, Joe A; Adams, Sam E; Waudby, Christopher A; de Souza, Vanessa K; Goodman, Jonathan M; Murray-Rust, Peter

    2004-11-21

    Automatically extracting chemical information from documents is a challenging task, but an essential one for dealing with the vast quantity of data that is available. The task is least difficult for structured documents, such as chemistry department web pages or the output of computational chemistry programs, but requires increasingly sophisticated approaches for less structured documents, such as chemical papers. The identification of key units of information, such as chemical names, makes the extraction of useful information from unstructured documents possible. PMID:15534707

  12. USING A COMMERCIAL DNA EXTRACTION KIT TO OBTAIN RNA FROM MATURE RICE KERNELS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Extraction of total RNA from starchy plant material such as common food grains is difficult, and especially so from dry rice (Oryza sativa L.) kernels. Most commercial RNA kits are not suited for starchy materials and traditional RNA extraction procedures leave hazardous organic wastes that have ex...

  13. Optimization of RNA Purification and Analysis for Automated, Pre-Symptomatic Disease Diagnostics

    SciTech Connect

    Vaidya, A; Nasarabadi, S; Milanovich, F

    2005-06-28

    When diagnosing disease, time is often a more formidable enemy than the pathogen itself. Current detection methods rely primarily on post-symptomatic protein production (i.e. antibodies), which does not occur in noticeable levels until several weeks after infection. As such, a major goal among researchers today is to expedite pre-symptomatic disease recognition and treatment. Since most pathogens are known to leave a unique signature on the genetic expression of the host, one potential diagnostic tool is host mRNA. In my experiments, I examined several methods of isolating RNA and reading its genetic sequence. I first used two types of reverse transcriptase polymerase chain reactions (using commercial RNA) and examined the resultant complementary DNA through gel electrophoresis. I then proceeded to isolate and purify whole RNA from actual human monocytes and THP-1 cells using several published methods, and examined gene expression on the RNA itself. I compared the two RT-PCR methods and concluded that a double step RT-PCR is superior to the single step method. I also compared the various techniques of RNA isolation by examining the yield and purity of the resultant RNA. Finally, I studied the level of cellular IL-8 and IL-1 gene expression, two genes involved in the human immune response, which can serve as a baseline for future genetic comparison with LPS-exposed cells. Based on the results, I have determined which conditions and procedures are optimal for RNA isolation, RT-PCR, and RNA yield assessment. The overall goal of my research is to develop a flow-through system of RNA analysis, whereby blood samples can be collected and analyzed for disease prior to the onset of symptoms. The Pathomics group hopes to automate this process by removing the human labor factor, thereby decreasing the procedure's cost and increasing its availability to the general population. Eventually, our aim is to have an autonomous diagnostic system based on RNA analysis that would

  14. Accurate transcription of homologous 5S rRNA and tRNA genes and splicing of tRNA in vitro by soluble extracts of Neurospora.

    PubMed Central

    Tyler, B M; Giles, N H

    1984-01-01

    We have developed soluble extracts from Neurospora crassa capable of accurately and efficiently transcribing homologous 5S rRNA and tRNA genes. The extracts also appear to quantitatively end-process and splice the primary tRNA transcripts. Although the extracts could not transcribe a heterologous (yeast) 5S rRNA gene, they did transcribe a yeast tRNALeu gene and slowly process the transcripts. In addition, we have developed a novel strategy for rapidly sequencing uniformly labelled RNAs using base-specific ribonucleases. We have used this procedure to verify the identity of the in vitro transcripts and processing products. Images PMID:6235482

  15. Prescription Extraction from Clinical Notes: Towards Automating EMR Medication Reconciliation

    PubMed Central

    Wang, Yajuan; Steinhubl, Steven R.; Defilippi, Chrisopher; Ng, Kenney; Ebadollahi, Shahram; Stewart, Walter F.; Byrd, Roy J

    2015-01-01

    Medication in for ma lion is one of [he most important clinical data types in electronic medical records (EMR) This study developed an NLP application (PredMED) to extract full prescriptions and their relevant components from a large corpus of unstructured ambulatory office visit clinical notes and the corresponding structured medication reconciliation (MED REC) data in the EMR. PredMED achieved an 84.4% F-score on office visit encounter notes and 95.0% on MED„REC data, outperforming two available medication extraction systems. To assess the potential for using automatically extracted prescriptions in the medication reconciliation task, we manually analyzed discrepancies between prescriptions found in clinical encounter notes and in matching MED_REC data for sample patient encounters. PMID:26306266

  16. Using a commercial DNA extraction kit to obtain RNA from mature rice kernels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Few RNA extraction protocols or commercial kits work well with the starchy endosperm of cereal grains. Standard RNA extraction protocols are time consuming, use large amounts of expensive chemicals, and leave behind hazardous wastes. However, there are numerous commercial DNA extraction kits that ...

  17. A New Method for Extraction of Double-Stranded RNA from Plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The presence of high molecular weight double-stranded RNA (dsRNA) in plants is associated with the presence of RNA viruses. DsRNA is stable and can be extracted easily from the majority of plant species and provides an excellent tool for characterization of novel viruses that are recalcitrant to pur...

  18. A New Protocol for Extraction of Double-Stranded RNA from Plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are several unidentified virus-like diseases of blueberry that have been recalcitrant to standard methods of virus characterization based on double-stranded RNA (dsRNA). Relatives of these viruses yield large amounts of dsRNA in hosts other than blueberry. Modifications in dsRNA extraction p...

  19. Automated Boundary-Extraction and Region-Growing Techniques Applied to Solar Magnetograms

    NASA Technical Reports Server (NTRS)

    McAteer, R. T. James; Gallagher, Peter; Ireland, Jack; Young, C Alex

    2005-01-01

    We present an automated approach to active region extraction from full disc MDI longitudinal magnetograms. This uses a region-growing technique in conjunction with boundary-extraction to define a number of enclosed contours as belonging to separate regions of magnetic significance on the solar disc. This provides an objective definition of active regions and areas of plage on the Sun. A number of parameters relating to the flare-potential of each region is discussed.

  20. Discovering Indicators of Successful Collaboration Using Tense: Automated Extraction of Patterns in Discourse

    ERIC Educational Resources Information Center

    Thompson, Kate; Kennedy-Clark, Shannon; Wheeler, Penny; Kelly, Nick

    2014-01-01

    This paper describes a technique for locating indicators of success within the data collected from complex learning environments, proposing an application of e-research to access learner processes and measure and track group progress. The technique combines automated extraction of tense and modality via parts-of-speech tagging with a visualisation…

  1. Automated extraction of pleural effusion in three-dimensional thoracic CT images

    NASA Astrophysics Data System (ADS)

    Kido, Shoji; Tsunomori, Akinori

    2009-02-01

    It is important for diagnosis of pulmonary diseases to measure volume of accumulating pleural effusion in threedimensional thoracic CT images quantitatively. However, automated extraction of pulmonary effusion correctly is difficult. Conventional extraction algorithm using a gray-level based threshold can not extract pleural effusion from thoracic wall or mediastinum correctly, because density of pleural effusion in CT images is similar to those of thoracic wall or mediastinum. So, we have developed an automated extraction method of pulmonary effusion by use of extracting lung area with pleural effusion. Our method used a template of lung obtained from a normal lung for segmentation of lungs with pleural effusions. Registration process consisted of two steps. First step was a global matching processing between normal and abnormal lungs of organs such as bronchi, bones (ribs, sternum and vertebrae) and upper surfaces of livers which were extracted using a region-growing algorithm. Second step was a local matching processing between normal and abnormal lungs which were deformed by the parameter obtained from the global matching processing. Finally, we segmented a lung with pleural effusion by use of the template which was deformed by two parameters obtained from the global matching processing and the local matching processing. We compared our method with a conventional extraction method using a gray-level based threshold and two published methods. The extraction rates of pleural effusions obtained from our method were much higher than those obtained from other methods. Automated extraction method of pulmonary effusion by use of extracting lung area with pleural effusion is promising for diagnosis of pulmonary diseases by providing quantitative volume of accumulating pleural effusion.

  2. Improved protocol for the extraction of bacterial mRNA from soils.

    PubMed

    Sharma, Shilpi; Mehta, Ravikumar; Gupta, Rashi; Schloter, Michael

    2012-10-01

    An improved protocol for extraction of prokaryotic mRNA from soil samples was developed by modifying the extraction procedure to obtain higher yields of mRNA and to reduce co-extraction of humic acids. The modified protocol was found to be more robust and efficient compared to the original protocol by Griffiths et al. (2000) without compromising with the quality and quantity of RNA. PMID:22841738

  3. Towards automated support for extraction of reusable components

    NASA Technical Reports Server (NTRS)

    Abd-El-hafiz, S. K.; Basili, Victor R.; Caldiera, Gianluigi

    1992-01-01

    A cost effective introduction of software reuse techniques requires the reuse of existing software developed in many cases without aiming at reusability. This paper discusses the problems related to the analysis and reengineering of existing software in order to reuse it. We introduce a process model for component extraction and focus on the problem of analyzing and qualifying software components which are candidates for reuse. A prototype tool for supporting the extraction of reusable components is presented. One of the components of this tool aids in understanding programs and is based on the functional model of correctness. It can assist software engineers in the process of finding correct formal specifications for programs. A detailed description of this component and an example to demonstrate a possible operational scenario are given.

  4. Towards automated support for extraction of reusable components

    NASA Technical Reports Server (NTRS)

    Abd-El-hafiz, S. K.; Basili, V. R.; Caldier, G.

    1991-01-01

    A cost effective introduction of software reuse techniques requires the reuse of existing software developed in many cases without aiming at reusability. This paper discusses the problems related to the analysis and reengineering of existing software in order to reuse it. We introduce a process model for component extraction and focus on the problem of analyzing and qualifying software components which are candidates for reuse. A prototype tool for supporting the extraction of reusable components is presented. One of the components of this tool aids in understanding programs and is based on the functional model of correctness. It can assist software engineers in the process of finding correct formal specifications for programs. A detailed description of this component and an example to demonstrate a possible operational scenario are given.

  5. Extraction of high-quality RNA from pancreatic tissues for gene expression studies.

    PubMed

    Augereau, Cécile; Lemaigre, Frédéric P; Jacquemin, Patrick

    2016-05-01

    Extracting RNA from pancreatic tissue is notoriously challenging because of the organ's high RNase content. Standard methods using TriPure or TRIzol classically yield RNA of sufficient quality for routine gene expression analysis but not for microarray or deep sequencing analysis. Here we developed a simple method to extract high-quality RNA from mouse pancreas. Our method uses an RNase inhibitor and combines different protocols using guanidium thiocyanate-phenol extraction. It enables reproducible isolation of RNA with an RNA integrity number around 9. PMID:26896683

  6. Data Mining: The Art of Automated Knowledge Extraction

    NASA Astrophysics Data System (ADS)

    Karimabadi, H.; Sipes, T.

    2012-12-01

    Data mining algorithms are used routinely in a wide variety of fields and they are gaining adoption in sciences. The realities of real world data analysis are that (a) data has flaws, and (b) the models and assumptions that we bring to the data are inevitably flawed, and/or biased and misspecified in some way. Data mining can improve data analysis by detecting anomalies in the data, check for consistency of the user model assumptions, and decipher complex patterns and relationships that would not be possible otherwise. The common form of data collected from in situ spacecraft measurements is multi-variate time series which represents one of the most challenging problems in data mining. We have successfully developed algorithms to deal with such data and have extended the algorithms to handle streaming data. In this talk, we illustrate the utility of our algorithms through several examples including automated detection of reconnection exhausts in the solar wind and flux ropes in the magnetotail. We also show examples from successful applications of our technique to analysis of 3D kinetic simulations. With an eye to the future, we provide an overview of our upcoming plans that include collaborative data mining, expert outsourcing data mining, computer vision for image analysis, among others. Finally, we discuss the integration of data mining algorithms with web-based services such as VxOs and other Heliophysics data centers and the resulting capabilities that it would enable.

  7. Multispectral Image Road Extraction Based Upon Automated Map Conflation

    NASA Astrophysics Data System (ADS)

    Chen, Bin

    Road network extraction from remotely sensed imagery enables many important and diverse applications such as vehicle tracking, drone navigation, and intelligent transportation studies. There are, however, a number of challenges to road detection from an image. Road pavement material, width, direction, and topology vary across a scene. Complete or partial occlusions caused by nearby buildings, trees, and the shadows cast by them, make maintaining road connectivity difficult. The problems posed by occlusions are exacerbated with the increasing use of oblique imagery from aerial and satellite platforms. Further, common objects such as rooftops and parking lots are made of materials similar or identical to road pavements. This problem of common materials is a classic case of a single land cover material existing for different land use scenarios. This work addresses these problems in road extraction from geo-referenced imagery by leveraging the OpenStreetMap digital road map to guide image-based road extraction. The crowd-sourced cartography has the advantages of worldwide coverage that is constantly updated. The derived road vectors follow only roads and so can serve to guide image-based road extraction with minimal confusion from occlusions and changes in road material. On the other hand, the vector road map has no information on road widths and misalignments between the vector map and the geo-referenced image are small but nonsystematic. Properly correcting misalignment between two geospatial datasets, also known as map conflation, is an essential step. A generic framework requiring minimal human intervention is described for multispectral image road extraction and automatic road map conflation. The approach relies on the road feature generation of a binary mask and a corresponding curvilinear image. A method for generating the binary road mask from the image by applying a spectral measure is presented. The spectral measure, called anisotropy-tunable distance (ATD

  8. Evaluation of Three Automated Nucleic Acid Extraction Systems for Identification of Respiratory Viruses in Clinical Specimens by Multiplex Real-Time PCR

    PubMed Central

    Kwon, Aerin; Lee, Kyung-A

    2014-01-01

    A total of 84 nasopharyngeal swab specimens were collected from 84 patients. Viral nucleic acid was extracted by three automated extraction systems: QIAcube (Qiagen, Germany), EZ1 Advanced XL (Qiagen), and MICROLAB Nimbus IVD (Hamilton, USA). Fourteen RNA viruses and two DNA viruses were detected using the Anyplex II RV16 Detection kit (Seegene, Republic of Korea). The EZ1 Advanced XL system demonstrated the best analytical sensitivity for all the three viral strains. The nucleic acids extracted by EZ1 Advanced XL showed higher positive rates for virus detection than the others. Meanwhile, the MICROLAB Nimbus IVD system was comprised of fully automated steps from nucleic extraction to PCR setup function that could reduce human errors. For the nucleic acids recovered from nasopharyngeal swab specimens, the QIAcube system showed the fewest false negative results and the best concordance rate, and it may be more suitable for detecting various viruses including RNA and DNA virus strains. Each system showed different sensitivity and specificity for detection of certain viral pathogens and demonstrated different characteristics such as turnaround time and sample capacity. Therefore, these factors should be considered when new nucleic acid extraction systems are introduced to the laboratory. PMID:24868527

  9. Fully Automated Electro Membrane Extraction Autosampler for LC-MS Systems Allowing Soft Extractions for High-Throughput Applications.

    PubMed

    Fuchs, David; Pedersen-Bjergaard, Stig; Jensen, Henrik; Rand, Kasper D; Honoré Hansen, Steen; Petersen, Nickolaj Jacob

    2016-07-01

    The current work describes the implementation of electro membrane extraction (EME) into an autosampler for high-throughput analysis of samples by EME-LC-MS. The extraction probe was built into a luer lock adapter connected to a HTC PAL autosampler syringe. As the autosampler drew sample solution, analytes were extracted into the lumen of the extraction probe and transferred to a LC-MS system for further analysis. Various parameters affecting extraction efficacy were investigated including syringe fill strokes, syringe pull up volume, pull up delay and volume in the sample vial. The system was optimized for soft extraction of analytes and high sample throughput. Further, it was demonstrated that by flushing the EME-syringe with acidic wash buffer and reverting the applied electric potential, carry-over between samples can be reduced to below 1%. Performance of the system was characterized (RSD, <10%; R(2), 0.994) and finally, the EME-autosampler was used to analyze in vitro conversion of methadone into its main metabolite by rat liver microsomes and for demonstrating the potential of known CYP3A4 inhibitors to prevent metabolism of methadone. By making use of the high extraction speed of EME, a complete analytical workflow of purification, separation, and analysis of sample could be achieved within only 5.5 min. With the developed system large sequences of samples could be analyzed in a completely automated manner. This high degree of automation makes the developed EME-autosampler a powerful tool for a wide range of applications where high-throughput extractions are required before sample analysis. PMID:27237618

  10. Rnnotator: an automated de novo transcriptome assembly pipeline from stranded RNA-Seq reads

    SciTech Connect

    Martin, Jeffrey; Bruno, Vincent M.; Fang, Zhide; Meng, Xiandong; Blow, Matthew; Zhang, Tao; Sherlock, Gavin; Snyder, Michael; Wang, Zhong

    2010-11-19

    Background: Comprehensive annotation and quantification of transcriptomes are outstanding problems in functional genomics. While high throughput mRNA sequencing (RNA-Seq) has emerged as a powerful tool for addressing these problems, its success is dependent upon the availability and quality of reference genome sequences, thus limiting the organisms to which it can be applied. Results: Here, we describe Rnnotator, an automated software pipeline that generates transcript models by de novo assembly of RNA-Seq data without the need for a reference genome. We have applied the Rnnotator assembly pipeline to two yeast transcriptomes and compared the results to the reference gene catalogs of these organisms. The contigs produced by Rnnotator are highly accurate (95percent) and reconstruct full-length genes for the majority of the existing gene models (54.3percent). Furthermore, our analyses revealed many novel transcribed regions that are absent from well annotated genomes, suggesting Rnnotator serves as a complementary approach to analysis based on a reference genome for comprehensive transcriptomics. Conclusions: These results demonstrate that the Rnnotator pipeline is able to reconstruct full-length transcripts in the absence of a complete reference genome.

  11. An automated approach for extracting Barrier Island morphology from digital elevation models

    NASA Astrophysics Data System (ADS)

    Wernette, Phillipe; Houser, Chris; Bishop, Michael P.

    2016-06-01

    The response and recovery of a barrier island to extreme storms depends on the elevation of the dune base and crest, both of which can vary considerably alongshore and through time. Quantifying the response to and recovery from storms requires that we can first identify and differentiate the dune(s) from the beach and back-barrier, which in turn depends on accurate identification and delineation of the dune toe, crest and heel. The purpose of this paper is to introduce a multi-scale automated approach for extracting beach, dune (dune toe, dune crest and dune heel), and barrier island morphology. The automated approach introduced here extracts the shoreline and back-barrier shoreline based on elevation thresholds, and extracts the dune toe, dune crest and dune heel based on the average relative relief (RR) across multiple spatial scales of analysis. The multi-scale automated RR approach to extracting dune toe, dune crest, and dune heel based upon relative relief is more objective than traditional approaches because every pixel is analyzed across multiple computational scales and the identification of features is based on the calculated RR values. The RR approach out-performed contemporary approaches and represents a fast objective means to define important beach and dune features for predicting barrier island response to storms. The RR method also does not require that the dune toe, crest, or heel are spatially continuous, which is important because dune morphology is likely naturally variable alongshore.

  12. High-quality RNA extraction from small cardamom tissues rich in polysaccharides and polyphenols.

    PubMed

    Nadiya, Fasiludeen; Anjali, Narayanannair; Gangaprasad, Appukuttannair; Sabu, Kalluvettankuzhy Krishnannair

    2015-09-15

    Due to the presence of a diverse array of metabolites, no standard method of RNA isolation is available for plants. We noted that polysaccharide and polyphenol contents of cardamom tissues critically hinder the RNA extraction procedure. Hence, we attempted several methods for obtaining intact mRNA and small RNA from various cardamom tissues. It was found that protocols involving a combination of commercial kits and conventional CTAB (cetyl trimethylammonium bromide) methods yielded RNA with good purity, higher yield, and good integrity. The total RNA isolated through this approach was found to be amenable for transcriptome and small RNA analysis through next-generation sequencing platforms. PMID:26048648

  13. Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives.

    PubMed

    Koopmans, M; Monroe, S S; Coffield, L M; Zaki, S R

    1993-07-01

    A method was developed for fast and efficient isolation of RNA from paraffin-embedded tissue sections for subsequent PCR analysis. This method is based on the binding of RNA to acid-treated glass beads in the presence of a high molarity of guanidinium salt. It can be completed within an hour, and obviates the need for dewaxing and phenol/chloroform extractions. The effect of various fixatives and fixation times was tested and the amplification of actin mRNA fragments ranging in length from 82 to 507 bp was used to demonstrate the presence of RNA in the extracts. The method was compared to existing extraction techniques by studying the quality of the templates for reverse-transcriptase polymerase chain reaction amplification (RT-PCR), using virus-infected and mock-infected paraffin-embedded cell pellets as a model. PCR amplification of cellular and viral RNA was successful for RNA isolated by use of all extraction techniques, although the glass bead method was preferred for its simplicity and rapidity. Specimens fixed with formalin were found to be suitable for PCR, but the best results were obtained with acetone-fixed paraffin-embedded material. Dewaxing of tissue sections had no effect on the yield and quality of RNA extractions, and further purification of the extracts using gel filtration did not improve the results. After the protocols were optimized, rotavirus-infected cell pellets were used to demonstrate that extraction and amplification of dsRNA was possible. The information obtained from the studies with the model system was used for extraction of toroviral and rotaviral RNA from archival intestinal material. These data indicate that paraffin-embedded archival tissue can be used for RT-PCR analysis, adding an important technique to diagnostic pathology and retrospective studies. PMID:8396155

  14. Automated segmentation and feature extraction of product inspection items

    NASA Astrophysics Data System (ADS)

    Talukder, Ashit; Casasent, David P.

    1997-03-01

    X-ray film and linescan images of pistachio nuts on conveyor trays for product inspection are considered. The final objective is the categorization of pistachios into good, blemished and infested nuts. A crucial step before classification is the separation of touching products and the extraction of features essential for classification. This paper addresses new detection and segmentation algorithms to isolate touching or overlapping items. These algorithms employ a new filter, a new watershed algorithm, and morphological processing to produce nutmeat-only images. Tests on a large database of x-ray film and real-time x-ray linescan images of around 2900 small, medium and large nuts showed excellent segmentation results. A new technique to detect and segment dark regions in nutmeat images is also presented and tested on approximately 300 x-ray film and approximately 300 real-time linescan x-ray images with 95-97 percent detection and correct segmentation. New algorithms are described that determine nutmeat fill ratio and locate splits in nutmeat. The techniques formulated in this paper are of general use in many different product inspection and computer vision problems.

  15. [Rapid method to extract high-quality RNA from activated sludge].

    PubMed

    Jin, Min; Zhao, Zu-Guo; Qiu, Zhi-Gang; Wang, Jing-Feng; Chen, Zhao-Li; Shen, Zhi-Qiang; Li, Chao; Wang, Xin-Wei; Dong, Yan; Li, Jun-Wen

    2010-01-01

    An effective and fast RNA isolation method of activated sludge was established and five different methods were compared based on RNA yield, purity, integrity, RT-PCR amplification of 16S rRNA genes and subsequent terminal restriction fragment length polymorphism (T-RFLP) analysis. That is, the precipitated activated sludge was washed with TENP and PBS buffer, followed by using lysozyme and TRIzol to direct lysis of microbial cells, chloroform to remove protein and most of the DNA from bacterial lysate, isopropanol to precipitate nucleic acid and DNase I to hydrolyze residual DNA. To further purify RNA, RNA purifying column was utilized. The results demonstrated that the extraction method, with the aid of TRIzol and RNA purification kit, can effectively extract high-quality RNA. It not only means low degradability and high quantity, purity and diversity, but also the genes of 16S rRNA and amoA can be amplified by RT-PCR. Compared with other methods, it showed great advantage of low cost and high efficiency and can be applied to RNA extraction of activated sludge in a large number. Furthermore, T-RFLP results indicated that the community composition as well as the abundance of individual members was affected by the kind of RNA extraction methods. This work established a rapid and effective method to extract high-quality RNA from activated sludge and would show great potential for monitoring microbial changes and studying metabolism and community array of activated sludge. PMID:20329549

  16. Comparative evaluation of different extraction and quantification methods for forensic RNA analysis.

    PubMed

    Grabmüller, Melanie; Madea, Burkhard; Courts, Cornelius

    2015-05-01

    Since about 2005, there is increasing interest in forensic RNA analysis whose versatility may very favorably complement traditional DNA profiling in forensic casework. There is, however, no method available specifically dedicated for extraction of RNA from forensically relevant sample material. In this study we compared five commercially available and commonly used RNA extraction kits and methods (mirVana™ miRNA Isolation Kit Ambion; Trizol® Reagent, Invitrogen; NucleoSpin® miRNA Kit Macherey-Nagel; AllPrep DNA/RNA Mini Kit and RNeasy® Mini Kit both Qiagen) to assess their relative effectiveness of yielding RNA of good quality and their compatibility with co-extraction of DNA amenable to STR profiling. We set up samples of small amounts of dried blood, liquid saliva, semen and buccal mucosa that were aged for different time intervals for co-extraction of RNA and DNA. RNA quality was assessed by determination of 'RNA integrity number' (RIN) and quantitative PCR based expression analysis. DNA quality was assessed via monitoring STR typing success rates. By comparison, the different methods exhibited considerable differences between RNA and DNA yields, RNA quality values and expression levels, and STR profiling success, with the AllPrep DNA/RNA Mini Kit and the NucleoSpin® miRNA Kit excelling at DNA co-extraction and RNA results, respectively. Overall, there was no 'best' method to satisfy all demands of comprehensible co-analysis of RNA and DNA and it appears that each method has specific merits and flaws. We recommend to cautiously choose from available methods and align its characteristics with the needs of the experimental setting at hand. PMID:25625965

  17. Extraction of Prostatic Lumina and Automated Recognition for Prostatic Calculus Image Using PCA-SVM

    PubMed Central

    Wang, Zhuocai; Xu, Xiangmin; Ding, Xiaojun; Xiao, Hui; Huang, Yusheng; Liu, Jian; Xing, Xiaofen; Wang, Hua; Liao, D. Joshua

    2011-01-01

    Identification of prostatic calculi is an important basis for determining the tissue origin. Computation-assistant diagnosis of prostatic calculi may have promising potential but is currently still less studied. We studied the extraction of prostatic lumina and automated recognition for calculus images. Extraction of lumina from prostate histology images was based on local entropy and Otsu threshold recognition using PCA-SVM and based on the texture features of prostatic calculus. The SVM classifier showed an average time 0.1432 second, an average training accuracy of 100%, an average test accuracy of 93.12%, a sensitivity of 87.74%, and a specificity of 94.82%. We concluded that the algorithm, based on texture features and PCA-SVM, can recognize the concentric structure and visualized features easily. Therefore, this method is effective for the automated recognition of prostatic calculi. PMID:21461364

  18. Specific initiation by RNA polymerase I in a whole-cell extract from yeast.

    PubMed Central

    Schultz, M C; Choe, S Y; Reeder, R H

    1991-01-01

    A protocol is described for making a soluble whole-cell extract from yeast (Saccharomyces cerevisiae) that supports active and specific transcription initiation by RNA polymerases I, II, and III. Specific initiation by polymerase I decreases in high-density cultures, paralleling the decrease in abundance of the endogenous 35S rRNA precursor. This extract should be useful for studying the molecular mechanisms that regulate rRNA transcription in yeast. Images PMID:1992452

  19. Automation of Extraction Chromatograhic and Ion Exchange Separations for Radiochemical Analysis and Monitoring

    SciTech Connect

    Grate, Jay W.; O'Hara, Matthew J.; Egorov, Oleg

    2009-08-19

    Radiochemical analysis, complete with the separation of radionuclides of interest from the sample matrix and from other interfering radionuclides, is often an essential step in the determination of the radiochemical composition of a nuclear sample or process stream. Although some radionuclides can be determined nondestructively by gamma spectroscopy, where the gamma rays penetrate significant distances in condensed media and the gamma ray energies are diagnostic for specific radionuclides, other radionuclides that may be of interest emit only alpha or beta particles. For these, samples must be taken for destructive analysis and radiochemical separations are required. For process monitoring purposes, the radiochemical separation and detection methods must be rapid so that the results will be timely. These results could be obtained by laboratory analysis or by radiochemical process analyzers operating on-line or at-site. In either case, there is a need for automated radiochemical analysis methods to provide speed, throughput, safety, and consistent analytical protocols. Classical methods of separation used during the development of nuclear technologies, namely manual precipitations, solvent extractions, and ion exchange, are slow and labor intensive. Fortunately, the convergence of digital instrumentation for preprogrammed fluid manipulation and the development of new separation materials for column-based isolation of radionuclides has enabled the development of automated radiochemical analysis methodology. The primary means for separating radionuclides in solution are liquid-liquid extraction and ion exchange. These processes are well known and have been reviewed in the past.1 Ion exchange is readily employed in column formats. Liquid-liquid extraction can also be implemented on column formats using solvent-impregnated resins as extraction chromatographic materials. The organic liquid extractant is immobilized in the pores of a microporous polymer material. Under

  20. Improved RNA extraction method using the BioMasher and BioMasher power-plus.

    PubMed

    Yamamoto, Takuji; Nakashima, Kentaro; Maruta, Yukio; Kiriyama, Tomomi; Sasaki, Michi; Sugiyama, Shunpei; Suzuki, Kana; Fujisaki, Hitomi; Sasaki, Jun; Kaku-Ushiki, Yuko; Tanida, Masatoshi; Irie, Shinkichi; Hattori, Shunji

    2012-12-01

    The BioMasher is a disposable homogenizer that was developed to homogenize bovine brain tissue for bovine spongiform encephalopathy diagnosis. Capable of preventing the biohazard risk from infectious samples, it also prevents cross-contamination among samples. The BioMasher is thus widely used in biochemical research, especially for RNA extraction. Here, we tested a novel BioMasher application for RNA extraction from animal and plant tissues. We also developed a grinding machine specific for the BioMasher, named the BioMasher Power-Plus. We developed RNA extraction protocols using the BioMasher combined with the BioMasher Power-Plus. We compared RNA extraction efficiency of the BioMasher with that of the FastPrep and the glass homogenizer. Though the RNA extraction efficiency by the BioMasher was nearly equivalent to that of the FastPrep and the glass homogenizer, sample preparation time was shorter for the BioMasher. The utility of RNA extraction by the BioMasher was examined in mouse, rat, and tomato tissue samples. In the rodent tissues, the highest extraction efficiency of total RNA was from liver, with lowest efficiency from fibrous tissues such as muscle. The quality of extracted total RNA was confirmed by agarose gel electrophoresis which produced highly visible clear bands of 18S and 28S rRNAs. Reproducibility among different operators in RNA extraction from tomato roots was improved by using the BioMasher Power-Plus. The BioMasher and BioMasher Power-Plus provide an effective and easy homogenization method for total RNA extraction from some rodent and plant tissues. PMID:22813946

  1. Evaluation of an automated hydrolysis and extraction method for quantification of total fat and lipid classess in cereal products.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The utility of an automated acid hydrolysis-extraction (AHE) system was evaluated for extraction of fat for the quantification of total, saturated, polyunsaturated, monounsaturated, and trans fat in cereal products. Oil extracted by the AHE system was assessed for total fat gravimetrically and by c...

  2. Automated extraction of acetylgestagens from kidney fat by matrix solid phase dispersion.

    PubMed

    Rosén, J; Hellenäs, K E; Törnqvist, P; Shearan, P

    1994-12-01

    A new extraction method for the acetylgestagens medroxyprogesterone acetate (MPA), chloromadinone acetate and megestrol acetate, from kidney fat, has been developed. The method is a combination of matrix solid phase dispersion and solid phase extraction and is simpler and safer than previous methods, especially as it can be automated. The recovery was estimated as 59 +/- 5% (mean +/- standard deviation) for MPA. For screening purposes detection can be achieved using a commercially available enzyme immunoassay kit giving detection limits in the range of 1.0-2.0 ng g-1. PMID:7533481

  3. Extraction of RNA from the plant Kalanchoë daigremontiana.

    PubMed

    Garcês, Helena; Sinha, Neelima

    2009-10-01

    This protocol describes how to isolate total RNA from several tissues of Kalanchoë daigremontiana. Total RNA can be isolated by using the TRI-reagent method, scaled up for processing 2 g of tissue, or by using the protocol described here, which gives higher concentrations of high quality RNA. The resulting RNA can be used for various applications including generation of cDNA for reverse transcriptase-PCR (RT-PCR), Northern blots, or other purposes. PMID:20147050

  4. Comparison of two methods for RNA extraction from the nucleus pulposus of intervertebral discs.

    PubMed

    Gan, M F; Yang, H L; Qian, J L; Wu, C S; Yuan, C X; Li, X F; Zou, J

    2016-01-01

    RNA extraction from the nucleus pulposus of intervertebral discs has been extensively used in orthopedic studies. We compared two methods for extracting RNA from the nucleus pulposus: liquid nitrogen grinding and enzyme digestion. The RNA was detected by agarose gel electrophoresis, and the purity was evaluated by absorbance ratio using a spectrophotometer. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR). Thirty human lumbar intervertebral discs were used in this study. The liquid nitrogen-grinding method was used for RNA extraction from 15 samples, and the mean RNA concentration was 491.04 ± 44.16 ng/mL. The enzyme digestion method was used on 15 samples, and the mean RNA concentration was 898.42 ± 38.64 ng/mL. The statistical analysis revealed that there was a significant difference in concentration between the different methods. Apparent 28S, 18S, and 5S bands were detectable in RNA extracted using the enzyme digestion method, whereas no 28S or 18S bands were detected in RNA extracted using the liquid nitrogen-grinding method. The GAPDH band was visible, and no non-specific band was detected in the RT-PCR assay by the enzyme digestion method. Therefore, the enzyme digestion method is an efficient and easy method for RNA extraction from the nucleus pulposus of intervertebral discs for further intervertebral disc degeneration-related studies. PMID:27323116

  5. Automated physics-based design of synthetic riboswitches from diverse RNA aptamers

    PubMed Central

    Espah Borujeni, Amin; Mishler, Dennis M.; Wang, Jingzhi; Huso, Walker; Salis, Howard M.

    2016-01-01

    Riboswitches are shape-changing regulatory RNAs that bind chemicals and regulate gene expression, directly coupling sensing to cellular actuation. However, it remains unclear how their sequence controls the physics of riboswitch switching and activation, particularly when changing the ligand-binding aptamer domain. We report the development of a statistical thermodynamic model that predicts the sequence-structure-function relationship for translation-regulating riboswitches that activate gene expression, characterized inside cells and within cell-free transcription–translation assays. Using the model, we carried out automated computational design of 62 synthetic riboswitches that used six different RNA aptamers to sense diverse chemicals (theophylline, tetramethylrosamine, fluoride, dopamine, thyroxine, 2,4-dinitrotoluene) and activated gene expression by up to 383-fold. The model explains how aptamer structure, ligand affinity, switching free energy and macromolecular crowding collectively control riboswitch activation. Our model-based approach for engineering riboswitches quantitatively confirms several physical mechanisms governing ligand-induced RNA shape-change and enables the development of cell-free and bacterial sensors for diverse applications. PMID:26621913

  6. Automated physics-based design of synthetic riboswitches from diverse RNA aptamers.

    PubMed

    Espah Borujeni, Amin; Mishler, Dennis M; Wang, Jingzhi; Huso, Walker; Salis, Howard M

    2016-01-01

    Riboswitches are shape-changing regulatory RNAs that bind chemicals and regulate gene expression, directly coupling sensing to cellular actuation. However, it remains unclear how their sequence controls the physics of riboswitch switching and activation, particularly when changing the ligand-binding aptamer domain. We report the development of a statistical thermodynamic model that predicts the sequence-structure-function relationship for translation-regulating riboswitches that activate gene expression, characterized inside cells and within cell-free transcription-translation assays. Using the model, we carried out automated computational design of 62 synthetic riboswitches that used six different RNA aptamers to sense diverse chemicals (theophylline, tetramethylrosamine, fluoride, dopamine, thyroxine, 2,4-dinitrotoluene) and activated gene expression by up to 383-fold. The model explains how aptamer structure, ligand affinity, switching free energy and macromolecular crowding collectively control riboswitch activation. Our model-based approach for engineering riboswitches quantitatively confirms several physical mechanisms governing ligand-induced RNA shape-change and enables the development of cell-free and bacterial sensors for diverse applications. PMID:26621913

  7. Knowledge-based automated road network extraction system using multispectral images

    NASA Astrophysics Data System (ADS)

    Sun, Weihua; Messinger, David W.

    2013-04-01

    A novel approach for automated road network extraction from multispectral WorldView-2 imagery using a knowledge-based system is presented. This approach uses a multispectral flood-fill technique to extract asphalt pixels from satellite images; it follows by identifying prominent curvilinear structures using template matching. The extracted curvilinear structures provide an initial estimate of the road network, which is refined by the knowledge-based system. This system breaks the curvilinear structures into small segments and then groups them using a set of well-defined rules; a saliency check is then performed to prune the road segments. As a final step, these segments, carrying road width and orientation information, can be reconstructed to generate a proper road map. The approach is shown to perform well with various urban and suburban scenes. It can also be deployed to extract the road network in large-scale scenes.

  8. Rapid and specific detection of tdh, trh1, and trh2 mRNA of Vibrio parahaemolyticus by transcription-reverse transcription concerted reaction with an automated system.

    PubMed

    Nakaguchi, Yoshitsugu; Ishizuka, Tetsuya; Ohnaka, Satoru; Hayashi, Toshinori; Yasukawa, Kiyoshi; Ishiguro, Takahiko; Nishibuchi, Mitsuaki

    2004-09-01

    Vibrio parahaemolyticus strains carrying the thermostable direct hemolysin (TDH) tdh gene, the TDH-related hemolysin (trh) gene, or both genes are considered virulent strains. We previously demonstrated that the transcription-reverse transcription concerted (TRC) method could be used to quantify the amount of mRNA transcribed from the tdh gene by using an automated detection system. In this study, we devised two TRC-based assays to quantify the mRNAs transcribed from the trh1 and trh2 genes, the two representative trh genes. The TRC-based detection assays for the tdh, trh1, and trh2 transcripts could specifically and quantitatively detect 10(3) to 10(7) copies of the corresponding calibrator RNAs. We examined by the three TRC assays the total RNA preparations extracted from 103 strains of Vibrio parahaemolyticus carrying the tdh, trh1, or trh2 gene in various combinations. The tdh, trh1, and trh2 mRNAs in the total RNA preparations were specifically quantified, and the time needed for detection ranged from 9 to 19 min, from 14 to 18 min, and from 9 to 12 min, respectively. The results showed that this automated TRC assays could detect the tdh, trh1, and trh2 mRNAs specifically, quantitatively, and rapidly. The relative levels of TDH determined by the immunological method and that of tdh mRNA determined by the TRC assays for most tdh-positive strains correlated. Interestingly, the levels of TDH produced from the strains carrying both tdh and trh genes were lower than those carrying only the tdh gene, whereas the levels of mRNA did not significantly differ between the two groups. PMID:15365024

  9. Comparative evaluation of total RNA extraction methods in Theobroma cacao using shoot apical meristems.

    PubMed

    Silva, D V; Branco, S M J; Holanda, I S A; Royaert, S; Motamayor, J C; Marelli, J P; Corrêa, R X

    2016-01-01

    Theobroma cacao is a species of great economic importance with its beans used for chocolate production. The tree has been a target of various molecular studies. It contains many polyphenols, which complicate the extraction of nucleic acids with the extraction protocols requiring a large amount of plant material. These issues, therefore, necessitate the optimization of the protocols. The aim of the present study was to evaluate different methods for extraction of total RNA from shoot apical meristems of T. cacao 'CCN 51' and to assess the influence of storage conditions for the meristems on the extraction. The study also aimed to identify the most efficient protocol for RNA extraction using a small amount of plant material. Four different protocols were evaluated for RNA extraction using one shoot apical meristem per sample. Among these protocols, one that was more efficient was then tested to extract RNA using four different numbers of shoot apical meristems, subjected to three different storage conditions. The best protocol was tested for cDNA amplification using reverse transcription-polymerase chain reaction; the cDNA quality was determined to be satisfactory for molecular analyses. The study revealed that with the best RNA extraction protocol, one shoot apical meristem was sufficient for extraction of high-quality total RNA. The results obtained might enable advances in genetic analyses and molecular studies using reduced amount of plant material. PMID:26985935

  10. High-quality RNA extraction from copepods for Next Generation Sequencing: A comparative study.

    PubMed

    Asai, Sneha; Ianora, Adrianna; Lauritano, Chiara; Lindeque, Penelope K; Carotenuto, Ylenia

    2015-12-01

    Despite the ecological importance of copepods, few Next Generation Sequencing studies (NGS) have been performed on small crustaceans, and a standard method for RNA extraction is lacking. In this study, we compared three commonly-used methods: TRIzol®, Aurum Total RNA Mini Kit and Qiagen RNeasy Micro Kit, in combination with preservation reagents TRIzol® or RNAlater®, to obtain high-quality and quantity of RNA from copepods for NGS. Total RNA was extracted from the copepods Calanus helgolandicus, Centropages typicus and Temora stylifera and its quantity and quality were evaluated using NanoDrop, agarose gel electrophoresis and Agilent Bioanalyzer. Our results demonstrate that preservation of copepods in RNAlater® and extraction with Qiagen RNeasy Micro Kit were the optimal isolation method for high-quality and quantity of RNA for NGS studies of C. helgolandicus. Intriguingly, C. helgolandicus 28S rRNA is formed by two subunits that separate after heat-denaturation and migrate along with 18S rRNA. This unique property of protostome RNA has never been reported in copepods. Overall, our comparative study on RNA extraction protocols will help increase gene expression studies on copepods using high-throughput applications, such as RNA-Seq and microarrays. PMID:25546577

  11. Brain MAPS: an automated, accurate and robust brain extraction technique using a template library

    PubMed Central

    Leung, Kelvin K.; Barnes, Josephine; Modat, Marc; Ridgway, Gerard R.; Bartlett, Jonathan W.; Fox, Nick C.; Ourselin, Sébastien

    2011-01-01

    Whole brain extraction is an important pre-processing step in neuro-image analysis. Manual or semi-automated brain delineations are labour-intensive and thus not desirable in large studies, meaning that automated techniques are preferable. The accuracy and robustness of automated methods are crucial because human expertise may be required to correct any sub-optimal results, which can be very time consuming. We compared the accuracy of four automated brain extraction methods: Brain Extraction Tool (BET), Brain Surface Extractor (BSE), Hybrid Watershed Algorithm (HWA) and a Multi-Atlas Propagation and Segmentation (MAPS) technique we have previously developed for hippocampal segmentation. The four methods were applied to extract whole brains from 682 1.5T and 157 3T T1-weighted MR baseline images from the Alzheimer’s Disease Neuroimaging Initiative database. Semi-automated brain segmentations with manual editing and checking were used as the gold-standard to compare with the results. The median Jaccard index of MAPS was higher than HWA, BET and BSE in 1.5T and 3T scans (p < 0.05, all tests), and the 1st-99th centile range of the Jaccard index of MAPS was smaller than HWA, BET and BSE in 1.5T and 3T scans (p < 0.05, all tests). HWA and MAPS were found to be best at including all brain tissues (median false negative rate ≤ 0.010% for 1.5T scans and ≤ 0.019% for 3T scans, both methods). The median Jaccard index of MAPS were similar in both 1.5T and 3T scans, whereas those of BET, BSE and HWA were higher in 1.5T scans than 3T scans (p < 0.05, all tests). We found that the diagnostic group had a small effect on the median Jaccard index of all four methods. In conclusion, MAPS had relatively high accuracy and low variability compared to HWA, BET and BSE in MR scans with and without atrophy. PMID:21195780

  12. Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays.

    PubMed

    Small, J; Call, D R; Brockman, F J; Straub, T M; Chandler, D P

    2001-10-01

    We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 microg of total RNA, representing approximately 7.5 x 10(6) Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR. PMID:11571176

  13. Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts

    PubMed Central

    Pelczar, Hélène; Woisard, Anne; Lemaître, Jean Marc; Chachou, Mohamed; Andéol, Yannick

    2010-01-01

    We have previously reported a post-transcriptional RNA amplification observed in vivo following injection of in vitro synthesized transcripts into axolotl oocytes, unfertilized (UFE) or fertilized eggs. To further characterize this phenomenon, low speed extracts (LSE) from axolotl and Xenopus UFE were prepared and tested in an RNA polymerization assay. The major conclusions are: i) the amphibian extracts catalyze the incorporation of radioactive ribonucleotide in RNase but not DNase sensitive products showing that these products correspond to RNA; ii) the phenomenon is resistant to α-amanitin, an inhibitor of RNA polymerases II and III and to cordycepin (3′dAMP), but sensitive to cordycepin 5′-triphosphate, an RNA elongation inhibitor, which supports the existence of an RNA polymerase activity different from polymerases II and III; the detection of radiolabelled RNA comigrating at the same length as the exogenous transcript added to the extracts allowed us to show that iii) the RNA polymerization is not a 3′ end labelling and that iv) the radiolabelled RNA is single rather than double stranded. In vitro cell-free systems derived from amphibian UFE therefore validate our previous in vivo results hypothesizing the existence of an evolutionary conserved enzymatic activity with the properties of an RNA dependent RNA polymerase (RdRp). PMID:21203452

  14. Automated extraction of natural drainage density patterns for the conterminous United States through high performance computing

    USGS Publications Warehouse

    Stanislawski, Larry V.; Falgout, Jeff T.; Buttenfield, Barbara P.

    2015-01-01

    Hydrographic networks form an important data foundation for cartographic base mapping and for hydrologic analysis. Drainage density patterns for these networks can be derived to characterize local landscape, bedrock and climate conditions, and further inform hydrologic and geomorphological analysis by indicating areas where too few headwater channels have been extracted. But natural drainage density patterns are not consistently available in existing hydrographic data for the United States because compilation and capture criteria historically varied, along with climate, during the period of data collection over the various terrain types throughout the country. This paper demonstrates an automated workflow that is being tested in a high-performance computing environment by the U.S. Geological Survey (USGS) to map natural drainage density patterns at the 1:24,000-scale (24K) for the conterminous United States. Hydrographic network drainage patterns may be extracted from elevation data to guide corrections for existing hydrographic network data. The paper describes three stages in this workflow including data pre-processing, natural channel extraction, and generation of drainage density patterns from extracted channels. The workflow is concurrently implemented by executing procedures on multiple subbasin watersheds within the U.S. National Hydrography Dataset (NHD). Pre-processing defines parameters that are needed for the extraction process. Extraction proceeds in standard fashion: filling sinks, developing flow direction and weighted flow accumulation rasters. Drainage channels with assigned Strahler stream order are extracted within a subbasin and simplified. Drainage density patterns are then estimated with 100-meter resolution and subsequently smoothed with a low-pass filter. The extraction process is found to be of better quality in higher slope terrains. Concurrent processing through the high performance computing environment is shown to facilitate and refine

  15. Extraction, identification, and functional characterization of a bioactive substance from automated compound-handling plastic tips.

    PubMed

    Watson, John; Greenough, Emily B; Leet, John E; Ford, Michael J; Drexler, Dieter M; Belcastro, James V; Herbst, John J; Chatterjee, Moneesh; Banks, Martyn

    2009-06-01

    Disposable plastic labware is ubiquitous in contemporary pharmaceutical research laboratories. Plastic labware is routinely used for chemical compound storage and during automated liquid-handling processes that support assay development, high-throughput screening, structure-activity determinations, and liability profiling. However, there is little information available in the literature on the contaminants released from plastic labware upon DMSO exposure and their resultant effects on specific biological assays. The authors report here the extraction, by simple DMSO washing, of a biologically active substance from one particular size of disposable plastic tips used in automated compound handling. The active contaminant was identified as erucamide ((Z)-docos-13-enamide), a long-chain mono-unsaturated fatty acid amide commonly used in plastics manufacturing, by gas chromatography/mass spectroscopy analysis of the DMSO-extracted material. Tip extracts prepared in DMSO, as well as a commercially obtained sample of erucamide, were active in a functional bioassay of a known G-protein-coupled fatty acid receptor. A sample of a different disposable tip product from the same vendor did not release detectable erucamide following solvent extraction, and DMSO extracts prepared from this product were inactive in the receptor functional assay. These results demonstrate that solvent-extractable contaminants from some plastic labware used in the contemporary pharmaceutical research and development (R&D) environment can be introduced into physical and biological assays during routine compound management liquid-handling processes. These contaminants may further possess biological activity and are therefore a potential source of assay-specific confounding artifacts. PMID:19470712

  16. Dynamic electromembrane extraction: Automated movement of donor and acceptor phases to improve extraction efficiency.

    PubMed

    Asl, Yousef Abdossalami; Yamini, Yadollah; Seidi, Shahram; Amanzadeh, Hatam

    2015-11-01

    In the present research, dynamic electromembrane extraction (DEME) was introduced for the first time for extraction and determination of ionizable species from different biological matrices. The setup proposed for DEME provides an efficient, stable, and reproducible method to increase extraction efficiency. This setup consists of a piece of hollow fiber mounted inside a glass flow cell by means of two plastics connector tubes. In this dynamic system, an organic solvent is impregnated into the pores of hollow fiber as supported liquid membrane (SLM); an aqueous acceptor solution is repeatedly pumped into the lumen of hollow fiber by a syringe pump whereas a peristaltic pump is used to move sample solution around the mounted hollow fiber into the flow cell. Two platinum electrodes connected to a power supply are used during extractions which are located into the lumen of the hollow fiber and glass flow cell, respectively. The method was applied for extraction of amitriptyline (AMI) and nortriptyline (NOR) as model analytes from biological fluids. Effective parameters on DEME of the model analytes were investigated and optimized. Under optimized conditions, the calibration curves were linear in the range of 2.0-100μgL(-1) with coefficient of determination (r(2)) more than 0.9902 for both of the analytes. The relative standard deviations (RSD %) were less than 8.4% based on four replicate measurements. LODs less than 1.0μgL(-1) were obtained for both AMI and NOR. The preconcentration factors higher than 83-fold were obtained for the extraction of AMI and NOR in various biological samples. PMID:26455283

  17. Stimulation of delta-Aminolevulinic Acid Formation in Algal Extracts by Heterologous RNA.

    PubMed

    Weinstein, J D; Mayer, S M; Beale, S I

    1986-12-01

    Formation of the chlorophyll and heme precursor delta-aminolevulinic acid (ALA) from glutamate in soluble extracts of Chlorella vulgaris, Euglena gracilis, and Cyanidium caldarium was stimulated by addition of low molecular weight RNA derived from greening algae or plant tissue. Enzyme extracts were prepared for the ALA formation assay by high-speed centrifugation, partial RNA depletion, and gel filtration through Sephadex G-25. RNA was extracted from greening barley epicotyls, greening cucumber cotyledon chloroplasts, and growing cells of Chlorella, Euglena, Chlamydomonas reinhardtii, and Anacystis nidulans, freed of protein, and fractionated on DEAE-cellulose to yield an active component corresponding to the tRNA-containing fraction. RNA from homologous and heterologous species stimulated ALA formation when added to enzyme extracts, and the degree of stimulation was proportional to the amount of RNA added. Algal enzyme extracts were stimulated by algal RNAs interchangeably, with the exception of RNA prepared from aplastidic Euglena, which did not stimulate ALA production. RNA from greening cucumber cotyledon chloroplasts and greening barley epicotyls stimulated ALA formation in algal enzyme incubations. In contrast, tRNA from Escherichia coli, both nonspecific and glutamate-specific, as well as wheat germ, bovine liver, and yeast tRNA, failed to reconstitute ALA formation. Moreover, E. coli tRNA inhibited ALA formation by algal extracts, both in the presence and absence of added algal RNA. Chlorella extracts were capable of catalyzing aminoacyl bond formation between glutamate and both the activity reconstituting and nonreconstituting RNAs, indicating that the inability of some RNAs to stimulate ALA formation was not due to their inability to serve as glutamyl acceptors. The first step in the ALA-forming reaction sequence has been proposed to be activation of glutamate via aminoacyl bond formation with a specific tRNA, analogous to the first step in peptide bond

  18. Application and evaluation of automated methods to extract neuroanatomical connectivity statements from free text

    PubMed Central

    Pavlidis, Paul

    2012-01-01

    Motivation: Automated annotation of neuroanatomical connectivity statements from the neuroscience literature would enable accessible and large-scale connectivity resources. Unfortunately, the connectivity findings are not formally encoded and occur as natural language text. This hinders aggregation, indexing, searching and integration of the reports. We annotated a set of 1377 abstracts for connectivity relations to facilitate automated extraction of connectivity relationships from neuroscience literature. We tested several baseline measures based on co-occurrence and lexical rules. We compare results from seven machine learning methods adapted from the protein interaction extraction domain that employ part-of-speech, dependency and syntax features. Results: Co-occurrence based methods provided high recall with weak precision. The shallow linguistic kernel recalled 70.1% of the sentence-level connectivity statements at 50.3% precision. Owing to its speed and simplicity, we applied the shallow linguistic kernel to a large set of new abstracts. To evaluate the results, we compared 2688 extracted connections with the Brain Architecture Management System (an existing database of rat connectivity). The extracted connections were connected in the Brain Architecture Management System at a rate of 63.5%, compared with 51.1% for co-occurring brain region pairs. We found that precision increases with the recency and frequency of the extracted relationships. Availability and implementation: The source code, evaluations, documentation and other supplementary materials are available at http://www.chibi.ubc.ca/WhiteText. Contact: paul@chibi.ubc.ca Supplementary information: Supplementary data are available at Bioinformatics Online. PMID:22954628

  19. Automated solid-phase extraction of herbicides from water for gas chromatographic-mass spectrometric analysis

    USGS Publications Warehouse

    Meyer, M.T.; Mills, M.S.; Thurman, E.M.

    1993-01-01

    An automated solid-phase extraction (SPE) method was developed for the pre-concentration of chloroacetanilide and triazine herbicides, and two triazine metabolites from 100-ml water samples. Breakthrough experiments for the C18 SPE cartridge show that the two triazine metabolites are not fully retained and that increasing flow-rate decreases their retention. Standard curve r2 values of 0.998-1.000 for each compound were consistently obtained and a quantitation level of 0.05 ??g/l was achieved for each compound tested. More than 10,000 surface and ground water samples have been analyzed by this method.

  20. Automated CO2 extraction from air for clumped isotope analysis in the atmo- and biosphere

    NASA Astrophysics Data System (ADS)

    Hofmann, Magdalena; Ziegler, Martin; Pons, Thijs; Lourens, Lucas; Röckmann, Thomas

    2015-04-01

    The conventional stable isotope ratios 13C/12C and 18O/16O in atmospheric CO2 are a powerful tool for unraveling the global carbon cycle. In recent years, it has been suggested that the abundance of the very rare isotopologue 13C18O16O on m/z 47 might be a promising tracer to complement conventional stable isotope analysis of atmospheric CO2 [Affek and Eiler, 2006; Affek et al. 2007; Eiler and Schauble, 2004; Yeung et al., 2009]. Here we present an automated analytical system that is designed for clumped isotope analysis of atmo- and biospheric CO2. The carbon dioxide gas is quantitatively extracted from about 1.5L of air (ATP). The automated stainless steel extraction and purification line consists of three main components: (i) a drying unit (a magnesium perchlorate unit and a cryogenic water trap), (ii) two CO2 traps cooled with liquid nitrogen [Werner et al., 2001] and (iii) a GC column packed with Porapak Q that can be cooled with liquid nitrogen to -30°C during purification and heated up to 230°C in-between two extraction runs. After CO2 extraction and purification, the CO2 is automatically transferred to the mass spectrometer. Mass spectrometric analysis of the 13C18O16O abundance is carried out in dual inlet mode on a MAT 253 mass spectrometer. Each analysis generally consists of 80 change-over-cycles. Three additional Faraday cups were added to the mass spectrometer for simultaneous analysis of the mass-to-charge ratios 44, 45, 46, 47, 48 and 49. The reproducibility for δ13C, δ18O and Δ47 for repeated CO2 extractions from air is in the range of 0.11o (SD), 0.18o (SD) and 0.02 (SD)o respectively. This automated CO2 extraction and purification system will be used to analyse the clumped isotopic signature in atmospheric CO2 (tall tower, Cabauw, Netherlands) and to study the clumped isotopic fractionation during photosynthesis (leaf chamber experiments) and soil respiration. References Affek, H. P., Xu, X. & Eiler, J. M., Geochim. Cosmochim. Acta 71, 5033

  1. Strategies for Medical Data Extraction and Presentation Part 3: Automated Context- and User-Specific Data Extraction.

    PubMed

    Reiner, Bruce

    2015-08-01

    In current medical practice, data extraction is limited by a number of factors including lack of information system integration, manual workflow, excessive workloads, and lack of standardized databases. The combined limitations result in clinically important data often being overlooked, which can adversely affect clinical outcomes through the introduction of medical error, diminished diagnostic confidence, excessive utilization of medical services, and delays in diagnosis and treatment planning. Current technology development is largely inflexible and static in nature, which adversely affects functionality and usage among the diverse and heterogeneous population of end users. In order to address existing limitations in medical data extraction, alternative technology development strategies need to be considered which incorporate the creation of end user profile groups (to account for occupational differences among end users), customization options (accounting for individual end user needs and preferences), and context specificity of data (taking into account both the task being performed and data subject matter). Creation of the proposed context- and user-specific data extraction and presentation templates offers a number of theoretical benefits including automation and improved workflow, completeness in data search, ability to track and verify data sources, creation of computerized decision support and learning tools, and establishment of data-driven best practice guidelines. PMID:25833768

  2. CHANNEL MORPHOLOGY TOOL (CMT): A GIS-BASED AUTOMATED EXTRACTION MODEL FOR CHANNEL GEOMETRY

    SciTech Connect

    JUDI, DAVID; KALYANAPU, ALFRED; MCPHERSON, TIMOTHY; BERSCHEID, ALAN

    2007-01-17

    This paper describes an automated Channel Morphology Tool (CMT) developed in ArcGIS 9.1 environment. The CMT creates cross-sections along a stream centerline and uses a digital elevation model (DEM) to create station points with elevations along each of the cross-sections. The generated cross-sections may then be exported into a hydraulic model. Along with the rapid cross-section generation the CMT also eliminates any cross-section overlaps that might occur due to the sinuosity of the channels using the Cross-section Overlap Correction Algorithm (COCoA). The CMT was tested by extracting cross-sections from a 5-m DEM for a 50-km channel length in Houston, Texas. The extracted cross-sections were compared directly with surveyed cross-sections in terms of the cross-section area. Results indicated that the CMT-generated cross-sections satisfactorily matched the surveyed data.

  3. Modelling and representation issues in automated feature extraction from aerial and satellite images

    NASA Astrophysics Data System (ADS)

    Sowmya, Arcot; Trinder, John

    New digital systems for the processing of photogrammetric and remote sensing images have led to new approaches to information extraction for mapping and Geographic Information System (GIS) applications, with the expectation that data can become more readily available at a lower cost and with greater currency. Demands for mapping and GIS data are increasing as well for environmental assessment and monitoring. Hence, researchers from the fields of photogrammetry and remote sensing, as well as computer vision and artificial intelligence, are bringing together their particular skills for automating these tasks of information extraction. The paper will review some of the approaches used in knowledge representation and modelling for machine vision, and give examples of their applications in research for image understanding of aerial and satellite imagery.

  4. Urinary extracellular vesicles for RNA extraction: optimization of a protocol devoid of prokaryote contamination

    PubMed Central

    Tataruch-Weinert, Dorota; Musante, Luca; Kretz, Oliver; Holthofer, Harry

    2016-01-01

    Background Urinary extracellular vesicles (UEVs) represent an ideal platform for biomarker discovery. They carry different types of RNA species, and reported profile discrepancies related to the presence/absence of 18s and 28s rRNA remain controversial. Moreover, sufficient urinary RNA yields and respective quality RNA profiles are still to be fully established. Methods UEVs were enriched by hydrostatic filtration dialysis, and RNA content was extracted using 7 different commercially available techniques. RNA quantity was assessed using spectrophotometry and fluorometry, whilst RNA quality was determined by capillary electrophoresis. Results The presence of prokaryotic transcriptome was stressed when cellular RNA, as a control, was spiked into the UEVs samples before RNA extraction. The presence of bacteria in hydrostatic filtration dialysis above 1,000 kDa molecular weight cut-off and in crude urine was confirmed with growth media plates. The efficiency in removing urinary bacteria was evaluated by differential centrifugation, filtration (0.22 µm filters) and chemical pretreatment (water purification tablet). For volumes of urine >200 ml, the chemical treatment provides ease of handling without affecting vesicle integrity, protein and RNA profiles. This protocol was selected to enrich RNA with 7 methods, and its respective quality and quantity were assessed. The results were given as follows: (a) Fluorometry gave more repeatability and reproducibility than spectrophotometry to assess the RNA yields, (b) UEVs were enriched with small RNA, (c) Ribosomal RNA peaks were not observed for any RNA extraction method used and (d) RNA yield was higher for column-based method designed for urinary exosome, whilst the highest relative microRNA presence was obtained using TRIzol method. Conclusion Our results show that the presence of bacteria can lead to misidentification in the electrophoresis peaks. Fluorometry is more reliable than spectrophotometry. RNA isolation method

  5. Automated extraction of fine features of kinetochore microtubules and plus-ends from electron tomography volume.

    PubMed

    Jiang, Ming; Ji, Qiang; McEwen, Bruce F

    2006-07-01

    Kinetochore microtubules (KMTs) and the associated plus-ends have been areas of intense investigation in both cell biology and molecular medicine. Though electron tomography opens up new possibilities in understanding their function by imaging their high-resolution structures, the interpretation of the acquired data remains an obstacle because of the complex and cluttered cellular environment. As a result, practical segmentation of the electron tomography data has been dominated by manual operation, which is time consuming and subjective. In this paper, we propose a model-based automated approach to extracting KMTs and the associated plus-ends with a coarse-to-fine scale scheme consisting of volume preprocessing, microtubule segmentation and plus-end tracing. In volume preprocessing, we first apply an anisotropic invariant wavelet transform and a tube-enhancing filter to enhance the microtubules at coarse level for localization. This is followed with a surface-enhancing filter to accentuate the fine microtubule boundary features. The microtubule body is then segmented using a modified active shape model method. Starting from the segmented microtubule body, the plus-ends are extracted with a probabilistic tracing method improved with rectangular window based feature detection and the integration of multiple cues. Experimental results demonstrate that our automated method produces results comparable to manual segmentation but using only a fraction of the manual segmentation time. PMID:16830922

  6. Model-based automated extraction of microtubules from electron tomography volume.

    PubMed

    Jiang, Ming; Ji, Qiang; McEwen, Bruce F

    2006-07-01

    We propose a model-based automated approach to extracting microtubules from noisy electron tomography volume. Our approach consists of volume enhancement, microtubule localization, and boundary segmentation to exploit the unique geometric and photometric properties of microtubules. The enhancement starts with an anisotropic invariant wavelet transform to enhance the microtubules globally, followed by a three-dimensional (3-D) tube-enhancing filter based on Weingarten matrix to further accentuate the tubular structures locally. The enhancement ends with a modified coherence-enhancing diffusion to complete the interruptions along the microtubules. The microtubules are then localized with a centerline extraction algorithm adapted for tubular objects. To perform segmentation, we novelly modify and extend active shape model method. We first use 3-D local surface enhancement to characterize the microtubule boundary and improve shape searching by relating the boundary strength with the weight matrix of the searching error. We then integrate the active shape model with Kalman filtering to utilize the longitudinal smoothness along the microtubules. The segmentation improved in this way is robust against missing boundaries and outliers that are often present in the tomography volume. Experimental results demonstrate that our automated method produces results close to those by manual process and uses only a fraction of the time of the latter. PMID:16871731

  7. Automated Detection and Extraction of Coronal Dimmings from SDO/AIA Data

    NASA Astrophysics Data System (ADS)

    Davey, Alisdair R.; Attrill, G. D. R.; Wills-Davey, M. J.

    2010-05-01

    The sheer volume of data anticipated from the Solar Dynamics Observatory/Atmospheric Imaging Assembly (SDO/AIA) highlights the necessity for the development of automatic detection methods for various types of solar activity. Initially recognised in the 1970s, it is now well established that coronal dimmings are closely associated with coronal mass ejections (CMEs), and are particularly recognised as an indicator of front-side (halo) CMEs, which can be difficult to detect in white-light coronagraph data. An automated coronal dimming region detection and extraction algorithm removes visual observer bias from determination of physical quantities such as spatial location, area and volume. This allows reproducible, quantifiable results to be mined from very large datasets. The information derived may facilitate more reliable early space weather detection, as well as offering the potential for conducting large-sample studies focused on determining the geoeffectiveness of CMEs, coupled with analysis of their associated coronal dimmings. We present examples of dimming events extracted using our algorithm from existing EUV data, demonstrating the potential for the anticipated application to SDO/AIA data. Metadata returned by our algorithm include: location, area, volume, mass and dynamics of coronal dimmings. As well as running on historic datasets, this algorithm is capable of detecting and extracting coronal dimmings in near real-time. The coronal dimming detection and extraction algorithm described in this poster is part of the SDO/Computer Vision Center effort hosted at SAO (Martens et al., 2009). We acknowledge NASA grant NNH07AB97C.

  8. Comparison and optimization of methods for the simultaneous extraction of DNA, RNA, proteins, and metabolites.

    PubMed

    Vorreiter, Fränze; Richter, Silke; Peter, Michel; Baumann, Sven; von Bergen, Martin; Tomm, Janina M

    2016-09-01

    The challenge of performing a time-resolved comprehensive analysis of molecular systems has led to the quest to optimize extraction methods. When the size of a biological sample is limited, there is demand for the simultaneous extraction of molecules representing the four areas of "omics": genomics, transcriptomics, proteomics, and metabolomics. Here we optimized a protocol for the simultaneous extraction of DNA, RNA, proteins, and metabolites and compared it with two existing protocols. Our optimization comprised the addition of a methanol/chloroform metabolite purification before the separation of DNA/RNA and proteins. Extracted DNA, RNA, proteins, and metabolites were quantitatively and/or qualitatively analyzed. Of the three methods, only the newly developed protocol yielded all biomolecule classes of adequate quantity and quality. PMID:27237373

  9. Extraction and fractionation of RNA and DNA from single cells using selective lysing and isotachophoresis

    NASA Astrophysics Data System (ADS)

    Shintaku, Hirofumi; Santiago, Juan G.

    2015-03-01

    Single cell analyses of RNA and DNA are crucial to understanding the heterogeneity of cell populations. The numbers of approaches to single cells analyses are expanding, but sequence specific measurements of nucleic acids have been mostly limited to studies of either DNA or RNA, and not both. This remains a challenge as RNA and DNA have very similar physical and biochemical properties, and cross-contamination with each other can introduce false positive results. We present an electrokinetic technique which creates the opportunity to fractionate and deliver cytoplasmic RNA and genomic DNA to independent downstream analyses. Our technique uses an on-chip system that enables selective lysing of cytoplasmic membrane, extraction of RNA (away from genomic DNA and nucleus), focusing, absolute quantification of cytoplasmic RNA mass. The absolute RNA mass quantification is performed using fluorescence observation without enzymatic amplification in < 5 min. The cell nucleus is left intact and the relative genomic DNA amount in the nucleus can be measured. We demonstrate the technique using single mouse B lymphocyte cells, for which we extracted an average of 14.1 pg total cytoplasmic RNA per cell. We also demonstrate correlation analysis between the absolute amount of cytoplasmic RNA and relative amount of genomic DNA, showing heterogeneity associated with cell cycle.

  10. A Novel Validation Algorithm Allows for Automated Cell Tracking and the Extraction of Biologically Meaningful Parameters

    PubMed Central

    Madany Mamlouk, Amir; Schicktanz, Simone; Kruse, Charli

    2011-01-01

    Automated microscopy is currently the only method to non-invasively and label-free observe complex multi-cellular processes, such as cell migration, cell cycle, and cell differentiation. Extracting biological information from a time-series of micrographs requires each cell to be recognized and followed through sequential microscopic snapshots. Although recent attempts to automatize this process resulted in ever improving cell detection rates, manual identification of identical cells is still the most reliable technique. However, its tedious and subjective nature prevented tracking from becoming a standardized tool for the investigation of cell cultures. Here, we present a novel method to accomplish automated cell tracking with a reliability comparable to manual tracking. Previously, automated cell tracking could not rival the reliability of manual tracking because, in contrast to the human way of solving this task, none of the algorithms had an independent quality control mechanism; they missed validation. Thus, instead of trying to improve the cell detection or tracking rates, we proceeded from the idea to automatically inspect the tracking results and accept only those of high trustworthiness, while rejecting all other results. This validation algorithm works independently of the quality of cell detection and tracking through a systematic search for tracking errors. It is based only on very general assumptions about the spatiotemporal contiguity of cell paths. While traditional tracking often aims to yield genealogic information about single cells, the natural outcome of a validated cell tracking algorithm turns out to be a set of complete, but often unconnected cell paths, i.e. records of cells from mitosis to mitosis. This is a consequence of the fact that the validation algorithm takes complete paths as the unit of rejection/acceptance. The resulting set of complete paths can be used to automatically extract important biological parameters with high

  11. A novel validation algorithm allows for automated cell tracking and the extraction of biologically meaningful parameters.

    PubMed

    Rapoport, Daniel H; Becker, Tim; Madany Mamlouk, Amir; Schicktanz, Simone; Kruse, Charli

    2011-01-01

    Automated microscopy is currently the only method to non-invasively and label-free observe complex multi-cellular processes, such as cell migration, cell cycle, and cell differentiation. Extracting biological information from a time-series of micrographs requires each cell to be recognized and followed through sequential microscopic snapshots. Although recent attempts to automatize this process resulted in ever improving cell detection rates, manual identification of identical cells is still the most reliable technique. However, its tedious and subjective nature prevented tracking from becoming a standardized tool for the investigation of cell cultures. Here, we present a novel method to accomplish automated cell tracking with a reliability comparable to manual tracking. Previously, automated cell tracking could not rival the reliability of manual tracking because, in contrast to the human way of solving this task, none of the algorithms had an independent quality control mechanism; they missed validation. Thus, instead of trying to improve the cell detection or tracking rates, we proceeded from the idea to automatically inspect the tracking results and accept only those of high trustworthiness, while rejecting all other results. This validation algorithm works independently of the quality of cell detection and tracking through a systematic search for tracking errors. It is based only on very general assumptions about the spatiotemporal contiguity of cell paths. While traditional tracking often aims to yield genealogic information about single cells, the natural outcome of a validated cell tracking algorithm turns out to be a set of complete, but often unconnected cell paths, i.e. records of cells from mitosis to mitosis. This is a consequence of the fact that the validation algorithm takes complete paths as the unit of rejection/acceptance. The resulting set of complete paths can be used to automatically extract important biological parameters with high

  12. Design and self-assembly of siRNA-functionalized RNA nanoparticles for use in automated nanomedicine

    PubMed Central

    Afonin, Kirill A; Grabow, Wade W; Walker, Faye M; Bindewald, Eckart; Dobrovolskaia, Marina A; Shapiro, Bruce A; Jaeger, Luc

    2012-01-01

    Individual genes can be targeted with siRNAs. The use of nucleic acid nanoparticles (NPs) is a convenient method for delivering combinations of specific siRNAs in an organized and programmable manner. We present three assembly protocols to produce two different types of RNA self-assembling functional NPs using processes that are fully automatable. These NPs are engineered based on two complementary nanoscaffold designs (nanoring and nanocube), which serve as carriers of multiple siRNAs. The NPs are functionalized by the extension of up to six scaffold strands with siRNA duplexes. The assembly protocols yield functionalized RNA NPs, and we show that they interact in vitro with human recombinant Dicer to produce siRNAs. Our design strategies allow for fast, economical and easily controlled production of endotoxin-free therapeutic RNA NPs that are suitable for preclinical development. PMID:22134126

  13. Comparison of three magnetic bead surface functionalities for RNA extraction and detection.

    PubMed

    Adams, Nicholas M; Bordelon, Hali; Wang, Kwo-Kwang A; Albert, Laura E; Wright, David W; Haselton, Frederick R

    2015-03-25

    Magnetic beads are convenient for extracting nucleic acid biomarkers from biological samples prior to molecular detection. These beads are available with a variety of surface functionalities designed to capture particular subsets of RNA. We hypothesized that bead surface functionality affects binding kinetics, processing simplicity, and compatibility with molecular detection strategies. In this report, three magnetic bead surface chemistries designed to bind nucleic acids, silica, oligo (dT), and a specific oligonucleotide sequence were evaluated. Commercially available silica-coated and oligo (dT) beads, as well as beads functionalized with oligonucleotides complementary to respiratory syncytial virus (RSV) nucleocapsid gene, respectively recovered ∼75, ∼71, and ∼7% target RSV mRNA after a 1 min of incubation time in a surrogate patient sample spiked with the target. RSV-specific beads required much longer incubation times to recover amounts of the target comparable to the other beads (∼77% at 180 min). As expected, silica-coated beads extracted total RNA, oligo (dT) beads selectively extracted total mRNA, and RSV-specific beads selectively extracted RSV N gene mRNA. The choice of bead functionality is generally dependent on the target detection strategy. The silica-coated beads are most suitable for applications that require nucleic acids other than mRNA, especially with detection strategies that are tolerant of a high concentration of nontarget background nucleic acids, such as RT-PCR. On the other hand, oligo (dT) beads are best-suited for mRNA targets, as they bind biomarkers rapidly, have relatively high recovery, and enable detection strategies to be performed directly on the bead surface. Sequence-specific beads may be best for applications that are not tolerant of a high concentration of nontarget nucleic acids that require short RNA sequences without poly(A) tails, such as microRNAs, or that perform RNA detection directly on the bead surface. PMID

  14. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  15. Evaluation of the semen swim-up method for bovine sperm RNA extraction.

    PubMed

    Han, C M; Chen, R; Li, T; Chen, X L; Zheng, Y F; Ma, M T; Gao, Q H

    2016-01-01

    Isolation of high-quality RNA is important for assessing sperm gene expression, and semen purification methods may affect the integrity of the isolated RNA. This study evaluated the effectiveness of the sperm swim-up method for seminal RNA isolation. Frozen semen samples in straws from three bulls of proven fertility were purified by the swim-up method. RNA extraction was carried out using the E.Z.N.A.(TM) Total RNA kit II, with non-swim-up sperm as a control. Total sperm RNA was analyzed by UV spectrophotometry, reverse transcription polymerase chain reaction (RT-PCR), and agarose gel electrophoresis, and expression of the sex-determining region on the Y chromosome (SRY), leptin (LEP), and ribosomal protein subunit 23 (RPS23) genes, were determined. 18S RNA was used as a positive control. Fewer somatic cells were found in sperm swim-up samples than in the non-swim-up counterparts (0 x 10(3) vs 17.33 ± 2.52 x 10(3) sperm, P < 0.05). In addition, high-quality RNA was obtained in about 2 h, with no significant difference between groups. Interestingly, the yields of RNA fragments containing ≥200 nucleotides were significantly reduced in sperm swim-up samples (0.92 ± 0.41 x 10(7) sperm) compared with the non-swim-up samples (1.36 ± 0.33 x 10(7) sperm, P < 0.05). After RT-PCR, clear bands representing SRY, LEP, and RPS23 in sperm cDNA were observed on agarose gel electrophoresis. Finally, no bands corresponding to 18S RNA were found in RNA samples from the sperm swim-up group. Our findings suggest that small amounts of sperm RNA can be efficiently extracted from frozen straw semen samples using the swim-up technique. PMID:27173315

  16. Automated multisyringe stir bar sorptive extraction using robust montmorillonite/epoxy-coated stir bars.

    PubMed

    Ghani, Milad; Saraji, Mohammad; Maya, Fernando; Cerdà, Víctor

    2016-05-01

    Herein we present a simple, rapid and low cost strategy for the preparation of robust stir bar coatings based on the combination of montmorillonite with epoxy resin. The composite stir bar was implemented in a novel automated multisyringe stir bar sorptive extraction system (MS-SBSE), and applied to the extraction of four chlorophenols (4-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol) as model compounds, followed by high performance liquid chromatography-diode array detection. The different experimental parameters of the MS-SBSE, such as sample volume, selection of the desorption solvent, desorption volume, desorption time, sample solution pH, salt effect and extraction time were studied. Under the optimum conditions, the detection limits were between 0.02 and 0.34μgL(-1). Relative standard deviations (RSD) of the method for the analytes at 10μgL(-1) concentration level ranged from 3.5% to 4.1% (as intra-day RSD) and from 3.9% to 4.3% (as inter-day RSD at 50μgL(-1) concentration level). Batch-to-batch reproducibility for three different stir bars was 4.6-5.1%. The enrichment factors were between 30 and 49. In order to investigate the capability of the developed technique for real sample analysis, well water, wastewater and leachates from a solid waste treatment plant were satisfactorily analyzed. PMID:27062720

  17. Automated extraction and classification of time-frequency contours in humpback vocalizations.

    PubMed

    Ou, Hui; Au, Whitlow W L; Zurk, Lisa M; Lammers, Marc O

    2013-01-01

    A time-frequency contour extraction and classification algorithm was created to analyze humpback whale vocalizations. The algorithm automatically extracted contours of whale vocalization units by searching for gray-level discontinuities in the spectrogram images. The unit-to-unit similarity was quantified by cross-correlating the contour lines. A library of distinctive humpback units was then generated by applying an unsupervised, cluster-based learning algorithm. The purpose of this study was to provide a fast and automated feature selection tool to describe the vocal signatures of animal groups. This approach could benefit a variety of applications such as species description, identification, and evolution of song structures. The algorithm was tested on humpback whale song data recorded at various locations in Hawaii from 2002 to 2003. Results presented in this paper showed low probability of false alarm (0%-4%) under noisy environments with small boat vessels and snapping shrimp. The classification algorithm was tested on a controlled set of 30 units forming six unit types, and all the units were correctly classified. In a case study on humpback data collected in the Auau Chanel, Hawaii, in 2002, the algorithm extracted 951 units, which were classified into 12 distinctive types. PMID:23297903

  18. Automated parallel synthesis of 5'-triphosphate oligonucleotides and preparation of chemically modified 5'-triphosphate small interfering RNA.

    PubMed

    Zlatev, Ivan; Lackey, Jeremy G; Zhang, Ligang; Dell, Amy; McRae, Kathy; Shaikh, Sarfraz; Duncan, Richard G; Rajeev, Kallanthottathil G; Manoharan, Muthiah

    2013-02-01

    A fully automated chemical method for the parallel and high-throughput solid-phase synthesis of 5'-triphosphate and 5'-diphosphate oligonucleotides is described. The desired full-length oligonucleotides were first constructed using standard automated DNA/RNA solid-phase synthesis procedures. Then, on the same column and instrument, efficient implementation of an uninterrupted sequential cycle afforded the corresponding unmodified or chemically modified 5'-triphosphates and 5'-diphosphates. The method was readily translated into a scalable and high-throughput synthesis protocol compatible with the current DNA/RNA synthesizers yielding a large variety of unique 5'-polyphosphorylated oligonucleotides. Using this approach, we accomplished the synthesis of chemically modified 5'-triphosphate oligonucleotides that were annealed to form small-interfering RNAs (ppp-siRNAs), a potentially interesting class of novel RNAi therapeutic tools. The attachment of the 5'-triphosphate group to the passenger strand of a siRNA construct did not induce a significant improvement in the in vitro RNAi-mediated gene silencing activity nor a strong specific in vitro RIG-I activation. The reported method will enable the screening of many chemically modified ppp-siRNAs, resulting in a novel bi-functional RNAi therapeutic platform. PMID:23260577

  19. Extraction of words from the national ID cards for automated recognition

    NASA Astrophysics Data System (ADS)

    Akhter, Md. Rezwan; Bhuiyan, Md. Hasanuzzaman; Uddin, Mohammad Shorif

    2011-10-01

    The government of Bangladesh introduced national ID cards in 2008 for all peoples of age 18 years and above. This card is now a de-facto identity document and finds diverse applications in vote casting, bank account opening, telephone subscribing as well as in many real life transactions and security checking. To get real fruits of this versatile ID card, automated retrieving and recognition of an independent person from this extra large national database is an ultimate necessity. This work is the first step to fill this gap in making the recognition in automated fashion. Here we have investigated an image analysis technique to extract the words that will be used in subsequent recognition steps. At first scanned ID card image is used as an input into the computer system and then the target text region is separated from the picture region. The text region is used for separation of lines and words on the basis of the vertical and horizontal projections of image intensity, respectively. Experimentation using real national ID cards confirms the effectiveness of our technique.

  20. Reference line extraction for automated data-entry system using wavelet transform

    NASA Astrophysics Data System (ADS)

    Chitwong, Sakreya; Phonsri, Seksan; Thitimajshima, Punya

    1999-12-01

    It is common that most document forms opt for the use of straight line as a reference position for filled information. The automated data-entry systems of such documents require an ability to search these reference lines so that the location of information in the forms can be known. This paper proposes a wavelet-based algorithm for extracting these reference lines in business forms. Stationary wavelet transform is used to transform a gray-level document image into different frequency-band images. The horizontal detail subband is then selected and passed through a post-processing to produce a binary bitmap of reference lines. The experimental results on synthetic and real document images will be given to illustrate the usefulness of such an algorithm.

  1. Improvement on the extraction method of RNA in mites and its quality test.

    PubMed

    Zhao, YaE; Hu, Li; Yang, Yuan Jun; Niu, Dong Ling; Wang, Rui Ling; Li, Wen Hao; Ma, Si Jia; Cheng, Juan

    2016-02-01

    To solve the long-existing difficult problems in extracting RNA and constructing a complementary DNA (cDNA) library for trace mites, we conducted a further comparative experiment among three RNA extraction methods (TRIzol method, Omega method, and Azanno method) based on our previous attempts at the construction of cDNA library of mites, with Psoroptes cuniculi still used as the experimental subject. By subsequently decreasing the number of mites, the least number of mites needed for RNA extraction of each method were found by criteria of completeness, concentration, and purity of the extracted RNA. Specific primers were designed according to the allergen Pso c1, Pso c2, and Actin gene sequences of Psoroptes to test the reliability of cDNA library. The results showed that Azanno method needed only 10 mites with sensitivity 204 times higher than previously used TRIzol method and 20 times higher than Omega method; clear RNA band was detected by agarose gel electrophoresis; and ultraviolet spectrophotometer determination showed that RNA concentration, 260/280, and 260/230 were in the range of 102 to 166 ng/μl, 1.83 to 1.99, and 1.49 to 1.72, respectively. Finally, specific primers detection showed that the amplified sequences had 98.33, 98.19, and 99.52% identities with those of P. cuniculi or Psoroptes ovis in GenBank, respectively, indicating that the cDNA library constructed using 10 mites was successful and it could meet the requirements for molecular biology research. Therefore, we concluded that Azanno method was more effective than TRIzol method and Omega method in RNA extraction and cDNA library construction of trace mites. PMID:26545909

  2. Automated concept-level information extraction to reduce the need for custom software and rules development

    PubMed Central

    Nguyen, Thien M; Goryachev, Sergey; Fiore, Louis D

    2011-01-01

    Objective Despite at least 40 years of promising empirical performance, very few clinical natural language processing (NLP) or information extraction systems currently contribute to medical science or care. The authors address this gap by reducing the need for custom software and rules development with a graphical user interface-driven, highly generalizable approach to concept-level retrieval. Materials and methods A ‘learn by example’ approach combines features derived from open-source NLP pipelines with open-source machine learning classifiers to automatically and iteratively evaluate top-performing configurations. The Fourth i2b2/VA Shared Task Challenge's concept extraction task provided the data sets and metrics used to evaluate performance. Results Top F-measure scores for each of the tasks were medical problems (0.83), treatments (0.82), and tests (0.83). Recall lagged precision in all experiments. Precision was near or above 0.90 in all tasks. Discussion With no customization for the tasks and less than 5 min of end-user time to configure and launch each experiment, the average F-measure was 0.83, one point behind the mean F-measure of the 22 entrants in the competition. Strong precision scores indicate the potential of applying the approach for more specific clinical information extraction tasks. There was not one best configuration, supporting an iterative approach to model creation. Conclusion Acceptable levels of performance can be achieved using fully automated and generalizable approaches to concept-level information extraction. The described implementation and related documentation is available for download. PMID:21697292

  3. In vitro RNA editing-like activity in a mitochondrial extract from Leishmania tarentolae.

    PubMed Central

    Frech, G C; Bakalara, N; Simpson, L; Simpson, A M

    1995-01-01

    A mitochondrial extract from Leishmania tarentolae directs the incorporation of uridylate (U) residues within the pre-edited domain of synthetic cytochrome b (CYb) and NADH dehydrogenase subunit 7 mRNA. This has several characteristics of an in vitro RNA editing activity, but no direct evidence for involvement of guide RNAs was obtained. Inhibition by micrococcal nuclease suggests a requirement for some type of endogenous RNA. The limitation of internal U-incorporation to the pre-edited region in the CYb mRNA and the inhibition by deletion or substitution of both mRNA anchor sequences for CYb gRNA-I and -II could be consistent either with a gRNA-mediated process or a secondary structure-mediated process. A low level of incorporation of [alpha-32P]CTP occurs at the same sites as UTP. Internal U-incorporation activity is selectively inhibited by heterologous RNAs, suggesting an involvement of low affinity RNA-binding proteins which can be competed by the added RNA. Images PMID:7828590

  4. Automated Extraction of Dose/Volume Statistics for Radiotherapy-Treatment-Plan Evaluation in Clinical-Trial Quality Assurance

    PubMed Central

    Gong, Yutao U. T.; Yu, Jialu; Pang, Dalong; Zhen, Heming; Galvin, James; Xiao, Ying

    2016-01-01

    Radiotherapy clinical-trial quality assurance is a crucial yet challenging process. This note presents a tool that automatically extracts dose/volume statistics for determining dosimetry compliance review with improved efficiency and accuracy. A major objective of this study is to develop an automated solution for clinical-trial radiotherapy dosimetry review. PMID:26973814

  5. How Severely Is DNA Quantification Hampered by RNA Co-extraction?

    PubMed

    Sanchez, Ignacio; Remm, Matthieu; Frasquilho, Sonia; Betsou, Fay; Mathieson, William

    2015-10-01

    The optional RNase digest that is part of many DNA extraction protocols is often omitted, either because RNase is not provided in the kit or because users do not want to risk contaminating their laboratory. Consequently, co-eluting RNA can become a "contaminant" of unknown magnitude in a DNA extraction. We extracted DNA from liver, lung, kidney, and heart tissues and established that 28-52% of the "DNA" as assessed by spectrophotometry is actually RNA (depending on tissue type). Including an RNase digest in the extraction protocol reduced 260:280 purity ratios. Co-eluting RNA drives an overestimation of DNA yield when quantification is carried out using OD 260 nm spectrophotometry, or becomes an unquantified contaminant when spectrofluorometry is used for DNA quantification. This situation is potentially incompatible with the best practice guidelines for biobanks issued by organizations such as the International Society for Biological and Environmental Repositories, which state that biospecimens should be accurately characterized in terms of their identity, purity, concentration, and integrity. Consequently, we conclude that an RNase digest must be included in DNA extractions if pure DNA is required. We also discuss the implications of unquantified RNA contamination in DNA samples in the context of laboratory accreditation schemes. PMID:26418169

  6. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.

    PubMed Central

    Jackson, D P; Lewis, F A; Taylor, G R; Boylston, A W; Quirke, P

    1990-01-01

    Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted. Images PMID:1696290

  7. Rapid and automated sample preparation for nucleic acid extraction on a microfluidic CD (compact disk)

    NASA Astrophysics Data System (ADS)

    Kim, Jitae; Kido, Horacio; Zoval, Jim V.; Gagné, Dominic; Peytavi, Régis; Picard, François J.; Bastien, Martine; Boissinot, Maurice; Bergeron, Michel G.; Madou, Marc J.

    2006-01-01

    Rapid and automated preparation of PCR (polymerase chain reaction)-ready genomic DNA was demonstrated on a multiplexed CD (compact disk) platform by using hard-to-lyse bacterial spores. Cell disruption is carried out while beadcell suspensions are pushed back and forth in center-tapered lysing chambers by angular oscillation of the disk - keystone effect. During this lysis period, the cell suspensions are securely held within the lysing chambers by heatactivated wax valves. Upon application of a remote heat to the disk in motion, the wax valves release lysate solutions into centrifuge chambers where cell debris are separated by an elevated rotation of the disk. Only debris-free DNA extract is then transferred to collection chambers by capillary-assisted siphon and collected for heating that inactivates PCR inhibitors. Lysing capacity was evaluated using a real-time PCR assay to monitor the efficiency of Bacillus globigii spore lysis. PCR analysis showed that 5 minutes' CD lysis run gave spore lysis efficiency similar to that obtained with a popular commercial DNA extraction kit (i.e., IDI-lysis kit from GeneOhm Sciences Inc.) which is highly efficient for microbial cell and spore lysis. This work will contribute to the development of an integrated CD-based assay for rapid diagnosis of infectious diseases.

  8. Automated data extraction from in situ protein-stable isotope probing studies.

    PubMed

    Slysz, Gordon W; Steinke, Laurey; Ward, David M; Klatt, Christian G; Clauss, Therese R W; Purvine, Samuel O; Payne, Samuel H; Anderson, Gordon A; Smith, Richard D; Lipton, Mary S

    2014-03-01

    Protein-stable isotope probing (protein-SIP) has strong potential for revealing key metabolizing taxa in complex microbial communities. While most protein-SIP work to date has been performed under controlled laboratory conditions to allow extensive isotope labeling of the target organism(s), a key application will be in situ studies of microbial communities for short periods of time under natural conditions that result in small degrees of partial labeling. One hurdle restricting large-scale in situ protein-SIP studies is the lack of algorithms and software for automated data processing of the massive data sets resulting from such studies. In response, we developed Stable Isotope Probing Protein Extraction Resources software (SIPPER) and applied it for large-scale extraction and visualization of data from short-term (3 h) protein-SIP experiments performed in situ on phototrophic bacterial mats isolated from Yellowstone National Park. Several metrics incorporated into the software allow it to support exhaustive analysis of the complex composite isotopic envelope observed as a result of low amounts of partial label incorporation. SIPPER also enables the detection of labeled molecular species without the need for any prior identification. PMID:24467184

  9. Automated data extraction from in situ protein stable isotope probing studies

    SciTech Connect

    Slysz, Gordon W.; Steinke, Laurey A.; Ward, David M.; Klatt, Christian G.; Clauss, Therese RW; Purvine, Samuel O.; Payne, Samuel H.; Anderson, Gordon A.; Smith, Richard D.; Lipton, Mary S.

    2014-01-27

    Protein stable isotope probing (protein-SIP) has strong potential for revealing key metabolizing taxa in complex microbial communities. While most protein-SIP work to date has been performed under controlled laboratory conditions to allow extensive isotope labeling of the target organism, a key application will be in situ studies of microbial communities under conditions that result in small degrees of partial labeling. One hurdle restricting large scale in situ protein-SIP studies is the lack of algorithms and software for automated data processing of the massive data sets resulting from such studies. In response, we developed Stable Isotope Probing Protein Extraction Resources software (SIPPER) and applied it for large scale extraction and visualization of data from short term (3 h) protein-SIP experiments performed in situ on Yellowstone phototrophic bacterial mats. Several metrics incorporated into the software allow it to support exhaustive analysis of the complex composite isotopic envelope observed as a result of low amounts of partial label incorporation. SIPPER also enables the detection of labeled molecular species without the need for any prior identification.

  10. Streamlining DNA Barcoding Protocols: Automated DNA Extraction and a New cox1 Primer in Arachnid Systematics

    PubMed Central

    Vidergar, Nina; Toplak, Nataša; Kuntner, Matjaž

    2014-01-01

    Background DNA barcoding is a popular tool in taxonomic and phylogenetic studies, but for most animal lineages protocols for obtaining the barcoding sequences—mitochondrial cytochrome C oxidase subunit I (cox1 AKA CO1)—are not standardized. Our aim was to explore an optimal strategy for arachnids, focusing on the species-richest lineage, spiders by (1) improving an automated DNA extraction protocol, (2) testing the performance of commonly used primer combinations, and (3) developing a new cox1 primer suitable for more efficient alignment and phylogenetic analyses. Methodology We used exemplars of 15 species from all major spider clades, processed a range of spider tissues of varying size and quality, optimized genomic DNA extraction using the MagMAX Express magnetic particle processor—an automated high throughput DNA extraction system—and tested cox1 amplification protocols emphasizing the standard barcoding region using ten routinely employed primer pairs. Results The best results were obtained with the commonly used Folmer primers (LCO1490/HCO2198) that capture the standard barcode region, and with the C1-J-2183/C1-N-2776 primer pair that amplifies its extension. However, C1-J-2183 is designed too close to HCO2198 for well-interpreted, continuous sequence data, and in practice the resulting sequences from the two primer pairs rarely overlap. We therefore designed a new forward primer C1-J-2123 60 base pairs upstream of the C1-J-2183 binding site. The success rate of this new primer (93%) matched that of C1-J-2183. Conclusions The use of C1-J-2123 allows full, indel-free overlap of sequences obtained with the standard Folmer primers and with C1-J-2123 primer pair. Our preliminary tests suggest that in addition to spiders, C1-J-2123 will also perform in other arachnids and several other invertebrates. We provide optimal PCR protocols for these primer sets, and recommend using them for systematic efforts beyond DNA barcoding. PMID:25415202

  11. Optimisation of DNA and RNA extraction from archival formalin-fixed tissue.

    PubMed Central

    Coombs, N J; Gough, A C; Primrose, J N

    1999-01-01

    Archival, formalin-fixed, paraffin-embedded tissue is an invaluable resource for molecular genetic studies but the extraction of high quality nucleic acid may be problematic. We have optimised DNA extraction by comparing 10 protocols, including a commercially available kit and a novel method that utilises a thermal cycler. The thermal cycler and Chelex-100 extraction method yielded DNA capable of amplification by PCR from every block and 61% of sections versus 54% using microwave and Chelex-100, 15% with classical xylene-based extraction and 60% of sections using the kit. Successful RNA extraction was observed, by beta-actin amplification, in 83.7% sections for samples treated by the thermal cycler and Chelex-100 method. Thermal cycler and Chelex-100 extraction of nucleic acid is reliable, quick and inexpensive. PMID:10454649

  12. Systematic analysis of mRNA 5' coding sequence incompleteness in Danio rerio: an automated EST-based approach

    PubMed Central

    Frabetti, Flavia; Casadei, Raffaella; Lenzi, Luca; Canaider, Silvia; Vitale, Lorenza; Facchin, Federica; Carinci, Paolo; Zannotti, Maria; Strippoli, Pierluigi

    2007-01-01

    Background All standard methods for cDNA cloning are affected by a potential inability to effectively clone the 5' region of mRNA. The aim of this work was to estimate mRNA open reading frame (ORF) 5' region sequence completeness in the model organism Danio rerio (zebrafish). Results We implemented a novel automated approach (5'_ORF_Extender) that systematically compares available expressed sequence tags (ESTs) with all the zebrafish experimentally determined mRNA sequences, identifies additional sequence stretches at 5' region and scans for the presence of all conditions needed to define a new, extended putative ORF. Our software was able to identify 285 (3.3%) mRNAs with putatively incomplete ORFs at 5' region and, in three example cases selected (selt1a, unc119.2, nppa), the extended coding region at 5' end was cloned by reverse transcription-polymerase chain reaction (RT-PCR). Conclusion The implemented method, which could also be useful for the analysis of other genomes, allowed us to describe the relevance of the "5' end mRNA artifact" problem for genomic annotation and functional genomic experiment design in zebrafish. Open peer review This article was reviewed by Alexey V. Kochetov (nominated by Mikhail Gelfand), Shamil Sunyaev, and Gáspár Jékely. For the full reviews, please go to the Reviewers' Comments section. PMID:18042283

  13. High quality RNA extraction from Maqui berry for its application in next-generation sequencing.

    PubMed

    Sánchez, Carolina; Villacreses, Javier; Blanc, Noelle; Espinoza, Loreto; Martinez, Camila; Pastor, Gabriela; Manque, Patricio; Undurraga, Soledad F; Polanco, Victor

    2016-01-01

    Maqui berry (Aristotelia chilensis) is a native Chilean species that produces berries that are exceptionally rich in anthocyanins and natural antioxidants. These natural compounds provide an array of health benefits for humans, making them very desirable in a fruit. At the same time, these substances also interfere with nucleic acid preparations, making RNA extraction from Maqui berry a major challenge. Our group established a method for RNA extraction of Maqui berry with a high quality RNA (good purity, good integrity and higher yield). This procedure is based on the adapted CTAB method using high concentrations of PVP (4 %) and β-mercaptoethanol (4 %) and spermidine in the extraction buffer. These reagents help to remove contaminants such as polysaccharides, proteins, phenols and also prevent the oxidation of phenolic compounds. The high quality of RNA isolated through this method allowed its uses with success in molecular applications for this endemic Chilean fruit, such as differential expression analysis of RNA-Seq data using next generation sequencing (NGS). Furthermore, we consider that our method could potentially be used for other plant species with extremely high levels of antioxidants and anthocyanins. PMID:27536526

  14. Californian demonstration and validation of automated agricultural field extraction from multi-temporal Landsat data

    NASA Astrophysics Data System (ADS)

    Yan, L.; Roy, D. P.

    2013-12-01

    The spatial distribution of agricultural fields is a fundamental description of rural landscapes and the location and extent of fields is important to establish the area of land utilized for agricultural yield prediction, resource allocation, and for economic planning. To date, field objects have not been extracted from satellite data over large areas because of computational constraints and because consistently processed appropriate resolution data have not been available or affordable. We present a fully automated computational methodology to extract agricultural fields from 30m Web Enabled Landsat data (WELD) time series and results for approximately 250,000 square kilometers (eleven 150 x 150 km WELD tiles) encompassing all the major agricultural areas of California. The extracted fields, including rectangular, circular, and irregularly shaped fields, are evaluated by comparison with manually interpreted Landsat field objects. Validation results are presented in terms of standard confusion matrix accuracy measures and also the degree of field object over-segmentation, under-segmentation, fragmentation and shape distortion. The apparent success of the presented field extraction methodology is due to several factors. First, the use of multi-temporal Landsat data, as opposed to single Landsat acquisitions, that enables crop rotations and inter-annual variability in the state of the vegetation to be accommodated for and provides more opportunities for cloud-free, non-missing and atmospherically uncontaminated surface observations. Second, the adoption of an object based approach, namely the variational region-based geometric active contour method that enables robust segmentation with only a small number of parameters and that requires no training data collection. Third, the use of a watershed algorithm to decompose connected segments belonging to multiple fields into coherent isolated field segments and a geometry based algorithm to detect and associate parts of

  15. Primerize: automated primer assembly for transcribing non-coding RNA domains.

    PubMed

    Tian, Siqi; Yesselman, Joseph D; Cordero, Pablo; Das, Rhiju

    2015-07-01

    Customized RNA synthesis is in demand for biological and biotechnological research. While chemical synthesis and gel or chromatographic purification of RNA is costly and difficult for sequences longer than tens of nucleotides, a pipeline of primer assembly of DNA templates, in vitro transcription by T7 RNA polymerase and kit-based purification provides a cost-effective and fast alternative for preparing RNA molecules. Nevertheless, designing template primers that optimize cost and avoid mispriming during polymerase chain reaction currently requires expert inspection, downloading specialized software or both. Online servers are currently not available or maintained for the task. We report here a server named Primerize that makes available an efficient algorithm for primer design developed and experimentally tested in our laboratory for RNA domains with lengths up to 300 nucleotides. Free access: http://primerize.stanford.edu. PMID:25999345

  16. Primerize: automated primer assembly for transcribing non-coding RNA domains

    PubMed Central

    Tian, Siqi; Yesselman, Joseph D.; Cordero, Pablo; Das, Rhiju

    2015-01-01

    Customized RNA synthesis is in demand for biological and biotechnological research. While chemical synthesis and gel or chromatographic purification of RNA is costly and difficult for sequences longer than tens of nucleotides, a pipeline of primer assembly of DNA templates, in vitro transcription by T7 RNA polymerase and kit-based purification provides a cost-effective and fast alternative for preparing RNA molecules. Nevertheless, designing template primers that optimize cost and avoid mispriming during polymerase chain reaction currently requires expert inspection, downloading specialized software or both. Online servers are currently not available or maintained for the task. We report here a server named Primerize that makes available an efficient algorithm for primer design developed and experimentally tested in our laboratory for RNA domains with lengths up to 300 nucleotides. Free access: http://primerize.stanford.edu. PMID:25999345

  17. Accurate initiation by RNA polymerase II in a whole cell extract from Saccharomyces cerevisiae.

    PubMed

    Woontner, M; Jaehning, J A

    1990-06-01

    We have developed a simple procedure for isolating a transcriptional extract from whole yeast cells which obviates the requirement for nuclear isolation. Detection of accurate mRNA initiation by RNA polymerase II in the extract requires the use of a sensitive assay, recently described by Kornberg and co-workers (Lue, N. F., Flanagan, P. M., Sugimoto, K., and Kornberg, R. D. (1989) Science 246, 661-664) that involves activation by a GAL4-VP16 fusion protein and a template lacking guanosine residues in the coding strand. The extract is prepared from fresh or frozen yeast cells by disruption with glass beads and fractionation of proteins by ammonium sulfate precipitation. The alpha-amanitin-sensitive transcripts synthesized in the assay were identical to those produced in a parallel assay using a yeast nuclear extract. The activity of the whole cell extract is lower per mg of protein than a nuclear extract but proportional to the volume of the nucleus relative to the whole cell. The optimal ranges for several reaction components including template, mono- and divalent cations, and nucleotide substrate concentration were determined. Under optimal conditions the whole cell extract produced a maximum of approximately 1 X 10(-2) transcripts/template molecule in 30 min. PMID:2188968

  18. miRTex: A Text Mining System for miRNA-Gene Relation Extraction

    PubMed Central

    Li, Gang; Ross, Karen E.; Arighi, Cecilia N.; Peng, Yifan; Wu, Cathy H.; Vijay-Shanker, K.

    2015-01-01

    MicroRNAs (miRNAs) regulate a wide range of cellular and developmental processes through gene expression suppression or mRNA degradation. Experimentally validated miRNA gene targets are often reported in the literature. In this paper, we describe miRTex, a text mining system that extracts miRNA-target relations, as well as miRNA-gene and gene-miRNA regulation relations. The system achieves good precision and recall when evaluated on a literature corpus of 150 abstracts with F-scores close to 0.90 on the three different types of relations. We conducted full-scale text mining using miRTex to process all the Medline abstracts and all the full-length articles in the PubMed Central Open Access Subset. The results for all the Medline abstracts are stored in a database for interactive query and file download via the website at http://proteininformationresource.org/mirtex. Using miRTex, we identified genes potentially regulated by miRNAs in Triple Negative Breast Cancer, as well as miRNA-gene relations that, in conjunction with kinase-substrate relations, regulate the response to abiotic stress in Arabidopsis thaliana. These two use cases demonstrate the usefulness of miRTex text mining in the analysis of miRNA-regulated biological processes. PMID:26407127

  19. Semi-automated procedures for shoreline extraction using single RADARSAT-1 SAR image

    NASA Astrophysics Data System (ADS)

    Al Fugura, A.'kif; Billa, Lawal; Pradhan, Biswajeet

    2011-12-01

    Coastline identification is important for surveying and mapping reasons. Coastline serves as the basic point of reference and is used on nautical charts for navigation purposes. Its delineation has become crucial and more important in the wake of the many recent earthquakes and tsunamis resulting in complete change and redraw of some shorelines. In a tropical country like Malaysia, presence of cloud cover hinders the application of optical remote sensing data. In this study a semi-automated technique and procedures are presented for shoreline delineation from RADARSAT-1 image. A scene of RADARSAT-1 satellite image was processed using enhanced filtering technique to identify and extract the shoreline coast of Kuala Terengganu, Malaysia. RADSARSAT image has many advantages over the optical data because of its ability to penetrate cloud cover and its night sensing capabilities. At first, speckles were removed from the image by using Lee sigma filter which was used to reduce random noise and to enhance the image and discriminate the boundary between land and water. The results showed an accurate and improved extraction and delineation of the entire coastline of Kuala Terrenganu. The study demonstrated the reliability of the image averaging filter in reducing random noise over the sea surface especially near the shoreline. It enhanced land-water boundary differentiation, enabling better delineation of the shoreline. Overall, the developed techniques showed the potential of radar imagery for accurate shoreline mapping and will be useful for monitoring shoreline changes during high and low tides as well as shoreline erosion in a tropical country like Malaysia.

  20. Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil.

    PubMed

    Fang, Changxun; Xu, Tiecheng; Ye, Changliang; Huang, Likun; Wang, Qingshui; Lin, Wenxiong

    2014-02-01

    Techniques to analyze the transcriptome of the soil rhizosphere are essential to reveal the interactions and communications between plants and microorganisms in the soil ecosystem. In this study, different volumes of Al₂(SO₄)₃ were added to rhizosphere soil samples to precipitate humic substances, which interfere with most procedures of RNA and DNA analyses. After humic substances were precipitated, cells of soil microorganisms were broken by vortexing with glass beads, and then DNA and RNA were recovered using Tris-HCl buffer with LiCl, SDS, and EDTA. The crude extract was precipitated and dissolved in RNAse-free water, and then separated by agarose gel electrophoresis. We determined the optimum volume of Al₂(SO₄)₃ for treating rhizosphere soil of rice, tobacco, sugarcane, Rehmannia glutinosa, and Pseudostellaria heterophylla. The crude nucleic acids extract from rice soil was treated with DNase I and then RNA was purified using a gel filtration column. The purified RNA was reverse-transcribed into single-strand cDNA and then ligated with an adaptor at each end before amplifying ds cDNA. The ds cDNA was sub-cloned for subsequent gene sequence analysis. We conducted qPCR to amplify 16S ribosomal DNA and observed highly efficient amplification. These results show that the extraction method can be optimized to isolate and obtain high-quality nucleic acids from microbes in different rhizosphere soils, suitable for genomic and post-genomic analyses. PMID:24078111

  1. Neuron Image Analyzer: Automated and Accurate Extraction of Neuronal Data from Low Quality Images.

    PubMed

    Kim, Kwang-Min; Son, Kilho; Palmore, G Tayhas R

    2015-01-01

    Image analysis software is an essential tool used in neuroscience and neural engineering to evaluate changes in neuronal structure following extracellular stimuli. Both manual and automated methods in current use are severely inadequate at detecting and quantifying changes in neuronal morphology when the images analyzed have a low signal-to-noise ratio (SNR). This inadequacy derives from the fact that these methods often include data from non-neuronal structures or artifacts by simply tracing pixels with high intensity. In this paper, we describe Neuron Image Analyzer (NIA), a novel algorithm that overcomes these inadequacies by employing Laplacian of Gaussian filter and graphical models (i.e., Hidden Markov Model, Fully Connected Chain Model) to specifically extract relational pixel information corresponding to neuronal structures (i.e., soma, neurite). As such, NIA that is based on vector representation is less likely to detect false signals (i.e., non-neuronal structures) or generate artifact signals (i.e., deformation of original structures) than current image analysis algorithms that are based on raster representation. We demonstrate that NIA enables precise quantification of neuronal processes (e.g., length and orientation of neurites) in low quality images with a significant increase in the accuracy of detecting neuronal changes post-stimulation. PMID:26593337

  2. Neuron Image Analyzer: Automated and Accurate Extraction of Neuronal Data from Low Quality Images

    PubMed Central

    Kim, Kwang-Min; Son, Kilho; Palmore, G. Tayhas R.

    2015-01-01

    Image analysis software is an essential tool used in neuroscience and neural engineering to evaluate changes in neuronal structure following extracellular stimuli. Both manual and automated methods in current use are severely inadequate at detecting and quantifying changes in neuronal morphology when the images analyzed have a low signal-to-noise ratio (SNR). This inadequacy derives from the fact that these methods often include data from non-neuronal structures or artifacts by simply tracing pixels with high intensity. In this paper, we describe Neuron Image Analyzer (NIA), a novel algorithm that overcomes these inadequacies by employing Laplacian of Gaussian filter and graphical models (i.e., Hidden Markov Model, Fully Connected Chain Model) to specifically extract relational pixel information corresponding to neuronal structures (i.e., soma, neurite). As such, NIA that is based on vector representation is less likely to detect false signals (i.e., non-neuronal structures) or generate artifact signals (i.e., deformation of original structures) than current image analysis algorithms that are based on raster representation. We demonstrate that NIA enables precise quantification of neuronal processes (e.g., length and orientation of neurites) in low quality images with a significant increase in the accuracy of detecting neuronal changes post-stimulation. PMID:26593337

  3. Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization.

    PubMed

    Anahtar, Melis N; Bowman, Brittany A; Kwon, Douglas S

    2016-01-01

    There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information. PMID:27168460

  4. Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

    PubMed Central

    Anahtar, Melis N.; Bowman, Brittany A.; Kwon, Douglas S.

    2016-01-01

    There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information. PMID:27168460

  5. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  6. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  7. The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma[S

    PubMed Central

    Löfgren, Lars; Ståhlman, Marcus; Forsberg, Gun-Britt; Saarinen, Sinikka; Nilsson, Ralf; Hansson, Göran I.

    2012-01-01

    Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10–100 µl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 µl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 µl heptane:ethyl acetate (3:1) using 300 µl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources. PMID:22645248

  8. A Multi-Atlas Based Method for Automated Anatomical Rat Brain MRI Segmentation and Extraction of PET Activity

    PubMed Central

    Lancelot, Sophie; Roche, Roxane; Slimen, Afifa; Bouillot, Caroline; Levigoureux, Elise; Langlois, Jean-Baptiste; Zimmer, Luc; Costes, Nicolas

    2014-01-01

    Introduction Preclinical in vivo imaging requires precise and reproducible delineation of brain structures. Manual segmentation is time consuming and operator dependent. Automated segmentation as usually performed via single atlas registration fails to account for anatomo-physiological variability. We present, evaluate, and make available a multi-atlas approach for automatically segmenting rat brain MRI and extracting PET activies. Methods High-resolution 7T 2DT2 MR images of 12 Sprague-Dawley rat brains were manually segmented into 27-VOI label volumes using detailed protocols. Automated methods were developed with 7/12 atlas datasets, i.e. the MRIs and their associated label volumes. MRIs were registered to a common space, where an MRI template and a maximum probability atlas were created. Three automated methods were tested: 1/registering individual MRIs to the template, and using a single atlas (SA), 2/using the maximum probability atlas (MP), and 3/registering the MRIs from the multi-atlas dataset to an individual MRI, propagating the label volumes and fusing them in individual MRI space (propagation & fusion, PF). Evaluation was performed on the five remaining rats which additionally underwent [18F]FDG PET. Automated and manual segmentations were compared for morphometric performance (assessed by comparing volume bias and Dice overlap index) and functional performance (evaluated by comparing extracted PET measures). Results Only the SA method showed volume bias. Dice indices were significantly different between methods (PF>MP>SA). PET regional measures were more accurate with multi-atlas methods than with SA method. Conclusions Multi-atlas methods outperform SA for automated anatomical brain segmentation and PET measure’s extraction. They perform comparably to manual segmentation for FDG-PET quantification. Multi-atlas methods are suitable for rapid reproducible VOI analyses. PMID:25330005

  9. A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.

    PubMed

    Majumdar, Gipsy; Vera, Santiago; Elam, Marshall B; Raghow, Rajendra

    2015-04-01

    We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at -80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues. PMID:25579785

  10. Rapid and Semi-Automated Extraction of Neuronal Cell Bodies and Nuclei from Electron Microscopy Image Stacks

    PubMed Central

    Holcomb, Paul S.; Morehead, Michael; Doretto, Gianfranco; Chen, Peter; Berg, Stuart; Plaza, Stephen; Spirou, George

    2016-01-01

    Connectomics—the study of how neurons wire together in the brain—is at the forefront of modern neuroscience research. However, many connectomics studies are limited by the time and precision needed to correctly segment large volumes of electron microscopy (EM) image data. We present here a semi-automated segmentation pipeline using freely available software that can significantly decrease segmentation time for extracting both nuclei and cell bodies from EM image volumes. PMID:27259933

  11. RNA sequencing using fluorescent-labeled dideoxynucleotides and automated fluorescence detection.

    PubMed Central

    Bauer, G J

    1990-01-01

    Although dideoxy terminated sequencing of RNA, using reverse transcriptase and oligodeoxynucleotide primers, is now a well established method, the accuracy is limited by sequence ambiguities due to unspecific chain termination events. A protocol is described which circumvents these ambiguities by using fluorescence labels tagged to dideoxynucleotides. Only chain terminations caused by dideoxynucleotides were detected while premature terminated cDNA's remain undetectable. In addition, the remaining multiple signals at nucleotide positions can be assigned to sequence heterogeneities within the RNA sequence to be determined. Images PMID:1690393

  12. Recovery of Mycobacterium avium subspecies paratuberculosis from the natural host for the extraction and analysis in vivo-derived RNA.

    PubMed

    Granger, Kathy; Moore, Robert J; Davies, John K; Vaughan, Jill A; Stiles, Paula L; Stewart, David J; Tizard, Mark L V

    2004-05-01

    RNA has been extracted and analysed from in vivo-derived Mycobacterium avium subspecies paratuberculosis recovered from the natural host. The bacteria were selectively extracted from the intestinal tissue of two goats exhibiting clinical signs of Johne's disease. Small intestine was rapidly removed, luminal contents washed away and the mucosa and submucosa harvested. Mycobacteria in this material were released from the macrophages by isotonic lysis and differential centrifugation. RNA was extracted and compared with RNA extracted from bacteria grown in vitro. Real-time polymerase chain reaction was used to analyse the katG gene from the bacterial messenger RNA. The katG mRNA encoding the putative catalase/peroxidase showed differential expression in the in vivo and in vitro-derived samples. We hypothesize that the increase in katG expression for in vivo-derived M. paratuberculosis may represent a response to the oxidative stress encountered within the intra-macrophage environment. PMID:15063064

  13. Transcriptator: An Automated Computational Pipeline to Annotate Assembled Reads and Identify Non Coding RNA

    PubMed Central

    Zuccaro, Antonio; Guarracino, Mario Rosario

    2015-01-01

    RNA-seq is a new tool to measure RNA transcript counts, using high-throughput sequencing at an extraordinary accuracy. It provides quantitative means to explore the transcriptome of an organism of interest. However, interpreting this extremely large data into biological knowledge is a problem, and biologist-friendly tools are lacking. In our lab, we developed Transcriptator, a web application based on a computational Python pipeline with a user-friendly Java interface. This pipeline uses the web services available for BLAST (Basis Local Search Alignment Tool), QuickGO and DAVID (Database for Annotation, Visualization and Integrated Discovery) tools. It offers a report on statistical analysis of functional and Gene Ontology (GO) annotation’s enrichment. It helps users to identify enriched biological themes, particularly GO terms, pathways, domains, gene/proteins features and protein—protein interactions related informations. It clusters the transcripts based on functional annotations and generates a tabular report for functional and gene ontology annotations for each submitted transcript to the web server. The implementation of QuickGo web-services in our pipeline enable the users to carry out GO-Slim analysis, whereas the integration of PORTRAIT (Prediction of transcriptomic non coding RNA (ncRNA) by ab initio methods) helps to identify the non coding RNAs and their regulatory role in transcriptome. In summary, Transcriptator is a useful software for both NGS and array data. It helps the users to characterize the de-novo assembled reads, obtained from NGS experiments for non-referenced organisms, while it also performs the functional enrichment analysis of differentially expressed transcripts/genes for both RNA-seq and micro-array experiments. It generates easy to read tables and interactive charts for better understanding of the data. The pipeline is modular in nature, and provides an opportunity to add new plugins in the future. Web application is freely

  14. Transcriptator: An Automated Computational Pipeline to Annotate Assembled Reads and Identify Non Coding RNA.

    PubMed

    Tripathi, Kumar Parijat; Evangelista, Daniela; Zuccaro, Antonio; Guarracino, Mario Rosario

    2015-01-01

    RNA-seq is a new tool to measure RNA transcript counts, using high-throughput sequencing at an extraordinary accuracy. It provides quantitative means to explore the transcriptome of an organism of interest. However, interpreting this extremely large data into biological knowledge is a problem, and biologist-friendly tools are lacking. In our lab, we developed Transcriptator, a web application based on a computational Python pipeline with a user-friendly Java interface. This pipeline uses the web services available for BLAST (Basis Local Search Alignment Tool), QuickGO and DAVID (Database for Annotation, Visualization and Integrated Discovery) tools. It offers a report on statistical analysis of functional and Gene Ontology (GO) annotation's enrichment. It helps users to identify enriched biological themes, particularly GO terms, pathways, domains, gene/proteins features and protein-protein interactions related informations. It clusters the transcripts based on functional annotations and generates a tabular report for functional and gene ontology annotations for each submitted transcript to the web server. The implementation of QuickGo web-services in our pipeline enable the users to carry out GO-Slim analysis, whereas the integration of PORTRAIT (Prediction of transcriptomic non coding RNA (ncRNA) by ab initio methods) helps to identify the non coding RNAs and their regulatory role in transcriptome. In summary, Transcriptator is a useful software for both NGS and array data. It helps the users to characterize the de-novo assembled reads, obtained from NGS experiments for non-referenced organisms, while it also performs the functional enrichment analysis of differentially expressed transcripts/genes for both RNA-seq and micro-array experiments. It generates easy to read tables and interactive charts for better understanding of the data. The pipeline is modular in nature, and provides an opportunity to add new plugins in the future. Web application is freely

  15. HIV Viral RNA Extraction in Wax Immiscible Filtration Assisted by Surface Tension (IFAST) Devices

    PubMed Central

    Berry, Scott M.; LaVanway, Alex J.; Pezzi, Hannah M.; Guckenberger, David J.; Anderson, Meghan A.; Loeb, Jennifer M.; Beebe, David J.

    2015-01-01

    The monitoring of viral load is critical for proper management of antiretroviral therapy for HIV-positive patients. Unfortunately, in the developing world, significant economic and geographical barriers exist, limiting access to this test. The complexity of current viral load assays makes them expensive and their access limited to advanced facilities. We attempted to address these limitations by replacing conventional RNA extraction, one of the essential processes in viral load quantitation, with a simplified technique known as immiscible filtration assisted by surface tension (IFAST). Furthermore, these devices were produced via the embossing of wax, enabling local populations to produce and dispose of their own devices with minimal training or infrastructure, potentially reducing the total assay cost. In addition, IFAST can be used to reduce cold chain dependence during transportation. Viral RNA extracted from raw samples stored at 37°C for 1 week exhibited nearly complete degradation. However, IFAST-purified RNA could be stored at 37°C for 1 week without significant loss. These data suggest that RNA isolated at the point of care (eg, in a rural clinic) via IFAST could be shipped to a central laboratory for quantitative RT-PCR without a cold chain. Using this technology, we have demonstrated accurate and repeatable measurements of viral load on samples with as low as 50 copies per milliliter of sample. PMID:24613822

  16. Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta1

    PubMed Central

    Jordon-Thaden, Ingrid E.; Chanderbali, Andre S.; Gitzendanner, Matthew A.; Soltis, Douglas E.

    2015-01-01

    Premise of the study: Here we present a series of protocols for RNA extraction across a diverse array of plants; we focus on woody, aromatic, aquatic, and other chemically complex taxa. Methods and Results: Ninety-one taxa were subjected to RNA extraction with three methods presented here: (1) TRIzol/TURBO DNA-free kits using the manufacturer’s protocol with the addition of sarkosyl; (2) a combination method using cetyltrimethylammonium bromide (CTAB) and TRIzol/sarkosyl/TURBO DNA-free; and (3) a combination of CTAB and QIAGEN RNeasy Plant Mini Kit. Bench-ready protocols are given. Conclusions: After an iterative process of working with chemically complex taxa, we conclude that the use of TRIzol supplemented with sarkosyl and the TURBO DNA-free kit is an effective, efficient, and robust method for obtaining RNA from 100 mg of leaf tissue of land plant species (Embryophyta) examined. Our protocols can be used to provide RNA of suitable stability, quantity, and quality for transcriptome sequencing. PMID:25995975

  17. Identification of Mushroom Species by Automated rRNA Intergenic Spacer Analysis (ARISA) and Its Application to a Suspected Case of Food Poisoning with Tricholoma ustale.

    PubMed

    Sugawara, Ryota; Yamada, Sayumi; Tu, Zhihao; Sugawara, Akiko; Hoshiba, Toshihiro; Eisaka, Sadao; Yamaguchi, Akihiro

    2016-01-01

    Automated rRNA intergenic spacer analysis (ARISA), a method of microbiome analysis, was evaluated for species identification of mushrooms based on the specific fragment sizes. We used 51 wild mushroom-fruiting bodies collected in the centre of Hokkaido and two cultivated mushrooms. Samples were hot-air-dried and DNA were extracted by a beads beating procedure. Sequencing analysis of portions of the rRNA gene (rDNA) provided 33 identifications of mushrooms by genus or species. The results of ARISA identification based on the combination of the fragment sizes corresponding to two inter spacer regions (ITS2 and ITS1) of rDNA within±0.1% accuracy showed that 27 out of the 33 species had specific fragment sizes differentiated from other species. The remaining 6 species formed 3 pairs that showed overlapping fragment sizes. In addition, within-species polymorphisms were observed as 1 bp differences among 32 samples of 13 species. ARISA was applied to investigate a case of suspected food poisoning in which the mushroom was thought to be a toxic Kakishimeji. The morphological identification of the mushroom was ambiguous since the remaining sample lacked a part of the fruiting body. Further, yeast colonies had grown on the surface of the fruiting body during storage. The ARISA fragment size of the mushroom showed 7 bp difference from that of the candidate toxic mushroom. Although ARISA could be a useful tools for estimation of mushroom species, especially in case where the fruiting bodies have deteriorated or been processed, further studies are necessary for reliable identification. For example, it may be necessary to adopt more informative genes which could provide clearer species-specific polymorphisms than the ITS regions. PMID:27211917

  18. Automated Agricultural Field Extraction from Multi-temporal Web Enabled Landsat Data

    NASA Astrophysics Data System (ADS)

    Yan, L.; Roy, D. P.

    2012-12-01

    Agriculture has caused significant anthropogenic surface change. In many regions agricultural field sizes may be increasing to maximize yields and reduce costs resulting in decreased landscape spatial complexity and increased homogenization of land uses with potential for significant biogeochemical and ecological effects. To date, studies of the incidence, drivers and impacts of changing field sizes have not been undertaken over large areas because of computational constraints and because consistently processed appropriate resolution data have not been available or affordable. The Landsat series of satellites provides near-global coverage, long term, and appropriate spatial resolution (30m) satellite data to document changing field sizes. The recent free availability of all the Landsat data in the U.S. Landsat archive now provides the opportunity to study field size changes in a global and consistent way. Commercial software can be used to extract fields from Landsat data but are inappropriate for large area application because they require considerable human interaction. This paper presents research to develop and validate an automated computational Geographic Object Based Image Analysis methodology to extract agricultural fields and derive field sizes from Web Enabled Landsat Data (WELD) (http://weld.cr.usgs.gov/). WELD weekly products (30m reflectance and brightness temperature) are classified into Satellite Image Automatic Mapper™ (SIAM™) spectral categories and an edge intensity map and a map of the probability of each pixel being agricultural are derived from five years of 52 weeks of WELD and corresponding SIAM™ data. These data are fused to derive candidate agriculture field segments using a variational region-based geometric active contour model. Geometry-based algorithms are used to decompose connected segments belonging to multiple fields into coherent isolated field objects with a divide and conquer strategy to detect and merge partial circle

  19. Characterization of the genus Bifidobacterium by automated ribotyping and 16S rRNA gene sequences.

    PubMed

    Sakata, Shinji; Ryu, Chun Sun; Kitahara, Maki; Sakamoto, Mitsuo; Hayashi, Hidenori; Fukuyama, Masafumi; Benno, Yoshimi

    2006-01-01

    In order to characterize the genus Bifidobacterium, ribopatterns and approximately 500 bp (Escherichia coli positions 27 to 520) of 16S rRNA gene sequences of 28 type strains and 64 reference strains of the genus Bifidobacterium were determined. Ribopatterns obtained from Bifidobacterium strains were divided into nine clusters (clusters I-IX) with a similarity of 60%. Cluster V, containing 17 species, was further subdivided into 22 subclusters with a similarity of 90%. In the genus Bifidobacterium, four groups were shown according to Miyake et al.: (i) the Bifidobacterium longum infantis-longum-suis type group, (ii) the B. catenulatum-pseudocatenulatum group, (iii) the B. gallinarum-saeculare-pullorum group, and (iv) the B. coryneforme-indicum group, which showed higher than 97% similarity of the 16S rRNA gene sequences in each group. Using ribotyping analysis, unique ribopatterns were obtained from these species, and they could be separated by cluster analysis. Ribopatterns of six B. adolescentis strains were separated into different clusters, and also showed diversity in 16S rRNA gene sequences. B. adolescentis consisted of heterogeneous strains. The nine strains of B. pseudolongum subsp. pseudolongum were divided into five subclusters. Each type strain of B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum and two intermediate groups, which were suggested by Yaeshima et al., consisted of individual clusters. B. animalis subsp. animalis and B. animalis subsp. lactis could not be separated by ribotyping using Eco RI. We conclude that ribotyping is able to provide another characteristic of Bifidobacterium strains in addition to 16S rRNA gene sequence phylogenetic analysis, and this information suggests that ribotyping analysis is a useful tool for the characterization of Bifidobacterium species in combination with other techniques for taxonomic characterization. PMID:16428867

  20. Validation of two real-time RT-PCR methods for foot-and-mouth disease diagnosis: RNA-extraction, matrix effect, uncertainty of measurement and precision.

    PubMed

    Goris, Nesya; Vandenbussche, Frank; Herr, Cécile; Villers, Jérôme; Van der Stede, Yves; De Clercq, Kris

    2009-09-01

    Real-time reverse transcription polymerase chain reaction (rRT-PCR) assays are being used routinely for diagnosing foot-and-mouth disease virus (FMDV). Although most laboratories determine analytical and diagnostic sensitivity and specificity, a thorough validation in terms of establishing optimal RNA-extraction conditions, matrix effect, uncertainty of measurement and precision is not performed or reported generally. In this study, different RNA-extraction procedures were compared for two FMDV rRT-PCRs. The NucleoSpin columns available commercially combined high extraction efficiency with ease-of-automation. Furthermore, six different FMDV-negative matrices were spiked with a dilution series of FMDV SAT1 ZIM 25/89. Compared to cell-culture-spiked viral control samples, no matrix effect on the analytical sensitivity was found for blood or foot epithelium. Approximately 1log(10) reduction in detection limit was noted for faecal and tongue epithelium samples, whereas a 3log(10) decrease was observed for spleen samples. By testing the same dilution series in duplicate on 10 different occasions, an estimation of uncertainty of measurement and precision was obtained using blood as matrix. Both rRT-PCRs produced highly precise results emphasising their potential to replace conventional virological methods. The uncertainty measurement, as described in this study, proved to be a useful tool to evaluate the probability of making a wrong decision. PMID:19447138

  1. Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.

    PubMed

    Patel, Palak G; Selvarajah, Shamini; Boursalie, Suzanne; How, Nathan E; Ejdelman, Joshua; Guerard, Karl-Philippe; Bartlett, John M; Lapointe, Jacques; Park, Paul C; Okello, John B A; Berman, David M

    2016-01-01

    Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation. PMID:27583817

  2. A novel automated device for rapid nucleic acid extraction utilizing a zigzag motion of magnetic silica beads.

    PubMed

    Yamaguchi, Akemi; Matsuda, Kazuyuki; Uehara, Masayuki; Honda, Takayuki; Saito, Yasunori

    2016-02-01

    We report a novel automated device for nucleic acid extraction, which consists of a mechanical control system and a disposable cassette. The cassette is composed of a bottle, a capillary tube, and a chamber. After sample injection in the bottle, the sample is lysed, and nucleic acids are adsorbed on the surface of magnetic silica beads. These magnetic beads are transported and are vibrated through the washing reagents in the capillary tube under the control of the mechanical control system, and thus, the nucleic acid is purified without centrifugation. The purified nucleic acid is automatically extracted in 3 min for the polymerase chain reaction (PCR). The nucleic acid extraction is dependent on the transport speed and the vibration frequency of the magnetic beads, and optimizing these two parameters provided better PCR efficiency than the conventional manual procedure. There was no difference between the detection limits of our novel device and that of the conventional manual procedure. We have already developed the droplet-PCR machine, which can amplify and detect specific nucleic acids rapidly and automatically. Connecting the droplet-PCR machine to our novel automated extraction device enables PCR analysis within 15 min, and this system can be made available as a point-of-care testing in clinics as well as general hospitals. PMID:26772121

  3. Mixed-mode isolation of triazine metabolites from soil and aquifer sediments using automated solid-phase extraction

    USGS Publications Warehouse

    Mills, M.S.; Thurman, E.M.

    1992-01-01

    Reversed-phase isolation and ion-exchange purification were combined in the automated solid-phase extraction of two polar s-triazine metabolites, 2-amino-4-chloro-6-(isopropylamino)-s-triazine (deethylatrazine) and 2-amino-4-chloro-6-(ethylamino)-s-triazine (deisopropylatrazine) from clay-loam and slit-loam soils and sandy aquifer sediments. First, methanol/ water (4/1, v/v) soil extracts were transferred to an automated workstation following evaporation of the methanol phase for the rapid reversed-phase isolation of the metabolites on an octadecylresin (C18). The retention of the triazine metabolites on C18 decreased substantially when trace methanol concentrations (1%) remained. Furthermore, the retention on C18 increased with decreasing aqueous solubility and increasing alkyl-chain length of the metabolites and parent herbicides, indicating a reversed-phase interaction. The analytes were eluted with ethyl acetate, which left much of the soil organic-matter impurities on the resin. Second, the small-volume organic eluate was purified on an anion-exchange resin (0.5 mL/min) to extract the remaining soil pigments that could foul the ion source of the GC/MS system. Recoveries of the analytes were 75%, using deuterated atrazine as a surrogate, and were comparable to recoveries by soxhlet extraction. The detection limit was 0.1 ??g/kg with a coefficient of variation of 15%. The ease and efficiency of this automated method makes it viable, practical technique for studying triazine metabolites in the environment.

  4. Olive Leaf Extract Elevates Hepatic PPAR α mRNA Expression and Improves Serum Lipid Profiles in Ovariectomized Rats.

    PubMed

    Yoon, Leena; Liu, Ya-Nan; Park, Hyunjin; Kim, Hyun-Sook

    2015-07-01

    We hypothesized that olive leaf extract might alleviate dyslipidemia resulting from estrogen deficiency. Serum lipid profile and mRNA expression of the related genes in the liver and adipose tissue were analyzed after providing olive leaf extract (200 or 400 mg/kg body weight; n=7 for each group) to ovariectomized rats for 10 weeks. After 10 weeks' administration, the rats in the olive leaf extract-administered groups showed significantly lower levels of serum triglyceride and very-low-density lipoprotein (VLDL)-cholesterol compared with the rats in the control group, whereas the administration of olive leaf extract did not significantly change the elevated low-density lipoprotein cholesterol levels. In addition, administration of high dose of olive leaf extract significantly decreased the liver triglyceride and increased serum estradiol levels. mRNA expressions of peroxisome proliferator-activated receptor alpha (PPAR α) and acyl-CoA oxidase (ACO) were not affected by ovariectomy, however, administration of olive leaf extract significantly increased both PPAR α and ACO mRNA expression. Expression of adiponectin mRNA in adipose tissue was significantly decreased in the ovariectomized control group. Rats administered low-dose olive leaf extract showed significantly elevated adiponectin mRNA expression compared with rats in the ovariectomized control group. Even though dose-dependent effects were not observed in most of the measurements, these results suggest that genes involved in lipid metabolism may be regulated by olive leaf extract administration in ovariectomized rats. PMID:25714618

  5. Olive Leaf Extract Elevates Hepatic PPAR α mRNA Expression and Improves Serum Lipid Profiles in Ovariectomized Rats

    PubMed Central

    Yoon, Leena; Liu, Ya-Nan; Park, Hyunjin

    2015-01-01

    Abstract We hypothesized that olive leaf extract might alleviate dyslipidemia resulting from estrogen deficiency. Serum lipid profile and mRNA expression of the related genes in the liver and adipose tissue were analyzed after providing olive leaf extract (200 or 400 mg/kg body weight; n=7 for each group) to ovariectomized rats for 10 weeks. After 10 weeks' administration, the rats in the olive leaf extract-administered groups showed significantly lower levels of serum triglyceride and very-low-density lipoprotein (VLDL)-cholesterol compared with the rats in the control group, whereas the administration of olive leaf extract did not significantly change the elevated low-density lipoprotein cholesterol levels. In addition, administration of high dose of olive leaf extract significantly decreased the liver triglyceride and increased serum estradiol levels. mRNA expressions of peroxisome proliferator-activated receptor alpha (PPAR α) and acyl-CoA oxidase (ACO) were not affected by ovariectomy, however, administration of olive leaf extract significantly increased both PPAR α and ACO mRNA expression. Expression of adiponectin mRNA in adipose tissue was significantly decreased in the ovariectomized control group. Rats administered low-dose olive leaf extract showed significantly elevated adiponectin mRNA expression compared with rats in the ovariectomized control group. Even though dose-dependent effects were not observed in most of the measurements, these results suggest that genes involved in lipid metabolism may be regulated by olive leaf extract administration in ovariectomized rats. PMID:25714618

  6. RNA Extraction from a Mycobacterium under Ultrahigh Electric Field Intensity in a Microfluidic Device.

    PubMed

    Ma, Sai; Bryson, Bryan D; Sun, Chen; Fortune, Sarah M; Lu, Chang

    2016-05-17

    Studies of transcriptomes are critical for understanding gene expression. Release of RNA molecules from cells is typically the first step for transcriptomic analysis. Effective cell lysis approaches that completely release intracellular materials are in high demand especially for cells that are structurally robust. In this report, we demonstrate a microfluidic electric lysis device that is effective for mRNA extraction from mycobacteria that have hydrophobic and waxy cell walls. We used a packed bed of microscale silica beads to filter M. smegmatis out of the suspension. 4000-8000 V/cm field intensity was used to lyse M. smegmatis with long pulses (i.e., up to 30 pulses that were 5 s long each). Our quantitative reverse transcription (qRT)-PCR results showed that our method yielded a factor of 10-20 higher extraction efficiency than the current state-of-the-art method (bead beating). We conclude that our electric lysis technique is an effective approach for mRNA release from hard-to-lyse cells and highly compatible with microfluidic molecular assays. PMID:27081872

  7. Semi-automated extraction of landslides in Taiwan based on SPOT imagery and DEMs

    NASA Astrophysics Data System (ADS)

    Eisank, Clemens; Hölbling, Daniel; Friedl, Barbara; Chen, Yi-Chin; Chang, Kang-Tsung

    2014-05-01

    The vast availability and improved quality of optical satellite data and digital elevation models (DEMs), as well as the need for complete and up-to-date landslide inventories at various spatial scales have fostered the development of semi-automated landslide recognition systems. Among the tested approaches for designing such systems, object-based image analysis (OBIA) stepped out to be a highly promising methodology. OBIA offers a flexible, spatially enabled framework for effective landslide mapping. Most object-based landslide mapping systems, however, have been tailored to specific, mainly small-scale study areas or even to single landslides only. Even though reported mapping accuracies tend to be higher than for pixel-based approaches, accuracy values are still relatively low and depend on the particular study. There is still room to improve the applicability and objectivity of object-based landslide mapping systems. The presented study aims at developing a knowledge-based landslide mapping system implemented in an OBIA environment, i.e. Trimble eCognition. In comparison to previous knowledge-based approaches, the classification of segmentation-derived multi-scale image objects relies on digital landslide signatures. These signatures hold the common operational knowledge on digital landslide mapping, as reported by 25 Taiwanese landslide experts during personal semi-structured interviews. Specifically, the signatures include information on commonly used data layers, spectral and spatial features, and feature thresholds. The signatures guide the selection and implementation of mapping rules that were finally encoded in Cognition Network Language (CNL). Multi-scale image segmentation is optimized by using the improved Estimation of Scale Parameter (ESP) tool. The approach described above is developed and tested for mapping landslides in a sub-region of the Baichi catchment in Northern Taiwan based on SPOT imagery and a high-resolution DEM. An object

  8. miRNAfe: A comprehensive tool for feature extraction in microRNA prediction.

    PubMed

    Yones, Cristian A; Stegmayer, Georgina; Kamenetzky, Laura; Milone, Diego H

    2015-12-01

    miRNAfe is a comprehensive tool to extract features from RNA sequences. It is freely available as a web service, allowing a single access point to almost all state-of-the-art feature extraction methods used today in a variety of works from different authors. It has a very simple user interface, where the user only needs to load a file containing the input sequences and select the features to extract. As a result, the user obtains a text file with the features extracted, which can be used to analyze the sequences or as input to a miRNA prediction software. The tool can calculate up to 80 features where many of them are multidimensional arrays. In order to simplify the web interface, the features have been divided into six pre-defined groups, each one providing information about: primary sequence, secondary structure, thermodynamic stability, statistical stability, conservation between genomes of different species and substrings analysis of the sequences. Additionally, pre-trained classifiers are provided for prediction in different species. All algorithms to extract the features have been validated, comparing the results with the ones obtained from software of the original authors. The source code is freely available for academic use under GPL license at http://sourceforge.net/projects/sourcesinc/files/mirnafe/0.90/. A user-friendly access is provided as web interface at http://fich.unl.edu.ar/sinc/web-demo/mirnafe/. A more configurable web interface can be accessed at http://fich.unl.edu.ar/sinc/web-demo/mirnafe-full/. PMID:26499212

  9. Post-extraction stabilization of HIV viral RNA for quantitative molecular tests.

    PubMed

    Stevens, Daniel S; Crudder, Christopher H; Domingo, Gonzalo J

    2012-06-01

    Two approaches to stabilize viral nucleic acid in processed clinical specimens were evaluated. HIV-1 RNA extracted from clinical specimens was stabilized in a dry matrix in a commercial product (RNAstable, Biomatrica, San Diego, CA, USA) and in a reverse-transcription reaction mixture in liquid form as cDNA. As few as 145 HIV-1 genome copies of viral RNA are reliably stabilized by RNAstable at 45°C for 92 days and in the cDNA format at 45°C for 7 days as determined by real-time PCR. With RNAstable the R(2) at days 1, 7, and 92 were 0.888, 0.871, and 0.943 when compared to baseline viral load values. The cDNA generated from the same clinical specimens was highly stable with an R(2) value of 0.762 when comparing viral load determinations at day 7 to baseline values. In conclusion viral RNA stabilized in a dry RNAstable matrix is highly stable for long periods of time at high temperatures across a substantial dynamic range. Viral RNA signal can also be stabilized in liquid in the form of cDNA for limited periods of time. Methods that reduce reliance on the cold chain and preserve specimen integrity are critical for extending the reach of molecular testing to low-resource settings. Products based on anhydrobiosis, such as the RNAstable should be evaluated further to support viral pathogen diagnosis. PMID:22433512

  10. Viral RNA testing and automation on the bead-based CBNE detection microsystem.

    SciTech Connect

    Galambos, Paul C.; Bourdon, Christopher Jay; Farrell, Cara M.; Rossito, Paul; McClain, Jaime L.; Derzon, Mark Steven; Cullor, James Sterling; Rahimian, Kamayar

    2008-09-01

    We developed prototype chemistry for nucleic acid hybridization on our bead-based diagnostics platform and we established an automatable bead handling protocol capable of 50 part-per-billion (ppb) sensitivity. We are working towards a platform capable of parallel, rapid (10 minute), raw sample testing for orthogonal (in this case nucleic acid and immunoassays) identification of biological (and other) threats in a single sensor microsystem. In this LDRD we developed the nucleic acid chemistry required for nucleic acid hybridization. Our goal is to place a non-cell associated RNA virus (Bovine Viral Diarrhea, BVD) on the beads for raw sample testing. This key pre-requisite to showing orthogonality (nucleic acid measurements can be performed in parallel with immunoassay measurements). Orthogonal detection dramatically reduces false positives. We chose BVD because our collaborators (UC-Davis) can supply samples from persistently infected animals; and because proof-of-concept field testing can be performed with modification of the current technology platform at the UC Davis research station. Since BVD is a cattle-prone disease this research dovetails with earlier immunoassay work on Botulinum toxin simulant testing in raw milk samples. Demonstration of BVD RNA detection expands the repertoire of biological macromolecules that can be adapted to our bead-based detection. The resources of this late start LDRD were adequate to partially demonstrate the conjugation of the beads to the nucleic acids. It was never expected to be adequate for a full live virus test but to motivate that additional investment. In addition, we were able to reduce the LOD (Limit of Detection) for the botulinum toxin stimulant to 50 ppb from the earlier LOD of 1 ppm. A low LOD combined with orthogonal detection provides both low false negatives and low false positives. The logical follow-on steps to this LDRD research are to perform live virus identification as well as concurrent nucleic acid and

  11. Identification of More Feasible MicroRNA-mRNA Interactions within Multiple Cancers Using Principal Component Analysis Based Unsupervised Feature Extraction.

    PubMed

    Taguchi, Y-H

    2016-01-01

    MicroRNA(miRNA)-mRNA interactions are important for understanding many biological processes, including development, differentiation and disease progression, but their identification is highly context-dependent. When computationally derived from sequence information alone, the identification should be verified by integrated analyses of mRNA and miRNA expression. The drawback of this strategy is the vast number of identified interactions, which prevents an experimental or detailed investigation of each pair. In this paper, we overcome this difficulty by the recently proposed principal component analysis (PCA)-based unsupervised feature extraction (FE), which reduces the number of identified miRNA-mRNA interactions that properly discriminate between patients and healthy controls without losing biological feasibility. The approach is applied to six cancers: hepatocellular carcinoma, non-small cell lung cancer, esophageal squamous cell carcinoma, prostate cancer, colorectal/colon cancer and breast cancer. In PCA-based unsupervised FE, the significance does not depend on the number of samples (as in the standard case) but on the number of features, which approximates the number of miRNAs/mRNAs. To our knowledge, we have newly identified miRNA-mRNA interactions in multiple cancers based on a single common (universal) criterion. Moreover, the number of identified interactions was sufficiently small to be sequentially curated by literature searches. PMID:27171078

  12. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  13. Automated solid-phase extraction coupled online with HPLC-FLD for the quantification of zearalenone in edible oil.

    PubMed

    Drzymala, Sarah S; Weiz, Stefan; Heinze, Julia; Marten, Silvia; Prinz, Carsten; Zimathies, Annett; Garbe, Leif-Alexander; Koch, Matthias

    2015-05-01

    Established maximum levels for the mycotoxin zearalenone (ZEN) in edible oil require monitoring by reliable analytical methods. Therefore, an automated SPE-HPLC online system based on dynamic covalent hydrazine chemistry has been developed. The SPE step comprises a reversible hydrazone formation by ZEN and a hydrazine moiety covalently attached to a solid phase. Seven hydrazine materials with different properties regarding the resin backbone, pore size, particle size, specific surface area, and loading have been evaluated. As a result, a hydrazine-functionalized silica gel was chosen. The final automated online method was validated and applied to the analysis of three maize germ oil samples including a provisionally certified reference material. Important performance criteria for the recovery (70-120 %) and precision (RSDr <25 %) as set by the Commission Regulation EC 401/2006 were fulfilled: The mean recovery was 78 % and RSDr did not exceed 8 %. The results of the SPE-HPLC online method were further compared to results obtained by liquid-liquid extraction with stable isotope dilution analysis LC-MS/MS and found to be in good agreement. The developed SPE-HPLC online system with fluorescence detection allows a reliable, accurate, and sensitive quantification (limit of quantification, 30 μg/kg) of ZEN in edible oils while significantly reducing the workload. To our knowledge, this is the first report on an automated SPE-HPLC method based on a covalent SPE approach. PMID:25709066

  14. Automated extraction of 11-nor-delta9-tetrahydrocannabinol carboxylic acid from urine samples using the ASPEC XL solid-phase extraction system.

    PubMed

    Langen, M C; de Bijl, G A; Egberts, A C

    2000-09-01

    The analysis of 11-nor-delta9-tetrahydrocannabinol-carboxylic acid (THCCOOH, the major metabolite of cannabis) in urine with gas chromatography and mass spectrometry (GC-MS) and solid-phase extraction (SPE) sample preparation is well documented. Automated SPE sample preparation of THCCOOH in urine, although potentially advantageous, is to our knowledge poorly investigated. The objective of the present study was to develop and validate an automated SPE sample-preparation step using ASPEC XL suited for GC-MS confirmation analysis of THCCOOH in urine drug control. The recoveries showed that it was not possible to transfer the protocol for the manual SPE procedure with the vacuum manifold to the ASPEC XL without loss of recovery. Making the sample more lipophilic by adding 1 mL 2-propanol after hydrolysis to the urine sample in order to overcome the problem of surface adsorption of THCCOOH led to an extraction efficiency (77%) comparable to that reached with the vacuum manifold (84%). The reproducibility of the automated SPE procedure was better (coefficient of variation 5%) than that of the manual procedure (coefficient of variation 12%). The limit of detection was 1 ng/mL, and the limit of quantitation was 4 ng/mL. Precision at the 12.5-ng/mL level was as follows: mean, 12.4 and coefficient of variation, 3.0%. Potential carryover was evaluated, but a carryover effect could not be detected. It was concluded that the proposed method is suited for GC-MS confirmation urinalysis of THCCOOH for prisons and detoxification centers. PMID:10999349

  15. High quality DNA obtained with an automated DNA extraction method with 70+ year old formalin-fixed celloidin-embedded (FFCE) blocks from the indiana medical history museum

    PubMed Central

    Niland, Erin E; McGuire, Audrey; Cox, Mary H; Sandusky, George E

    2012-01-01

    DNA and RNA have been used as markers of tissue quality and integrity throughout the last few decades. In this research study, genomic quality DNA of kidney, liver, heart, lung, spleen, and brain were analyzed in tissues from post-mortem patients and surgical cancer cases spanning the past century. DNA extraction was performed on over 180 samples from: 70+ year old formalin-fixed celloidin-embedded (FFCE) tissues, formalin-fixed paraffin-embedded (FFPE) tissue samples from surgical cases and post-mortem cases from the 1970’s, 1980’s, 1990’s, and 2000’s, tissues fixed in 10% neutral buffered formalin/stored in 70% ethanol from the 1990’s, 70+ year old tissues fixed in unbuffered formalin of various concentrations, and fresh tissue as a control. To extract DNA from FFCE samples and ethanol-soaked samples, a modified standard operating procedure was used in which all tissues were homogenized, digested with a proteinase K solution for a long period of time (24-48 hours), and DNA was extracted using the Autogen Flexstar automated extraction machine. To extract DNA from FFPE, all tissues were soaked in xylene to remove the paraffin from the tissue prior to digestion, and FFPE tissues were not homogenized. The results were as follows: celloidin-embedded and paraffin-embedded tissues yielded the highest DNA concentration and greatest DNA quality, while the formalin in various concentrations, and long term formalin/ethanol-stored tissue yielded both the lowest DNA concentration and quality of the tissues tested. The average DNA yield for the various fixatives was: 367.77 μg/ mL FFCE, 590.7 μg/mL FFPE, 53.74 μg/mL formalin-fixed/70% ethanol-stored and 33.2 μg/mL unbuffered formalin tissues. The average OD readings for FFCE, FFPE, formalin-fixed/70% ethanol-stored tissues, and tissues fixed in unbuffered formalin were 1.86, 1.87, 1.43, and 1.48 respectively. The results show that usable DNA can be extracted from tissue fixed in formalin and embedded in celloidin

  16. Whole-Cell versus Total RNA Extraction for Analysis of Microbial Community Structure with 16S rRNA-Targeted Oligonucleotide Probes in Salt Marsh Sediments

    PubMed Central

    Frischer, Marc E.; Danforth, Jean M.; Healy, Michele A. Newton; Saunders, F. Michael

    2000-01-01

    rRNA-targeted oligonucleotide probes have become powerful tools for describing microbial communities, but their use in sediments remains difficult. Here we describe a simple technique involving homogenization, detergents, and dispersants that allows the quantitative extraction of cells from formalin-preserved salt marsh sediments. Resulting cell extracts are amenable to membrane blotting and hybridization protocols. Using this procedure, the efficiency of cell extraction was high (95.7% ± 3.7% [mean ± standard deviation]) relative to direct DAPI (4′,6′-diamidino-2-phenylindole) epifluorescence cell counts for a variety of salt marsh sediments. To test the hypothesis that cells were extracted without phylogenetic bias, the relative abundance (depth distribution) of five major divisions of the gram-negative mesophilic sulfate-reducing delta proteobacteria were determined in sediments maintained in a tidal mesocosm system. A suite of six 16S rRNA-targeted oligonucleotide probes were utilized. The apparent structure of sulfate-reducing bacteria communities determined from whole-cell and RNA extracts were consistent with each other (r2 = 0.60), indicating that the whole-cell extraction and RNA extraction hybridization approaches for describing sediment microbial communities are equally robust. However, the variability associated with both methods was high and appeared to be a result of the natural heterogeneity of sediment microbial communities and methodological artifacts. The relative distribution of sulfate-reducing bacteria was similar to that observed in natural marsh systems, providing preliminary evidence that the mesocosm systems accurately simulate native marsh systems. PMID:10877803

  17. INVESTIGATION OF ARSENIC SPECIATION ON DRINKING WATER TREATMENT MEDIA UTILIZING AUTOMATED SEQUENTIAL CONTINUOUS FLOW EXTRACTION WITH IC-ICP-MS DETECTION

    EPA Science Inventory

    Three treatment media, used for the removal of arsenic from drinking water, were sequentially extracted using 10mM MgCl2 (pH 8), 10mM NaH2PO4 (pH 7) followed by 10mM (NH4)2C2O4 (pH 3). The media were extracted using an on-line automated continuous extraction system which allowed...

  18. Direct Sampling and Analysis from Solid Phase Extraction Cards using an Automated Liquid Extraction Surface Analysis Nanoelectrospray Mass Spectrometry System

    SciTech Connect

    Walworth, Matthew J; ElNaggar, Mariam S; Stankovich, Joseph J; WitkowskiII, Charles E.; Norris, Jeremy L; Van Berkel, Gary J

    2011-01-01

    Direct liquid extraction based surface sampling, a technique previously demonstrated with continuous flow and autonomous pipette liquid microjunction surface sampling probes, has recently been implemented as the Liquid Extraction Surface Analysis (LESA) mode on the commercially available Advion NanoMate chip-based infusion nanoelectrospray ionization system. In the present paper, the LESA mode was applied to the analysis of 96-well format custom solid phase extraction (SPE) cards, with each well consisting of either a 1 or 2 mm diameter monolithic hydrophobic stationary phase. These substrate wells were conditioned, loaded with either single or multi-component aqueous mixtures, and read out using the LESA mode of a TriVersa NanoMate or a Nanomate 100 coupled to an ABI/Sciex 4000QTRAPTM hybrid triple quadrupole/linear ion trap mass spectrometer and a Thermo LTQ XL linear ion trap mass spectrometer. Extraction conditions, including extraction/nanoESI solvent composition, volume, and dwell times, were optimized in the analysis of targeted compounds. Limit of detection and quantitation as well as analysis reproducibility figures of merit were measured. Calibration data was obtained for propranolol using a deuterated internal standard which demonstrated linearity and reproducibility. A 10x increase in signal and cleanup of micromolar Angiotensin II from a concentrated salt solution was demonstrated. Additionally, a multicomponent herbicide mixture at ppb concentration levels was analyzed using MS3 spectra for compound identification in the presence of isobaric interferences.

  19. Sample preparation for avian and porcine influenza virus cDNA amplification simplified: Boiling vs. conventional RNA extraction.

    PubMed

    Fereidouni, Sasan R; Starick, Elke; Ziller, Mario; Harder, Timm C; Unger, Hermann; Hamilton, Keith; Globig, Anja

    2015-09-01

    RNA extraction and purification is a fundamental step that allows for highly sensitive amplification of specific RNA targets in PCR applications. However, commercial extraction kits that are broadly used because of their robustness and high yield of purified RNA are expensive and labor-intensive. In this study, boiling in distilled water or a commercial lysis buffer of different sample matrices containing avian or porcine influenza viruses was tested as an alternative. Real-time PCR (RTqPCR) for nucleoprotein gene fragment was used as read out. Results were compared with freshly extracted RNA by use of a commercial extraction kit. Different batches of virus containing materials, including diluted virus positive allantoic fluid or cell culture supernatant, and avian faecal, cloacal or oropharyngeal swab samples were used in this study. Simple boiling of samples without any additional purification steps can be used as an alternative RNA preparation method to detect influenza A virus nucleoprotein RNA in oropharyngeal swab samples, allantoic fluid or cell-culture supernatant. The boiling method is not applicable for sample matrices containing faecal material. PMID:25929989

  20. High performance liquid chromatography for quantification of gatifloxacin in rat plasma following automated on-line solid phase extraction.

    PubMed

    Tasso, Leandro; Dalla Costa, Teresa

    2007-05-01

    An automated system using on-line solid phase extraction and HPLC with fluorimetric detection was developed and validated for quantification of gatifloxacin in rat plasma. The extraction was carried out using C(18) cartridges (BondElut), with a high extraction yield. After washing, gatifloxacin was eluted from the cartridge with mobile phase onto a C(18) HPLC column. The mobile phase consisted of a mixture of phosphoric acid (2.5mM), methanol, acetonitrile and triethylamine (64.8:15:20:0.2, v/v/v/v, apparent pH(app.) 2.8). All samples and standard solutions were chromatographed at 28 degrees C. The method developed was selective and linear for drug concentrations ranging between 20 and 600 ng/ml. Gatifloxacin recovery ranged from 95.6 to 99.7%, and the limit of quantification was 20 ng/ml. The intra and inter-assay accuracy were up to 94.3%. The precision determined not exceed 5.8% of the CV. High extraction yield up to 95% was obtained. Drug stability in plasma was shown in freezer at -20 degrees C up to 1 month, after three freeze-thaw cycles and for 24h in the autosampler after processing. The assay has been successfully applied to measure gatifloxacin plasma concentrations in pharmacokinetic study in rats. PMID:17403594

  1. Automated on-line liquid-liquid extraction system for temporal mass spectrometric analysis of dynamic samples.

    PubMed

    Hsieh, Kai-Ta; Liu, Pei-Han; Urban, Pawel L

    2015-09-24

    Most real samples cannot directly be infused to mass spectrometers because they could contaminate delicate parts of ion source and guides, or cause ion suppression. Conventional sample preparation procedures limit temporal resolution of analysis. We have developed an automated liquid-liquid extraction system that enables unsupervised repetitive treatment of dynamic samples and instantaneous analysis by mass spectrometry (MS). It incorporates inexpensive open-source microcontroller boards (Arduino and Netduino) to guide the extraction and analysis process. Duration of every extraction cycle is 17 min. The system enables monitoring of dynamic processes over many hours. The extracts are automatically transferred to the ion source incorporating a Venturi pump. Operation of the device has been characterized (repeatability, RSD = 15%, n = 20; concentration range for ibuprofen, 0.053-2.000 mM; LOD for ibuprofen, ∼0.005 mM; including extraction and detection). To exemplify its usefulness in real-world applications, we implemented this device in chemical profiling of pharmaceutical formulation dissolution process. Temporal dissolution profiles of commercial ibuprofen and acetaminophen tablets were recorded during 10 h. The extraction-MS datasets were fitted with exponential functions to characterize the rates of release of the main and auxiliary ingredients (e.g. ibuprofen, k = 0.43 ± 0.01 h(-1)). The electronic control unit of this system interacts with the operator via touch screen, internet, voice, and short text messages sent to the mobile phone, which is helpful when launching long-term (e.g. overnight) measurements. Due to these interactive features, the platform brings the concept of the Internet-of-Things (IoT) to the chemistry laboratory environment. PMID:26423626

  2. Comparison of RNA extraction methods for the detection of porcine reproductive and respiratory syndrome virus from boar semen.

    PubMed

    Christopher-Hennings, Jane; Dammen, Matthew; Nelson, Eric; Rowland, Raymond; Oberst, Richard

    2006-09-01

    To detect Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in semen, various RNA extraction techniques have been utilized for RT-PCR, but rarely compared, to determine an optimized extraction protocol. Due to the viscosity, non-homogeneity, high cellularity and large volume of boar semen produced, difficulties can be encountered in obtaining RNA from the seminal cell fraction. This study compared six RNA extractions, five which used a commercially available kit (RNeasy, Qiagen Inc.) for use on highly cellular samples and a traditional phenol/chloroform procedure. All extractions were compared on serially diluted PRRSV "spiked" seminal cell fractions. The two methods resulting in recovery of the highest amount of RNA, which included a Qiashredder (Qiagen Inc.) (protocol 1) or cell lysis/centrifugation technique (protocol 3) preceding the RNeasy procedure were then compared using naturally infected semen samples from experimentally infected boars. Both protocols detected similar amounts of virus in "spiked" samples, but protocol 1 detected eight additional PRRSV-positive semen samples in naturally infected semen. This study demonstrated that semen "spiked" with PRRSV (cell-free virus) may not be representative of naturally infected semen samples (cell associated virus) for comparing extraction protocols, but did identify a useful extraction technique for boar semen. PMID:16621036

  3. Comparative Assessment of Automated Nucleic Acid Sample Extraction Equipment for Biothreat Agents

    PubMed Central

    Kalina, Warren Vincent; Douglas, Christina Elizabeth; Coyne, Susan Rajnik

    2014-01-01

    Magnetic beads offer superior impurity removal and nucleic acid selection over older extraction methods. The performances of nucleic acid extraction of biothreat agents in blood or buffer by easyMAG, MagNA Pure, EZ1 Advanced XL, and Nordiag Arrow were evaluated. All instruments showed excellent performance in blood; however, the easyMAG had the best precision and versatility. PMID:24452173

  4. Accurate and efficient 3' processing of U2 small nuclear RNA precursor in a fractionated cytoplasmic extract.

    PubMed Central

    Kleinschmidt, A M; Pederson, T

    1987-01-01

    The small nuclear RNAs U1, U2, U4, and U5 are cofactors in mRNA splicing and, like the pre-mRNAs with which they interact, are transcribed by RNA polymerase II. Also like mRNAs, mature U1 and U2 RNAs are generated by 3' processing of their primary transcripts. In this study we have investigated the in vitro processing of an SP6-transcribed human U2 RNA precursor, the 3' end of which matches that of authentic human U2 RNA precursor molecules. Although the SP6-U2 RNA precursor was efficiently processed in an ammonium sulfate-fractionated HeLa cytoplasmic S100 extract, the product RNA was unstable. Further purification of the processing activity on glycerol gradients resolved a 7S activity that nonspecifically cleaved all RNAs tested and a 15S activity that efficiently processed the 3' end of pre-U2 RNA. The 15S activity did not process the 3' end of a tRNA precursor molecule. As demonstrated by RNase protection, the processed 3' end of the SP6-U2 RNA maps to the same nucleotides as does mature HeLa U2 RNA. Images PMID:3670307

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

    PubMed Central

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

    2016-01-01

    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included. PMID:27341629

  6. A novel approach on fluid dispensing for a DNA/RNA extraction chip package

    NASA Astrophysics Data System (ADS)

    Xie, Ling; Premachandran, C. S.; Chew, Michelle; Yao, Qiang; Xu, Diao; Pinjala, D.

    2008-02-01

    Micro fluidic package with integrated reservoirs has been developed for DNA /RNA extraction application. A membrane based pump which consists of a reservoir to store reagents and a pin valve to control the fluid is developed to dispense the reagents into the chip. A programmable external actuator is fabricated to dispense the fluid from the membrane pump into the DNA chip. An elastic and high elongation thin rubber membrane is used to seal the membrane pump and at the same time prevent actuator from mixing with different reagents in the micro fluidic package. Break displacement during actuation of membrane pump sealing material is studied with different ratios of PDMS and other types of rubber materials. The fluid flow from the reservoir to the chip is controlled by a pin valve which is activated during the external actuation. A CFD simulation is performed to study the pumping action dusting the external actuation and is validated with experimental results.

  7. Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes.

    PubMed

    Yamagishi, Junya; Sato, Yukuto; Shinozaki, Natsuko; Ye, Bin; Tsuboi, Akito; Nagasaki, Masao; Yamashita, Riu

    2016-01-01

    The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no "gold standard" for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study. PMID:27104353

  8. Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes

    PubMed Central

    Shinozaki, Natsuko; Ye, Bin; Tsuboi, Akito; Nagasaki, Masao; Yamashita, Riu

    2016-01-01

    The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no “gold standard” for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study. PMID:27104353

  9. Toward automated parasitic extraction of silicon photonics using layout physical verifications

    NASA Astrophysics Data System (ADS)

    Ismail, Mohamed; El Shamy, Raghi S.; Madkour, Kareem; Hammouda, Sherif; Swillam, Mohamed A.

    2016-08-01

    A physical verification flow of the layout of silicon photonic circuits is suggested. Simple empirical models are developed to estimate the bend power loss and coupled power in photonic integrated circuits fabricated using SOI standard wafers. These models are utilized in physical verification flow of the circuit layout to verify reliable fabrication using any electronic design automation tool. The models are accurate compared with electromagnetic solvers. The models are closed form and circumvent the need to utilize any EM solver for the verification process. Hence, it dramatically reduces the time of the verification process.

  10. CAMUR: Knowledge extraction from RNA-seq cancer data through equivalent classification rules

    PubMed Central

    Cestarelli, Valerio; Fiscon, Giulia; Felici, Giovanni; Bertolazzi, Paola; Weitschek, Emanuel

    2016-01-01

    Motivation: Nowadays, knowledge extraction methods from Next Generation Sequencing data are highly requested. In this work, we focus on RNA-seq gene expression analysis and specifically on case–control studies with rule-based supervised classification algorithms that build a model able to discriminate cases from controls. State of the art algorithms compute a single classification model that contains few features (genes). On the contrary, our goal is to elicit a higher amount of knowledge by computing many classification models, and therefore to identify most of the genes related to the predicted class. Results: We propose CAMUR, a new method that extracts multiple and equivalent classification models. CAMUR iteratively computes a rule-based classification model, calculates the power set of the genes present in the rules, iteratively eliminates those combinations from the data set, and performs again the classification procedure until a stopping criterion is verified. CAMUR includes an ad-hoc knowledge repository (database) and a querying tool. We analyze three different types of RNA-seq data sets (Breast, Head and Neck, and Stomach Cancer) from The Cancer Genome Atlas (TCGA) and we validate CAMUR and its models also on non-TCGA data. Our experimental results show the efficacy of CAMUR: we obtain several reliable equivalent classification models, from which the most frequent genes, their relationships, and the relation with a particular cancer are deduced. Availability and implementation: dmb.iasi.cnr.it/camur.php Contact: emanuel@iasi.cnr.it Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26519501

  11. Automated Development of Feature Extraction Tools for Planetary Science Image Datasets

    NASA Astrophysics Data System (ADS)

    Plesko, C.; Brumby, S.; Asphaug, E.

    2003-03-01

    We explore development of feature extraction algorithms for Mars Orbiter Camera narrow angle data using GENIE machine learning software. The algorithms are successful at detecting craters within the images, and generalize well to a new image.

  12. World-to-digital-microfluidic interface enabling extraction and purification of RNA from human whole blood.

    PubMed

    Jebrail, Mais J; Sinha, Anupama; Vellucci, Samantha; Renzi, Ronald F; Ambriz, Cesar; Gondhalekar, Carmen; Schoeniger, Joseph S; Patel, Kamlesh D; Branda, Steven S

    2014-04-15

    Digital microfluidics (DMF) is a powerful technique for simple and precise manipulation of microscale droplets of fluid. This technique enables processing and analysis of a wide variety of samples and reagents and has proven useful in a broad range of chemical, biological, and medical applications. Handling of "real-world" samples has been a challenge, however, because typically their volumes are greater than those easily accommodated by DMF devices and contain analytes of interest at low concentration. To address this challenge, we have developed a novel "world-to-DMF" interface in which an integrated companion module drives the large-volume sample through a 10 μL droplet region on the DMF device, enabling magnet-mediated recovery of bead-bound analytes onto the device as they pass through the region. To demonstrate its utility, we use this system for extraction of RNA from human whole blood lysates (110-380 μL) and further purification in microscale volumes (5-15 μL) on the DMF device itself. Processing by the system was >2-fold faster and consumed 12-fold less reagents, yet produced RNA yields and quality fully comparable to conventional preparations and supporting qRT-PCR and RNA-Seq analyses. The world-to-DMF system is designed for flexibility in accommodating different sample types and volumes, as well as for facile integration of additional modules to enable execution of more complex protocols for sample processing and analysis. As the first technology of its kind, this innovation represents an important step forward for DMF, further enhancing its utility for a wide range of applications. PMID:24479881

  13. Automated Large Scale Parameter Extraction of Road-Side Trees Sampled by a Laser Mobile Mapping System

    NASA Astrophysics Data System (ADS)

    Lindenbergh, R. C.; Berthold, D.; Sirmacek, B.; Herrero-Huerta, M.; Wang, J.; Ebersbach, D.

    2015-08-01

    In urbanized Western Europe trees are considered an important component of the built-up environment. This also means that there is an increasing demand for tree inventories. Laser mobile mapping systems provide an efficient and accurate way to sample the 3D road surrounding including notable roadside trees. Indeed, at, say, 50 km/h such systems collect point clouds consisting of half a million points per 100m. Method exists that extract tree parameters from relatively small patches of such data, but a remaining challenge is to operationally extract roadside tree parameters at regional level. For this purpose a workflow is presented as follows: The input point clouds are consecutively downsampled, retiled, classified, segmented into individual trees and upsampled to enable automated extraction of tree location, tree height, canopy diameter and trunk diameter at breast height (DBH). The workflow is implemented to work on a laser mobile mapping data set sampling 100 km of road in Sachsen, Germany and is tested on a stretch of road of 7km long. Along this road, the method detected 315 trees that were considered well detected and 56 clusters of tree points were no individual trees could be identified. Using voxels, the data volume could be reduced by about 97 % in a default scenario. Processing the results of this scenario took ~2500 seconds, corresponding to about 10 km/h, which is getting close to but is still below the acquisition rate which is estimated at 50 km/h.

  14. A possible role for the guide RNA U-tail as a specificity determinant in formation of guide RNA-messenger RNA chimeras in mitochondrial extracts of Crithidia fasciculata.

    PubMed

    Arts, G J; Sloof, P; Benne, R

    1995-07-01

    Chimeric g(uide) RNA:pre-mRNA molecules are potential intermediates of the RNA editing process in kinetoplastid mitochondria. We have studied the characteristics of chimeric molecules formed in mitochondrial extracts of the insect trypanosomatid Crithidia fasciculata which had been supplied with synthetic NADH dehydrogenase (ND) subunit-7 gRNA and pre-mRNA variants. The ability of a gRNA to participate in chimera formation in this system depends on the possibility of base pairing with the pre-mRNA via the anchor sequence, but not on the presence of a U-tail or a full-length informational part. Chimeras formed with a specific gRNA:pre-mRNA pair displayed a large variation in length, due to variably sized 3' end truncations of the gRNA moieties and variation in the sites in the pre-mRNA to which the gRNAs were attached. Surprisingly, the presence of a U-tail in the gRNA for a large part determined the specificity of the linkage. In 60% of the cases gRNAs possessing a U-tail of at least one residue were attached to an editing site, whereas 75% of the gRNAs without Us were attached to non-editing sites. Furthermore, the chimera forming activity was greatly stimulated by the addition of ATP but not by AMP-CPP, an ATP-analogue with a non-hydrolyzable alpha-beta phosphate bond. This suggests the involvement in the chimera formation of an RNA ligase. PMID:8577329

  15. Advancing forensic RNA typing: On non-target secretions, a nasal mucosa marker, a differential co-extraction protocol and the sensitivity of DNA and RNA profiling.

    PubMed

    van den Berge, Margreet; Bhoelai, Bryan; Harteveld, Joyce; Matai, Anuska; Sijen, Titia

    2016-01-01

    The forensic identification of human body fluids and tissues by means of messenger RNA (mRNA) profiling is a long studied methodology that is increasingly applied to casework samples. Previously, we have described an mRNA multiplex system that targets blood, saliva, semen, menstrual secretion, vaginal mucosa and skin (Lindenbergh et al. and van den Berge et al.). In this study we consider various topics to improve this mRNA profiling system or its use and adapt the method accordingly. Bodily secretions that may be encountered at a crime scene whilst not targeted by the multiplex-id est nasal mucosa, sweat, tears, faeces and urine-were examined for false positive signals. The results prompted us to identify a nasal mucosa marker that allows the discrimination of nasal mucosa from saliva or vaginal mucosa and nosebleed blood from peripheral blood. An updated version of the multiplex was prepared to which the nasal mucosa marker was added and in which markers for semen, vaginal mucosa and blood were replaced. Lactobacillus markers were regarded unsuitable as replacement for vaginal mucosa mRNA markers because of background signals on penile swabs that appeared devoid of female DNA. Furthermore, we provide approaches to deal with highly unbalanced mixtures. First, a differential extraction protocol was incorporated into a co-extraction protocol to allow DNA and RNA analysis of separated non-sperm and sperm fractions. In a second approach, besides the standard multiplex, a customized multiplex is used which excludes markers for prevailing cell types. This allows the use of lower cDNA inputs for the prevailing cell types and higher inputs for cell types that appear masked. Additionally, we assessed the relation between the percentage of alleles or markers detected in DNA or RNA profiles when decreasing sample amounts are analysed. While blood, saliva, semen and menstrual secretion show the trend that DNA profiling is more sensitive than RNA profiling, the reverse is seen

  16. RNA Extraction from Xenopus Auditory and Vestibular Organs for Molecular Cloning and Expression Profiling with RNA-Seq and Microarrays.

    PubMed

    Trujillo-Provencio, Casilda; Powers, TuShun R; Sultemeier, David R; Ramirez-Gordillo, Daniel; Serrano, Elba E

    2016-01-01

    The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from inner ear tissue. Methods presented here facilitate preparation of high-quality inner ear RNA from larval and post-metamorphic Xenopus specimens that can be used for a variety of purposes. We demonstrate that RNA isolated with these protocols is suitable for microarray analysis and Illumina-Solexa sequencing (RNA-Seq) of inner ear organs, and for cloning of large transcripts, such as those for ion channels. Genetic sequences cloned with these procedures can be used for transient transfection of Xenopus kidney cell lines with fluorescent protein fusion constructs. PMID:27259922

  17. Technical Note: Semi-automated effective width extraction from time-lapse RGB imagery of a remote, braided Greenlandic river

    NASA Astrophysics Data System (ADS)

    Gleason, C. J.; Smith, L. C.; Finnegan, D. C.; LeWinter, A. L.; Pitcher, L. H.; Chu, V. W.

    2015-06-01

    River systems in remote environments are often challenging to monitor and understand where traditional gauging apparatus are difficult to install or where safety concerns prohibit field measurements. In such cases, remote sensing, especially terrestrial time-lapse imaging platforms, offer a means to better understand these fluvial systems. One such environment is found at the proglacial Isortoq River in southwestern Greenland, a river with a constantly shifting floodplain and remote Arctic location that make gauging and in situ measurements all but impossible. In order to derive relevant hydraulic parameters for this river, two true color (RGB) cameras were installed in July 2011, and these cameras collected over 10 000 half hourly time-lapse images of the river by September of 2012. Existing approaches for extracting hydraulic parameters from RGB imagery require manual or supervised classification of images into water and non-water areas, a task that was impractical for the volume of data in this study. As such, automated image filters were developed that removed images with environmental obstacles (e.g., shadows, sun glint, snow) from the processing stream. Further image filtering was accomplished via a novel automated histogram similarity filtering process. This similarity filtering allowed successful (mean accuracy 79.6 %) supervised classification of filtered images from training data collected from just 10 % of those images. Effective width, a hydraulic parameter highly correlated with discharge in braided rivers, was extracted from these classified images, producing a hydrograph proxy for the Isortoq River between 2011 and 2012. This hydrograph proxy shows agreement with historic flooding observed in other parts of Greenland in July 2012 and offers promise that the imaging platform and processing methodology presented here will be useful for future monitoring studies of remote rivers.

  18. [Corrected Title: Solid-Phase Extraction of Polar Compounds from Water] Automated Electrostatics Environmental Chamber

    NASA Technical Reports Server (NTRS)

    Sauer, Richard; Rutz, Jeffrey; Schultz, John

    2005-01-01

    A solid-phase extraction (SPE) process has been developed for removing alcohols, carboxylic acids, aldehydes, ketones, amines, and other polar organic compounds from water. This process can be either a subprocess of a water-reclamation process or a means of extracting organic compounds from water samples for gas-chromatographic analysis. This SPE process is an attractive alternative to an Environmental Protection Administration liquid-liquid extraction process that generates some pollution and does not work in a microgravitational environment. In this SPE process, one forces a water sample through a resin bed by use of positive pressure on the upstream side and/or suction on the downstream side, thereby causing organic compounds from the water to be adsorbed onto the resin. If gas-chromatographic analysis is to be done, the resin is dried by use of a suitable gas, then the adsorbed compounds are extracted from the resin by use of a solvent. Unlike the liquid-liquid process, the SPE process works in both microgravity and Earth gravity. In comparison with the liquid-liquid process, the SPE process is more efficient, extracts a wider range of organic compounds, generates less pollution, and costs less.

  19. Automated extraction of urban trees from mobile LiDAR point clouds

    NASA Astrophysics Data System (ADS)

    Fan, W.; Chenglu, W.; Jonathan, L.

    2016-03-01

    This paper presents an automatic algorithm to localize and extract urban trees from mobile LiDAR point clouds. First, in order to reduce the number of points to be processed, the ground points are filtered out from the raw point clouds, and the un-ground points are segmented into supervoxels. Then, a novel localization method is proposed to locate the urban trees accurately. Next, a segmentation method by localization is proposed to achieve objects. Finally, the features of objects are extracted, and the feature vectors are classified by random forests trained on manually labeled objects. The proposed method has been tested on a point cloud dataset. The results prove that our algorithm efficiently extracts the urban trees.

  20. Application of a novel and automated branched DNA in situ hybridization method for the rapid and sensitive localization of mRNA molecules in plant tissues1

    PubMed Central

    Bowling, Andrew J.; Pence, Heather E.; Church, Jeffrey B.

    2014-01-01

    • Premise of the study: A novel branched DNA detection technology, RNAscope in situ hybridization (ISH), originally developed for use on human clinical and animal tissues, was adapted for use in plant tissue in an attempt to overcome some of the limitations associated with traditional ISH assays. • Methods and Results: Zea mays leaf tissue was formaldehyde fixed and paraffin embedded (FFPE) and then probed with the RNAscope ISH assay for two endogenous genes, phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PEPCK). Results from both manual and automated methods showed tissue- and cell-specific mRNA localization patterns expected from these well-studied genes. • Conclusions: RNAscope ISH is a sensitive method that generates high-quality, easily interpretable results from FFPE plant tissues. Automation of the RNAscope method on the Ventana Discovery Ultra platform allows significant advantages for repeatability, reduction in variability, and flexibility of workflow processes. PMID:25202621

  1. Large Scale Application of Neural Network Based Semantic Role Labeling for Automated Relation Extraction from Biomedical Texts

    PubMed Central

    Barnickel, Thorsten; Weston, Jason; Collobert, Ronan; Mewes, Hans-Werner; Stümpflen, Volker

    2009-01-01

    To reduce the increasing amount of time spent on literature search in the life sciences, several methods for automated knowledge extraction have been developed. Co-occurrence based approaches can deal with large text corpora like MEDLINE in an acceptable time but are not able to extract any specific type of semantic relation. Semantic relation extraction methods based on syntax trees, on the other hand, are computationally expensive and the interpretation of the generated trees is difficult. Several natural language processing (NLP) approaches for the biomedical domain exist focusing specifically on the detection of a limited set of relation types. For systems biology, generic approaches for the detection of a multitude of relation types which in addition are able to process large text corpora are needed but the number of systems meeting both requirements is very limited. We introduce the use of SENNA (“Semantic Extraction using a Neural Network Architecture”), a fast and accurate neural network based Semantic Role Labeling (SRL) program, for the large scale extraction of semantic relations from the biomedical literature. A comparison of processing times of SENNA and other SRL systems or syntactical parsers used in the biomedical domain revealed that SENNA is the fastest Proposition Bank (PropBank) conforming SRL program currently available. 89 million biomedical sentences were tagged with SENNA on a 100 node cluster within three days. The accuracy of the presented relation extraction approach was evaluated on two test sets of annotated sentences resulting in precision/recall values of 0.71/0.43. We show that the accuracy as well as processing speed of the proposed semantic relation extraction approach is sufficient for its large scale application on biomedical text. The proposed approach is highly generalizable regarding the supported relation types and appears to be especially suited for general-purpose, broad-scale text mining systems. The presented approach

  2. Automated Identification of Medically Important Bacteria by 16S rRNA Gene Sequencing Using a Novel Comprehensive Database, 16SpathDB▿

    PubMed Central

    Woo, Patrick C. Y.; Teng, Jade L. L.; Yeung, Juilian M. Y.; Tse, Herman; Lau, Susanna K. P.; Yuen, Kwok-Yung

    2011-01-01

    Despite the increasing use of 16S rRNA gene sequencing, interpretation of 16S rRNA gene sequence results is one of the most difficult problems faced by clinical microbiologists and technicians. To overcome the problems we encountered in the existing databases during 16S rRNA gene sequence interpretation, we built a comprehensive database, 16SpathDB (http://147.8.74.24/16SpathDB) based on the 16S rRNA gene sequences of all medically important bacteria listed in the Manual of Clinical Microbiology and evaluated its use for automated identification of these bacteria. Among 91 nonduplicated bacterial isolates collected in our clinical microbiology laboratory, 71 (78%) were reported by 16SpathDB as a single bacterial species having >98.0% nucleotide identity with the query sequence, 19 (20.9%) were reported as more than one bacterial species having >98.0% nucleotide identity with the query sequence, and 1 (1.1%) was reported as no match. For the 71 bacterial isolates reported as a single bacterial species, all results were identical to their true identities as determined by a polyphasic approach. For the 19 bacterial isolates reported as more than one bacterial species, all results contained their true identities as determined by a polyphasic approach and all of them had their true identities as the “best match in 16SpathDB.” For the isolate (Gordonibacter pamelaeae) reported as no match, the bacterium has never been reported to be associated with human disease and was not included in the Manual of Clinical Microbiology. 16SpathDB is an automated, user-friendly, efficient, accurate, and regularly updated database for 16S rRNA gene sequence interpretation in clinical microbiology laboratories. PMID:21389154

  3. An automated algorithm for extracting road edges from terrestrial mobile LiDAR data

    NASA Astrophysics Data System (ADS)

    Kumar, Pankaj; McElhinney, Conor P.; Lewis, Paul; McCarthy, Timothy

    2013-11-01

    Terrestrial mobile laser scanning systems provide rapid and cost effective 3D point cloud data which can be used for extracting features such as the road edge along a route corridor. This information can assist road authorities in carrying out safety risk assessment studies along road networks. The knowledge of the road edge is also a prerequisite for the automatic estimation of most other road features. In this paper, we present an algorithm which has been developed for extracting left and right road edges from terrestrial mobile LiDAR data. The algorithm is based on a novel combination of two modified versions of the parametric active contour or snake model. The parameters involved in the algorithm are selected empirically and are fixed for all the road sections. We have developed a novel way of initialising the snake model based on the navigation information obtained from the mobile mapping vehicle. We tested our algorithm on different types of road sections representing rural, urban and national primary road sections. The successful extraction of road edges from these multiple road section environments validates our algorithm. These findings and knowledge provide valuable insights as well as a prototype road edge extraction tool-set, for both national road authorities and survey companies.

  4. Using mobile laser scanning data for automated extraction of road markings

    NASA Astrophysics Data System (ADS)

    Guan, Haiyan; Li, Jonathan; Yu, Yongtao; Wang, Cheng; Chapman, Michael; Yang, Bisheng

    2014-01-01

    A mobile laser scanning (MLS) system allows direct collection of accurate 3D point information in unprecedented detail at highway speeds and at less than traditional survey costs, which serves the fast growing demands of transportation-related road surveying including road surface geometry and road environment. As one type of road feature in traffic management systems, road markings on paved roadways have important functions in providing guidance and information to drivers and pedestrians. This paper presents a stepwise procedure to recognize road markings from MLS point clouds. To improve computational efficiency, we first propose a curb-based method for road surface extraction. This method first partitions the raw MLS data into a set of profiles according to vehicle trajectory data, and then extracts small height jumps caused by curbs in the profiles via slope and elevation-difference thresholds. Next, points belonging to the extracted road surface are interpolated into a geo-referenced intensity image using an extended inverse-distance-weighted (IDW) approach. Finally, we dynamically segment the geo-referenced intensity image into road-marking candidates with multiple thresholds that correspond to different ranges determined by point-density appropriate normality. A morphological closing operation with a linear structuring element is finally used to refine the road-marking candidates by removing noise and improving completeness. This road-marking extraction algorithm is comprehensively discussed in the analysis of parameter sensitivity and overall performance. An experimental study performed on a set of road markings with ground-truth shows that the proposed algorithm provides a promising solution to the road-marking extraction from MLS data.

  5. EFFECTS OF STORAGE, RNA EXTRACTION, GENECHIP TYPE, AND DONOR SEX ON GENE EXPRESSION PROFILING OF HUMAN WHOLE BLOOD

    EPA Science Inventory

    Background: Gene expression profiling of whole blood may be useful for monitoring toxicological exposure and for diagnosis and monitoring of various diseases. Several methods are available that can be used to transport, store, and extract RNA from whole blood, but it is not clear...

  6. Extraction of high-quality RNA from germinating barley (Hordeum vulgare L.) seeds containing high levels of starch.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparative evaluation of gene expression levels can lead to improved understanding of the gene networks underlying traits of economic importance. Extraction of high-quality RNA from germinating barley seeds that contain high levels of starch is of vital importance for analysing the expression of ca...

  7. Evaluation of Four Different Systems for Extraction of RNA from Stool Suspensions Using MS-2 Coliphage as an Exogenous Control for RT-PCR Inhibition

    PubMed Central

    Shulman, Lester M.; Hindiyeh, Musa; Muhsen, Khitam; Cohen, Dani; Mendelson, Ella; Sofer, Danit

    2012-01-01

    Knowing when, and to what extent co-extracted inhibitors interfere with molecular RNA diagnostic assays is of utmost importance. The QIAamp Viral RNA Mini Kit (A); MagNA Pure LC2.0 Automatic extractor (B); KingFisher (C); and NucliSENS EasyMag (D) RNA extraction systems were evaluated for extraction efficiency and co-purification of inhibitors from stool suspensions. Real-Time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) of MS-2 coliphage spiked into each system’s lysis buffer served as an external control for both. Cycle thresholds (Cts) of the MS2 were determined for RNA extracted from stool suspensions containing unknown (n = 93) or varying amounts of inhibitors (n = 92). Stool suspensions from the latter group were also used to determine whether MS-2 and enterovirus rRT-PCR inhibitions were correlated. Specifically 23 RNA extracts from stool suspensions were spiked with enterovirus RNA after extraction and 13 of these stool suspension were spiked with intact enterovirus before extraction. MS2 rRT-PCR inhibition varied for RNAs extracted by the different systems. Inhibition was noted in 12 (13.0%), 26 (28.3%), 7 (7.6%), and 7 (7.6%) of the first 93 RNA extracts, and 58 (63.0%), 55 (59.8%), 37 (40.2%) and 30 (32.6%) of the second 92 extracts for A, B, C, and D, respectively. Furthermore, enterovirus rRT-PCR inhibition correlated with MS2 rRT-PCR inhibition for added enterovirus RNA or virus particles. In conclusion, rRT-PCR for MS-2 RNA is a good predictor of inhibition of enterovirus RNA extracted from stool suspensions. EasyMag performed the best, however all four extraction methods were suitable provided that external controls identified problematic samples. PMID:22815706

  8. Automation of ⁹⁹Tc extraction by LOV prior ICP-MS detection: application to environmental samples.

    PubMed

    Rodríguez, Rogelio; Leal, Luz; Miranda, Silvia; Ferrer, Laura; Avivar, Jessica; García, Ariel; Cerdà, Víctor

    2015-02-01

    A new, fast, automated and inexpensive sample pre-treatment method for (99)Tc determination by inductively coupled plasma-mass spectrometry (ICP-MS) detection is presented. The miniaturized approach is based on a lab-on-valve (LOV) system, allowing automatic separation and preconcentration of (99)Tc. Selectivity is provided by the solid phase extraction system used (TEVA resin) which retains selectively pertechnetate ion in diluted nitric acid solution. The proposed system has some advantages such as minimization of sample handling, reduction of reagents volume, improvement of intermediate precision and sample throughput, offering a significant decrease of both time and cost per analysis in comparison to other flow techniques and batch methods. The proposed LOV system has been successfully applied to different samples of environmental interest (water and soil) with satisfactory recoveries, between 94% and 98%. The detection limit (LOD) of the developed method is 0.005 ng. The high durability of the resin and its low amount (32 mg), its good intermediate precision (RSD 3.8%) and repeatability (RSD 2%) and its high extraction frequency (up to 5 h(-1)) makes this method an inexpensive, high precision and fast tool for monitoring (99)Tc in environmental samples. PMID:25435232

  9. Fully automated Liquid Extraction-Based Surface Sampling and Ionization Using a Chip-Based Robotic Nanoelectrospray Platform

    SciTech Connect

    Kertesz, Vilmos; Van Berkel, Gary J

    2010-01-01

    A fully automated liquid extraction-based surface sampling device utilizing an Advion NanoMate chip-based infusion nanoelectrospray ionization system is reported. Analyses were enabled for discrete spot sampling by using the Advanced User Interface of the current commercial control software. This software interface provided the parameter control necessary for the NanoMate robotic pipettor to both form and withdraw a liquid microjunction for sampling from a surface. The system was tested with three types of analytically important sample surface types, viz., spotted sample arrays on a MALDI plate, dried blood spots on paper, and whole-body thin tissue sections from drug dosed mice. The qualitative and quantitative data were consistent with previous studies employing other liquid extraction-based surface sampling techniques. The successful analyses performed here utilized the hardware and software elements already present in the NanoMate system developed to handle and analyze liquid samples. Implementation of an appropriate sample (surface) holder, a solvent reservoir, faster movement of the robotic arm, finer control over solvent flow rate when dispensing and retrieving the solution at the surface, and the ability to select any location on a surface to sample from would improve the analytical performance and utility of the platform.

  10. Sequential automated fusion/extraction chromatography methodology for the dissolution of uranium in environmental samples for mass spectrometric determination.

    PubMed

    Milliard, Alex; Durand-Jézéquel, Myriam; Larivière, Dominic

    2011-01-17

    An improved methodology has been developed, based on dissolution by automated fusion followed by extraction chromatography for the detection and quantification of uranium in environmental matrices by mass spectrometry. A rapid fusion protocol (<8 min) was investigated for the complete dissolution of various samples. It could be preceded, if required, by an effective ashing procedure using the M4 fluxer and a newly designed platinum lid. Complete dissolution of the sample was observed and measured using standard reference materials (SRMs) and experimental data show no evidence of cross-contamination of crucibles when LiBO(2)/LiBr melts were used. The use of a M4 fusion unit also improved repeatability in sample preparation over muffle furnace fusion. Instrumental issues originating from the presence of high salt concentrations in the digestate after lithium metaborate fusion was also mitigated using an extraction chromatography (EXC) protocol aimed at removing lithium and interfering matrix constituants prior to the elution of uranium. The sequential methodology, which can be performed simultaneously on three samples, requires less than 20 min per sample for fusion and separation. It was successfully coupled to inductively coupled plasma mass spectrometry (ICP-MS) achieving detection limits below 100 pg kg(-1) for 5-300 mg of sample. PMID:21167982

  11. Quantitative analysis of ex vivo colorectal epithelium using an automated feature extraction algorithm for microendoscopy image data.

    PubMed

    Prieto, Sandra P; Lai, Keith K; Laryea, Jonathan A; Mizell, Jason S; Muldoon, Timothy J

    2016-04-01

    Qualitative screening for colorectal polyps via fiber bundle microendoscopy imaging has shown promising results, with studies reporting high rates of sensitivity and specificity, as well as low interobserver variability with trained clinicians. A quantitative image quality control and image feature extraction algorithm (QFEA) was designed to lessen the burden of training and provide objective data for improved clinical efficacy of this method. After a quantitative image quality control step, QFEA extracts field-of-view area, crypt area, crypt circularity, and crypt number per image. To develop and validate this QFEA, a training set of microendoscopy images was collected from freshly resected porcine colon epithelium. The algorithm was then further validated on ex vivo image data collected from eight human subjects, selected from clinically normal appearing regions distant from grossly visible tumor in surgically resected colorectal tissue. QFEA has proven flexible in application to both mosaics and individual images, and its automated crypt detection sensitivity ranges from 71 to 94% despite intensity and contrast variation within the field of view. It also demonstrates the ability to detect and quantify differences in grossly normal regions among different subjects, suggesting the potential efficacy of this approach in detecting occult regions of dysplasia. PMID:27335893

  12. Progress in automated extraction and purification of in situ 14C from quartz: Results from the Purdue in situ 14C laboratory

    NASA Astrophysics Data System (ADS)

    Lifton, Nathaniel; Goehring, Brent; Wilson, Jim; Kubley, Thomas; Caffee, Marc

    2015-10-01

    Current extraction methods for in situ 14C from quartz [e.g., Lifton et al., (2001), Pigati et al., (2010), Hippe et al., (2013)] are time-consuming and repetitive, making them an attractive target for automation. We report on the status of in situ 14C extraction and purification systems originally automated at the University of Arizona that have now been reconstructed and upgraded at the Purdue Rare Isotope Measurement Laboratory (PRIME Lab). The Purdue in situ 14C laboratory builds on the flow-through extraction system design of Pigati et al. (2010), automating most of the procedure by retrofitting existing valves with external servo-controlled actuators, regulating the pressure of research purity O2 inside the furnace tube via a PID-based pressure controller in concert with an inlet mass flow controller, and installing an automated liquid N2 distribution system, all driven by LabView® software. A separate system for cryogenic CO2 purification, dilution, and splitting is also fully automated, ensuring a highly repeatable process regardless of the operator. We present results from procedural blanks and an intercomparison material (CRONUS-A), as well as results of experiments to increase the amount of material used in extraction, from the standard 5 g to 10 g or above. Results thus far are quite promising with procedural blanks comparable to previous work and significant improvements in reproducibility for CRONUS-A measurements. The latter analyses also demonstrate the feasibility of quantitative extraction of in situ 14C from sample masses up to 10 g. Our lab is now analyzing unknowns routinely, but lowering overall blank levels is the focus of ongoing research.

  13. Short Communication An efficient method for simultaneous extraction of high-quality RNA and DNA from various plant tissues.

    PubMed

    Oliveira, R R; Viana, A J C; Reátegui, A C E; Vincentz, M G A

    2015-01-01

    Determination of gene expression is an important tool to study biological processes and relies on the quality of the extracted RNA. Changes in gene expression profiles may be directly related to mutations in regulatory DNA sequences or alterations in DNA cytosine methylation, which is an epigenetic mark. Correlation of gene expression with DNA sequence or epigenetic mark polymorphism is often desirable; for this, a robust protocol to isolate high-quality RNA and DNA simultaneously from the same sample is required. Although commercial kits and protocols are available, they are mainly optimized for animal tissues and, in general, restricted to RNA or DNA extraction, not both. In the present study, we describe an efficient and accessible method to extract both RNA and DNA simultaneously from the same sample of various plant tissues, using small amounts of starting material. The protocol was efficient in the extraction of high-quality nucleic acids from several Arabidopsis thaliana tissues (e.g., leaf, inflorescence stem, flower, fruit, cotyledon, seedlings, root, and embryo) and from other tissues of non-model plants, such as Avicennia schaueriana (Acanthaceae), Theobroma cacao (Malvaceae), Paspalum notatum (Poaceae), and Sorghum bicolor (Poaceae). The obtained nucleic acids were used as templates for downstream analyses, such as mRNA sequencing, quantitative real time-polymerase chain reaction, bisulfite treatment, and others; the results were comparable to those obtained with commercial kits. We believe that this protocol could be applied to a broad range of plant species, help avoid technical and sampling biases, and facilitate several RNA- and DNA-dependent analyses. PMID:26782533

  14. Detecting and extracting clusters in atom probe data: a simple, automated method using Voronoi cells.

    PubMed

    Felfer, P; Ceguerra, A V; Ringer, S P; Cairney, J M

    2015-03-01

    The analysis of the formation of clusters in solid solutions is one of the most common uses of atom probe tomography. Here, we present a method where we use the Voronoi tessellation of the solute atoms and its geometric dual, the Delaunay triangulation to test for spatial/chemical randomness of the solid solution as well as extracting the clusters themselves. We show how the parameters necessary for cluster extraction can be determined automatically, i.e. without user interaction, making it an ideal tool for the screening of datasets and the pre-filtering of structures for other spatial analysis techniques. Since the Voronoi volumes are closely related to atomic concentrations, the parameters resulting from this analysis can also be used for other concentration based methods such as iso-surfaces. PMID:25497494

  15. Quantification of lung tumor rotation with automated landmark extraction using orthogonal cine MRI images

    NASA Astrophysics Data System (ADS)

    Paganelli, Chiara; Lee, Danny; Greer, Peter B.; Baroni, Guido; Riboldi, Marco; Keall, Paul

    2015-09-01

    The quantification of tumor motion in sites affected by respiratory motion is of primary importance to improve treatment accuracy. To account for motion, different studies analyzed the translational component only, without focusing on the rotational component, which was quantified in a few studies on the prostate with implanted markers. The aim of our study was to propose a tool able to quantify lung tumor rotation without the use of internal markers, thus providing accurate motion detection close to critical structures such as the heart or liver. Specifically, we propose the use of an automatic feature extraction method in combination with the acquisition of fast orthogonal cine MRI images of nine lung patients. As a preliminary test, we evaluated the performance of the feature extraction method by applying it on regions of interest around (i) the diaphragm and (ii) the tumor and comparing the estimated motion with that obtained by (i) the extraction of the diaphragm profile and (ii) the segmentation of the tumor, respectively. The results confirmed the capability of the proposed method in quantifying tumor motion. Then, a point-based rigid registration was applied to the extracted tumor features between all frames to account for rotation. The median lung rotation values were  -0.6   ±   2.3° and  -1.5   ±   2.7° in the sagittal and coronal planes respectively, confirming the need to account for tumor rotation along with translation to improve radiotherapy treatment.

  16. Automated Extraction of Buildings and Roads in a Graph Partitioning Framework

    NASA Astrophysics Data System (ADS)

    Ok, A. O.

    2013-10-01

    This paper presents an original unsupervised framework to identify regions belonging to buildings and roads from monocular very high resolution (VHR) satellite images. The proposed framework consists of three main stages. In the first stage, we extract information only related to building regions using shadow evidence and probabilistic fuzzy landscapes. Firstly, the shadow areas cast by building objects are detected and the directional spatial relationship between buildings and their shadows is modelled with the knowledge of illumination direction. Thereafter, each shadow region is handled separately and initial building regions are identified by iterative graph-cuts designed in a two-label partitioning. The second stage of the framework automatically classifies the image into four classes: building, shadow, vegetation, and others. In this step, the previously labelled building regions as well as the shadow and vegetation areas are involved in a four-label graph optimization performed in the entire image domain to achieve the unsupervised classification result. The final stage aims to extend this classification to five classes in which the class road is involved. For that purpose, we extract the regions that might belong to road segments and utilize that information in a final graph optimization. This final stage eventually characterizes the regions belonging to buildings and roads. Experiments performed on seven test images selected from GeoEye-1 VHR datasets show that the presented approach has ability to extract the regions belonging to buildings and roads in a single graph theory framework.

  17. Automated extraction of clinical traits of multiple sclerosis in electronic medical records

    PubMed Central

    Davis, Mary F; Sriram, Subramaniam; Bush, William S; Denny, Joshua C; Haines, Jonathan L

    2013-01-01

    Objectives The clinical course of multiple sclerosis (MS) is highly variable, and research data collection is costly and time consuming. We evaluated natural language processing techniques applied to electronic medical records (EMR) to identify MS patients and the key clinical traits of their disease course. Materials and methods We used four algorithms based on ICD-9 codes, text keywords, and medications to identify individuals with MS from a de-identified, research version of the EMR at Vanderbilt University. Using a training dataset of the records of 899 individuals, algorithms were constructed to identify and extract detailed information regarding the clinical course of MS from the text of the medical records, including clinical subtype, presence of oligoclonal bands, year of diagnosis, year and origin of first symptom, Expanded Disability Status Scale (EDSS) scores, timed 25-foot walk scores, and MS medications. Algorithms were evaluated on a test set validated by two independent reviewers. Results We identified 5789 individuals with MS. For all clinical traits extracted, precision was at least 87% and specificity was greater than 80%. Recall values for clinical subtype, EDSS scores, and timed 25-foot walk scores were greater than 80%. Discussion and conclusion This collection of clinical data represents one of the largest databases of detailed, clinical traits available for research on MS. This work demonstrates that detailed clinical information is recorded in the EMR and can be extracted for research purposes with high reliability. PMID:24148554

  18. Comprehensive automation of the solid phase extraction gas chromatographic mass spectrometric analysis (SPE-GC/MS) of opioids, cocaine, and metabolites from serum and other matrices.

    PubMed

    Lerch, Oliver; Temme, Oliver; Daldrup, Thomas

    2014-07-01

    The analysis of opioids, cocaine, and metabolites from blood serum is a routine task in forensic laboratories. Commonly, the employed methods include many manual or partly automated steps like protein precipitation, dilution, solid phase extraction, evaporation, and derivatization preceding a gas chromatography (GC)/mass spectrometry (MS) or liquid chromatography (LC)/MS analysis. In this study, a comprehensively automated method was developed from a validated, partly automated routine method. This was possible by replicating method parameters on the automated system. Only marginal optimization of parameters was necessary. The automation relying on an x-y-z robot after manual protein precipitation includes the solid phase extraction, evaporation of the eluate, derivatization (silylation with N-methyl-N-trimethylsilyltrifluoroacetamide, MSTFA), and injection into a GC/MS. A quantitative analysis of almost 170 authentic serum samples and more than 50 authentic samples of other matrices like urine, different tissues, and heart blood on cocaine, benzoylecgonine, methadone, morphine, codeine, 6-monoacetylmorphine, dihydrocodeine, and 7-aminoflunitrazepam was conducted with both methods proving that the analytical results are equivalent even near the limits of quantification (low ng/ml range). To our best knowledge, this application is the first one reported in the literature employing this sample preparation system. PMID:24788888

  19. Evaluation of an Automated Information Extraction Tool for Imaging Data Elements to Populate a Breast Cancer Screening Registry.

    PubMed

    Lacson, Ronilda; Harris, Kimberly; Brawarsky, Phyllis; Tosteson, Tor D; Onega, Tracy; Tosteson, Anna N A; Kaye, Abby; Gonzalez, Irina; Birdwell, Robyn; Haas, Jennifer S

    2015-10-01

    Breast cancer screening is central to early breast cancer detection. Identifying and monitoring process measures for screening is a focus of the National Cancer Institute's Population-based Research Optimizing Screening through Personalized Regimens (PROSPR) initiative, which requires participating centers to report structured data across the cancer screening continuum. We evaluate the accuracy of automated information extraction of imaging findings from radiology reports, which are available as unstructured text. We present prevalence estimates of imaging findings for breast imaging received by women who obtained care in a primary care network participating in PROSPR (n = 139,953 radiology reports) and compared automatically extracted data elements to a "gold standard" based on manual review for a validation sample of 941 randomly selected radiology reports, including mammograms, digital breast tomosynthesis, ultrasound, and magnetic resonance imaging (MRI). The prevalence of imaging findings vary by data element and modality (e.g., suspicious calcification noted in 2.6% of screening mammograms, 12.1% of diagnostic mammograms, and 9.4% of tomosynthesis exams). In the validation sample, the accuracy of identifying imaging findings, including suspicious calcifications, masses, and architectural distortion (on mammogram and tomosynthesis); masses, cysts, non-mass enhancement, and enhancing foci (on MRI); and masses and cysts (on ultrasound), range from 0.8 to1.0 for recall, precision, and F-measure. Information extraction tools can be used for accurate documentation of imaging findings as structured data elements from text reports for a variety of breast imaging modalities. These data can be used to populate screening registries to help elucidate more effective breast cancer screening processes. PMID:25561069

  20. An energy minimization approach to automated extraction of regular building footprints from airborne LiDAR data

    NASA Astrophysics Data System (ADS)

    He, Y.; Zhang, C.; Fraser, C. S.

    2014-08-01

    This paper presents an automated approach to the extraction of building footprints from airborne LiDAR data based on energy minimization. Automated 3D building reconstruction in complex urban scenes has been a long-standing challenge in photogrammetry and computer vision. Building footprints constitute a fundamental component of a 3D building model and they are useful for a variety of applications. Airborne LiDAR provides large-scale elevation representation of urban scene and as such is an important data source for object reconstruction in spatial information systems. However, LiDAR points on building edges often exhibit a jagged pattern, partially due to either occlusion from neighbouring objects, such as overhanging trees, or to the nature of the data itself, including unavoidable noise and irregular point distributions. The explicit 3D reconstruction may thus result in irregular or incomplete building polygons. In the presented work, a vertex-driven Douglas-Peucker method is developed to generate polygonal hypotheses from points forming initial building outlines. The energy function is adopted to examine and evaluate each hypothesis and the optimal polygon is determined through energy minimization. The energy minimization also plays a key role in bridging gaps, where the building outlines are ambiguous due to insufficient LiDAR points. In formulating the energy function, hard constraints such as parallelism and perpendicularity of building edges are imposed, and local and global adjustments are applied. The developed approach has been extensively tested and evaluated on datasets with varying point cloud density over different terrain types. Results are presented and analysed. The successful reconstruction of building footprints, of varying structural complexity, along with a quantitative assessment employing accurate reference data, demonstrate the practical potential of the proposed approach.

  1. Automated identification and geometrical features extraction of individual trees from Mobile Laser Scanning data in Budapest

    NASA Astrophysics Data System (ADS)

    Koma, Zsófia; Székely, Balázs; Folly-Ritvay, Zoltán; Skobrák, Ferenc; Koenig, Kristina; Höfle, Bernhard

    2016-04-01

    Mobile Laser Scanning (MLS) is an evolving operational measurement technique for urban environment providing large amounts of high resolution information about trees, street features, pole-like objects on the street sides or near to motorways. In this study we investigate a robust segmentation method to extract the individual trees automatically in order to build an object-based tree database system. We focused on the large urban parks in Budapest (Margitsziget and Városliget; KARESZ project) which contained large diversity of different kind of tree species. The MLS data contained high density point cloud data with 1-8 cm mean absolute accuracy 80-100 meter distance from streets. The robust segmentation method contained following steps: The ground points are determined first. As a second step cylinders are fitted in vertical slice 1-1.5 meter relative height above ground, which is used to determine the potential location of each single trees trunk and cylinder-like object. Finally, residual values are calculated as deviation of each point from a vertically expanded fitted cylinder; these residual values are used to separate cylinder-like object from individual trees. After successful parameterization, the model parameters and the corresponding residual values of the fitted object are extracted and imported into the tree database. Additionally, geometric features are calculated for each segmented individual tree like crown base, crown width, crown length, diameter of trunk, volume of the individual trees. In case of incompletely scanned trees, the extraction of geometric features is based on fitted circles. The result of the study is a tree database containing detailed information about urban trees, which can be a valuable dataset for ecologist, city planners, planting and mapping purposes. Furthermore, the established database will be the initial point for classification trees into single species. MLS data used in this project had been measured in the framework of

  2. Linearly Supporting Feature Extraction for Automated Estimation of Stellar Atmospheric Parameters

    NASA Astrophysics Data System (ADS)

    Li, Xiangru; Lu, Yu; Comte, Georges; Luo, Ali; Zhao, Yongheng; Wang, Yongjun

    2015-05-01

    We describe a scheme to extract linearly supporting (LSU) features from stellar spectra to automatically estimate the atmospheric parameters {{T}{\\tt{eff} }}, log g, and [Fe/H]. “Linearly supporting” means that the atmospheric parameters can be accurately estimated from the extracted features through a linear model. The successive steps of the process are as follow: first, decompose the spectrum using a wavelet packet (WP) and represent it by the derived decomposition coefficients; second, detect representative spectral features from the decomposition coefficients using the proposed method Least Absolute Shrinkage and Selection Operator (LARS)bs; third, estimate the atmospheric parameters {{T}{\\tt{eff} }}, log g, and [Fe/H] from the detected features using a linear regression method. One prominent characteristic of this scheme is its ability to evaluate quantitatively the contribution of each detected feature to the atmospheric parameter estimate and also to trace back the physical significance of that feature. This work also shows that the usefulness of a component depends on both the wavelength and frequency. The proposed scheme has been evaluated on both real spectra from the Sloan Digital Sky Survey (SDSS)/SEGUE and synthetic spectra calculated from Kurucz's NEWODF models. On real spectra, we extracted 23 features to estimate {{T}{\\tt{eff} }}, 62 features for log g, and 68 features for [Fe/H]. Test consistencies between our estimates and those provided by the Spectroscopic Parameter Pipeline of SDSS show that the mean absolute errors (MAEs) are 0.0062 dex for log {{T}{\\tt{eff} }} (83 K for {{T}{\\tt{eff} }}), 0.2345 dex for log g, and 0.1564 dex for [Fe/H]. For the synthetic spectra, the MAE test accuracies are 0.0022 dex for log {{T}{\\tt{eff} }} (32 K for {{T}{\\tt{eff} }}), 0.0337 dex for log g, and 0.0268 dex for [Fe/H].

  3. Pedestrian detection in thermal images: An automated scale based region extraction with curvelet space validation

    NASA Astrophysics Data System (ADS)

    Lakshmi, A.; Faheema, A. G. J.; Deodhare, Dipti

    2016-05-01

    Pedestrian detection is a key problem in night vision processing with a dozen of applications that will positively impact the performance of autonomous systems. Despite significant progress, our study shows that performance of state-of-the-art thermal image pedestrian detectors still has much room for improvement. The purpose of this paper is to overcome the challenge faced by the thermal image pedestrian detectors, which employ intensity based Region Of Interest (ROI) extraction followed by feature based validation. The most striking disadvantage faced by the first module, ROI extraction, is the failed detection of cloth insulted parts. To overcome this setback, this paper employs an algorithm and a principle of region growing pursuit tuned to the scale of the pedestrian. The statistics subtended by the pedestrian drastically vary with the scale and deviation from normality approach facilitates scale detection. Further, the paper offers an adaptive mathematical threshold to resolve the problem of subtracting the background while extracting cloth insulated parts as well. The inherent false positives of the ROI extraction module are limited by the choice of good features in pedestrian validation step. One such feature is curvelet feature, which has found its use extensively in optical images, but has as yet no reported results in thermal images. This has been used to arrive at a pedestrian detector with a reduced false positive rate. This work is the first venture made to scrutinize the utility of curvelet for characterizing pedestrians in thermal images. Attempt has also been made to improve the speed of curvelet transform computation. The classification task is realized through the use of the well known methodology of Support Vector Machines (SVMs). The proposed method is substantiated with qualified evaluation methodologies that permits us to carry out probing and informative comparisons across state-of-the-art features, including deep learning methods, with six

  4. Automated DICOM metadata and volumetric anatomical information extraction for radiation dosimetry

    NASA Astrophysics Data System (ADS)

    Papamichail, D.; Ploussi, A.; Kordolaimi, S.; Karavasilis, E.; Papadimitroulas, P.; Syrgiamiotis, V.; Efstathopoulos, E.

    2015-09-01

    Patient-specific dosimetry calculations based on simulation techniques have as a prerequisite the modeling of the modality system and the creation of voxelized phantoms. This procedure requires the knowledge of scanning parameters and patients’ information included in a DICOM file as well as image segmentation. However, the extraction of this information is complicated and time-consuming. The objective of this study was to develop a simple graphical user interface (GUI) to (i) automatically extract metadata from every slice image of a DICOM file in a single query and (ii) interactively specify the regions of interest (ROI) without explicit access to the radiology information system. The user-friendly application developed in Matlab environment. The user can select a series of DICOM files and manage their text and graphical data. The metadata are automatically formatted and presented to the user as a Microsoft Excel file. The volumetric maps are formed by interactively specifying the ROIs and by assigning a specific value in every ROI. The result is stored in DICOM format, for data and trend analysis. The developed GUI is easy, fast and and constitutes a very useful tool for individualized dosimetry. One of the future goals is to incorporate a remote access to a PACS server functionality.

  5. Automated feature extraction and spatial organization of seafloor pockmarks, Belfast Bay, Maine, USA

    USGS Publications Warehouse

    Andrews, B.D.; Brothers, L.L.; Barnhardt, W.A.

    2010-01-01

    Seafloor pockmarks occur worldwide and may represent millions of m3 of continental shelf erosion, but few numerical analyses of their morphology and spatial distribution of pockmarks exist. We introduce a quantitative definition of pockmark morphology and, based on this definition, propose a three-step geomorphometric method to identify and extract pockmarks from high-resolution swath bathymetry. We apply this GIS-implemented approach to 25km2 of bathymetry collected in the Belfast Bay, Maine USA pockmark field. Our model extracted 1767 pockmarks and found a linear pockmark depth-to-diameter ratio for pockmarks field-wide. Mean pockmark depth is 7.6m and mean diameter is 84.8m. Pockmark distribution is non-random, and nearly half of the field's pockmarks occur in chains. The most prominent chains are oriented semi-normal to the steepest gradient in Holocene sediment thickness. A descriptive model yields field-wide spatial statistics indicating that pockmarks are distributed in non-random clusters. Results enable quantitative comparison of pockmarks in fields worldwide as well as similar concave features, such as impact craters, dolines, or salt pools. ?? 2010.

  6. Stability and extractability of double-stranded RNA of pangola stunt and sugarcane Fiji disease viruses in dried plant tissues.

    PubMed

    Karan, M; Hicks, S; Harding, R M; Teakle, D S

    1991-06-01

    When leaves infected with pangola stunt virus (PaSV) were dried at 23, 37, 50, 70 or 105 degrees C, the dsRNA was stable and could be extracted after aerobic storage at room temperature for 1 month, although at 105 degrees C the amount obtained was reduced. The dsRNA was also recovered after leaves were freeze dried and stored in vacuo at room temperature for 6 months, or were dried and stored aerobically at room temperature for 10.5 months. dsRNA of sugarcane Fiji disease virus (FDV) was also stable when infected leaves were dried at 23, 37, 50 or 105 degrees C and stored aerobically for 3 months or for at least 6 months when infected leaves were dried at 70 degrees C. The unexpected high stability and extractability of both PaSV and FDV dsRNA when dried in leaves at low or high temperatures and stored at room temperature indicate that these, and probably other plant-infecting reoviruses, can be transported readily in desiccated host tissue between different countries for later extraction and comparison of their dsRNAs. PMID:1939508

  7. A novel method for RNA extraction from Andosols using casein and its application to amoA gene expression study in soil.

    PubMed

    Wang, Yong; Nagaoka, Kazunari; Hayatsu, Masahito; Sakai, Yoriko; Tago, Kanako; Asakawa, Susumu; Fujii, Takeshi

    2012-11-01

    The lack of a universal method to extract RNA from soil hinders the progress of studies related to nitrification in soil, which is an important step in the nitrogen cycle. It is particularly difficult to extract RNA from certain types of soils such as Andosols (volcanic ash soils), which is the dominant agricultural soil in Japan, because of RNA adsorption by soil. To obtain RNA from these challenging soils to study the bacteria involved in nitrification, we developed a soil RNA extraction method for gene expression analysis. Autoclaved casein was added to an RNA extraction buffer to recover RNA from soil, and high-quality RNA was successfully extracted from eight types of agricultural soils that were significantly different in their physicochemical characteristics. To detect bacterial ammonia monooxygenase subunit A gene (amoA) transcripts, bacterial genomic DNA and messenger RNA were co-extracted from two different types of Andosols during incubation with ammonium sulfate. Polymerase chain reaction-denaturing gradient gel electrophoresis and reverse transcription polymerase chain reaction-denaturing gradient gel electrophoresis analyses of amoA in soil microcosms revealed that only few amoA, which had the highest similarities to those in Nitrosospira multiformis, were expressed in these soils after treatment with ammonium sulfate, although multiple amoA genes were present in the soil microcosms examined. PMID:22993110

  8. Extractions of High Quality RNA from the Seeds of Jerusalem Artichoke and Other Plant Species with High Levels of Starch and Lipid

    PubMed Central

    Mornkham, Tanupat; Puangsomlee Wangsomnuk, Preeya; Fu, Yong-Bi; Wangsomnuk, Pinich; Jogloy, Sanun; Patanothai, Aran

    2013-01-01

    Jerusalem artichoke (Helianthus tuberosus L.) is an important tuber crop. However, Jerusalem artichoke seeds contain high levels of starch and lipid, making the extraction of high-quality RNA extremely difficult and the gene expression analysis challenging. This study was aimed to improve existing methods for extracting total RNA from Jerusalem artichoke dry seeds and to assess the applicability of the improved method in other plant species. Five RNA extraction methods were evaluated on Jerusalem artichoke seeds and two were modified. One modified method with the significant improvement was applied to assay seeds of diverse Jerusalem artichoke accessions, sunflower, rice, maize, peanut and marigold. The effectiveness of the improved method to extract total RNA from seeds was assessed using qPCR analysis of four selected genes. The improved method of Ma and Yang (2011) yielded a maximum RNA solubility and removed most interfering substances. The improved protocol generated 29 to 41 µg RNA/30 mg fresh weight. An A260/A280 ratio of 1.79 to 2.22 showed their RNA purity. Extracted RNA was effective for downstream applications such as first-stranded cDNA synthesis, cDNA cloning and qPCR. The improved method was also effective to extract total RNA from seeds of sunflower, rice, maize and peanut that are rich in polyphenols, lipids and polysaccharides. PMID:27137377

  9. Extractions of High Quality RNA from the Seeds of Jerusalem Artichoke and Other Plant Species with High Levels of Starch and Lipid.

    PubMed

    Mornkham, Tanupat; Wangsomnuk, Preeya Puangsomlee; Fu, Yong-Bi; Wangsomnuk, Pinich; Jogloy, Sanun; Patanothai, Aran

    2013-01-01

    Jerusalem artichoke (Helianthus tuberosus L.) is an important tuber crop. However, Jerusalem artichoke seeds contain high levels of starch and lipid, making the extraction of high-quality RNA extremely difficult and the gene expression analysis challenging. This study was aimed to improve existing methods for extracting total RNA from Jerusalem artichoke dry seeds and to assess the applicability of the improved method in other plant species. Five RNA extraction methods were evaluated on Jerusalem artichoke seeds and two were modified. One modified method with the significant improvement was applied to assay seeds of diverse Jerusalem artichoke accessions, sunflower, rice, maize, peanut and marigold. The effectiveness of the improved method to extract total RNA from seeds was assessed using qPCR analysis of four selected genes. The improved method of Ma and Yang (2011) yielded a maximum RNA solubility and removed most interfering substances. The improved protocol generated 29 to 41 µg RNA/30 mg fresh weight. An A260/A280 ratio of 1.79 to 2.22 showed their RNA purity. Extracted RNA was effective for downstream applications such as first-stranded cDNA synthesis, cDNA cloning and qPCR. The improved method was also effective to extract total RNA from seeds of sunflower, rice, maize and peanut that are rich in polyphenols, lipids and polysaccharides. PMID:27137377

  10. A novel approach for automated shoreline extraction from remote sensing images using low level programming

    NASA Astrophysics Data System (ADS)

    Rigos, Anastasios; Vaiopoulos, Aristidis; Skianis, George; Tsekouras, George; Drakopoulos, Panos

    2015-04-01

    Tracking coastline changes is a crucial task in the context of coastal management and synoptic remotely sensed data has become an essential tool for this purpose. In this work, and within the framework of BeachTour project, we introduce a new method for shoreline extraction from high resolution satellite images. It was applied on two images taken by the WorldView-2 satellite (7 channels, 2m resolution) during July 2011 and August 2014. The location is the well-known tourist destination of Laganas beach spanning 5 km along the southern part of Zakynthos Island, Greece. The atmospheric correction was performed with the ENVI FLAASH procedure and the final images were validated against hyperspectral field measurements. Using three channels (CH2=blue, CH3=green and CH7=near infrared) the Modified Redness Index image was calculated according to: MRI=(CH7)2/[CH2x(CH3)3]. MRI has the property that its value keeps increasing as the water becomes shallower. This is followed by an abrupt reduction trend at the location of the wet sand up to the point where the dry shore face begins. After that it remains low-valued throughout the beach zone. Images based on this index were used for the shoreline extraction process that included the following steps: a) On the MRI based image, only an area near the shoreline was kept (this process is known as image masking). b) On the masked image the Canny edge detector operator was applied. c) Of all edges discovered on step (b) only the biggest was kept. d) If the line revealed on step (c) was unacceptable, i.e. not defining the shoreline or defining only part of it, then either more than one areas on step (c) were kept or on the MRI image the pixel values were bound in a particular interval [Blow, Bhigh] and only the ones belonging in this interval were kept. Then, steps (a)-(d) were repeated. Using this method, which is still under development, we were able to extract the shoreline position and reveal its changes during the 3-year period

  11. Automated structure extraction and XML conversion of life science database flat files.

    PubMed

    Philippi, Stephan; Köhler, Jacob

    2006-10-01

    In the light of the increasing number of biological databases, their integration is a fundamental prerequisite for answering complex biological questions. Database integration, therefore, is an important area of research in bioinformatics. Since most of the publicly available life science databases are still exclusively exchanged by means of proprietary flat files, database integration requires parsers for very different flat file formats. Unfortunately, the development and maintenance of database specific flat file parsers is a nontrivial and time-consuming task, which takes considerable effort in large-scale integration scenarios. This paper introduces heuristically based concepts for automatic structure extraction from life science database flat files. On the basis of these concepts the FlatEx prototype is developed for the automatic conversion of flat files into XML representations. PMID:17044405

  12. Robust semi-automated path extraction for visualising stenosis of the coronary arteries.

    PubMed

    Mueller, Daniel; Maeder, Anthony

    2008-09-01

    Computed tomography angiography (CTA) is useful for diagnosing and planning treatment of heart disease. However, contrast agent in surrounding structures (such as the aorta and left ventricle) makes 3D visualisation of the coronary arteries difficult. This paper presents a composite method employing segmentation and volume rendering to overcome this issue. A key contribution is a novel Fast Marching minimal path cost function for vessel centreline extraction. The resultant centreline is used to compute a measure of vessel lumen, which indicates the degree of stenosis (narrowing of a vessel). Two volume visualisation techniques are presented which utilise the segmented arteries and lumen measure. The system is evaluated and demonstrated using synthetic and clinically obtained datasets. PMID:18603408

  13. Automated extraction of absorption features from Airborne Visible/Infrared Imaging Spectrometer (AVIRIS) and Geophysical and Environmental Research Imaging Spectrometer (GERIS) data

    NASA Technical Reports Server (NTRS)

    Kruse, Fred A.; Calvin, Wendy M.; Seznec, Olivier

    1988-01-01

    Automated techniques were developed for the extraction and characterization of absorption features from reflectance spectra. The absorption feature extraction algorithms were successfully tested on laboratory, field, and aircraft imaging spectrometer data. A suite of laboratory spectra of the most common minerals was analyzed and absorption band characteristics tabulated. A prototype expert system was designed, implemented, and successfully tested to allow identification of minerals based on the extracted absorption band characteristics. AVIRIS spectra for a site in the northern Grapevine Mountains, Nevada, have been characterized and the minerals sericite (fine grained muscovite) and dolomite were identified. The minerals kaolinite, alunite, and buddingtonite were identified and mapped for a site at Cuprite, Nevada, using the feature extraction algorithms on the new Geophysical and Environmental Research 64 channel imaging spectrometer (GERIS) data. The feature extraction routines (written in FORTRAN and C) were interfaced to the expert system (written in PROLOG) to allow both efficient processing of numerical data and logical spectrum analysis.

  14. Accurate initiation of mRNA synthesis in extracts from Schizosaccharomyces pombe, Kluyveromyces lactis and Candida glabrata.

    PubMed

    Woontner, M; Jaehning, J A

    1993-12-01

    We demonstrate the successful adaptation to other yeast species of a protocol previously described for production of transcriptionally active whole cell extracts from Saccharomyces cerevisiae (Woontner and Jaehning, 1990, J. Biol. Chem. 265, 8979-8982). Extracts prepared from Schizosaccharomyces pombe, Kluyveromyces lactis and Candida glabrata were all capable of initiating transcription from a template containing the S. cerevisiae CYC1 TATA box fused to a G-less cassette. Transcription in all of the extracts was sensitive to inhibition by alpha-amanitin, indicating that it was catalysed by RNA polymerase II, and was dramatically stimulated by the chimeric activator GAL4/VP16. The different extracts used different subsets of a group of three initiation sites. PMID:8154183

  15. Screening for plant viruses by next generation sequencing using a modified double strand RNA extraction protocol with an internal amplification control.

    PubMed

    Kesanakurti, Prasad; Belton, Mark; Saeed, Hanaa; Rast, Heidi; Boyes, Ian; Rott, Michael

    2016-10-01

    The majority of plant viruses contain RNA genomes. Detection of viral RNA genomes in infected plant material by next generation sequencing (NGS) is possible through the extraction and sequencing of total RNA, total RNA devoid of ribosomal RNA, small RNA interference (RNAi) molecules, or double stranded RNA (dsRNA). Plants do not typically produce high molecular weight dsRNA, therefore the presence of dsRNA makes it an attractive target for plant virus diagnostics. The sensitivity of NGS as a diagnostic method demands an effective dsRNA protocol that is both representative of the sample and minimizes sample cross contamination. We have developed a modified dsRNA extraction protocol that is more efficient compared to traditional protocols, requiring reduced amounts of starting material, that is less prone to sample cross contamination. This was accomplished by using bead based homogenization of plant material in closed, disposable 50ml tubes. To assess the quality of extraction, we also developed an internal control by designing a real-time (quantitative) PCR (qPCR) assay that targets endornaviruses present in Phaseolus vulgaris cultivar Black Turtle Soup (BTS). PMID:27387642

  16. Quantification of rosuvastatin in human plasma by automated solid-phase extraction using tandem mass spectrometric detection.

    PubMed

    Hull, C K; Penman, A D; Smith, C K; Martin, P D

    2002-06-01

    An assay employing automated solid-phase extraction (SPE) followed by high-performance liquid chromatography with positive ion TurboIonspray tandem mass spectrometry (LC-MS-MS) was developed and validated for the quantification of rosuvastatin (Crestor) in human plasma. Rosuvastatin is a hydroxy-methyl glutaryl coenzyme A reductase inhibitor currently under development by AstraZeneca. The standard curve range in human plasma was 0.1-30 ng/ml with a lower limit of quantification (LLOQ) verified at 0.1 ng/ml. Inaccuracy was less than 8% and imprecision less than +/-15% at all concentration levels. There was no interference from endogenous substances. The analyte was stable in human plasma following three freeze/thaw cycles and for up to 6 months following storage at both -20 and -70 degrees C. The assay was successfully applied to the analysis of rosuvastatin in human plasma samples derived from clinical trials, allowing the pharmacokinetics of the compound to be determined. PMID:12007766

  17. Automated and portable solid phase extraction platform for immuno-detection of 17β-estradiol in water.

    PubMed

    Heub, Sarah; Tscharner, Noe; Monnier, Véronique; Kehl, Florian; Dittrich, Petra S; Follonier, Stéphane; Barbe, Laurent

    2015-02-13

    A fully automated and portable system for solid phase extraction (SPE) has been developed for the analysis of the natural hormone 17β-estradiol (E2) in environmental water by enzyme linked immuno-sorbent assay (ELISA). The system has been validated with de-ionized and artificial sea water as model samples and allowed for pre-concentration of E2 at levels of 1, 10 and 100 ng/L with only 100 ml of sample. Recoveries ranged from 24±3% to 107±6% depending on the concentration and sample matrix. The method successfully allowed us to determine the concentration of two seawater samples. A concentration of 15.1±0.3 ng/L of E2 was measured in a sample obtained from a food production process, and 8.8±0.7 ng/L in a sample from the Adriatic Sea. The system would be suitable for continuous monitoring of water quality as it is user friendly, and as the method is reproducible and totally compatible with the analysis of water sample by simple immunoassays and other detection methods such as biosensors. PMID:25604269

  18. Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

    NASA Astrophysics Data System (ADS)

    Martin, Nicholas J.; Bunch, Josephine; Cooper, Helen J.

    2013-08-01

    Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

  19. Automated Feature Extraction in Brain Tumor by Magnetic Resonance Imaging Using Gaussian Mixture Models

    PubMed Central

    Chaddad, Ahmad

    2015-01-01

    This paper presents a novel method for Glioblastoma (GBM) feature extraction based on Gaussian mixture model (GMM) features using MRI. We addressed the task of the new features to identify GBM using T1 and T2 weighted images (T1-WI, T2-WI) and Fluid-Attenuated Inversion Recovery (FLAIR) MR images. A pathologic area was detected using multithresholding segmentation with morphological operations of MR images. Multiclassifier techniques were considered to evaluate the performance of the feature based scheme in terms of its capability to discriminate GBM and normal tissue. GMM features demonstrated the best performance by the comparative study using principal component analysis (PCA) and wavelet based features. For the T1-WI, the accuracy performance was 97.05% (AUC = 92.73%) with 0.00% missed detection and 2.95% false alarm. In the T2-WI, the same accuracy (97.05%, AUC = 91.70%) value was achieved with 2.95% missed detection and 0.00% false alarm. In FLAIR mode the accuracy decreased to 94.11% (AUC = 95.85%) with 0.00% missed detection and 5.89% false alarm. These experimental results are promising to enhance the characteristics of heterogeneity and hence early treatment of GBM. PMID:26136774

  20. Towards the automated geomorphometric extraction of talus slopes in Martian landscapes

    NASA Astrophysics Data System (ADS)

    Podobnikar, Tomaž; Székely, Balázs

    2015-01-01

    Terrestrial talus slopes are a common feature of mountainous environments. Their geomorphic form is determined by their being constituted of scree, or similar loose and often poorly sorted material. Martian talus slopes are governed by the different nature of the Martian environment, namely: weaker gravity, the wide availability of loose material, the lack of fluvial erosion and the typicality of large escarpments; all these factors make talus slopes a more striking areomorphic feature on Mars than on Earth. This paper concerns the development of a numerical geomorphometric analysis, parameterization and detection of the talus slopes method. We design inventive variables, a multidirectional visibility index (MVI) and a relief above (RA) and propose two techniques of talus slope extraction: ISOcluster and progressive Boolean overlay. Our Martian digital terrain model (DTM) was derived from the ESA Mars Express HRSC imagery, with a resolution of 50 m. The method was tested in the study areas of Nanedi Valles and West Candor Chasma. The major challenge concerned the quality of the DTM. The selection of robust variables was therefore crucial. Our final model is to a certain degree DTM-error tolerant. The results show that the method is selective concerning those slopes that can be considered to constitute a talus slopes area, according to the visual interpretation of HRSC images. Based on an analysis of the DTM, it is possible to infer various geological properties and geophysical processes of the Martian and terrestrial environments; this has a range of applications, such as natural hazard risk management.

  1. Integrated expression profiles of mRNA and microRNA in the liver of Fructus Meliae Toosendan water extract injured mice

    PubMed Central

    Zheng, Jie; Ji, Cai; Lu, Xiaoyan; Tong, Wei; Fan, Xiaohui; Gao, Yue

    2015-01-01

    Liver toxicity is a severe problem associated with Traditional Chinese Medicine (TCM). Fructus Meliae Toosendan (FMT) is a known hepatotoxic TCM, however, the toxicological mechanisms of liver injury caused by FMT treatment still remain largely unknown. In this study, we aimed to reveal possible mechanisms of FMT water extract-induced liver injury using a systemic approach. After three consecutive daily dosing of FMT water extract, significant increases of alanine transaminase, aspartate transaminase, and alkaline phosphatase activities, along with elevated total bilirubin and total cholesterol levels and a decrease of triglyceride level, were detected in mice serum. Moreover, hydropic degeneration was observed in hepatocytes, suggesting the presence of FMT-induced liver injury. mRNA and microRNA expression profiles of liver samples from injured mice were analyzed and revealed 8 miRNAs and 1,723 mRNAs were significantly changed after FMT water extract treatment. For the eight differentially expressed miRNAs, their predicted target genes were collected and a final set of 125 genes and 4 miRNAs (miR-139-5p, miR-199a-5p, miR-2861, and miR-3960) was selected to investigate important processes involved in FMT hepatotoxicity. Our results demonstrated several cellular functions were disordered after FMT treatment, such as cellular growth and proliferation, gene expression and cellular development. We hypothesized that liver cell necrosis was the main liver toxicity of FMT water extract, which was possibly caused by oxidative stress responses. PMID:26539117

  2. Automated extraction and analysis of rock discontinuity characteristics from 3D point clouds

    NASA Astrophysics Data System (ADS)

    Bianchetti, Matteo; Villa, Alberto; Agliardi, Federico; Crosta, Giovanni B.

    2016-04-01

    A reliable characterization of fractured rock masses requires an exhaustive geometrical description of discontinuities, including orientation, spacing, and size. These are required to describe discontinuum rock mass structure, perform Discrete Fracture Network and DEM modelling, or provide input for rock mass classification or equivalent continuum estimate of rock mass properties. Although several advanced methodologies have been developed in the last decades, a complete characterization of discontinuity geometry in practice is still challenging, due to scale-dependent variability of fracture patterns and difficult accessibility to large outcrops. Recent advances in remote survey techniques, such as terrestrial laser scanning and digital photogrammetry, allow a fast and accurate acquisition of dense 3D point clouds, which promoted the development of several semi-automatic approaches to extract discontinuity features. Nevertheless, these often need user supervision on algorithm parameters which can be difficult to assess. To overcome this problem, we developed an original Matlab tool, allowing fast, fully automatic extraction and analysis of discontinuity features with no requirements on point cloud accuracy, density and homogeneity. The tool consists of a set of algorithms which: (i) process raw 3D point clouds, (ii) automatically characterize discontinuity sets, (iii) identify individual discontinuity surfaces, and (iv) analyse their spacing and persistence. The tool operates in either a supervised or unsupervised mode, starting from an automatic preliminary exploration data analysis. The identification and geometrical characterization of discontinuity features is divided in steps. First, coplanar surfaces are identified in the whole point cloud using K-Nearest Neighbor and Principal Component Analysis algorithms optimized on point cloud accuracy and specified typical facet size. Then, discontinuity set orientation is calculated using Kernel Density Estimation and

  3. Development of an automated method for Folin-Ciocalteu total phenolic assay in artichoke extracts.

    PubMed

    Yoo, Kil Sun; Lee, Eun Jin; Leskovar, Daniel; Patil, Bhimanagouda S

    2012-12-01

    We developed a system to run the Folin-Ciocalteu (F-C) total phenolic assay, in artichoke extract samples, which is fully automatic, consistent, and fast. The system uses 2 high performance liquid chromatography (HPLC) pumps, an autosampler, a column heater, a UV/Vis detector, and a data collection system. To test the system, a pump delivered 10-fold diluted F-C reagent solution at a rate of 0.7 mL/min, and 0.4 g/mL sodium carbonate at a rate of 2.1 mL/min. The autosampler injected 10 μL per 1.2 min, which was mixed with the F-C reagent and heated to 65 °C while it passed through the column heater. The heated reactant was mixed with sodium carbonate and color intensity was measured by the detector at 600 nm. The data collection system recorded the color intensity, and peak area of each sample was calculated as the concentration of the total phenolic content, expressed in μg/mL as either chlorogenic acid or gallic acid. This new method had superb repeatability (0.7% CV) and a high correlation with both the manual method (r(2) = 0.93) and the HPLC method (r(2) = 0.78). Ascorbic acid and quercetin showed variable antioxidant activity, but sugars did not. This method can be efficiently applied to research that needs to test many numbers of antioxidant capacity samples with speed and accuracy. PMID:23163965

  4. A unique method for isolation and solubilization of proteins after extraction of RNA from tumor tissue using trizol.

    PubMed

    Likhite, Neah; Warawdekar, Ujjwala M

    2011-04-01

    The aim of this study was to develop a systems approach to study tumor tissue. The importance of concurrent extraction of RNA, DNA, and protein is evident when genetic aberrations and the differences in the proteome and transcriptome have to be correlated. The need is magnified, as the tissue available for study is miniscule, is shared amongst investigators, and needs to support the holistic approach. Trizol is a monophasic solution of phenol and guanidine isothiocyanate and can be used to isolate the three biomolecules simultaneously. Trizol solution was used for RNA extraction in an ongoing study about expression of molecular markers in non-small cell lung carcinoma (NSCLC) and breast tumor tissue. After isolation of RNA, the remaining Trizol fraction was stored at -80°C for over 6 months. We have shown the extraction of protein from 17 tumor and adjacent, normal tissue samples and PBMC obtained from four blood samples. The isolation and solubilization of the protein fraction were done according to the product information using isopropanol for precipitation and guanidine hydrochloride and SDS for washing and solubilization, respectively, modifying the time of solubilization. The protein was estimated by the bicinchoninic acid (BCA) method and analyzed on polyacrylamide gels. Staining showed a wide repertoire, and Western blotting confirmed extraction of cytokeratins (CK) and DNA repair proteins. Whereas tissue samples in which the RNA was degraded could be assessed by the presence of the protein salvaging the marker analysis, it was seen that nuclear proteins cannot be retrieved and are probably lost with the DNA fraction. PMID:21455480

  5. A Unique Method for Isolation and Solubilization of Proteins after Extraction of RNA from Tumor Tissue Using Trizol

    PubMed Central

    Likhite, Neah; Warawdekar, Ujjwala M.

    2011-01-01

    The aim of this study was to develop a systems approach to study tumor tissue. The importance of concurrent extraction of RNA, DNA, and protein is evident when genetic aberrations and the differences in the proteome and transcriptome have to be correlated. The need is magnified, as the tissue available for study is miniscule, is shared amongst investigators, and needs to support the holistic approach. Trizol is a monophasic solution of phenol and guanidine isothiocyanate and can be used to isolate the three biomolecules simultaneously. Trizol solution was used for RNA extraction in an ongoing study about expression of molecular markers in non-small cell lung carcinoma (NSCLC) and breast tumor tissue. After isolation of RNA, the remaining Trizol fraction was stored at −80°C for over 6 months. We have shown the extraction of protein from 17 tumor and adjacent, normal tissue samples and PBMC obtained from four blood samples. The isolation and solubilization of the protein fraction were done according to the product information using isopropanol for precipitation and guanidine hydrochloride and SDS for washing and solubilization, respectively, modifying the time of solubilization. The protein was estimated by the bicinchoninic acid (BCA) method and analyzed on polyacrylamide gels. Staining showed a wide repertoire, and Western blotting confirmed extraction of cytokeratins (CK) and DNA repair proteins. Whereas tissue samples in which the RNA was degraded could be assessed by the presence of the protein salvaging the marker analysis, it was seen that nuclear proteins cannot be retrieved and are probably lost with the DNA fraction. PMID:21455480

  6. Single-Step RNA Extraction from Different Hydrogel-Embedded Mesenchymal Stem Cells for Quantitative Reverse Transcription-Polymerase Chain Reaction Analysis.

    PubMed

    Köster, Natascha; Schmiermund, Alexandra; Grubelnig, Stefan; Leber, Jasmin; Ehlicke, Franziska; Czermak, Peter; Salzig, Denise

    2016-06-01

    For many tissue engineering applications, cells such as human mesenchymal stem cells (hMSCs) must be embedded in hydrogels. The analysis of embedded hMSCs requires RNA extraction, but common extraction procedures often produce low yields and/or poor quality RNA. We systematically investigated four homogenization methods combined with eight RNA extraction protocols for hMSCs embedded in three common hydrogel types (alginate, agarose, and gelatin). We found for all three hydrogel types that using liquid nitrogen or a rotor-stator produced low RNA yields, whereas using a microhomogenizer or enzymatic/chemical hydrogel digestion achieved better yields regardless of which extraction protocol was subsequently applied. The hot phenol extraction protocol generally achieved the highest A260 values (representing up to 40.8 μg RNA per 10(6) cells), but the cetyltrimethylammonium bromide (CTAB) method produced RNA of better quality, with A260/A280 and A260/A230 ratios and UV spectra similar to the pure RNA control. The RNA produced by this method was also suitable as a template for endpoint and quantitative reverse transcription-PCR (qRT-PCR), achieving low Ct values of ∼20. The prudent choice of hydrogel homogenization and RNA extraction methods can ensure the preparation of high-quality RNA that generates reliable endpoint and quantitative RT-PCR data. We therefore propose a universal method that is suitable for the extraction of RNA from cells embedded in all three hydrogel types commonly used for tissue engineering. PMID:27094052

  7. Automation of static and dynamic non-dispersive liquid phase microextraction. Part 1: Approaches based on extractant drop-, plug-, film- and microflow-formation.

    PubMed

    Alexovič, Michal; Horstkotte, Burkhard; Solich, Petr; Sabo, Ján

    2016-02-01

    Simplicity, effectiveness, swiftness, and environmental friendliness - these are the typical requirements for the state of the art development of green analytical techniques. Liquid phase microextraction (LPME) stands for a family of elegant sample pretreatment and analyte preconcentration techniques preserving these principles in numerous applications. By using only fractions of solvent and sample compared to classical liquid-liquid extraction, the extraction kinetics, the preconcentration factor, and the cost efficiency can be increased. Moreover, significant improvements can be made by automation, which is still a hot topic in analytical chemistry. This review surveys comprehensively and in two parts the developments of automation of non-dispersive LPME methodologies performed in static and dynamic modes. Their advantages and limitations and the reported analytical performances are discussed and put into perspective with the corresponding manual procedures. The automation strategies, techniques, and their operation advantages as well as their potentials are further described and discussed. In this first part, an introduction to LPME and their static and dynamic operation modes as well as their automation methodologies is given. The LPME techniques are classified according to the different approaches of protection of the extraction solvent using either a tip-like (needle/tube/rod) support (drop-based approaches), a wall support (film-based approaches), or microfluidic devices. In the second part, the LPME techniques based on porous supports for the extraction solvent such as membranes and porous media are overviewed. An outlook on future demands and perspectives in this promising area of analytical chemistry is finally given. PMID:26772123

  8. Automated liquid-liquid extraction workstation for library synthesis and its use in the parallel and chromatography-free synthesis of 2-alkyl-3-alkyl-4-(3H)-quinazolinones.

    PubMed

    Carpintero, Mercedes; Cifuentes, Marta; Ferritto, Rafael; Haro, Rubén; Toledo, Miguel A

    2007-01-01

    An automated liquid-liquid extraction workstation has been developed. This module processes up to 96 samples in an automated and parallel mode avoiding the time-consuming and intensive sample manipulation during the workup process. To validate the workstation, a highly automated and chromatography-free synthesis of differentially substituted quinazolin-4(3H)-ones with two diversity points has been carried out using isatoic anhydride as starting material. PMID:17645313

  9. Optimization of the RNA extraction method for transcriptome studies of Salmonella inoculated on commercial raw chicken breast samples

    PubMed Central

    2011-01-01

    Background There has been increased interest in the study of molecular survival mechanisms expressed by foodborne pathogens present on food surfaces. Determining genomic responses of these pathogens to antimicrobials is of particular interest since this helps to understand antimicrobial effects at the molecular level. Assessment of bacterial gene expression by transcriptomic analysis in response to these antimicrobials would aid prediction of the phenotypic behavior of the bacteria in the presence of antimicrobials. However, before transcriptional profiling approaches can be implemented routinely, it is important to develop an optimal method to consistently recover pathogens from the food surface and ensure optimal quality RNA so that the corresponding gene expression analysis represents the current response of the organism. Another consideration is to confirm that there is no interference from the "background" food or meat matrix that could mask the bacterial response. Findings Our study involved developing a food model system using chicken breast meat inoculated with mid-log Salmonella cells. First, we tested the optimum number of Salmonella cells required on the poultry meat in order to extract high quality RNA. This was analyzed by inoculating 10-fold dilutions of Salmonella on the chicken samples followed by RNA extraction. Secondly, we tested the effect of two different bacterial cell recovery solutions namely 0.1% peptone water and RNAprotect (Qiagen Inc.) on the RNA yield and purity. In addition, we compared the efficiency of sonication and bead beater methods to break the cells for RNA extraction. To check chicken nucleic acid interference on downstream Salmonella microarray experiments both chicken and Salmonella cDNA labeled with different fluorescent dyes were mixed together and hybridized on a single Salmonella array. Results of this experiment did not show any cross-hybridization signal from the chicken nucleic acids. In addition, we demonstrated the

  10. Performance verification of the Maxwell 16 Instrument and DNA IQ Reference Sample Kit for automated DNA extraction of known reference samples.

    PubMed

    Krnajski, Z; Geering, S; Steadman, S

    2007-12-01

    Advances in automation have been made for a number of processes conducted in the forensic DNA laboratory. However, because most robotic systems are designed for high-throughput laboratories batching large numbers of samples, smaller laboratories are left with a limited number of cost-effective options for employing automation. The Maxwell 16 Instrument and DNA IQ Reference Sample Kit marketed by Promega are designed for rapid, automated purification of DNA extracts from sample sets consisting of sixteen or fewer samples. Because the system is based on DNA capture by paramagnetic particles with maximum binding capacity, it is designed to generate extracts with yield consistency. The studies herein enabled evaluation of STR profile concordance, consistency of yield, and cross-contamination performance for the Maxwell 16 Instrument. Results indicate that the system performs suitably for streamlining the process of extracting known reference samples generally used for forensic DNA analysis and has many advantages in a small or moderate-sized laboratory environment. PMID:25869266

  11. AUTOMATED ANALYSIS OF AQUEOUS SAMPLES CONTAINING PESTICIDES, ACIDIC/BASIC/NEUTRAL SEMIVOLATILES AND VOLATILE ORGANIC COMPOUNDS BY SOLID PHASE EXTRACTION COUPLED IN-LINE TO LARGE VOLUME INJECTION GC/MS

    EPA Science Inventory

    Data is presented on the development of a new automated system combining solid phase extraction (SPE) with GC/MS spectrometry for the single-run analysis of water samples containing a broad range of organic compounds. The system uses commercially available automated in-line 10-m...

  12. An improved method for extraction of high-quality total RNA from oil seeds.

    PubMed

    Rayani, Azadeh; Dehghan Nayeri, Fatemeh

    2015-04-01

    Seeds of oilseed plants that contain large amounts of oil, polysaccharides, proteins and polyphenols are not amenable to conventional RNA isolation protocols. The presence of these substances affects the quality and quantity of isolated nucleic acids. Here, a rapid and efficient RNA isolation protocol that, in contrast to other methods tested, allows high purify, integrity and yield of total RNA from seeds of sesame, corn, sunflower, flax and rapeseed was developed. The average yields of total RNA from 70 mg oil seeds ranged from 84 to 310 µg with A260/A280 between 1.9 and 2.08. The RNA isolated with this protocol was verified to be suitable for PCR, quantitative real-time PCR, semi-quantitative RT-PCR, cDNA synthesis and expression analysis. PMID:25534638

  13. Preparation and characterization of yeast nuclear extracts for efficient RNA polymerase B (II)-dependent transcription in vitro.

    PubMed Central

    Verdier, J M; Stalder, R; Roberge, M; Amati, B; Sentenac, A; Gasser, S M

    1990-01-01

    We present a reproducible method for the preparation of nuclear extracts from the yeast Saccharomyces cerevisiae that support efficient RNA polymerase B (II)-dependent transcription. Extracts from both a crude nuclear fraction and Percoll-purified nuclei are highly active for site-specific initiation and transcription of a G-free cassette under the Adenovirus major late promoter. At optimal extract concentrations transcription is at least 5 times more efficient with the yeast extracts than with HeLa whole cell extracts. We show that the transcriptional activity is sensitive to alpha-amanitin and to depletion of factor(s) recognizing the TATA-box of the promoter. The in vitro reaction showed maximal activity after 45 min, was very sensitive to Cl-, but was not affected by high concentrations of potassium. We find that the efficiency of in vitro transcription in nuclear extracts is reproducibly high when spheroplasting is performed with a partially purified beta 1,3-glucanase (lyticase). Therefore a simplified method to isolate the lyticase from the supernatant of Oerskovia xanthineolytica is also presented. Images PMID:2263463

  14. Submicrometric Magnetic Nanoporous Carbons Derived from Metal-Organic Frameworks Enabling Automated Electromagnet-Assisted Online Solid-Phase Extraction.

    PubMed

    Frizzarin, Rejane M; Palomino Cabello, Carlos; Bauzà, Maria Del Mar; Portugal, Lindomar A; Maya, Fernando; Cerdà, Víctor; Estela, José M; Turnes Palomino, Gemma

    2016-07-19

    We present the first application of submicrometric magnetic nanoporous carbons (μMNPCs) as sorbents for automated solid-phase extraction (SPE). Small zeolitic imidazolate framework-67 crystals are obtained at room temperature and directly carbonized under an inert atmosphere to obtain submicrometric nanoporous carbons containing magnetic cobalt nanoparticles. The μMNPCs have a high contact area, high stability, and their preparation is simple and cost-effective. The prepared μMNPCs are exploited as sorbents in a microcolumn format in a sequential injection analysis (SIA) system with online spectrophotometric detection, which includes a specially designed three-dimensional (3D)-printed holder containing an automatically actuated electromagnet. The combined action of permanent magnets and an automatically actuated electromagnet enabled the movement of the solid bed of particles inside the microcolumn, preventing their aggregation, increasing the versatility of the system, and increasing the preconcentration efficiency. The method was optimized using a full factorial design and Doehlert Matrix. The developed system was applied to the determination of anionic surfactants, exploiting the retention of the ion-pairs formed with Methylene Blue on the μMNPC. Using sodium dodecyl sulfate as a model analyte, quantification was linear from 50 to 1000 μg L(-1), and the detection limit was equal to 17.5 μg L(-1), the coefficient of variation (n = 8; 100 μg L(-1)) was 2.7%, and the analysis throughput was 13 h(-1). The developed approach was applied to the determination of anionic surfactants in water samples (natural water, groundwater, and wastewater), yielding recoveries of 93% to 110% (95% confidence level). PMID:27336802

  15. Satellite mapping and automated feature extraction: Geographic information system-based change detection of the Antarctic coast

    NASA Astrophysics Data System (ADS)

    Kim, Kee-Tae

    Declassified Intelligence Satellite Photograph (DISP) data are important resources for measuring the geometry of the coastline of Antarctica. By using the state-of-art digital imaging technology, bundle block triangulation based on tie points and control points derived from a RADARSAT-1 Synthetic Aperture Radar (SAR) image mosaic and Ohio State University (OSU) Antarctic digital elevation model (DEM), the individual DISP images were accurately assembled into a map quality mosaic of Antarctica as it appeared in 1963. The new map is one of important benchmarks for gauging the response of the Antarctic coastline to changing climate. Automated coastline extraction algorithm design is the second theme of this dissertation. At the pre-processing stage, an adaptive neighborhood filtering was used to remove the film-grain noise while preserving edge features. At the segmentation stage, an adaptive Bayesian approach to image segmentation was used to split the DISP imagery into its homogenous regions, in which the fuzzy c-means clustering (FCM) technique and Gibbs random field (GRF) model were introduced to estimate the conditional and prior probability density functions. A Gaussian mixture model was used to estimate the reliable initial values for the FCM technique. At the post-processing stage, image object formation and labeling, removal of noisy image objects, and vectorization algorithms were sequentially applied to segmented images for extracting a vector representation of coastlines. Results were presented that demonstrate the effectiveness of the algorithm in segmenting the DISP data. In the cases of cloud cover and little contrast scenes, manual editing was carried out based on intermediate image processing and visual inspection in comparison of old paper maps. Through a geographic information system (GIS), the derived DISP coastline data were integrated with earlier and later data to assess continental scale changes in the Antarctic coast. Computing the area of

  16. Full Design Automation of Multi-State RNA Devices to Program Gene Expression Using Energy-Based Optimization

    PubMed Central

    Majer, Eszter; Daròs, José-Antonio; Jaramillo, Alfonso

    2013-01-01

    Small RNAs (sRNAs) can operate as regulatory agents to control protein expression by interaction with the 5′ untranslated region of the mRNA. We have developed a physicochemical framework, relying on base pair interaction energies, to design multi-state sRNA devices by solving an optimization problem with an objective function accounting for the stability of the transition and final intermolecular states. Contrary to the analysis of the reaction kinetics of an ensemble of sRNAs, we solve the inverse problem of finding sequences satisfying targeted reactions. We show here that our objective function correlates well with measured riboregulatory activity of a set of mutants. This has enabled the application of the methodology for an extended design of RNA devices with specified behavior, assuming different molecular interaction models based on Watson-Crick interaction. We designed several YES, NOT, AND, and OR logic gates, including the design of combinatorial riboregulators. In sum, our de novo approach provides a new paradigm in synthetic biology to design molecular interaction mechanisms facilitating future high-throughput functional sRNA design. PMID:23935479

  17. Hydroethanolic Pistacia atlantica hulls extract improved wound healing process; evidence for mast cells infiltration, angiogenesis and RNA stability.

    PubMed

    Farahpour, Mohammad Reza; Mirzakhani, Navideh; Doostmohammadi, Jamal; Ebrahimzadeh, Mahmood

    2015-05-01

    In Iranian traditional therapy folk, the Pistacia is used for treatment of wound inflammation. Here in the present study, the In vivo effect of Pistacia atlantica hulls ointment (PAO) on the wound healing process was assessed. Excision and incision wounds were induced in rats. Three different doses of PAO were administrated. Following 3, 7, 14 and 21 days, the tissue samples were obtained and skin irritation ratio, hydroxyproline content, as well as immune cells, fibroblasts, fibrocytes distribution and collagen density were analyzed. Moreover, the cellular RNA damage examined using epi-fluorescent microscope. Hydroethanolic extract of PAO significantly (P < 0.05) increased wound contraction percentage and up-regulated hydroxyproline content. The animals in medium and high dose PAO-treated groups exhibited remarkably (P < 0.05) higher fibroblast distribution and significantly (P < 0.05) lower immune cells infiltration. PAO up-regulated mast cells distribution on day 7 and elevated neovascularization in a dose dependent manner. Significantly lower RNA damage was revealed in PAO-treated animals. Our data showed that, PAO shortened the inflammation phase by provoking the fibroblast proliferation. Moreover, PAO enhanced mast cells distribution and infiltration, which in turn promoted the neovascularization. Ultimately, promoted angiogenesis increased RNA stability in different cell types. Thus, Hydroethanolic extract of PAO can be considered as an appropriate compound for wound healing medicine. PMID:25849027

  18. Evaluation of commercial kits for dual extraction of DNA and RNA from human body fluids.

    PubMed

    Schweighardt, Andrew J; Tate, Courtney M; Scott, Kristina A; Harper, Kathryn A; Robertson, James M

    2015-01-01

    STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body-fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR-Duet(™) kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand-alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification. PMID:25284026

  19. RNA Polymerase II Second Largest Subunit Molecular Identification of Boletus griseipurpureus Corner From Thailand and Antibacterial Activity of Basidiocarp Extracts

    PubMed Central

    Aung-aud-chariya, Amornrat; Bangrak, Phuwadol; Lumyong, Saisamorn; Phupong, Worrapong; Aggangan, Nelly Siababa; Kamlangdee, Niyom

    2015-01-01

    Background: Boletus griseipurpureus Corner, an edible mushroom, is a putative ectomycorrhizal fungus. Currently, the taxonomic boundary of this mushroom is unclear and its bitter taste makes it interesting for evaluating its antibacterial properties. Objectives: The purpose of this study was to identify the genetic variation of this mushroom and also to evaluate any antibacterial activities. Materials and Methods: Basidiocarps were collected from 2 north-eastern provinces, Roi Et and Ubon Ratchathani, and from 2 southern provinces, Songkhla and Surat Thani, in Thailand. Genomic DNA was extracted and molecular structure was examined using the RNA polymerase II (RPB2) analysis. Antibacterial activities of basidiocarp extracts were conducted with Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29523 and methicillin-resistant Staphylococcus aureus (MRSA) 189 using the agar-well diffusion method. Results: All the samples collected for this study constituted a monophyletic clade, which was closely related with the Boletus group of polypore fungi. For the antibacterial study, it was found that the crude methanol extract of basidiomes inhibited the growth of all bacteria in vitro more than the crude ethyl acetate extract. Conclusions: Basidomes collected from four locations in Thailand had low genetic variation and their extracts inhibited the growth of all tested bacteria. The health benefits of this edible species should be evaluated further. PMID:25834720

  20. Determination of Low Concentrations of Acetochlor in Water by Automated Solid-Phase Extraction and Gas Chromatography with Mass-Selective Detection

    USGS Publications Warehouse

    Lindley, C.E.; Stewart, J.T.; Sandstrom, M.W.

    1996-01-01

    A sensitive and reliable gas chromatographic/mass spectrometric (GC/MS) method for determining acetochlor in environmental water samples was developed. The method involves automated extraction of the herbicide from a filtered 1 L water sample through a C18 solid-phase extraction column, elution from the column with hexane-isopropyl alcohol (3 + 1), and concentration of the extract with nitrogen gas. The herbicide is quantitated by capillary/column GC/MS with selected-ion monitoring of 3 characteristic ions. The single-operator method detection limit for reagent water samples is 0.0015 ??g/L. Mean recoveries ranged from about 92 to 115% for 3 water matrixes fortified at 0.05 and 0.5 ??g/L. Average single-operator precision, over the course of 1 week, was better than 5%.

  1. Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

    PubMed Central

    Gulliksen, Anja; Keegan, Helen; Martin, Cara; O'Leary, John; Solli, Lars A.; Falang, Inger Marie; Grønn, Petter; Karlgård, Aina; Mielnik, Michal M.; Johansen, Ib-Rune; Tofteberg, Terje R.; Baier, Tobias; Gransee, Rainer; Drese, Klaus; Hansen-Hagge, Thomas; Riegger, Lutz; Koltay, Peter; Zengerle, Roland; Karlsen, Frank; Ausen, Dag; Furuberg, Liv

    2012-01-01

    The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n = 28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection. PMID:22235204

  2. In situ, real-time catabolic gene expression: Extraction and characterization of naphthalene dioxygenase mRNA transcripts from groundwater

    SciTech Connect

    Wilson, M.S.; Bakermans, C.; Madsen, E.L.

    1999-01-01

    The authors developed procedures for isolating and characterizing in situ-transcribed mRNA from groundwater microorganisms catabolizing naphthalene at a coal tar waste-contaminated site. Groundwater was pumped through 0.22-{micro}m-pore-size filters, which were then frozen to dry ice-ethanol. RNA was extracted from the frozen filters by boiling sodium dodecyl sulfate lysis and acidic phenol-chloroform extraction. Transcript characterization was performed with a series of PCR primers designed to amplify nahAc homologs. Several primer pairs were found to amplify nahAc homologs representing the entire diversity of the naphthalene-degrading genes. The environmental RNA extract was reverse transcribed, and the resultant mixture of cDNAs was amplified by PCR. A digoxigenin-labeled probe mixture was produced by PCR amplification of groundwater cDNA. This probe mixture hybridized under stringent conditions with the corresponding PCR products from naphthalene-degrading bacteria carrying a variety of nahAc homologs, indicating that diverse dioxygenase transcripts had been retrieved from groundwater. Diluted and undiluted cDNA preparations were independently amplified, and 28 of the resulting PCR products were cloned and sequenced. Sequence comparisons revealed two major groups related to the dioxygenase genes ndoB and dntAc, previously cloned from Pseudomonas putida NCIB 9816-4 and Burkholderia sp. strain DNT, respectively. A distinctive subgroup of sequences was found only in experiments performed with the undiluted cDNA preparation. To the authors` knowledge, these results are the first to directly document in situ transcription of genes encoding naphthalene catabolism at a contaminated site by indigenous microorganisms. The retrieved sequences represent greater diversity than has been detected at the study site by culture-based approaches.

  3. Structure of a Neurospora RNA polymerase I promoter defined by transcription in vitro with homologous extracts.

    PubMed Central

    Tyler, B M; Giles, N H

    1985-01-01

    A Neurospora in vitro transcription system has been developed which specifically and efficiently initiates transcription of a cloned Neurospora crassa ribosomal RNA gene by RNA polymerase I. The initiation site of transcription (both in vitro and in vivo) appears to be located about 850 bp from the 5' end of mature 17S rRNA. However, the primary rRNA transcripts are normally cleaved very rapidly at a site 120-125 nt from the 5' end in vitro and in vivo. The nucleotide sequence surrounding the initiation site has been determined. The region from -16 to +9 exhibits partial homology to the corresponding sequences from a wide variety of organisms including yeast, but the most striking similarity is to the initiation region from Dictyostelium discoideum which displays 73% homology to the Neurospora sequence from -23 to +47. The Neurospora sequences from -96 to +97 have been shown to be sufficient for transcription. This region contains two sequences displaying 8/9 bp matches to elements of the 5S rDNA promoter. Images PMID:2989792

  4. Development of a Nucleic Acid Extraction Procedure for Simultaneous Recovery of DNA and RNA from Diverse Microbes in Water

    PubMed Central

    Hill, Vincent R.; Narayanan, Jothikumar; Gallen, Rachel R.; Ferdinand, Karen L.; Cromeans, Theresa; Vinjé, Jan

    2015-01-01

    Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT) values were used as the metrics for evaluating relative performance. Experimental results were used to develop a guanidinium isothiocyanate-based lysis buffer (UNEX buffer) that enabled effective simultaneous extraction and recovery of DNA and RNA from the suite of study microbes. Procedures for bead beating, nucleic acid purification, and PCR facilitation were also developed and integrated in the protocol. The final lysis buffer and sample preparation procedure was found to be effective for a panel of drinking water and source water concentrates when compared to commercial nucleic acid extraction kits. The UNEX buffer-based extraction protocol enabled PCR detection of six study microbes, in 100 L finished water samples from four drinking water treatment facilities, within three CT values (i.e., within 90% difference) of the reagent-grade water control. The results from this study indicate that this newly formulated lysis buffer and sample preparation procedure can be useful for standardized molecular testing of drinking and environmental waters. PMID:26016775

  5. Arsenic fractionation in agricultural soil using an automated three-step sequential extraction method coupled to hydride generation-atomic fluorescence spectrometry.

    PubMed

    Rosas-Castor, J M; Portugal, L; Ferrer, L; Guzmán-Mar, J L; Hernández-Ramírez, A; Cerdà, V; Hinojosa-Reyes, L

    2015-05-18

    A fully automated modified three-step BCR flow-through sequential extraction method was developed for the fractionation of the arsenic (As) content from agricultural soil based on a multi-syringe flow injection analysis (MSFIA) system coupled to hydride generation-atomic fluorescence spectrometry (HG-AFS). Critical parameters that affect the performance of the automated system were optimized by exploiting a multivariate approach using a Doehlert design. The validation of the flow-based modified-BCR method was carried out by comparison with the conventional BCR method. Thus, the total As content was determined in the following three fractions: fraction 1 (F1), the acid-soluble or interchangeable fraction; fraction 2 (F2), the reducible fraction; and fraction 3 (F3), the oxidizable fraction. The limits of detection (LOD) were 4.0, 3.4, and 23.6 μg L(-1) for F1, F2, and F3, respectively. A wide working concentration range was obtained for the analysis of each fraction, i.e., 0.013-0.800, 0.011-0.900 and 0.079-1.400 mg L(-1) for F1, F2, and F3, respectively. The precision of the automated MSFIA-HG-AFS system, expressed as the relative standard deviation (RSD), was evaluated for a 200 μg L(-1) As standard solution, and RSD values between 5 and 8% were achieved for the three BCR fractions. The new modified three-step BCR flow-based sequential extraction method was satisfactorily applied for arsenic fractionation in real agricultural soil samples from an arsenic-contaminated mining zone to evaluate its extractability. The frequency of analysis of the proposed method was eight times higher than that of the conventional BCR method (6 vs 48 h), and the kinetics of lixiviation were established for each fraction. PMID:25910440

  6. Time-resolved Characterization of Particle Associated Polycyclic Aromatic Hydrocarbons using a newly-developed Sequential Spot Sampler with Automated Extraction and Analysis

    PubMed Central

    Lewis, Gregory S.; Spielman, Steven R.; Hering, Susanne V.

    2014-01-01

    A versatile and compact sampling system, the Sequential Spot Sampler (S3) has been developed for pre-concentrated, time-resolved, dry collection of fine and ultrafine particles. Using a temperature-moderated laminar flow water condensation method, ambient particles as small as 6 nm are deposited within a dry, 1-mm diameter spot. Sequential samples are collected on a multiwell plate. Chemical analyses are laboratory-based, but automated. The sample preparation, extraction and chemical analysis steps are all handled through a commercially-available, needle-based autosampler coupled to a liquid chromatography system. This automation is enabled by the small deposition area of the collection. The entire sample is extracted into 50–100μl volume of solvent, providing quantifiable samples with small collected air volumes. A pair of S3 units was deployed in Stockton (CA) from November 2011 to February 2012. PM2.5 samples were collected every 12 hrs, and analyzed for polycyclic aromatic hydrocarbons (PAHs). In parallel, conventional filter samples were collected for 48 hrs and used to assess the new system’s performance. An automated sample preparation and extraction was developed for samples collected using the S3. Collocated data from the two sequential spot samplers were highly correlated for all measured compounds, with a regression slope of 1.1 and r2=0.9 for all measured concentrations. S3/filter ratios for the mean concentration of each individual PAH vary between 0.82 and 1.33, with the larger variability observed for the semivolatile components. Ratio for total PAH concentrations was 1.08. Total PAH concentrations showed similar temporal trend as ambient PM2.5 concentrations. Source apportionment analysis estimated a significant contribution of biomass burning to ambient PAH concentrations during winter. PMID:25574151

  7. Time-resolved characterization of particle associated polycyclic aromatic hydrocarbons using a newly-developed sequential spot sampler with automated extraction and analysis

    NASA Astrophysics Data System (ADS)

    Eiguren-Fernandez, Arantzazu; Lewis, Gregory S.; Spielman, Steven R.; Hering, Susanne V.

    2014-10-01

    A versatile and compact sampling system, the Sequential Spot Sampler (S3) has been developed for pre-concentrated, time-resolved, dry collection of fine and ultrafine particles. Using a temperature-moderated laminar flow water condensation method, ambient particles as small as 6 nm are deposited within a dry, 1-mm diameter spot. Sequential samples are collected on a multiwell plate. Chemical analyses are laboratory-based, but automated. The sample preparation, extraction and chemical analysis steps are all handled through a commercially-available, needle-based autosampler coupled to a liquid chromatography system. This automation is enabled by the small deposition area of the collection. The entire sample is extracted into 50-100 μL volume of solvent, providing quantifiable samples with small collected air volumes. A pair of S3 units was deployed in Stockton (CA) from November 2011 to February 2012. PM2.5 samples were collected every 12 h, and analyzed for polycyclic aromatic hydrocarbons (PAHs). In parallel, conventional filter samples were collected for 48 h and used to assess the new system's performance. An automated sample preparation and extraction was developed for samples collected using the S3. Collocated data from the two sequential spot samplers were highly correlated for all measured compounds, with a regression slope of 1.1 and r2 = 0.9 for all measured concentrations. S3/filter ratios for the mean concentration of each individual PAH vary between 0.82 and 1.33, with the larger variability observed for the semivolatile components. Ratio for total PAH concentrations was 1.08. Total PAH concentrations showed similar temporal trend as ambient PM2.5 concentrations. Source apportionment analysis estimated a significant contribution of biomass burning to ambient PAH concentrations during winter.

  8. Automated mini-column solid-phase extraction cleanup for high-throughput analysis of chemical contaminants in foods by low-pressure gas chromatography – tandem mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study demonstrated the application of an automated high-throughput mini-cartridge solid-phase extraction (mini-SPE) cleanup for the rapid low-pressure gas chromatography – tandem mass spectrometry (LPGC-MS/MS) analysis of pesticides and environmental contaminants in QuEChERS extracts of foods. ...

  9. Affinity chromatography using 2' fluoro-substituted RNAs for detection of RNA-protein interactions in RNase-rich or RNase-treated extracts.

    PubMed

    Hovhannisyan, Ruben; Carstens, Russ

    2009-02-01

    Use of RNA affinity chromatography is commonly used to identify RNA binding proteins that interact with specific RNA cis-elements that function in post-transcriptional gene regulation. These purifications can be complicated by residual RNase activity in cellular extracts that can degrade the RNAs on these affinity columns. Furthermore, some proteins may associate indirectly with the column as a component of multi-protein complexes that are "tethered" through the binding of cellular RNAs. We present a protocol for an RNA affinity procedure that can be used in conjunction with RNase-rich or RNase-treated extracts by using RNAs synthesized with 2' fluoro-substituted cytidine triphosphate (CTP) and uridine triphosphate (UTP). The resulting RNAs are shown to be RNase A-resistant and capable of direct coupling to adipic acid dihydrazide agarose beads. Using an RNA cis-element previously shown to bind hnRNP M, we demonstrated that the substituted RNAs preserve binding capability by a common class of RNA binding proteins. Our results provide a method that may be used more generally for RNA affinity purification or as a validation step to verify more direct binding of a given RNA binding protein to a target RNA. PMID:19317654

  10. A fully automated method for simultaneous determination of aflatoxins and ochratoxin A in dried fruits by pressurized liquid extraction and online solid-phase extraction cleanup coupled to ultra-high-pressure liquid chromatography-tandem mass spectrometry.

    PubMed

    Campone, Luca; Piccinelli, Anna Lisa; Celano, Rita; Russo, Mariateresa; Valdés, Alberto; Ibáñez, Clara; Rastrelli, Luca

    2015-04-01

    According to current demands and future perspectives in food safety, this study reports a fast and fully automated analytical method for the simultaneous analysis of the mycotoxins with high toxicity and wide spread, aflatoxins (AFs) and ochratoxin A (OTA) in dried fruits, a high-risk foodstuff. The method is based on pressurized liquid extraction (PLE), with aqueous methanol (30%) at 110 °C, of the slurried dried fruit and online solid-phase extraction (online SPE) cleanup of the PLE extracts with a C18 cartridge. The purified sample was directly analysed by ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for sensitive and selective determination of AFs and OTA. The proposed analytical procedure was validated for different dried fruits (vine fruit, fig and apricot), providing method detection and quantification limits much lower than the AFs and OTA maximum levels imposed by EU regulation in dried fruit for direct human consumption. Also, recoveries (83-103%) and repeatability (RSD < 8, n = 3) meet the performance criteria required by EU regulation for the determination of the levels of mycotoxins in foodstuffs. The main advantage of the proposed method is full automation of the whole analytical procedure that reduces the time and cost of the analysis, sample manipulation and solvent consumption, enabling high-throughput analysis and highly accurate and precise results. PMID:25694147

  11. Automated flow-based anion-exchange method for high-throughput isolation and real-time monitoring of RuBisCO in plant extracts.

    PubMed

    Suárez, Ruth; Miró, Manuel; Cerdà, Víctor; Perdomo, Juan Alejandro; Galmés, Jeroni

    2011-06-15

    In this work, a miniaturized, completely enclosed multisyringe-flow system is proposed for high-throughput purification of RuBisCO from Triticum aestivum extracts. The automated method capitalizes on the uptake of the target protein at 4°C onto Q-Sepharose Fast Flow strong anion-exchanger packed in a cylindrical microcolumn (105 × 4 mm) followed by a stepwise ionic-strength gradient elution (0-0.8 mol/L NaCl) to eliminate concomitant extract components and retrieve highly purified RuBisCO. The manifold is furnished downstream with a flow-through diode-array UV/vis spectrophotometer for real-time monitoring of the column effluent at the protein-specific wavelength of 280 nm to detect the elution of RuBisCO. Quantitation of RuBisCO and total soluble proteins in the eluate fractions were undertaken using polyacrylamide gel electrophoresis (PAGE) and the spectrophotometric Bradford assay, respectively. A comprehensive investigation of the effect of distinct concentration gradients on the isolation of RuBisCO and experimental conditions (namely, type of resin, column dimensions and mobile-phase flow rate) upon column capacity and analyte breakthrough was effected. The assembled set-up was aimed to critically ascertain the efficiency of preliminary batchwise pre-treatments of crude plant extracts (viz., polyethylenglycol (PEG) precipitation, ammonium sulphate precipitation and sucrose gradient centrifugation) in terms of RuBisCO purification and absolute recovery prior to automated anion-exchange column separation. Under the optimum physical and chemical conditions, the flow-through column system is able to admit crude plant extracts and gives rise to RuBisCO purification yields better than 75%, which might be increased up to 96 ± 9% with a prior PEG fractionation followed by sucrose gradient step. PMID:21641435

  12. Automated extraction method for the center line of spinal canal and its application to the spinal curvature quantification in torso X-ray CT images

    NASA Astrophysics Data System (ADS)

    Hayashi, Tatsuro; Zhou, Xiangrong; Chen, Huayue; Hara, Takeshi; Miyamoto, Kei; Kobayashi, Tatsunori; Yokoyama, Ryujiro; Kanematsu, Masayuki; Hoshi, Hiroaki; Fujita, Hiroshi

    2010-03-01

    X-ray CT images have been widely used in clinical routine in recent years. CT images scanned by a modern CT scanner can show the details of various organs and tissues. This means various organs and tissues can be simultaneously interpreted on CT images. However, CT image interpretation requires a lot of time and energy. Therefore, support for interpreting CT images based on image-processing techniques is expected. The interpretation of the spinal curvature is important for clinicians because spinal curvature is associated with various spinal disorders. We propose a quantification scheme of the spinal curvature based on the center line of spinal canal on CT images. The proposed scheme consists of four steps: (1) Automated extraction of the skeletal region based on CT number thresholding. (2) Automated extraction of the center line of spinal canal. (3) Generation of the median plane image of spine, which is reformatted based on the spinal canal. (4) Quantification of the spinal curvature. The proposed scheme was applied to 10 cases, and compared with the Cobb angle that is commonly used by clinicians. We found that a high-correlation (for the 95% confidence interval, lumbar lordosis: 0.81-0.99) between values obtained by the proposed (vector) method and Cobb angle. Also, the proposed method can provide the reproducible result (inter- and intra-observer variability: within 2°). These experimental results suggested a possibility that the proposed method was efficient for quantifying the spinal curvature on CT images.

  13. Determination of amlodipine in human plasma using automated online solid-phase extraction HPLC-tandem mass spectrometry: application to a bioequivalence study of Chinese volunteers.

    PubMed

    Shentu, Jianzhong; Fu, Lizhi; Zhou, Huili; Hu, Xing Jiang; Liu, Jian; Chen, Junchun; Wu, Guolan

    2012-11-01

    An automated method (XLC-MS/MS) that uses online solid-phase extraction coupled with HPLC-tandem mass spectrometry was reported here for the first time to quantify amlodipine in human plasma. Automated pre-purification of plasma was performed using 10 mm × 2 mm HySphere C8 EC-SE online solid-phase extraction cartridges. After being eluted from the cartridge, the analyte and the internal standard were separated by HPLC and detected by tandem mass spectrometry. Mass spectrometric detection was achieved in the multiple reaction monitoring mode using a quadrupole tandem mass spectrometer in the positive electrospray ionization mode. The XLC-MS/MS method was validated and yielded excellent specificity. The calibration curve ranged from 0.10 to 10.22 ng/mL, and both the intra- and inter-day precision and accuracy values were within 8%. This method proved to be less laborious and was faster per analysis (high-throughput) than offline sample preparation methods. This method has been successfully applied in clinical pharmacokinetic and bioequivalence analyses. PMID:22770846

  14. High-throughput method of dioxin analysis in aqueous samples using consecutive solid phase extraction steps with the new C18 Ultraflow™ pressurized liquid extraction and automated clean-up.

    PubMed

    Youn, Yeu-Young; Park, Deok Hie; Lee, Yeon Hwa; Lim, Young Hee; Cho, Hye Sung

    2015-01-01

    A high-throughput analytical method has been developed for the determination of seventeen 2,3,7,8-substituted congeners of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in aqueous samples. A recently introduced octadecyl (C18) disk for semi-automated solid-phase extraction of PCDD/Fs in water samples with a high level of particulate material has been tested for the analysis of dioxins. A new type of C18 disk specially designed for the analysis of hexane extractable material (HEM), but never previously reported for use in PCDD/Fs analysis. This kind of disk allows a higher filtration flow, and therefore the time of analysis is reduced. The solid-phase extraction technique is used to change samples from liquid to solid, and therefore pressurized liquid extraction (PLE) can be used in the pre-treatment. In order to achieve efficient purification, extracts from the PLE are purified using an automated Power-prep system with disposable silica, alumina, and carbon columns. Quantitative analyses of PCDD/Fs were performed by GC-HRMS using multi-ion detection (MID) mode. The method was successfully applied to the analysis of water samples from the wastewater treatment system of a vinyl chloride monomer plant. The entire procedure is in agreement with EPA1613 recommendations regarding the blank control, MDLs (method detection limits), accuracy, and precision. The high-throughput method not only meets the requirements of international standards, but also shortens the required analysis time from 2 weeks to 3d. PMID:25112208

  15. Evaluation of automated cell disruptor methods for oomycetous and ascomycetous model organisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two automated cell disruptor-based methods for RNA extraction; disruption of thawed cells submerged in TRIzol Reagent (method QP), and direct disruption of frozen cells on dry ice (method CP), were optimized for a model oomycete, Phytophthora capsici, and compared with grinding in a mortar and pestl...

  16. Activated charcoal-mediated RNA extraction method for Azadirachta indica and plants highly rich in polyphenolics, polysaccharides and other complex secondary compounds

    PubMed Central

    2013-01-01

    Background High quality RNA is a primary requisite for numerous molecular biological applications but is difficult to isolate from several plants rich in polysaccharides, polyphenolics and other secondary metabolites. These compounds either bind with nucleic acids or often co-precipitate at the final step and many times cannot be removed by conventional methods and kits. Addition of vinyl-pyrollidone polymers in extraction buffer efficiently removes polyphenolics to some extent, but, it failed in case of Azadirachta indica and several other medicinal and aromatic plants. Findings Here we report the use of adsorption property of activated charcoal (0.03%–0.1%) in RNA isolation procedures to remove complex secondary metabolites and polyphenolics to yield good quality RNA from Azadirachta indica. We tested and validated our modified RNA isolation method across 21 different plants including Andrographis paniculata, Aloe vera, Rosa damascena, Pelargonium graveolens, Phyllanthus amarus etc. from 13 other different families, many of which are considered as tough system for isolating RNA. The A260/280 ratio of the extracted RNA ranged between 1.8-2.0 and distinct 28S and 18S ribosomal RNA bands were observed in denaturing agarose gel electrophoresis. Analysis using Agilent 2100 Bioanalyzer revealed intact total RNA yield with very good RNA Integrity Number. Conclusions The RNA isolated by our modified method was found to be of high quality and amenable for sensitive downstream molecular applications like subtractive library construction and RT-PCR. This modified RNA isolation procedure would aid and accelerate the biotechnological studies in complex medicinal and aromatic plants which are extremely rich in secondary metabolic compounds. PMID:23537338

  17. Accurate and efficient N-6-adenosine methylation in spliceosomal U6 small nuclear RNA by HeLa cell extract in vitro.

    PubMed

    Shimba, S; Bokar, J A; Rottman, F; Reddy, R

    1995-07-11

    Human U6 small nuclear RNA (U6 snRNA), an abundant snRNA required for splicing of pre-mRNAs, contains several post-transcriptional modifications including a single m6A (N-6-methyladenosine) at position 43. This A-43 residue is critical for the function of U6 snRNA in splicing of pre-mRNAs. Yeast and plant U6 snRNAs also contain m6A in the corresponding position showing that this modification is evolutionarily conserved. In this study, we show that upon incubation of an unmodified U6 RNA with HeLa cell extract, A-43 residue in human U6 snRNA was rapidly converted to m6A-43. This conversion was detectable as early as 3 min after incubation and was nearly complete in 60 min; no other A residue in U6 snRNA was converted to m6A. Deletion studies showed that the stem-loop structure near the 5' end of U6 snRNA is dispensable for m6A formation; however, the integrity of the 3' stem-loop was necessary for efficient m6A formation. These data show that a short stretch of primary sequence flanking the methylation site is not sufficient for U6 m6A methyltransferase recognition and the enzyme probably recognizes secondary and/or tertiary structural features in U6 snRNA. The enzyme that catalyzes m6A formation in U6 snRNA appears to be distinct from the prolactin mRNA methyltransferase which is also present in HeLa nuclear extracts. PMID:7630720

  18. Automated extraction of aorta and pulmonary artery in mediastinum from 3D chest x-ray CT images without contrast medium

    NASA Astrophysics Data System (ADS)

    Kitasaka, Takayuki; Mori, Kensaku; Hasegawa, Jun-ichi; Toriwaki, Jun-ichiro; Katada, Kazuhiro

    2002-05-01

    This paper proposes a method for automated extraction of the aorta and pulmonary artery (PA) in the mediastinum of the chest from uncontrasted chest X-ray CT images. The proposed method employs a model fitting technique to use shape features of blood vessels for extraction. First, edge voxels are detected based on the standard deviation of CT values. A likelihood image, which shows the degree of likelihood on medial axes of vessels, are calculated by applying the Euclidean distance transformation to non-edge voxels. Second, the medial axis of each vessel is obtained by fitting the model. This is done by referring the likelihood image. Finally, the aorta and PA areas are recovered from the medial axes by executing the reverse Euclidean distance transformation. We applied the proposed method to seven cases of uncontrasted chest X-ray CT images and evaluated the results by calculating the coincidence index computed from the extracted regions and the regions manually traced. Experimental results showed that the extracted aorta and the PA areas coincides with manually input regions with the coincidence indexes values 90% and 80-90%,respectively.

  19. Demonstration and validation of automated agricultural field extraction from multi-temporal Landsat data for the majority of United States harvested cropland

    NASA Astrophysics Data System (ADS)

    Yan, L.; Roy, D. P.

    2014-12-01

    The spatial distribution of agricultural fields is a fundamental description of rural landscapes and the location and extent of fields is important to establish the area of land utilized for agricultural yield prediction, resource allocation, and for economic planning, and may be indicative of the degree of agricultural capital investment, mechanization, and labor intensity. To date, field objects have not been extracted from satellite data over large areas because of computational constraints, the complexity of the extraction task, and because consistently processed appropriate resolution data have not been available or affordable. A recently published automated methodology to extract agricultural crop fields from weekly 30 m Web Enabled Landsat data (WELD) time series was refined and applied to 14 states that cover 70% of harvested U.S. cropland (USDA 2012 Census). The methodology was applied to 2010 combined weekly Landsat 5 and 7 WELD data. The field extraction and quantitative validation results are presented for the following 14 states: Iowa, North Dakota, Illinois, Kansas, Minnesota, Nebraska, Texas, South Dakota, Missouri, Indiana, Ohio, Wisconsin, Oklahoma and Michigan (sorted by area of harvested cropland). These states include the top 11 U.S states by harvested cropland area. Implications and recommendations for systematic application to global coverage Landsat data are discussed.

  20. A Comprehensive Automated 3D Approach for Building Extraction, Reconstruction, and Regularization from Airborne Laser Scanning Point Clouds

    PubMed Central

    Dorninger, Peter; Pfeifer, Norbert

    2008-01-01

    Three dimensional city models are necessary for supporting numerous management applications. For the determination of city models for visualization purposes, several standardized workflows do exist. They are either based on photogrammetry or on LiDAR or on a combination of both data acquisition techniques. However, the automated determination of reliable and highly accurate city models is still a challenging task, requiring a workflow comprising several processing steps. The most relevant are building detection, building outline generation, building modeling, and finally, building quality analysis. Commercial software tools for building modeling require, generally, a high degree of human interaction and most automated approaches described in literature stress the steps of such a workflow individually. In this article, we propose a comprehensive approach for automated determination of 3D city models from airborne acquired point cloud data. It is based on the assumption that individual buildings can be modeled properly by a composition of a set of planar faces. Hence, it is based on a reliable 3D segmentation algorithm, detecting planar faces in a point cloud. This segmentation is of crucial importance for the outline detection and for the modeling approach. We describe the theoretical background, the segmentation algorithm, the outline detection, and the modeling approach, and we present and discuss several actual projects.

  1. Multiple automated headspace in-tube extraction for the accurate analysis of relevant wine aroma compounds and for the estimation of their relative liquid-gas transfer rates.

    PubMed

    Zapata, Julián; Lopez, Ricardo; Herrero, Paula; Ferreira, Vicente

    2012-11-30

    An automated headspace in-tube extraction (ITEX) method combined with multiple headspace extraction (MHE) has been developed to provide simultaneously information about the accurate wine content in 20 relevant aroma compounds and about their relative transfer rates to the headspace and hence about the relative strength of their interactions with the matrix. In the method, 5 μL (for alcohols, acetates and carbonyl alcohols) or 200 μL (for ethyl esters) of wine sample were introduced in a 2 mL vial, heated at 35°C and extracted with 32 (for alcohols, acetates and carbonyl alcohols) or 16 (for ethyl esters) 0.5 mL pumping strokes in four consecutive extraction and analysis cycles. The application of the classical theory of Multiple Extractions makes it possible to obtain a highly reliable estimate of the total amount of volatile compound present in the sample and a second parameter, β, which is simply the proportion of volatile not transferred to the trap in one extraction cycle, but that seems to be a reliable indicator of the actual volatility of the compound in that particular wine. A study with 20 wines of different types and 1 synthetic sample has revealed the existence of significant differences in the relative volatility of 15 out of 20 odorants. Differences are particularly intense for acetaldehyde and other carbonyls, but are also notable for alcohols and long chain fatty acid ethyl esters. It is expected that these differences, linked likely to sulphur dioxide and some unknown specific compositional aspects of the wine matrix, can be responsible for relevant sensory changes, and may even be the cause explaining why the same aroma composition can produce different aroma perceptions in two different wines. PMID:23102525

  2. Simultaneous analysis of organochlorinated pesticides (OCPs) and polychlorinated biphenyls (PCBs) from marine samples using automated pressurized liquid extraction (PLE) and Power Prep™ clean-up.

    PubMed

    Helaleh, Murad I H; Al-Rashdan, Amal; Ibtisam, A

    2012-05-30

    An automated pressurized liquid extraction (PLE) method followed by Power Prep™ clean-up was developed for organochlorinated pesticide (OCP) and polychlorinated biphenyl (PCB) analysis in environmental marine samples of fish, squid, bivalves, shells, octopus and shrimp. OCPs and PCBs were simultaneously determined in a single chromatographic run using gas chromatography-mass spectrometry-negative chemical ionization (GC-MS-NCI). About 5 g of each biological marine sample was mixed with anhydrous sodium sulphate and placed in the extraction cell of the PLE system. PLE is controlled by means of a PC using DMS 6000 software. Purification of the extract was accomplished using automated Power Prep™ clean-up with a pre-packed disposable silica column (6 g) supplied by Fluid Management Systems (FMS). All OCPs and PCBs were eluted from the silica column using two types of solvent: 80 mL of hexane and a 50 mL mixture of hexane and dichloromethane (1:1). A wide variety of fish and shellfish were collected from the fish market and analyzed using this method. The total PCB concentrations were 2.53, 0.25, 0.24, 0.24, 0.17 and 1.38 ng g(-1) (w/w) for fish, squid, bivalves, shells, octopus and shrimp, respectively, and the corresponding total OCP concentrations were 30.47, 2.86, 0.92, 10.72, 5.13 and 18.39 ng g(-1) (w/w). Lipids were removed using an SX-3 Bio-Beads gel permeation chromatography (GPC) column. Analytical criteria such as recovery, reproducibility and repeatability were evaluated through a range of biological matrices. PMID:22608412

  3. Quantitative radiology: automated measurement of polyp volume in computed tomography colonography using Hessian matrix-based shape extraction and volume growing

    PubMed Central

    Epstein, Mark L.; Obara, Piotr R.; Chen, Yisong; Liu, Junchi; Zarshenas, Amin; Makkinejad, Nazanin; Dachman, Abraham H.

    2015-01-01

    Background Current measurement of the single longest dimension of a polyp is subjective and has variations among radiologists. Our purpose was to develop a computerized measurement of polyp volume in computed tomography colonography (CTC). Methods We developed a 3D automated scheme for measuring polyp volume at CTC. Our scheme consisted of segmentation of colon wall to confine polyp segmentation to the colon wall, extraction of a highly polyp-like seed region based on the Hessian matrix, a 3D volume growing technique under the minimum surface expansion criterion for segmentation of polyps, and sub-voxel refinement and surface smoothing for obtaining a smooth polyp surface. Our database consisted of 30 polyp views (15 polyps) in CTC scans from 13 patients. Each patient was scanned in the supine and prone positions. Polyp sizes measured in optical colonoscopy (OC) ranged from 6-18 mm with a mean of 10 mm. A radiologist outlined polyps in each slice and calculated volumes by summation of volumes in each slice. The measurement study was repeated 3 times at least 1 week apart for minimizing a memory effect bias. We used the mean volume of the three studies as “gold standard”. Results Our measurement scheme yielded a mean polyp volume of 0.38 cc (range, 0.15-1.24 cc), whereas a mean “gold standard” manual volume was 0.40 cc (range, 0.15-1.08 cc). The “gold-standard” manual and computer volumetric reached excellent agreement (intra-class correlation coefficient =0.80), with no statistically significant difference [P (F≤f) =0.42]. Conclusions We developed an automated scheme for measuring polyp volume at CTC based on Hessian matrix-based shape extraction and volume growing. Polyp volumes obtained by our automated scheme agreed excellently with “gold standard” manual volumes. Our fully automated scheme can efficiently provide accurate polyp volumes for radiologists; thus, it would help radiologists improve the accuracy and efficiency of polyp volume

  4. A simple and efficient Triton X-100 boiling and chloroform extraction method of RNA isolation from Gram-positive and Gram-negative bacteria.

    PubMed

    Sung, Kidon; Khan, Saeed A; Nawaz, Mohamed S; Khan, Ashraf A

    2003-12-01

    A fast, reliable, and inexpensive Triton X-100 boiling procedure for RNA isolation from both the Gram-positive and Gram-negative bacteria was developed. The yield of RNA was 0.2-2 mg per 10 ml bacterial culture. The method was tested on Gram-positive and Gram-negative bacteria of eight genera and nine species and yielded reproducible results. In parallel experiments, the Qiagen and hot phenol extraction methods both yielded RNA that contained contaminating 16S and 23S rRNA. The Triton X-100 boiling method reported here yielded RNA that was free from 16S and 23S rRNA, contained full-length transcripts and did not require additional purification. The presence of specific mRNA in one of the RNA samples obtained by this procedure was demonstrated by partial amplification of a 732 bp vancomycin resistance gene, vanA, by reverse transcription-polymerase chain reaction (RT-PCR). The presence of a full-length transcript (1031 bases) of the vanA gene was verified by Northern hybridization and probing with a digoxigenin (DIG)-labeled vanA PCR partial product. The method provides a rapid, reliable, and simple tool for the isolation of good quality RNA suitable for various molecular biology experiments. PMID:14659548

  5. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on.

    PubMed

    Chomczynski, Piotr; Sacchi, Nicoletta

    2006-01-01

    Since its introduction, the 'single-step' method has become widely used for isolating total RNA from biological samples of different sources. The principle at the basis of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol and can be used for several applications. The original protocol, enabling the isolation of RNA from cells and tissues in less than 4 hours, greatly advanced the analysis of gene expression in plant and animal models as well as in pathological samples, as demonstrated by the overwhelming number of citations the paper gained over 20 years. PMID:17406285

  6. Poly(rC) binding protein 2 binds to stem-loop IV of the poliovirus RNA 5' noncoding region: identification by automated liquid chromatography-tandem mass spectrometry.

    PubMed Central

    Blyn, L B; Swiderek, K M; Richards, O; Stahl, D C; Semler, B L; Ehrenfeld, E

    1996-01-01

    The 5' noncoding region of poliovirus RNA contains an internal ribosome entry site (IRES) for cap-independent initiation of translation. Utilization of the IRES requires the participation of one or more cellular proteins that mediate events in the translation initiation reaction, but whose biochemical roles have not been defined. In this report, we identify a cellular RNA binding protein isolated from the ribosomal salt wash of uninfected HeLa cells that specifically binds to stem-loop IV, a domain located in the central part of the poliovirus IRES. The protein was isolated by specific RNA affinity chromatography, and 55% of its sequence was determined by automated liquid chromatography-tandem mass spectrometry. The sequence obtained matched that of poly(rC) binding protein 2 (PCBP2), previously identified as an RNA binding protein from human cells. PCBP2, as well as a related protein, PCBP1, was over-expressed in Escherichia coli after cloning the cDNAs into an expression plasmid to produce a histidine-tagged fusion protein. Specific interaction between recombinant PCBP2 and poliovirus stem-loop IV was demonstrated by RNA mobility shift analysis. The closely related PCBP1 showed no stable interaction with the RNA. Stem-loop IV RNA containing a three nucleotide insertion that abrogates translation activity and virus viability was unable to bind PCBP2. Images Fig. 1 Fig. 2 Fig. 5 Fig. 6 PMID:8855318

  7. SnapShot-Seq: A Method for Extracting Genome-Wide, In Vivo mRNA Dynamics from a Single Total RNA Sample

    PubMed Central

    Boswell, Sarah A.; Cloonan, Nicole; Mullen, Thomas E.; Ling, Joseph J.; Miller, Nimrod; Kuersten, Scott; Ma, Yong-Chao; McCarroll, Steven A.; Grimmond, Sean M.; Springer, Michael

    2014-01-01

    mRNA synthesis, processing, and destruction involve a complex series of molecular steps that are incompletely understood. Because the RNA intermediates in each of these steps have finite lifetimes, extensive mechanistic and dynamical information is encoded in total cellular RNA. Here we report the development of SnapShot-Seq, a set of computational methods that allow the determination of in vivo rates of pre-mRNA synthesis, splicing, intron degradation, and mRNA decay from a single RNA-Seq snapshot of total cellular RNA. SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA. We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors. SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment. PMID:24586954

  8. Facile semi-automated forensic body fluid identification by multiplex solution hybridization of NanoString® barcode probes to specific mRNA targets.

    PubMed

    Danaher, Patrick; White, Robin Lynn; Hanson, Erin K; Ballantyne, Jack

    2015-01-01

    A DNA profile from the perpetrator does not reveal, per se, the circumstances by which it was transferred. Body fluid identification by mRNA profiling may allow extraction of contextual 'activity level' information from forensic samples. Here we describe the development of a prototype multiplex digital gene expression (DGE) method for forensic body fluid/tissue identification based upon solution hybridization of color-coded NanoString(®) probes to 23 mRNA targets. The method identifies peripheral blood, semen, saliva, vaginal secretions, menstrual blood and skin. We showed that a simple 5 min room temperature cellular lysis protocol gave equivalent results to standard RNA isolation from the same source material, greatly enhancing the ease-of-use of this method in forensic sample processing. We first describe a model for gene expression in a sample from a single body fluid and then extend that model to mixtures of body fluids. We then describe calculation of maximum likelihood estimates (MLEs) of body fluid quantities in a sample, and we describe the use of likelihood ratios to test for the presence of each body fluid in a sample. Known single source samples of blood, semen, vaginal secretions, menstrual blood and skin all demonstrated the expected tissue-specific gene expression for at least two of the chosen biomarkers. Saliva samples were more problematic, with their previously identified characteristic genes exhibiting poor specificity. Nonetheless the most specific saliva biomarker, HTN3, was expressed at a higher level in saliva than in any of the other tissues. Crucially, our algorithm produced zero false positives across this study's 89 unique samples. As a preliminary indication of the ability of the method to discern admixtures of body fluids, five mixtures were prepared. The identities of the component fluids were evident from the gene expression profiles of four of the five mixtures. Further optimization of the biomarker 'CodeSet' will be required

  9. Development of an Automated Column Solid-Phase Extraction Cleanup of QuEChERS Extracts, Using a Zirconia-Based Sorbent, for Pesticide Residue Analyses by LC-MS/MS.

    PubMed

    Morris, Bruce D; Schriner, Richard B

    2015-06-01

    A new, automated, high-throughput, mini-column solid-phase extraction (c-SPE) cleanup method for QuEChERS extracts was developed, using a robotic X-Y-Z instrument autosampler, for analysis of pesticide residues in fruits and vegetables by LC-MS/MS. Removal of avocado matrix and recoveries of 263 pesticides and metabolites were studied, using various stationary phase mixtures, including zirconia-based sorbents, and elution with acetonitrile. These experiments allowed selection of a sorbent mixture consisting of zirconia, C18, and carbon-coated silica, that effectively retained avocado matrix but also retained 53 pesticides with <70% recoveries. Addition of MeOH to the elution solvent improved pesticide recoveries from zirconia, as did citrate ions in CEN QuEChERS extracts. Finally, formate buffer in acetonitrile/MeOH (1:1) was required to give >70% recoveries of all 263 pesticides. Analysis of avocado extracts by LC-Q-Orbitrap-MS showed that the method developed was removing >90% of di- and triacylglycerols. The method was validated for 269 pesticides (including homologues and metabolites) in avocado and citrus. Spike recoveries were within 70-120% and 20% RSD for 243 of these analytes in avocado and 254 in citrus, when calibrated against solvent-only standards, indicating effective matrix removal and minimal electrospray ionization suppression. PMID:25702899

  10. Automated detection of feeding strikes by larval fish using continuous high-speed digital video: a novel method to extract quantitative data from fast, sparse kinematic events.

    PubMed

    Shamur, Eyal; Zilka, Miri; Hassner, Tal; China, Victor; Liberzon, Alex; Holzman, Roi

    2016-06-01

    Using videography to extract quantitative data on animal movement and kinematics constitutes a major tool in biomechanics and behavioral ecology. Advanced recording technologies now enable acquisition of long video sequences encompassing sparse and unpredictable events. Although such events may be ecologically important, analysis of sparse data can be extremely time-consuming and potentially biased; data quality is often strongly dependent on the training level of the observer and subject to contamination by observer-dependent biases. These constraints often limit our ability to study animal performance and fitness. Using long videos of foraging fish larvae, we provide a framework for the automated detection of prey acquisition strikes, a behavior that is infrequent yet critical for larval survival. We compared the performance of four video descriptors and their combinations against manually identified feeding events. For our data, the best single descriptor provided a classification accuracy of 77-95% and detection accuracy of 88-98%, depending on fish species and size. Using a combination of descriptors improved the accuracy of classification by ∼2%, but did not improve detection accuracy. Our results indicate that the effort required by an expert to manually label videos can be greatly reduced to examining only the potential feeding detections in order to filter false detections. Thus, using automated descriptors reduces the amount of manual work needed to identify events of interest from weeks to hours, enabling the assembly of an unbiased large dataset of ecologically relevant behaviors. PMID:26994179

  11. A fully automated system for analysis of pesticides in water: on-line extraction followed by liquid chromatography-tandem photodiode array/postcolumn derivatization/fluorescence detection.

    PubMed

    Patsias, J; Papadopoulou-Mourkidou, E

    1999-01-01

    A fully automated system for on-line solid phase extraction (SPE) followed by high-performance liquid chromatography (HPLC) with tandem detection with a photodiode array detector and a fluorescence detector (after postcolumn derivatization) was developed for analysis of many chemical classes of pesticides and their major conversion products in aquatic systems. An automated on-line-SPE system (Prospekt) operated with reversed-phase cartridges (PRP-1) extracts analytes from 100 mL acidified (pH = 3) filtered water sample. On-line HPLC analysis is performed with a 15 cm C18 analytical column eluted with a mobile phase of phosphate (pH = 3)-acetonitrile in 25 min linear gradient mode. Solutes are detected by tandem diode array/derivatization/fluorescence detection. The system is controlled and monitored by a single computer operated with Millenium software. Recoveries of most analytes in samples fortified at 1 microgram/L are > 90%, with relative standard deviation values of < 5%. For a few very polar analytes, mostly N-methylcarbamoyloximes (i.e., aldicarb sulfone, methomyl, and oxamyl), recoveries are < 20%. However, for these compounds, as well as for the rest of the N-methylcarbamates except for aldicarb sulfoxide and butoxycarboxim, the limits of detection (LODs) are 0.005-0.05 microgram/L. LODs for aldicarb sulfoxide and butoxycarboxim are 0.2 and 0.1 microgram, respectively. LODs for the rest of the analytes except 4-nitrophenol, bentazone, captan, decamethrin, and MCPA are 0.05-0.1 microgram/L. LODs for the latter compounds are 0.2-1.0 microgram/L. The system can be operated unattended. PMID:10444834

  12. Comparison of three magnetic-bead-based RNA extraction methods for detection of cucumber green mottle mosaic virus by real-time RT-PCR.

    PubMed

    Zhao, Xiaoli; Zhou, Qi; Zhang, Lijie; Yan, Wenlong; Sun, Ning; Liang, Xinmiao; Deng, Congliang

    2015-07-01

    To determine the efficiency of RNA extraction methods based on magnetic beads, three different bead-based methods (one using silica-coated magnetic beads [SMNP], one using immunomagnetic beads conjugated to a specific antibody [IMB], and one using magnetic beads to nonspecifically adsorb virions [MNP]) were compared with the TRIzol method for the extraction of cucumber green mottle mosaic virus (CGMMV) RNA from cucumber leaves by real-time RT-PCR. The results indicated that the extraction efficiency of the SMNP method was 10 times higher than those of the IMB and MNP methods and 100 times higher than that of the TRIzol method. Therefore, the SMNP method could be considered for use in quarantine measures for the prevention and control of the disease caused by CGMMV. PMID:25951973

  13. A METHOD FOR AUTOMATED ANALYSIS OF 10 ML WATER SAMPLES CONTAINING ACIDIC, BASIC, AND NEUTRAL SEMIVOLATILE COMPOUNDS LISTED IN USEPA METHOD 8270 BY SOLID PHASE EXTRACTION COUPLED IN-LINE TO LARGE VOLUME INJECTION GAS CHROMATOGRAPHY/MASS SPECTROMETRY

    EPA Science Inventory

    Data is presented showing the progress made towards the development of a new automated system combining solid phase extraction (SPE) with gas chromatography/mass spectrometry for the single run analysis of water samples containing a broad range of acid, base and neutral compounds...

  14. Identification of More Feasible MicroRNA–mRNA Interactions within Multiple Cancers Using Principal Component Analysis Based Unsupervised Feature Extraction

    PubMed Central

    Taguchi, Y-h.

    2016-01-01

    MicroRNA(miRNA)–mRNA interactions are important for understanding many biological processes, including development, differentiation and disease progression, but their identification is highly context-dependent. When computationally derived from sequence information alone, the identification should be verified by integrated analyses of mRNA and miRNA expression. The drawback of this strategy is the vast number of identified interactions, which prevents an experimental or detailed investigation of each pair. In this paper, we overcome this difficulty by the recently proposed principal component analysis (PCA)-based unsupervised feature extraction (FE), which reduces the number of identified miRNA–mRNA interactions that properly discriminate between patients and healthy controls without losing biological feasibility. The approach is applied to six cancers: hepatocellular carcinoma, non-small cell lung cancer, esophageal squamous cell carcinoma, prostate cancer, colorectal/colon cancer and breast cancer. In PCA-based unsupervised FE, the significance does not depend on the number of samples (as in the standard case) but on the number of features, which approximates the number of miRNAs/mRNAs. To our knowledge, we have newly identified miRNA–mRNA interactions in multiple cancers based on a single common (universal) criterion. Moreover, the number of identified interactions was sufficiently small to be sequentially curated by literature searches. PMID:27171078

  15. An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7

    PubMed Central

    Holmes, Ashleigh; Birse, Louise; Jackson, Robert W.; Holden, Nicola J.

    2014-01-01

    Analysis of microbial gene expression during host colonization provides valuable information on the nature of interaction, beneficial or pathogenic, and the adaptive processes involved. Isolation of bacterial mRNA for in planta analysis can be challenging where host nucleic acid may dominate the preparation, or inhibitory compounds affect downstream analysis, e.g., quantitative reverse transcriptase PCR (qPCR), microarray, or RNA-seq. The goal of this work was to optimize the isolation of bacterial mRNA of food-borne pathogens from living plants. Reported methods for recovery of phytopathogen-infected plant material, using hot phenol extraction and high concentration of bacterial inoculation or large amounts of infected tissues, were found to be inappropriate for plant roots inoculated with Escherichia coli O157:H7. The bacterial RNA yields were too low and increased plant material resulted in a dominance of plant RNA in the sample. To improve the yield of bacterial RNA and reduce the number of plants required, an optimized method was developed which combines bead beating with directed bacterial lysis using SDS and lysozyme. Inhibitory plant compounds, such as phenolics and polysaccharides, were counteracted with the addition of high-molecular-weight polyethylene glycol and hexadecyltrimethyl ammonium bromide. The new method increased the total yield of bacterial mRNA substantially and allowed assessment of gene expression by qPCR. This method can be applied to other bacterial species associated with plant roots, and also in the wider context of food safety. PMID:25018749

  16. Automated solid-phase extraction and liquid chromatography-electrospray ionization-mass spectrometry for the determination of flunitrazepam and its metabolites in human urine and plasma samples.

    PubMed

    Jourdil, N; Bessard, J; Vincent, F; Eysseric, H; Bessard, G

    2003-05-25

    A sensitive and specific method using reversed-phase liquid chromatography coupled with electrospray ionization-mass spectrometry (LC-ESI-MS) has been developed for the quantitative determination of flunitrazepam (F) and its metabolites 7-aminoflunitrazepam (7-AF), N-desmethylflunitrazepam (N-DMF) and 3-hydroxyflunitrazepam (3-OHF) in biological fluids. After the addition of deuterium labelled standards of F,7-AF and N-DMF, the drugs were isolated from urine or plasma by automated solid-phase extraction, then chromatographed in an isocratic elution mode with a salt-free eluent. The quantification was performed using selected ion monitoring of protonated molecular ions (M+H(+)). Experiments were carried out to improve the extraction recovery (81-100%) and the sensitivity (limit of detection 0.025 ng/ml for F and 7-AF, 0.040 ng/ml for N-DMF and 0.200 ng/ml for 3-OHF). The method was applied to the determination of F and metabolites in drug addicts including withdrawal urine samples and in one date-rape plasma and urine sample. PMID:12705961

  17. Comparison of Two Commercial Automated Nucleic Acid Extraction and Integrated Quantitation Real-Time PCR Platforms for the Detection of Cytomegalovirus in Plasma

    PubMed Central

    Tsai, Huey-Pin; Tsai, You-Yuan; Lin, I-Ting; Kuo, Pin-Hwa; Chen, Tsai-Yun; Chang, Kung-Chao; Wang, Jen-Ren

    2016-01-01

    Quantitation of cytomegalovirus (CMV) viral load in the transplant patients has become a standard practice for monitoring the response to antiviral therapy. The cut-off values of CMV viral load assays for preemptive therapy are different due to the various assay designs employed. To establish a sensitive and reliable diagnostic assay for preemptive therapy of CMV infection, two commercial automated platforms including m2000sp extraction system integrated the Abbott RealTime (m2000rt) and the Roche COBAS AmpliPrep for extraction integrated COBAS Taqman (CAP/CTM) were evaluated using WHO international CMV standards and 110 plasma specimens from transplant patients. The performance characteristics, correlation, and workflow of the two platforms were investigated. The Abbott RealTime assay correlated well with the Roche CAP/CTM assay (R2 = 0.9379, P<0.01). The Abbott RealTime assay exhibited higher sensitivity for the detection of CMV viral load, and viral load values measured with Abbott RealTime assay were on average 0.76 log10 IU/mL higher than those measured with the Roche CAP/CTM assay (P<0.0001). Workflow analysis on a small batch size at one time, using the Roche CAP/CTM platform had a shorter hands-on time than the Abbott RealTime platform. In conclusion, these two assays can provide reliable data for different purpose in a clinical virology laboratory setting. PMID:27494707

  18. Comparison of Two Automated Solid Phase Extractions for the Detection of Ten Fentanyl Analogs and Metabolites in Human Urine Using Liquid Chromatography Tandem Mass Spectrometry

    PubMed Central

    Shaner, Rebecca L.; Kaplan, Pearl; Hamelin, Elizabeth I.; Bragg, William; Johnson, Rudolph C.

    2016-01-01

    Two types of automated solid phase extraction (SPE) were assessed for the determination of human exposure to fentanyls in urine. High sensitivity is required to detect these compounds following exposure because of the low dose required for therapeutic effect and the rapid clearance from the body for these compounds. To achieve this sensitivity, two acceptable methods for the detection of human exposure to seven fentanyl analogs and three metabolites were developed using either off-line 96-well plate SPE or on-line SPE. Each system offers different advantages: off-line 96-well plate SPE allows for high throughput analysis of many samples, which is needed for large sample numbers, while on-line SPE removes almost all analyst manipulation of the samples, minimizing the analyst time needed for sample preparation. Both sample preparations were coupled with reversed phase liquid chromatography and isotope dilution tandem mass spectrometry (LC-MS/MS) for analyte detection. For both methods, the resulting precision was within 15%, the accuracy within 25%, and the sensitivity was comparable with the limits of detection ranging from 0.002-0.041ng/mL. Additionally, matrix effects were substantially decreased from previous reports for both extraction protocols. The results of this comparison showed that both methods were acceptable for the detection of exposures to fentanyl analogs and metabolites in urine. PMID:24893271

  19. Regenerable immuno-biochip for screening ochratoxin A in green coffee extract using an automated microarray chip reader with chemiluminescence detection.

    PubMed

    Sauceda-Friebe, Jimena C; Karsunke, Xaver Y Z; Vazac, Susanna; Biselli, Scarlett; Niessner, Reinhard; Knopp, Dietmar

    2011-03-18

    Ochratoxin A (OTA) can contaminate foodstuffs in the ppb to ppm range and once formed, it is difficult to remove. Because of its toxicity and potential risks to human health, the need exists for rapid, efficient detection methods that comply with legal maximum residual limits. In this work we have synthesized an OTA conjugate functionalized with a water-soluble peptide for covalent immobilization on a glass biochip by means of contact spotting. The chip was used for OTA determination with an indirect competitive immunoassay format with flow-through reagent addition and chemiluminescence detection, carried out with the stand-alone automated Munich Chip Reader 3 (MCR 3) platform. A buffer model and real green coffee extracts were used for this purpose. At the present, covalent conjugate immobilization allowed for at least 20 assay-regeneration cycles of the biochip surface. The total analysis time for a single sample, including measurement and surface regeneration, was 12 min and the LOQ of OTA in green coffee extract was 0.3 μg L(-1) which corresponds to 7 μg kg(-1). PMID:21397079

  20. An automated flow injection system for metal determination by flame atomic absorption spectrometry involving on-line fabric disk sorptive extraction technique.

    PubMed

    Anthemidis, A; Kazantzi, V; Samanidou, V; Kabir, A; Furton, K G

    2016-08-15

    A novel flow injection-fabric disk sorptive extraction (FI-FDSE) system was developed for automated determination of trace metals. The platform was based on a minicolumn packed with sol-gel coated fabric media in the form of disks, incorporated into an on-line solid-phase extraction system, coupled with flame atomic absorption spectrometry (FAAS). This configuration provides minor backpressure, resulting in high loading flow rates and shorter analytical cycles. The potentials of this technique were demonstrated for trace lead and cadmium determination in environmental water samples. The applicability of different sol-gel coated FPSE media was investigated. The on-line formed complex of metal with ammonium pyrrolidine dithiocarbamate (APDC) was retained onto the fabric surface and methyl isobutyl ketone (MIBK) was used to elute the analytes prior to atomization. For 90s preconcentration time, enrichment factors of 140 and 38 and detection limits (3σ) of 1.8 and 0.4μgL(-1) were achieved for lead and cadmium determination, respectively, with a sampling frequency of 30h(-1). The accuracy of the proposed method was estimated by analyzing standard reference materials and spiked water samples. PMID:27260436

  1. Automated evaluation of electronic discharge notes to assess quality of care for cardiovascular diseases using Medical Language Extraction and Encoding System (MedLEE)

    PubMed Central

    Lin, Jou-Wei; Yang, Chen-Wei

    2010-01-01

    The objective of this study was to develop and validate an automated acquisition system to assess quality of care (QC) measures for cardiovascular diseases. This system combining searching and retrieval algorithms was designed to extract QC measures from electronic discharge notes and to estimate the attainment rates to the current standards of care. It was developed on the patients with ST-segment elevation myocardial infarction and tested on the patients with unstable angina/non-ST-segment elevation myocardial infarction, both diseases sharing almost the same QC measures. The system was able to reach a reasonable agreement (κ value) with medical experts from 0.65 (early reperfusion rate) to 0.97 (β-blockers and lipid-lowering agents before discharge) for different QC measures in the test set, and then applied to evaluate QC in the patients who underwent coronary artery bypass grafting surgery. The result has validated a new tool to reliably extract QC measures for cardiovascular diseases. PMID:20442141

  2. Establishment of a rapid, inexpensive protocol for extraction of high quality RNA from small amounts of strawberry plant tissues and other recalcitrant fruit crops.

    PubMed

    Christou, Anastasis; Georgiadou, Egli C; Filippou, Panagiota; Manganaris, George A; Fotopoulos, Vasileios

    2014-03-01

    Strawberry plant tissues and particularly fruit material are rich in polysaccharides and polyphenolic compounds, thus rendering the isolation of nucleic acids a difficult task. This work describes the successful modification of a total RNA extraction protocol, which enables the isolation of high quantity and quality of total RNA from small amounts of strawberry leaf, root and fruit tissues. Reverse-transcription polymerase chain reaction (RT-PCR) amplification of GAPDH housekeeping gene from isolated RNA further supports the proposed protocol efficiency and its use for downstream molecular applications. This novel procedure was also successfully followed using other fruit tissues, such as olive and kiwifruit. In addition, optional treatment with RNase A following initial nucleic acid extraction can provide sufficient quality and quality of genomic DNA for subsequent PCR analyses, as evidenced from PCR amplification of housekeeping genes using extracted genomic DNA as template. Overall, this optimized protocol allows easy, rapid and economic isolation of high quality RNA from small amounts of an important fruit crop, such as strawberry, with extended applicability to other recalcitrant fruit crops. PMID:24321691

  3. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types

    PubMed Central

    Lever, Mark A.; Torti, Andrea; Eickenbusch, Philip; Michaud, Alexander B.; Šantl-Temkiv, Tina; Jørgensen, Bo Barker

    2015-01-01

    A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world's oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample types and research goals. PMID:26042110

  4. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types.

    PubMed

    Lever, Mark A; Torti, Andrea; Eickenbusch, Philip; Michaud, Alexander B; Šantl-Temkiv, Tina; Jørgensen, Bo Barker

    2015-01-01

    A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world's oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample types and research goals. PMID:26042110

  5. Semi-automated extraction and delineation of 3D roads of street scene from mobile laser scanning point clouds

    NASA Astrophysics Data System (ADS)

    Yang, Bisheng; Fang, Lina; Li, Jonathan

    2013-05-01

    Accurate 3D road information is important for applications such as road maintenance and virtual 3D modeling. Mobile laser scanning (MLS) is an efficient technique for capturing dense point clouds that can be used to construct detailed road models for large areas. This paper presents a method for extracting and delineating roads from large-scale MLS point clouds. The proposed method partitions MLS point clouds into a set of consecutive "scanning lines", which each consists of a road cross section. A moving window operator is used to filter out non-ground points line by line, and curb points are detected based on curb patterns. The detected curb points are tracked and refined so that they are both globally consistent and locally similar. To evaluate the validity of the proposed method, experiments were conducted using two types of street-scene point clouds captured by Optech's Lynx Mobile Mapper System. The completeness, correctness, and quality of the extracted roads are over 94.42%, 91.13%, and 91.3%, respectively, which proves the proposed method is a promising solution for extracting 3D roads from MLS point clouds.

  6. Increased interleukin-1beta mRNA expression in skin biopsies of horses with Culicoides hypersensitivity following challenge with Culicoides nubeculosus extract.

    PubMed

    Kolm, Gabriela; Knapp, Elzbieta; Wagner, Regina; Klein, Dieter

    2006-09-15

    Interleukin-1beta (IL-1beta) is a primary cytokine of the skin that has a pivotal role in keratinocyte differentiation, epidermal wound healing and host defense. Pathological increase of cutaneous IL-1beta is associated with edema formation, epidermal hyperproliferation and atopic dermatitis in humans. However, in horses the role of cutaneous IL-1beta in edema formation and allergic skin disease has not been characterised so far. Particularly in Culicoides hypersensitivity (CHS), intradermal injection of Culicoides extract may be associated with enhanced transcription of local IL-1beta. To examine the mRNA expression of IL-1beta and its receptor antagonist IL-1RA in the skin of horses, biopsy specimens of horses affected and non-affected by CHS prior and following intradermal challenge with a commercial C. nubeculosus extract were examined. Our hypothesis was that cutaneous IL-1beta mRNA was significantly upregulated in horses with CHS in response to Culicoides allergen. Biopsies were taken from sites prior to and 4 h following intradermal challenge with C. nubeculosus extract. In order to obtain reliable data, real time PCR was performed and genes of interest were normalized using three different housekeeping genes, beta-actin, GAPDH, beta-2-microglobulin. No significant difference was detected in non-challenged cutaneous IL-1beta mRNA and IL-1RA mRNA levels between CHS affected and non-affected horses. Intradermal injection of C. nubeculosus extract resulted in local upregulation of IL-1beta mRNA both in horses with typical history, characteristic clinical signs for CHS and a positive intradermal skin test (IDT), and non-affected horses with a negative IDT. However, the difference in prior and post challenged site IL-1beta mRNA levels only reached statistical significance in the affected horses (p=0.01 versus 0.7). In contrast, IL-1RA mRNA levels did not demonstrate any modification following intradermal injection with C. nubeculosus in either group. In contrast

  7. Automated on-line renewable solid-phase extraction-liquid chromatography exploiting multisyringe flow injection-bead injection lab-on-valve analysis.

    PubMed

    Quintana, José Benito; Miró, Manuel; Estela, José Manuel; Cerdà, Víctor

    2006-04-15

    In this paper, the third generation of flow injection analysis, also named the lab-on-valve (LOV) approach, is proposed for the first time as a front end to high-performance liquid chromatography (HPLC) for on-line solid-phase extraction (SPE) sample processing by exploiting the bead injection (BI) concept. The proposed microanalytical system based on discontinuous programmable flow features automated packing (and withdrawal after single use) of a small amount of sorbent (<5 mg) into the microconduits of the flow network and quantitative elution of sorbed species into a narrow band (150 microL of 95% MeOH). The hyphenation of multisyringe flow injection analysis (MSFIA) with BI-LOV prior to HPLC analysis is utilized for on-line postextraction treatment to ensure chemical compatibility between the eluate medium and the initial HPLC gradient conditions. This circumvents the band-broadening effect commonly observed in conventional on-line SPE-based sample processors due to the low eluting strength of the mobile phase. The potential of the novel MSFI-BI-LOV hyphenation for on-line handling of complex environmental and biological samples prior to reversed-phase chromatographic separations was assessed for the expeditious determination of five acidic pharmaceutical residues (viz., ketoprofen, naproxen, bezafibrate, diclofenac, and ibuprofen) and one metabolite (viz., salicylic acid) in surface water, urban wastewater, and urine. To this end, the copolymeric divinylbenzene-co-n-vinylpyrrolidone beads (Oasis HLB) were utilized as renewable sorptive entities in the micromachined unit. The automated analytical method features relative recovery percentages of >88%, limits of detection within the range 0.02-0.67 ng mL(-1), and coefficients of variation <11% for the column renewable mode and gives rise to a drastic reduction in operation costs ( approximately 25-fold) as compared to on-line column switching systems. PMID:16615800

  8. Fully automated analysis of beta-lactams in bovine milk by online solid phase extraction-liquid chromatography-electrospray-tandem mass spectrometry.

    PubMed

    Kantiani, Lina; Farré, Marinella; Sibum, Martin; Postigo, Cristina; López de Alda, Miren; Barceló, Damiá

    2009-06-01

    A fully automated method for the detection of beta-lactam antibiotics, including six penicillins (amoxicillin, ampicillin, cloxacillin, dicloxacillin, oxacillin, and penicillin G) and four cephalosporins (cefazolin, ceftiofur, cefoperazone, and cefalexin) in bovine milk samples has been developed. The outlined method is based on online solid-phase extraction-liquid chromatography/electrospray-tandem mass spectrometry (SPE-LC/ESI-MS-MS). Target compounds were concentrated from 500 microL of centrifuged milk samples using an online SPE procedure with C18 HD cartridges. Target analytes were eluted with a gradient mobile phase (water + 0.1% formic acid/methanol + 0.1% formic acid) at a flow rate of 0.7 mL/min. Chromatographic separation was achieved within 10 min using a C-12 reversed phase analytical column. For unequivocal identification and confirmation, two multiple reaction monitoring (MRM) transitions were acquired for each analyte in the positive electrospray ionization mode (ESI(+)). Method limits of detection (LODs) in milk were well below the maximum residue limits (MRLs) set by the European Union for all compounds. Limits of quantification in milk were between 0.09 ng/mL and 1.44 ng/mL. The developed method was validated according to EU's requirements, and accuracy results ranged from 80 to 116%. Finally, the method was applied to the analysis of twenty real samples previously screened by the inhibition of microbial growth test Eclipse 100. This new developed method offers high sensitivity and accuracy of results, minimum sample pre-treatment, and uses for the first time an automated online SPE offering a high throughput analysis. Because of all these characteristics, the proposed method is applicable and could be deemed necessary within the field of food control and safety. PMID:19402673

  9. A molecular exploration of human DNA/RNA co-extracted from the palmar surface of the hands and fingers.

    PubMed

    Lacerenza, D; Aneli, S; Omedei, M; Gino, S; Pasino, S; Berchialla, P; Robino, C

    2016-05-01

    "Touch DNA" refers to the DNA that is left behind when a person touches or comes into contact with an item. However, the source of touch DNA is still debated and the large variability in DNA yield from casework samples suggests that, besides skin, various body fluids can be transferred through contact. Another important issue concerning touch DNA is the possible occurrence of secondary transfer, but the data published in the literature in relation to the background levels of foreign DNA present on the hand surfaces of the general population are very limited. As the present study aimed at better understanding the nature and characteristics of touch DNA, samples were collected from the palmar surface of the hands and fingers ("PHF" samples) of 30 male and 30 female donors by tape-lifting/swabbing and subjected to DNA/RNA co-extraction. Multiplex mRNA profiling showed that cellular material different from skin could be observed in 15% of the PHF samples. The total amount of DNA recovered from these samples (median 5.1ng) was significantly higher than that obtained from samples containing skin cells only (median 1.6ng). The integrity of the DNA isolated from the donors' hands and fingers as well as the prevalence of DNA mixtures were evaluated by STR typing and compared with reference STR profiles from buccal swabs. DNA integrity appeared significantly higher in the male rather than in the female subsample, as the average percentage of the donors' alleles effectively detected in PHF profiles was 75.1% and 60.1%, respectively. The prevalence of mixtures with a foreign DNA contribution ≥20% was 19.2% (30.0% in the female PHF samples and 8.3% in the male PHF samples). The obtained results support the hypothesis that transfer of cellular material different from skin may underlie the occasional recovery of quality STR profiles from handled items. These results also suggest that gender may represent an important factor influencing the propensity of individuals to carry and

  10. Automated data analysis.

    NASA Astrophysics Data System (ADS)

    Teuber, D.

    Automated data analysis assists the astronomer in the decision making processes applied for extracting astronomical information from data. Automated data analysis is the step between image processing and model interpretation. Tools developed in AI are applied (classification, expert system). Programming languages and computers are chosen to fulfil the increasing requirements. Expert systems have begun in astronomy. Data banks permit the astronomical community to share the large body of resulting information.

  11. Determination of pyronaridine in whole blood by automated solid phase extraction and high-performance liquid chromatography.

    PubMed

    Blessborn, Daniel; Lindegårdh, Niklas; Ericsson, Orjan; Hellgren, Urban; Bergqvist, Yngve

    2003-06-01

    A new extraction procedure for the analysis of pyronaridine in whole blood is presented. A weak cation exchanger with a carboxylic acid (CBA) sorbent was found to be a suitable solid phase sorbent for the extraction of pyronaridine. High-performance liquid chromatography with UV detection at 278 nm and an electrochemical detector at +0.75 V is used. The electrochemical detector gives higher selectivity than the UV detector. The separation was performed using a C18 reversed phase column with mobile phase of acetonitrile-phosphate buffer (0.01 mol/L, pH 2.5)- sodium perchlorate (1.0 mol/L; 22:77:1, v/v/v). The within-day RSDs were below 5% at all concentration levels between 75 nmol/L and 1500 nmol/L, and the between-day RSDs were below 14% at all concentration levels. The limit of quantification was about 50 nmol/L in 1000 microL whole blood with an RSD of 20% or less on a day-to-day basis. The stability of pyronaridine is increased if the pH is less than 3 in water solutions. In whole blood, the concentration decreases by about 10% for each freeze-thaw cycle performed. At room temperature (about 22 degrees C), pyronaridine concentration in whole blood decreases by about 10% within 12 to 24 hours. PMID:12766551

  12. Revealing Dimensions of Thinking in Open-Ended Self-Descriptions: An Automated Meaning Extraction Method for Natural Language

    PubMed Central

    2008-01-01

    A new method for extracting common themes from written text is introduced and applied to 1,165 open-ended self-descriptive narratives. Drawing on a lexical approach to personality, the most commonly-used adjectives within narratives written by college students were identified using computerized text analytic tools. A factor analysis on the use of these adjectives in the self-descriptions produced a 7-factor solution consisting of psychologically meaningful dimensions. Some dimensions were unipolar (e.g., Negativity factor, wherein most loaded items were negatively valenced adjectives); others were dimensional in that semantically opposite words clustered together (e.g., Sociability factor, wherein terms such as shy, outgoing, reserved, and loud all loaded in the same direction). The factors exhibited modest reliability across different types of writ writing samples and were correlated with self-reports and behaviors consistent with the dimensions. Similar analyses with additional content words (adjectives, adverbs, nouns, and verbs) yielded additional psychological dimensions associated with physical appearance, school, relationships, etc. in which people contextualize their self-concepts. The results suggest that the meaning extraction method is a promising strategy that determines the dimensions along which people think about themselves. PMID:18802499

  13. Isolation of DNA after Extraction of RNA To Detect the Presence of Borrelia burgdorferi and Expression of Host Cellular Genes from the Same Tissue Sample

    PubMed Central

    Amemiya, Kei; Schaefer, Henry; Pachner, Andrew R.

    1999-01-01

    We are investigating the neuropathogenesis of Lyme disease caused by Borrelia burgdorferi in a nonhuman primate model. In the past, two separate pieces of tissue had to be used when both analyzing for the presence of the spirochete and examining the host response to infection. We have modified a procedure to purify DNA from the same sample after the extraction of RNA. The remaining material containing the DNA was precipitated, and residual organic reagent was removed prior to deproteinization and extraction of the DNA. This procedure now allows us to both assay for the presence of the Lyme microorganism and analyze the host response in the same tissue preparation. PMID:10325389

  14. Protocol: high throughput silica-based purification of RNA from Arabidopsis seedlings in a 96-well format

    PubMed Central

    2011-01-01

    The increasing popularity of systems-based approaches to plant research has resulted in a demand for high throughput (HTP) methods to be developed. RNA extraction from multiple samples in an experiment is a significant bottleneck in performing systems-level genomic studies. Therefore we have established a high throughput method of RNA extraction from Arabidopsis thaliana to facilitate gene expression studies in this widely used plant model. We present optimised manual and automated protocols for the extraction of total RNA from 9-day-old Arabidopsis seedlings in a 96 well plate format using silica membrane-based methodology. Consistent and reproducible yields of high quality RNA are isolated averaging 8.9 μg total RNA per sample (~20 mg plant tissue). The purified RNA is suitable for subsequent qPCR analysis of the expression of over 500 genes in triplicate from each sample. Using the automated procedure, 192 samples (2 × 96 well plates) can easily be fully processed (samples homogenised, RNA purified and quantified) in less than half a day. Additionally we demonstrate that plant samples can be stored in RNAlater at -20°C (but not 4°C) for 10 months prior to extraction with no significant effect on RNA yield or quality. Additionally, disrupted samples can be stored in the lysis buffer at -20°C for at least 6 months prior to completion of the extraction procedure providing a flexible sampling and storage scheme to facilitate complex time series experiments. PMID:22136293

  15. Antioxidant Activity and Induction of mRNA Expressions of Antioxidant Enzymes in HEK-293 Cells of Moringa oleifera Leaf Extract.

    PubMed

    Vongsak, Boonyadist; Mangmool, Supachoke; Gritsanapan, Wandee

    2015-08-01

    The leaves of Moringa oleifera, collected in different provinces in Thailand, were determined for the contents of total phenolics, total flavonoids, major components, and antioxidant activity. The extract and its major active components were investigated for the inhibition of H2O2-induced reactive oxygen species production and the effects on antioxidant enzymes mRNA expression. The extract, crypto-chlorogenic acid, isoquercetin and astragalin, significantly reduced the reactive oxygen species production inducing by H2O2 in HEK-293 cells. Treatment with isoquercetin significantly increased the mRNA expression levels of antioxidant enzymes such as superoxide dismutase, catalase and heme oxygenase 1. These results confirm that M. oleifera leaves are good sources of natural antioxidant with isoquercetin as an active compound. PMID:26166137

  16. Automated and quantitative headspace in-tube extraction for the accurate determination of highly volatile compounds from wines and beers.

    PubMed

    Zapata, Julián; Mateo-Vivaracho, Laura; Lopez, Ricardo; Ferreira, Vicente

    2012-03-23

    An automatic headspace in-tube extraction (ITEX) method for the accurate determination of acetaldehyde, ethyl acetate, diacetyl and other volatile compounds from wine and beer has been developed and validated. Method accuracy is based on the nearly quantitative transference of volatile compounds from the sample to the ITEX trap. For achieving that goal most methodological aspects and parameters have been carefully examined. The vial and sample sizes and the trapping materials were found to be critical due to the pernicious saturation effects of ethanol. Small 2 mL vials containing very small amounts of sample (20 μL of 1:10 diluted sample) and a trap filled with 22 mg of Bond Elut ENV resins could guarantee a complete trapping of sample vapors. The complete extraction requires 100 × 0.5 mL pumping strokes at 60 °C and takes 24 min. Analytes are further desorbed at 240 °C into the GC injector under a 1:5 split ratio. The proportion of analytes finally transferred to the trap ranged from 85 to 99%. The validation of the method showed satisfactory figures of merit. Determination coefficients were better than 0.995 in all cases and good repeatability was also obtained (better than 7% in all cases). Reproducibility was better than 8.3% except for acetaldehyde (13.1%). Detection limits were below the odor detection thresholds of these target compounds in wine and beer and well below the normal ranges of occurrence. Recoveries were not significantly different to 100%, except in the case of acetaldehyde. In such a case it could be determined that the method is not able to break some of the adducts that this compound forms with sulfites. However, such problem was avoided after incubating the sample with glyoxal. The method can constitute a general and reliable alternative for the analysis of very volatile compounds in other difficult matrixes. PMID:22340891

  17. Protein–RNA specificity by high-throughput principal component analysis of NMR spectra

    PubMed Central

    Collins, Katherine M.; Oregioni, Alain; Robertson, Laura E.; Kelly, Geoff; Ramos, Andres

    2015-01-01

    Defining the RNA target selectivity of the proteins regulating mRNA metabolism is a key issue in RNA biology. Here we present a novel use of principal component analysis (PCA) to extract the RNA sequence preference of RNA binding proteins. We show that PCA can be used to compare the changes in the nuclear magnetic resonance (NMR) spectrum of a protein upon binding a set of quasi-degenerate RNAs and define the nucleobase specificity. We couple this application of PCA to an automated NMR spectra recording and processing protocol and obtain an unbiased and high-throughput NMR method for the analysis of nucleobase preference in protein–RNA interactions. We test the method on the RNA binding domains of three important regulators of RNA metabolism. PMID:25586222

  18. The ValleyMorph Tool: An automated extraction tool for transverse topographic symmetry (T-) factor and valley width to valley height (Vf-) ratio

    NASA Astrophysics Data System (ADS)

    Daxberger, Heidi; Dalumpines, Ron; Scott, Darren M.; Riller, Ulrich

    2014-09-01

    In tectonically active regions on Earth, shallow-crustal deformation associated with seismic hazards may pose a threat to human life and property. The study of landform development, such as analysis of the valley width to valley height ratio (Vf-ratio) and the Transverse Topographic Symmetry Factor (T-factor), delineating drainage basin symmetry, can be used as a relative measure of tectonic activity along fault-bound mountain fronts. The fast evolution of digital elevation models (DEM) provides an ideal base for remotely-sensed tectonomorphic studies of large areas using Geographical Information Systems (GIS). However, a manual extraction of the above mentioned morphologic parameters may be tedious and very time consuming. Moreover, basic GIS software suites do not provide the necessary built-in functions. Therefore, we present a newly developed, Python based, ESRI ArcGIS compatible tool and stand-alone script, the ValleyMorph Tool. This tool facilitates an automated extraction of the Vf-ratio and the T-factor data for large regions. Using a digital elevation raster and watershed polygon files as input, the tool provides output in the form of several ArcGIS data tables and shapefiles, ideal for further data manipulation and computation. This coding enables an easy application among the ArcGIS user community and code conversion to earlier ArcGIS versions. The ValleyMorph Tool is easy to use due to a simple graphical user interface. The tool is tested for the southern Central Andes using a total of 3366 watersheds.

  19. Semi-automated Technique to Extract Boundary of Valley/mountain Glaciers using Glacio-morphological Information from Digital Elevation Model

    NASA Astrophysics Data System (ADS)

    Chakraborty, M.; Panigrahy, S.; Kundu, S.

    2014-11-01

    A semi automated technique has been developed to extract the spatial extension of valleys and mountain glaciers. The method is based on morphological properties of glaciated area extracted from Digital Elevation Model (DEM). Identification of glacial boundary based on spectral information from optical remote sensing imageries produces errors due to misclassification of debris-covered ablation area with surrounding rocky terrain and perennially snow-covered slope with debris free glaciated area. Elevation information DEM of Shuttle Radar Topography Mission (SRTM), CartoDEM and ASTER DEM have been used. A part of western Himalayas was selected as the study area that contains large glaciated basins, e.g., Bhagirathi, Baspa, Chandra basin. First order derivatives, slope aspect, and second order derivatives like, profile and plan curvatures are computed from the DEM. The derivatives are used to quantify and characterise the morphological aspects of the glaciated area and used in the decision rule models to generate the glacial boundaries. The ridge lines of the study areas are also generated from the plan curvature and used in the model to delineate the catchments areas of the glaciers. The slope based boundary is checked for consistency with the boundary from profile curvature and combined manually to generate the final glacier boundary. Area and length under the derived boundary of Gangotri glacier of Bhagirathi catchments are 90.25 sq km and 30.5 km. The result has been checked with high resolution optical data. This objective approach is important to delineate glaciated area, measure the length, width and area and generate glacial hypsometry, concentration factor of the glaciers. Accuracy of the result depends up on the quality of the DEM. DEM generated by SAR interferometric technique is found superior over DEM generated from other interpolation techniques.

  20. Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples

    PubMed Central

    Carroll, Paul; Donatello, Simona; Connolly, Elizabeth; Griffin, Mairead; Dunne, Barbara; Burke, Louise; Flavin, Richard; Rizkalla, Hala; Ryan, Ciara; Hayes, Brian; D'Adhemar, Charles; Banville, Niamh; Faheem, Nazia; Muldoon, Cian; Gaffney, Eoin F.

    2011-01-01

    The Saint James's Hospital Biobank was established in 2008, to develop a high-quality breast tissue BioResource, as a part of the breast cancer clinical care pathway. The aims of this work were: (1) to ascertain the quality of RNA, DNA, and protein in biobanked carcinomas and normal breast tissues, (2) to assess the efficacy of AllPrep® (Qiagen) in isolating RNA, DNA, and protein simultaneously, (3) to compare AllPrep with RNEasy® and QIAamp® (both Qiagen), and (4) to examine the effectiveness of Allprotect® (Qiagen), a new tissue stabilization medium in preserving DNA, RNA, and proteins. One hundred eleven frozen samples of carcinoma and normal breast tissue were analyzed. Tumor and normal tissue morphology were confirmed by frozen sections. Tissue type, tissue treatment (Allprotect vs. no Allprotect), extraction kit, and nucleic acid quantification were analyzed by utilizing a 4 factorial design (SPSS PASW 18 Statistics Software®). QIAamp (DNA isolation), AllPrep (DNA, RNA, and Protein isolation), and RNeasy (RNA isolation) kits were assessed and compared. Mean DNA yield and A260/280 values using QIAamp were 33.2 ng/μL and 1.86, respectively, and using AllPrep were 23.2 ng/μL and 1.94. Mean RNA yield and RNA Integrity Number (RIN) values with RNeasy were 73.4 ng/μL and 8.16, respectively, and with AllPrep were 74.8 ng/μL and 7.92. Allprotect-treated tissues produced higher RIN values of borderline significance (P=0.055). No discernible loss of RNA stability was detected after 6 h incubation of stabilized or nonstabilized tissues at room temperature or 4°C or in 9 freeze-thaw cycles. Allprotect requires further detailed evaluation, but we consider AllPrep to be an excellent option for the simultaneous extraction of RNA, DNA, and protein from tumor and normal breast tissues. The essential presampling procedures that maintain the diagnostic integrity of pathology specimens do not appear to compromise the quality of molecular isolates. PMID

  1. Extraction of total nucleic acid based on silica-coated magnetic particles for RT-qPCR detection of plant RNA virus/viroid.

    PubMed

    Sun, Ning; Deng, Congliang; Zhao, Xiaoli; Zhou, Qi; Ge, Guanglu; Liu, Yi; Yan, Wenlong; Xia, Qiang

    2014-02-01

    In this study, a nucleic acid extraction method based on silica-coated magnetic particles (SMPs) and RT-qPCR assay was developed to detect Arabis mosaic virus (ArMV), Lily symptomless virus (LSV), Hop stunt viroid (HSVd) and grape yellow speckle viroid 1 (GYSVd-1). The amplification sequences of RT-qPCR were reversely transcribed in vitro as RNA standard templates. The standard curves covered six or seven orders of magnitude with a detection limit of 100 copies per each assay. Extraction efficiency of the SMPs method was evaluated by recovering spiked ssRNAs from plant samples and compared to two commercial kits (TRIzol and RNeasy Plant mini kit). Results showed that the recovery rate of SMPs method was comparable to the commercial kits when spiked ssRNAs were extracted from lily leaves, whereas it was two or three times higher than commercial kits when spiked ssRNAs were extracted from grapevine leaves. SMPs method was also used to extract viral nucleic acid from15 ArMV-positive lily leaf samples and 15 LSV-positive lily leaf samples. SMPs method did not show statistically significant difference from other methods on detecting ArMV, but LSV. The SMPs method has the same level of virus load as the TRIzol, and its mean virus load of was 0.5log10 lower than the RNeasy Plant mini kit. Nucleic acid was extracted from 19 grapevine-leaf samples with SMPs and the two commercial kits and subsequently screened for HSVd and GYSVd-1 by RT-qPCR. Regardless of HSVd or GYSVd-1, SMPs method outperforms other methods on both positive rate and the viroid load. In conclusion, SMPs method was able to efficiently extract the nucleic acid of RNA viruses or viroids, especially grapevine viroids, from lily-leaf or grapevine-leaf samples for RT-qPCR detection. PMID:24291163

  2. A new adaptive algorithm for automated feature extraction in exponentially damped signals for health monitoring of smart structures

    NASA Astrophysics Data System (ADS)

    Qarib, Hossein; Adeli, Hojjat

    2015-12-01

    In this paper authors introduce a new adaptive signal processing technique for feature extraction and parameter estimation in noisy exponentially damped signals. The iterative 3-stage method is based on the adroit integration of the strengths of parametric and nonparametric methods such as multiple signal categorization, matrix pencil, and empirical mode decomposition algorithms. The first stage is a new adaptive filtration or noise removal scheme. The second stage is a hybrid parametric-nonparametric signal parameter estimation technique based on an output-only system identification technique. The third stage is optimization of estimated parameters using a combination of the primal-dual path-following interior point algorithm and genetic algorithm. The methodology is evaluated using a synthetic signal and a signal obtained experimentally from transverse vibrations of a steel cantilever beam. The method is successful in estimating the frequencies accurately. Further, it estimates the damping exponents. The proposed adaptive filtration method does not include any frequency domain manipulation. Consequently, the time domain signal is not affected as a result of frequency domain and inverse transformations.

  3. Automated extraction of typing information for bacterial pathogens from whole genome sequence data: Neisseria meningitidis as an exemplar.

    PubMed

    Jolley, K A; Maiden, M C

    2013-01-01

    Whole genome sequence (WGS) data are increasingly used to characterise bacterial pathogens. These data provide detailed information on the genotypes and likely phenotypes of aetiological agents, enabling the relationships of samples from potential disease outbreaks to be established precisely. However, the generation of increasing quantities of sequence data does not, in itself, resolve the problems that many microbiological typing methods have addressed over the last 100 years or so; indeed, providing large volumes of unstructured data can confuse rather than resolve these issues. Here we review the nascent field of storage of WGS data for clinical application and show how curated sequence-based typing schemes on websites have generated an infrastructure that can exploit WGS for bacterial typing efficiently. We review the tools that have been implemented within the PubMLST website to extract clinically useful, strain-characterisation information that can be provided to physicians and public health professionals in a timely, concise and understandable way. These data can be used to inform medical decisions such as how to treat a patient, whether to instigate public health action, and what action might be appropriate. The information is compatible both with previous sequence-based typing data and also with data obtained in the absence of WGS, providing a flexible infrastructure for WGS-based clinical microbiology. PMID:23369391

  4. Development and validation of an automated liquid-liquid extraction GC/MS method for the determination of THC, 11-OH-THC, and free THC-carboxylic acid (THC-COOH) from blood serum.

    PubMed

    Purschke, Kirsten; Heinl, Sonja; Lerch, Oliver; Erdmann, Freidoon; Veit, Florian

    2016-06-01

    The analysis of Δ(9)-tetrahydrocannabinol (THC) and its metabolites 11-hydroxy-Δ(9)-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH) from blood serum is a routine task in forensic toxicology laboratories. For examination of consumption habits, the concentration of the phase I metabolite THC-COOH is used. Recommendations for interpretation of analysis values in medical-psychological assessments (regranting of driver's licenses, Germany) include threshold values for the free, unconjugated THC-COOH. Using a fully automated two-step liquid-liquid extraction, THC, 11-OH-THC, and free, unconjugated THC-COOH were extracted from blood serum, silylated with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), and analyzed by GC/MS. The automation was carried out by an x-y-z sample robot equipped with modules for shaking, centrifugation, and solvent evaporation. This method was based on a previously developed manual sample preparation method. Validation guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh) were fulfilled for both methods, at which the focus of this article is the automated one. Limits of detection and quantification for THC were 0.3 and 0.6 μg/L, for 11-OH-THC were 0.1 and 0.8 μg/L, and for THC-COOH were 0.3 and 1.1 μg/L, when extracting only 0.5 mL of blood serum. Therefore, the required limit of quantification for THC of 1 μg/L in driving under the influence of cannabis cases in Germany (and other countries) can be reached and the method can be employed in that context. Real and external control samples were analyzed, and a round robin test was passed successfully. To date, the method is employed in the Institute of Legal Medicine in Giessen, Germany, in daily routine. Automation helps in avoiding errors during sample preparation and reduces the workload of the laboratory personnel. Due to its flexibility, the analysis system can be employed for other liquid-liquid extractions as

  5. Evaluation of FTA(®) card for the rescue of infectious foot-and-mouth disease virus by chemical transfection of extracted RNA in cultured cells.

    PubMed

    Biswal, Jitendra K; Subramaniam, Saravanan; Ranjan, Rajeev; Pattnaik, Bramhadev

    2016-08-01

    Foot-and-mouth disease (FMD) is a highly contagious epidemic disease of transboundary importance. Inadequate storage and shipment of suspected clinical samples can compromise the ability to detect and characterise FMD virus (FMDV) in endemic countries, thereby, leading to the loss of valuable virological and epidemiological data. This study, investigates the potential of using FTA(®) cards for dry transportation of clinical samples and subsequent recovery of infectious FMDV by chemical transfection of FTA(®) card fixed RNA as an alternative to the conventional cell culture based virus isolation method. A higher proportion of infectious FMDV was rescued from clinical samples (cell culture isolates, tongue epithelial suspension and impression smears) by the FTA(®) card fixed RNA transfection method (76%) compared to the conventional cell culture based virus isolation (56%), suggesting a better performance of the current RNA transfection procedure. Furthermore, it was possible to rescue live virus by the transfection of RNA extracted from FTA(®) card impregnated with clinical samples that had been stored at varying temperature (4-37 °C) up to a period of six weeks. The VP1 sequence data and antigenic relationships with the vaccine strains, between viruses rescued by FTA(®) card fixed RNA transfection and conventional cell culture, were comparable. Therefore, these results support the use of the FTA(®) card for the economic, dry, non-hazardous transport of FMD suspected clinical samples from the site of collection to national/international reference laboratories. PMID:27321701

  6. Epithelial-mesenchymal transition and cancer stem cells, mediated by a long non-coding RNA, HOTAIR, are involved in cell malignant transformation induced by cigarette smoke extract

    SciTech Connect

    Liu, Yi; Luo, Fei; Xu, Yuan; Wang, Bairu; Zhao, Yue; Xu, Wenchao; Shi, Le; Lu, Xiaolin; Liu, Qizhan

    2015-01-01

    The incidence of lung diseases, including cancer, caused by cigarette smoke is increasing, but the molecular mechanisms of gene regulation induced by cigarette smoke remain unclear. This report describes a long noncoding RNA (lncRNA) that is induced by cigarette smoke extract (CSE) and experiments utilizing lncRNAs to integrate inflammation with the epithelial-mesenchymal transition (EMT) in human bronchial epithelial (HBE) cells. The present study shows that, induced by CSE, IL-6, a pro-inflammatory cytokine, leads to activation of STAT3, a transcription activator. A ChIP assay determined that the interaction of STAT3 with the promoter regions of HOX transcript antisense RNA (HOTAIR) increased levels of HOTAIR. Blocking of IL-6 with anti-IL-6 antibody, decreasing STAT3, and inhibiting STAT3 activation reduced HOTAIR expression. Moreover, for HBE cells cultured in the presence of HOTAIR siRNA for 24 h, the CSE-induced EMT, formation of cancer stem cells (CSCs), and malignant transformation were reversed. Thus, IL-6, acting on STAT3 signaling, which up-regulates HOTAIR in an autocrine manner, contributes to the EMT and to CSCs induced by CSE. These data define a link between inflammation and EMT, processes involved in the malignant transformation of cells caused by CSE. This link, mediated through lncRNAs, establishes a mechanism for CSE-induced lung carcinogenesis. - Highlights: • STAT3 directly regulates the levels of LncRNA HOTAIR. • LncRNA HOTAIR mediates the link between inflammation and EMT. • LncRNA HOTAIR is involved in the malignant transformation of cells caused by CSE.

  7. Progress Toward Automated Cost Estimation

    NASA Technical Reports Server (NTRS)

    Brown, Joseph A.

    1992-01-01

    Report discusses efforts to develop standard system of automated cost estimation (ACE) and computer-aided design (CAD). Advantage of system is time saved and accuracy enhanced by automating extraction of quantities from design drawings, consultation of price lists, and application of cost and markup formulas.

  8. Analysis of betamethasone in rat plasma using automated solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry. Determination of plasma concentrations in rat following oral and intravenous administration.

    PubMed

    Tamvakopoulos, C S; Neugebauer, J M; Donnelly, M; Griffin, P R

    2002-09-01

    A method is described for the determination of betamethasone in rat plasma by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The analyte was recovered from plasma by solid-phase extraction and subsequently analyzed by LC-MS-MS. A Packard Multiprobe II, an automated liquid handling system, was employed for the preparation and extraction of a 96-well plate containing unknown plasma samples, standards and quality control samples in an automated fashion. Prednisolone, a structurally related steroid, was used as an internal standard. Using the described approach, a limit of quantitation of 2 ng/ml was achieved with a 50 microl aliquot of rat plasma. The described level of sensitivity allowed the determination of betamethasone concentrations and subsequent measurement of kinetic parameters of betamethasone in rat. Combination of automated plasma extraction and the sensitivity and selectivity of LC-MS-MS offers a valuable alternative to the methodologies currently used for the quantitation of steroids in biological fluids. PMID:12137997

  9. Systematic review automation technologies

    PubMed Central

    2014-01-01

    Systematic reviews, a cornerstone of evidence-based medicine, are not produced quickly enough to support clinical practice. The cost of production, availability of the requisite expertise and timeliness are often quoted as major contributors for the delay. This detailed survey of the state of the art of information systems designed to support or automate individual tasks in the systematic review, and in particular systematic reviews of randomized controlled clinical trials, reveals trends that see the convergence of several parallel research projects. We surveyed literature describing informatics systems that support or automate the processes of systematic review or each of the tasks of the systematic review. Several projects focus on automating, simplifying and/or streamlining specific tasks of the systematic review. Some tasks are already fully automated while others are still largely manual. In this review, we describe each task and the effect that its automation would have on the entire systematic review process, summarize the existing information system support for each task, and highlight where further research is needed for realizing automation for the task. Integration of the systems that automate systematic review tasks may lead to a revised systematic review workflow. We envisage the optimized workflow will lead to system in which each systematic review is described as a computer program that automatically retrieves relevant trials, appraises them, extracts and synthesizes data, evaluates the risk of bias, performs meta-analysis calculations, and produces a report in real time. PMID:25005128

  10. Automated solvent concentrator

    NASA Technical Reports Server (NTRS)

    Griffith, J. S.; Stuart, J. L.

    1976-01-01

    Designed for automated drug identification system (AUDRI), device increases concentration by 100. Sample is first filtered, removing particulate contaminants and reducing water content of sample. Sample is extracted from filtered residue by specific solvent. Concentrator provides input material to analysis subsystem.

  11. Determination of talinolol in human plasma using automated on-line solid phase extraction combined with atmospheric pressure chemical ionization tandem mass spectrometry.

    PubMed

    Bourgogne, Emmanuel; Grivet, Chantal; Hopfgartner, Gérard

    2005-06-01

    A specific LC-MS/MS assay was developed for the automated determination of talinolol in human plasma, using on-line solid phase extraction system (prospekt 2) combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry. The method involved simple precipitation of plasma proteins with perchloric acid (contained propranolol) as the internal standard (IS) and injection of the supernatant onto a C8 End Capped (10 mmx2 mm) cartridge without any evaporation step. Using the back-flush mode, the analytes were transferred onto an analytical column (XTerra C18, 50 mmx4.6 mm) for chromatographic separation and mass spectrometry detection. One of the particularities of the assay is that the SPE cartridge is used as a column switching device and not as an SPE cartridge. Therefore, the same SPE cartridge could be used more than 28 times, significantly reducing the analysis cost. APCI ionization was selected to overcome any potential matrix suppression effects because the analyte and IS co-eluted. The mean precision and accuracy in the concentration range 2.5-200 ng/mL was found to be 103% and 7.4%, respectively. The data was assessed from QC samples during the validation phase of the assay. The lower limit of quantification was 2.5 ng/mL, using a 250 microL plasma aliquot. The LC-MS/MS method provided the requisite selectivity, sensitivity, robustness accuracy and precision to assess pharmacokinetics of the compound in several hundred human plasma samples. PMID:15866498

  12. Epigenetic silencing of p21 by long non-coding RNA HOTAIR is involved in the cell cycle disorder induced by cigarette smoke extract.

    PubMed

    Liu, Yi; Wang, Bairu; Liu, Xinlu; Lu, Lu; Luo, Fei; Lu, Xiaolin; Shi, Le; Xu, Wenchao; Liu, Qizhan

    2016-01-01

    Long noncoding RNAs (lncRNAs), which are epigenetic regulators, are involved in human malignancies. Little is known, however, about the molecular mechanisms for lncRNA regulation of genes induced by cigarette smoke. We recently found that, in human bronchial epithelial (HBE) cells, the lncRNA, Hox transcript antisense intergenic RNA (HOTAIR), is associated with changes in the cell cycle caused by cigarette smoke extract (CSE). In the present study, we report that increased expression of HOTAIR and enhancer of zeste homolog 2 (EZH2), and tri-methylation of Lys 27 of histone H3 (H3K27me3), affect cell cycle progression during CSE-induced transformation of HBE cells. Inhibition of HOTAIR and EZH2 by siRNAs attenuated CSE-induced decreases of p21 levels. Further, ChIP assays verified that HOTAIR and EZH2 were needed to maintain the interaction of H3K27me3 with the promoter regions of p21; combined use of a HOTAIR plasmid and EZH2 siRNA supported this observation. Thus, HOTAIR epigenetic silencing of p21 via EZH2-mediated H3K27 trimethylation contributes to changes in the cell cycle induced by CSE. These observations provide further understanding of the regulation of CSE-induced lung carcinogenesis and identify new therapeutic targets. PMID:26506537

  13. Validation of an automated solid-phase extraction method for the analysis of 23 opioids, cocaine, and metabolites in urine with ultra-performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Ramírez Fernández, María del Mar; Van Durme, Filip; Wille, Sarah M R; di Fazio, Vincent; Kummer, Natalie; Samyn, Nele

    2014-06-01

    The aim of this work was to automate a sample preparation procedure extracting morphine, hydromorphone, oxymorphone, norcodeine, codeine, dihydrocodeine, oxycodone, 6-monoacetyl-morphine, hydrocodone, ethylmorphine, benzoylecgonine, cocaine, cocaethylene, tramadol, meperidine, pentazocine, fentanyl, norfentanyl, buprenorphine, norbuprenorphine, propoxyphene, methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine from urine samples. Samples were extracted by solid-phase extraction (SPE) with cation exchange cartridges using a TECAN Freedom Evo 100 base robotic system, including a hydrolysis step previous extraction when required. Block modules were carefully selected in order to use the same consumable material as in manual procedures to reduce cost and/or manual sample transfers. Moreover, the present configuration included pressure monitoring pipetting increasing pipetting accuracy and detecting sampling errors. The compounds were then separated in a chromatographic run of 9 min using a BEH Phenyl analytical column on a ultra-performance liquid chromatography-tandem mass spectrometry system. Optimization of the SPE was performed with different wash conditions and elution solvents. Intra- and inter-day relative standard deviations (RSDs) were within ±15% and bias was within ±15% for most of the compounds. Recovery was >69% (RSD < 11%) and matrix effects ranged from 1 to 26% when compensated with the internal standard. The limits of quantification ranged from 3 to 25 ng/mL depending on the compound. No cross-contamination in the automated SPE system was observed. The extracted samples were stable for 72 h in the autosampler (4°C). This method was applied to authentic samples (from forensic and toxicology cases) and to proficiency testing schemes containing cocaine, heroin, buprenorphine and methadone, offering fast and reliable results. Automation resulted in improved precision and accuracy, and a minimum operator intervention, leading to safer sample

  14. Automated solid-phase extraction for the determination of polybrominated diphenyl ethers and polychlorinated biphenyls in serum--application on archived Norwegian samples from 1977 to 2003.

    PubMed

    Thomsen, Cathrine; Liane, Veronica Horpestad; Becher, Georg

    2007-02-01

    An analytical method comprised of automated solid-phase extraction and determination using gas chromatography mass spectrometry (single quadrupole) has been developed for the determination of 12 polybrominated diphenyl ethers (PBDEs), 26 polychlorinated biphenyls (PCBs), two organochlorine compounds (OCs) (hexachlorobenzene and octachlorostyrene) and two brominated phenols (pentabromophenol, and tetrabromobisphenol-A (TBBP-A)). The analytes were extracted using a sorbent of polystyrene-divinylbenzene and an additional clean-up was performed on a sulphuric acid-silica column to remove lipids. The method has been validated by spiking horse serum at five levels. The mean accuracy given as recovery relative to internal standards was 95%, 99%, 93% and 109% for the PBDEs PCBs, OCs and brominated phenols, respectively. The mean repeatability given as RSDs was respectively 6.9%, 8.7%, 7.5% and 15%. Estimated limits of detection (S/N=3) were in the range 0.2-1.8 pg/g serum for the PBDEs and phenols, and from 0.1 pg/g to 56 pg/g serum for the PCBs and OCs. The validated method has been used to investigate the levels of PBDEs and PCBs in 21 pooled serum samples from the general Norwegian population. In serum from men (age 40-50 years) the sum of seven PBDE congeners (IUPAC No. 28, 47, 99, 100, 153, 154 and 183) increased from 1977 (0.5 ng/g lipids) to 1998 (4.8 ng/g lipids). From 1999 to 2003 the concentration of PBDEs seems to have stabilised. On the other hand, the sum of five PCBs (IUPAC No. 101, 118, 138, 153 and 180) in these samples decreased steadily from 1977 (666 ng/g lipids) to 2003 (176 ng/g lipids). Tetrabromobisphenol-A and BDE-209 were detected in almost all samples, but no similar temporal trends to that seen for the PBDEs were observed for these compounds, which might be due to the short half-lives of these brominated flame retardants (FR) in humans. PMID:17023223

  15. Dispersive liquid-liquid microextraction combined with semi-automated in-syringe back extraction as a new approach for the sample preparation of ionizable organic compounds prior to liquid chromatography.

    PubMed

    Melwanki, Mahaveer B; Fuh, Ming-Ren

    2008-07-11

    Dispersive liquid-liquid microextraction (DLLME) followed by a newly designed semi-automated in-syringe back extraction technique has been developed as an extraction methodology for the extraction of polar organic compounds prior to liquid chromatography (LC) measurement. The method is based on the formation of tiny droplets of the extractant in the sample solution using water-immiscible organic solvent (extractant) dissolved in a water-miscible organic dispersive solvent. Extraction of the analytes from aqueous sample into the dispersed organic droplets took place. The extracting organic phase was separated by centrifuging and the sedimented phase was withdrawn into a syringe. Then in-syringe back extraction was utilized to extract the analytes into an aqueous solution prior to LC analysis. Clenbuterol (CB), a basic organic compound used as a model, was extracted from a basified aqueous sample using 25 microL tetrachloroethylene (TCE, extraction solvent) dissolved in 500 microL acetone (as a dispersive solvent). After separation of the organic extracting phase by centrifuging, CB enriched in TCE phase was back extracted into 10 microL of 1% aqueous formic acid (FA) within the syringe. Back extraction was facilitated by repeatedly moving the plunger back and forth within the barrel of syringe, assisted by a syringe pump. Due to the plunger movement, a thin organic film is formed on the inner layer of the syringe that comes in contact with the acidic aqueous phase. Here, CB, a basic analyte, will be protonated and back extracted into FA. Various parameters affecting the extraction efficiency, viz., choice of extraction and dispersive solvent, salt effect, speed of syringe pump, back extraction time period, effect of concentration of base and acid, were evaluated. Under optimum conditions, precision, linearity (correlation coefficient, r(2)=0.9966 over the concentration range of 10-1000 ng mL(-1) CB), detection limit (4.9 ng mL(-1)), enrichment factor (175), relative

  16. A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpus siliculosus, the model brown alga.

    PubMed

    Greco, Maria; Sáez, Claudio A; Brown, Murray T; Bitonti, Maria Beatrice

    2014-01-01

    The brown seaweed Ectocarpus siliculosus is an emerging model species distributed worldwide in temperate coastal ecosystems. Over 1500 strains of E. siliculosus are available in culture from a broad range of geographic locations and ecological niches. To elucidate the molecular mechanisms underlying its capacity to cope with different environmental and biotic stressors, genomic and transcriptomic studies are necessary; this requires the co-isolation of genomic DNA and total RNA. In brown algae, extraction of nucleic acids is hindered by high concentrations of secondary metabolites that co-precipitate with nucleic acids. Here, we propose a reliable, rapid and cost-effective procedure for the co-isolation of high-quality nucleic acids using small quantities of biomass (25-, 50- and 100 mg) from strains of E. siliculosus (RHO12; LIA4A; EC524 and REP10-11) isolated from sites with different environmental conditions. The procedure employs a high pH extraction buffer (pH 9.5) which contains 100 mM Tris-HCl and 150 mM NaCl, with the addition of 5 mM DTT and 1% sarkosyl to ensure maximum solubility of nucleic acids, effective inhibition of nuclease activity and removal of interfering contaminants (e.g. polysaccharides, polyphenols). The use of sodium acetate together with isopropanol shortened precipitation time and enhanced the yields of DNA/RNA. A phenol:chlorophorm:isoamyl alcohol step was subsequently used to purify the nucleic acids. The present protocol produces high yields of nucleic acids from only 25 mg of fresh algal biomass (0.195 and 0.284 µg mg(-1) fresh weigh of RNA and DNA, respectively) and the high quality of the extracted nucleic acids was confirmed through spectrophotometric and electrophoretic analyses. The isolated RNA can be used directly in downstream applications such as RT-PCR and the genomic DNA was suitable for PCR, producing reliable restriction enzyme digestion patterns. Co-isolation of DNA/RNA from different strains indicates that this method

  17. Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas.

    PubMed Central

    Kapur, V; Li, L L; Iordanescu, S; Hamrick, M R; Wanger, A; Kreiswirth, B N; Musser, J M

    1994-01-01

    Automated DNA sequencing was used to characterize mutations associated with rifampin resistance in a 69-bp region of the gene, rpoB, encoding the beta subunit of RNA polymerase in Mycobacterium tuberculosis. The data confirmed that greater than 90% of rifampin-resistant strains have sequence alterations in this region and showed that most are missense mutations. The analysis also identified several mutant rpoB alleles not previously associated with resistant organisms and one short region of rpoB that had an unusually high frequency of insertions and deletions. Although many strains with an identical IS6110 restriction fragment length polymorphism pattern have the same variant rpoB allele, some do not, a result that suggests the occurrence of evolutionary divergence at the clone level. PMID:8027320

  18. Internal Transcribed Spacer 2 (nu ITS2 rRNA) Sequence-Structure Phylogenetics: Towards an Automated Reconstruction of the Green Algal Tree of Life

    PubMed Central

    Buchheim, Mark A.; Keller, Alexander; Koetschan, Christian; Förster, Frank; Merget, Benjamin; Wolf, Matthias

    2011-01-01

    Background Chloroplast-encoded genes (matK and rbcL) have been formally proposed for use in DNA barcoding efforts targeting embryophytes. Extending such a protocol to chlorophytan green algae, though, is fraught with problems including non homology (matK) and heterogeneity that prevents the creation of a universal PCR toolkit (rbcL). Some have advocated the use of the nuclear-encoded, internal transcribed spacer two (ITS2) as an alternative to the traditional chloroplast markers. However, the ITS2 is broadly perceived to be insufficiently conserved or to be confounded by introgression or biparental inheritance patterns, precluding its broad use in phylogenetic reconstruction or as a DNA barcode. A growing body of evidence has shown that simultaneous analysis of nucleotide data with secondary structure information can overcome at least some of the limitations of ITS2. The goal of this investigation was to assess the feasibility of an automated, sequence-structure approach for analysis of IT2 data from a large sampling of phylum Chlorophyta. Methodology/Principal Findings Sequences and secondary structures from 591 chlorophycean, 741 trebouxiophycean and 938 ulvophycean algae, all obtained from the ITS2 Database, were aligned using a sequence structure-specific scoring matrix. Phylogenetic relationships were reconstructed by Profile Neighbor-Joining coupled with a sequence structure-specific, general time reversible substitution model. Results from analyses of the ITS2 data were robust at multiple nodes and showed considerable congruence with results from published phylogenetic analyses. Conclusions/Significance Our observations on the power of automated, sequence-structure analyses of ITS2 to reconstruct phylum-level phylogenies of the green algae validate this approach to assessing diversity for large sets of chlorophytan taxa. Moreover, our results indicate that objections to the use of ITS2 for DNA barcoding should be weighed against the utility of an automated

  19. The Impact of Different DNA Extraction Kits and Laboratories upon the Assessment of Human Gut Microbiota Composition by 16S rRNA Gene Sequencing

    PubMed Central

    Kennedy, Nicholas A.; Walker, Alan W.; Berry, Susan H.; Duncan, Sylvia H.; Farquarson, Freda M.; Louis, Petra; Thomson, John M.; Ahmad, T; Anderson, CA; Barrett, JC; Drummond, H; Edwards, C; Hart, A; Hawkey, C; Henderson, P; Khan, M; Lamb, CA; Lee, JC; Mansfield, JC; Mathew, CG; Mowat, C; Newman, WG; Prescott, NJ; Simmons, A; Simpson, P; Taylor, K; Taylor, K; Wilson, DC; Satsangi, Jack; Flint, Harry J.; Parkhill, Julian

    2014-01-01

    Introduction Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories. Methods Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient. Results DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/µL respectively (p<0.0001). Hierarchical clustering of sequence data by Yue and Clayton coefficient revealed four clusters. Samples from individuals H3 and I2 clustered by patient; however, samples from patient I1 extracted with the MoBio kit clustered with samples from patient H4 rather than the other I1 samples. Linear modelling on relative abundance of common bacterial families revealed significant differences between kits; samples extracted with MoBio Powersoil showed significantly increased Bacteroidaceae, Ruminococcaceae and Porphyromonadaceae, and lower Enterobacteriaceae, Lachnospiraceae, Clostridiaceae, and Erysipelotrichaceae (p<0.05). Conclusion This study demonstrates significant differences in DNA yield and bacterial DNA composition when comparing DNA extracted from the same fecal sample with different extraction kits. This highlights

  20. RAID: a comprehensive resource for human RNA-associated (RNA-RNA/RNA-protein) interaction.

    PubMed

    Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

    2014-07-01

    Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA-RNA/RNA-protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA-RNA interactions and 1619 RNA-protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA-RNA/RNA-protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA-RNA/RNA-protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network. PMID:24803509

  1. Single-Point Mutation Detection in RNA Extracts using Gold Nanoparticles Modified with Hydrophobic Molecular Beacon-Like Structures

    PubMed Central

    Latorre, Alfonso; Posch, Christian; Garcimartín, Yolanda; Ortiz-Urda, Susana; Somoza, Álvaro

    2015-01-01

    Gold nanoparticles functionalized with oligonucleotides that bear a cholesterol group are used as gene sensors. The hydrophobic molecule is buried inside the nanostructure but when the complementary RNA sequence is present the structure unfolds exposing the cholesterol group to the water. This rearrangement leads to the aggregation of the nanostructures. PMID:24496380

  2. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

    DOE PAGESBeta

    None

    2014-12-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illuminamore » 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.« less

  3. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

    SciTech Connect

    2014-12-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.

  4. Effects of DNA extraction and universal primers on 16S rRNA gene-based DGGE analysis of a bacterial community from fish farming water

    NASA Astrophysics Data System (ADS)

    Luo, Peng; Hu, Chaoqun; Zhang, Lüping; Ren, Chunhua; Shen, Qi

    2007-07-01

    Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore, the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied. It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE.

  5. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

    PubMed Central

    Rubin, Benjamin E R; Sanders, Jon G; Hampton-Marcell, Jarrad; Owens, Sarah M; Gilbert, Jack A; Moreau, Corrie S

    2014-01-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied. PMID:25257543

  6. Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification.

    PubMed

    Yang, Qi; Franco, Christopher M M; Zhang, Wei

    2015-10-01

    Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity. PMID:26245685

  7. Polyphenolic extracts from cowpea (Vigna unguiculata) protect colonic myofibroblasts (CCD18Co cells) from lipopolysaccharide (LPS)-induced inflammation--modulation of microRNA 126.

    PubMed

    Ojwang, Leonnard O; Banerjee, Nivedita; Noratto, Giuliana D; Angel-Morales, Gabriela; Hachibamba, Twambo; Awika, Joseph M; Mertens-Talcott, Susanne U

    2015-01-01

    Cowpea (Vigna unguiculata) is a drought tolerant crop with several agronomic advantages over other legumes. This study evaluated varieties from four major cowpea phenotypes (black, red, light brown and white) containing different phenolic profiles for their anti-inflammatory property on non-malignant colonic myofibroblasts (CCD18Co) cells challenged with an endotoxin (lipopolysaccharide, LPS). Intracellular reactive oxygen species (ROS) assay on the LPS-stimulated cells revealed antioxidative potential of black and red cowpea varieties. Real-time qRT-PCR analysis in LPS-stimulated cells revealed down-regulation of proinflammatory cytokines (IL-8, TNF-α, VCAM-1), transcription factor NF-κB and modulation of microRNA-126 (specific post-transcriptional regulator of VCAM-1) by cowpea polyphenolics. The ability of cowpea polyphenols to modulate miR-126 signaling and its target gene VCAM-1 were studied in LPS-stimulated endothelial cells transfected with a specific inhibitor of miR-126, and treated with 10 mg GAE/L black cowpea extract where the extract in part reversed the effect of the miR-126 inhibitor. This suggests that cowpea may exert their anti-inflammatory activities at least in part through induction of miR-126 that then down-regulate VCAM-1 mRNA and protein expressions. Overall, Cowpea therefore is promising as an anti-inflammatory dietary component. PMID:25300227

  8. An Automated Approach to Agricultural Tile Drain Detection and Extraction Utilizing High Resolution Aerial Imagery and Object-Based Image Analysis

    NASA Astrophysics Data System (ADS)

    Johansen, Richard A.

    Subsurface drainage from agricultural fields in the Maumee River watershed is suspected to adversely impact the water quality and contribute to the formation of harmful algal blooms (HABs) in Lake Erie. In early August of 2014, a HAB developed in the western Lake Erie Basin that resulted in over 400,000 people being unable to drink their tap water due to the presence of a toxin from the bloom. HAB development in Lake Erie is aided by excess nutrients from agricultural fields, which are transported through subsurface tile and enter the watershed. Compounding the issue within the Maumee watershed, the trend within the watershed has been to increase the installation of tile drains in both total extent and density. Due to the immense area of drained fields, there is a need to establish an accurate and effective technique to monitor subsurface farmland tile installations and their associated impacts. This thesis aimed at developing an automated method in order to identify subsurface tile locations from high resolution aerial imagery by applying an object-based image analysis (OBIA) approach utilizing eCognition. This process was accomplished through a set of algorithms and image filters, which segment and classify image objects by their spectral and geometric characteristics. The algorithms utilized were based on the relative location of image objects and pixels, in order to maximize the robustness and transferability of the final rule-set. These algorithms were coupled with convolution and histogram image filters to generate results for a 10km2 study area located within Clay Township in Ottawa County, Ohio. The eCognition results were compared to previously collected tile locations from an associated project that applied heads-up digitizing of aerial photography to map field tile. The heads-up digitized locations were used as a baseline for the accuracy assessment. The accuracy assessment generated a range of agreement values from 67.20% - 71.20%, and an average

  9. Biofunction-assisted DNA detection through RNase H-enhanced 3' processing of a premature tRNA probe in a wheat germ extract.

    PubMed

    Ogawa, Atsushi; Tabuchi, Junichiro; Doi, Yasunori; Takamatsu, Masashi

    2016-08-01

    We have developed a novel type of biofunction-assisted, signal-turn-on sensor for simply and homogenously detecting DNA. This sensor system is composed of two types of in vitro-transcribed label-free RNAs (a 3' premature amber suppressor tRNA probe and an amber-mutated mRNA encoding a reporter protein), RNase H, and a wheat germ extract (WGE). A target DNA induces the 3' end maturation of the tRNA probe, which is enhanced by RNase H and leads to the expression of a full-length reporter protein through amber suppression in WGE, while there is almost no expression without the target due to the inactivity of the premature probe. Therefore, the target can be readily detected with the activity of the translated reporter. The catalytic reuse of the target with the help of RNase H in addition to various bioprocesses in WGE enables this sensor system to exhibit relatively high selectivity and sensitivity. PMID:27289318

  10. Olive leaf extract suppresses messenger RNA expression of proinflammatory cytokines and enhances insulin receptor substrate 1 expression in the rats with streptozotocin and high-fat diet-induced diabetes.

    PubMed

    Liu, Ya-Nan; Jung, Ji-Hye; Park, Hyunjin; Kim, HyunSook

    2014-05-01

    Type 2 diabetes, characterized by hyperglycemia and hyperlipidemia, is a metabolic disease resulting from defects in both insulin secretion and insulin resistance. Recently, olive leaf has been reported as an anti-inflammatory, antioxidant, and antidiabetic agent. This study sought to investigate whether olive leaf extract can improve the insulin resistance and inflammation response in rats with type 2 diabetes induced by high-fat diet and streptozotocin. After administering olive leaf extract for 8 weeks (200 and 400 mg/kg body weight), rats given the higher dose showed significantly lower blood glucose, serum total cholesterol, and triglyceride levels compared with those of diabetic control rats (P < .05). Results of oral glucose tolerance tests, homeostasis model assessment of insulin resistance, and messenger RNA (mRNA) expression of tumor necrosis factor α and interleukin (IL) 6 in the liver show significantly decreased glucose level in rats given either dose of olive leaf extract (P < .05). Both olive leaf extract-treated groups showed significantly increased insulin receptor substrate 1 expression (P < .05). Tumor necrosis factor α, IL-6 and IL-1β mRNA expressions in epididymis adipose tissue were significantly lower in rats that received higher dose of olive leaf extract (P < .05). Lymphocyte infiltration was not observed in these rats. The results suggest that olive leaf extract may attenuate insulin resistance by suppressing mRNA expression of proinflammatory cytokines and elevating of insulin receptor substrate 1 expression. PMID:24916559

  11. Automation or De-automation

    NASA Astrophysics Data System (ADS)

    Gorlach, Igor; Wessel, Oliver

    2008-09-01

    In the global automotive industry, for decades, vehicle manufacturers have continually increased the level of automation of production systems in order to be competitive. However, there is a new trend to decrease the level of automation, especially in final car assembly, for reasons of economy and flexibility. In this research, the final car assembly lines at three production sites of Volkswagen are analysed in order to determine the best level of automation for each, in terms of manufacturing costs, productivity, quality and flexibility. The case study is based on the methodology proposed by the Fraunhofer Institute. The results of the analysis indicate that fully automated assembly systems are not necessarily the best option in terms of cost, productivity and quality combined, which is attributed to high complexity of final car assembly systems; some de-automation is therefore recommended. On the other hand, the analysis shows that low automation can result in poor product quality due to reasons related to plant location, such as inadequate workers' skills, motivation, etc. Hence, the automation strategy should be formulated on the basis of analysis of all relevant aspects of the manufacturing process, such as costs, quality, productivity and flexibility in relation to the local context. A more balanced combination of automated and manual assembly operations provides better utilisation of equipment, reduces production costs and improves throughput.

  12. Process automation

    SciTech Connect

    Moser, D.R.

    1986-01-01

    Process automation technology has been pursued in the chemical processing industries and to a very limited extent in nuclear fuel reprocessing. Its effective use has been restricted in the past by the lack of diverse and reliable process instrumentation and the unavailability of sophisticated software designed for process control. The Integrated Equipment Test (IET) facility was developed by the Consolidated Fuel Reprocessing Program (CFRP) in part to demonstrate new concepts for control of advanced nuclear fuel reprocessing plants. A demonstration of fuel reprocessing equipment automation using advanced instrumentation and a modern, microprocessor-based control system is nearing completion in the facility. This facility provides for the synergistic testing of all chemical process features of a prototypical fuel reprocessing plant that can be attained with unirradiated uranium-bearing feed materials. The unique equipment and mission of the IET facility make it an ideal test bed for automation studies. This effort will provide for the demonstration of the plant automation concept and for the development of techniques for similar applications in a full-scale plant. A set of preliminary recommendations for implementing process automation has been compiled. Some of these concepts are not generally recognized or accepted. The automation work now under way in the IET facility should be useful to others in helping avoid costly mistakes because of the underutilization or misapplication of process automation. 6 figs.

  13. Automated Cooperative Trajectories

    NASA Technical Reports Server (NTRS)

    Hanson, Curt; Pahle, Joseph; Brown, Nelson

    2015-01-01

    This presentation is an overview of the Automated Cooperative Trajectories project. An introduction to the phenomena of wake vortices is given, along with a summary of past research into the possibility of extracting energy from the wake by flying close parallel trajectories. Challenges and barriers to adoption of civilian automatic wake surfing technology are identified. A hardware-in-the-loop simulation is described that will support future research. Finally, a roadmap for future research and technology transition is proposed.

  14. Semi-automated fault system extraction and displacement analysis of an excavated oyster reef using high-resolution laser scanned data

    NASA Astrophysics Data System (ADS)

    Molnár, Gábor; Székely, Balázs; Harzhauser, Mathias; Djuricic, Ana; Mandic, Oleg; Dorninger, Peter; Nothegger, Clemens; Exner, Ulrike; Pfeifer, Norbert

    2015-04-01

    In this contribution we present a semi-automated method for reconstructing the brittle deformation field of an excavated Miocene oyster reef, in Stetten, Korneuburg Basin, Lower Austria. Oyster shells up to 80 cm in size were scattered in a shallow estuarine bay forming a continuous and almost isochronous layer as a consequence of a catastrophic event in the Miocene. This shell bed was preserved by burial of several hundred meters of sandy to silty sediments. Later the layers were tilted westward, uplifted and erosion almost exhumed them. An excavation revealed a 27 by 17 meters area of the oyster covered layer. During the tectonic processes the sediment volume suffered brittle deformation. Faults mostly with some centimeter normal component and NW-SE striking affected the oyster covered volume, dissecting many shells and the surrounding matrix as well. Faults and displacements due to them can be traced along the site typically at several meters long, and as fossil oysters are broken and parts are displaced due to the faulting, along some faults it is possible to follow these displacements in 3D. In order to quantify these varying displacements and to map the undulating fault traces high-resolution scanning of the excavated and cleaned surface of the oyster bed has been carried out using a terrestrial laser scanner. The resulting point clouds have been co-georeferenced at mm accuracy and a 1mm resolution 3D point cloud of the surface has been created. As the faults are well-represented in the point cloud, this enables us to measure the dislocations of the dissected shell parts along the fault lines. We used a semi-automatic method to quantify these dislocations. First we manually digitized the fault lines in 2D as an initial model. In the next step we estimated the vertical (i.e. perpendicular to the layer) component of the dislocation along these fault lines comparing the elevations on two sides of the faults with moving averaging windows. To estimate the strike

  15. Automated design of probes for rRNA-targeted fluorescence in situ hybridization reveals the advantages of using dual probes for accurate identification.

    PubMed

    Wright, Erik S; Yilmaz, L Safak; Corcoran, Andrew M; Ökten, Hatice E; Noguera, Daniel R

    2014-08-01

    Fluorescence in situ hybridization (FISH) is a common technique for identifying cells in their natural environment and is often used to complement next-generation sequencing approaches as an integral part of the full-cycle rRNA approach. A major challenge in FISH is the design of oligonucleotide probes with high sensitivity and specificity to their target group. The rapidly expanding number of rRNA sequences has increased awareness of the number of potential nontargets for every FISH probe, making the design of new FISH probes challenging using traditional methods. In this study, we conducted a systematic analysis of published probes that revealed that many have insufficient coverage or specificity for their intended target group. Therefore, we developed an improved thermodynamic model of FISH that can be applied at any taxonomic level, used the model to systematically design probes for all recognized genera of bacteria and archaea, and identified potential cross-hybridizations for the selected probes. This analysis resulted in high-specificity probes for 35.6% of the genera when a single probe was used in the absence of competitor probes and for 60.9% when up to two competitor probes were used. Requiring the hybridization of two independent probes for positive identification further increased specificity. In this case, we could design highly specific probe sets for up to 68.5% of the genera without the use of competitor probes and 87.7% when up to two competitor probes were used. The probes designed in this study, as well as tools for designing new probes, are available online (http://DECIPHER.cee.wisc.edu). PMID:24928876

  16. Quantification of 31 illicit and medicinal drugs and metabolites in whole blood by fully automated solid-phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Bjørk, Marie Kjærgaard; Simonsen, Kirsten Wiese; Andersen, David Wederkinck; Dalsgaard, Petur Weihe; Sigurðardóttir, Stella Rögn; Linnet, Kristian; Rasmussen, Brian Schou

    2013-03-01

    An efficient method for analyzing illegal and medicinal drugs in whole blood using fully automated sample preparation and short ultra-high-performance liquid chromatography-tandem mass spectrometry (MS/MS) run time is presented. A selection of 31 drugs, including amphetamines, cocaine, opioids, and benzodiazepines, was used. In order to increase the efficiency of routine analysis, a robotic system based on automated liquid handling and capable of handling all unit operation for sample preparation was built on a Freedom Evo 200 platform with several add-ons from Tecan and third-party vendors. Solid-phase extraction was performed using Strata X-C plates. Extraction time for 96 samples was less than 3 h. Chromatography was performed using an ACQUITY UPLC system (Waters Corporation, Milford, USA). Analytes were separated on a 100 mm × 2.1 mm, 1.7 μm Acquity UPLC CSH C(18) column using a 6.5 min 0.1 % ammonia (25 %) in water/0.1 % ammonia (25 %) in methanol gradient and quantified by MS/MS (Waters Quattro Premier XE) in multiple-reaction monitoring mode. Full validation, including linearity, precision and trueness, matrix effect, ion suppression/enhancement of co-eluting analytes, recovery, and specificity, was performed. The method was employed successfully in the laboratory and used for routine analysis of forensic material. In combination with tetrahydrocannabinol analysis, the method covered 96 % of cases involving driving under the influence of drugs. The manual labor involved in preparing blood samples, solvents, etc., was reduced to a half an hour per batch. The automated sample preparation setup also minimized human exposure to hazardous materials, provided highly improved ergonomics, and eliminated manual pipetting. PMID:23292043

  17. Ginkgo biloba extract and its flavonol and terpenelactone fractions do not affect beta-secretase mRNA and enzyme activity levels in cultured neurons and in mice.

    PubMed

    Augustin, Sabine; Huebbe, Patricia; Matzner, Nicole; Augustin, Kay; Schliebs, Reinhard; Cermak, Rainer; Wolffram, Siegfried; Rimbach, Gerald

    2008-01-01

    Numerous clinical trials have reported beneficial effects of the Ginkgo biloba extract EGb761 in the prevention and therapy of cognitive disorders including Alzheimer's disease (AD). Although neuroprotective properties of EGb761 have been consistently reported, the molecular mechanisms of EGb761 and the specific role of its major constituents, the flavonols and terpenlactones, are largely unknown. One major hallmark of AD is the deposition of amyloid-beta (A beta) as amyloid plaques in the brain. A beta is a cleavage product of amyloid precursor protein (APP). Certain proteases, called beta-secretases (BACE), are crucial in the formation of A beta. The purpose of the present study was to investigate the efficacy of EGb761 and its flavonol and terpenelactone fraction to modulate BACE-1 enzyme activity and mRNA levels in vitro and in vivo. Neither EGb761 nor its fractions affected BACE-1 activity in vitro. Furthermore, also in Neuro-2a cells and wild-type as well as transgenic (Tg2576) laboratory mice, no significant effect of EGb761 on BACE-1 enzyme activity and mRNA levels were observed. Current findings suggest that BACE-1 may not be a major molecular target of EGb761 and its flavonol and terpenelactone fraction. PMID:18186016

  18. Automation pilot

    NASA Technical Reports Server (NTRS)

    1983-01-01

    An important concept of the Action Information Management System (AIMS) approach is to evaluate office automation technology in the context of hands on use by technical program managers in the conduct of human acceptance difficulties which may accompany the transition to a significantly changing work environment. The improved productivity and communications which result from application of office automation technology are already well established for general office environments, but benefits unique to NASA are anticipated and these will be explored in detail.

  19. Automated Urinalysis

    NASA Technical Reports Server (NTRS)

    1994-01-01

    Information from NASA Tech Briefs assisted DiaSys Corporation in the development of the R/S 2000 which automates urinalysis, eliminating most manual procedures. An automatic aspirator is inserted into a standard specimen tube, the "Sample" button is pressed, and within three seconds a consistent amount of urine sediment is transferred to a microscope. The instrument speeds up, standardizes, automates and makes urine analysis safer. Additional products based on the same technology are anticipated.

  20. Automated analysis of siRNA screens of cells infected by hepatitis C and dengue viruses based on immunofluorescence microscopy images

    NASA Astrophysics Data System (ADS)

    Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

    2008-03-01

    We present an image analysis approach as part of a high-throughput microscopy siRNA-based screening system using cell arrays for the identification of cellular genes involved in hepatitis C and dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in the neighborhood of segmented cell nuclei, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment and single images. In particular, we propose a novel approach for the localization of regions of transfected cells within cell array images, which combines model-based circle fitting and grid fitting. By this scheme we integrate information from single cell array images and knowledge from the complete cell arrays. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behaviour of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

  1. Quantification of five compounds with heterogeneous physicochemical properties (morphine, 6-monoacetylmorphine, cyamemazine, meprobamate and caffeine) in 11 fluids and tissues, using automated solid-phase extraction and gas chromatography-tandem mass spectrometry.

    PubMed

    Bévalot, Fabien; Bottinelli, Charline; Cartiser, Nathalie; Fanton, Laurent; Guitton, Jérôme

    2014-06-01

    An automated solid-phase extraction (SPE) protocol followed by gas chromatography coupled with tandem mass spectrometry was developed for quantification of caffeine, cyamemazine, meprobamate, morphine and 6-monoacetylmorphine (6-MAM) in 11 biological matrices [blood, urine, bile, vitreous humor, liver, kidney, lung and skeletal muscle, brain, adipose tissue and bone marrow (BM)]. The assay was validated for linearity, within- and between-day precision and accuracy, limits of quantification, selectivity, extraction recovery (ER), sample dilution and autosampler stability on BM. For the other matrices, partial validation was performed (limits of quantification, linearity, within-day precision, accuracy, selectivity and ER). The lower limits of quantification were 12.5 ng/mL(ng/g) for 6-MAM, morphine and cyamemazine, 100 ng/mL(ng/g) for meprobamate and 50 ng/mL(ng/g) for caffeine. Analysis of real-case samples demonstrated the performance of the assay in forensic toxicology to investigate challenging cases in which, for example, blood is not available or in which analysis in alternative matrices could be relevant. The SPE protocol was also assessed as an extraction procedure that could target other relevant analytes of interest. The extraction procedure was applied to 12 molecules of forensic interest with various physicochemical properties (alimemazine, alprazolam, amitriptyline, citalopram, cocaine, diazepam, levomepromazine, nordazepam, tramadol, venlafaxine, pentobarbital and phenobarbital). All drugs were able to be detected at therapeutic concentrations in blood and in the alternate matrices. PMID:24790060

  2. Analysis of premature termination in c-myc during transcription by RNA polymerase II in a HeLa nuclear extract.

    PubMed Central

    London, L; Keene, R G; Landick, R

    1991-01-01

    Transcriptional regulation of the human c-myc gene, an important aspect of cellular differentiation, occurs in part at the level of transcript elongation. In vivo, transcriptional arrest, due to either pausing or termination, occurs near the junction between the first exon and first intron and varies with the growth state of the cell. We have tested the transcription of c-myc templates in HeLa nuclear extracts. We did not observe significant arrest under standard conditions, but we found that a considerable fraction of transcription complexes stopped at the c-myc TII site (just past the first exon-intron junction) when the KCl concentration was raised to 400 mM during elongation. Transcriptional arrest at TII also was observed at KCl concentrations as low as 130 mM and when potassium acetate or potassium glutamate was substituted for KCl. Under these conditions, arrest occurred at the TII site when transcription was initiated at either the c-myc P2 promoter or the adenovirus 2 major late promoter. Further, the TII sequence itself, in forward but not reverse orientation, was sufficient to stop transcription in a HeLa nuclear extract. By separating the TII RNA from active transcription complexes by using gel filtration, we found that arrest at TII at 400 mM KCl resulted in transcript release and thus true transcriptional termination. The efficiency of termination at TII depended on the growth state of the cells from which the extracts were made, suggesting that some factor or factors control premature termination in c-myc. Images PMID:1715021

  3. Habitat automation

    NASA Technical Reports Server (NTRS)

    Swab, Rodney E.

    1992-01-01

    A habitat, on either the surface of the Moon or Mars, will be designed and built with the proven technologies of that day. These technologies will be mature and readily available to the habitat designer. We believe an acceleration of the normal pace of automation would allow a habitat to be safer and more easily maintained than would be the case otherwise. This document examines the operation of a habitat and describes elements of that operation which may benefit from an increased use of automation. Research topics within the automation realm are then defined and discussed with respect to the role they can have in the design of the habitat. Problems associated with the integration of advanced technologies into real-world projects at NASA are also addressed.

  4. Application of Brown Planthopper Salivary Gland Extract to Rice Plants Induces Systemic Host mRNA Patterns Associated with Nutrient Remobilization

    PubMed Central

    Petrova, Adelina; Smith, Charles Michael

    2015-01-01

    Insect saliva plays an important role in modulation of plant-insect interactions. Although this area of research has generated much attention in recent years, mechanisms of how saliva affects plant responses remain poorly understood. To address this void, the present study investigated the impact of the brown planthopper (Nilaparvata lugens, Stål; hereafter BPH) salivary gland extract (SGE) on rice (Oryza sativa) systemic responses at the mRNA level. Differentially expressed rice mRNAs were generated through suppression subtractive hybridization (SSH) and classified into six functional groups. Those with the most representatives were from the primary metabolism (28%), signaling-defense (22%) and transcription-translation-regulation group (16%). To validate SSH library results, six genes were further analyzed by One-Step Real-Time Reverse Transcriptase-PCR. Five of these genes exhibited up-regulation levels of more than 150% of those in the control group in at least one post-application time point. Results of this study allow assignment of at least two putative roles of BPH saliva: First, application of SGE induces immediate systemic responses at the mRNA level, suggesting that altering of the rice transcriptome at sites distant to hoppers feeding locations may play an important role in BPH-rice interactions. Second, 58% of SGE-responsive up-regulated genes have a secondary function associated with senescence, a process characterized by remobilization of nutrients. This suggests that BPH salivary secretions may reprogram the rice transcriptome for nutritional enhancement. When these findings are translated onto ‘whole plant’ scale, they indicate that BPH saliva may play the ‘wise investment’ role of ‘minimum input today, maximum output tomorrow’. PMID:26641488

  5. MicroRNA-19a/b mediates grape seed procyanidin extract-induced anti-neoplastic effects against lung cancer.

    PubMed

    Mao, Jenny T; Xue, Bingye; Smoake, Jane; Lu, Qing-Yi; Park, Heesung; Henning, Susanne M; Burns, Windie; Bernabei, Alvise; Elashoff, David; Serio, Kenneth J; Massie, Larry

    2016-08-01

    Oncomirs are microRNAs (miRNA) associated with carcinogenesis and malignant transformation. They have emerged as potential molecular targets for anti-cancer therapy. We hypothesize that grape seed procyanidin extract (GSE) exerts antineoplastic effects through modulations of oncomirs and their downstream targets. We found that GSE significantly down-regulated oncomirs miR-19a and -19b in a variety of lung neoplastic cells. GSE also increased mRNA and protein levels of insulin-like growth factor II receptor (IGF-2R) and phosphatase and tensin homolog (PTEN), both predicted targets of miR-19a and -19b. Furthermore, GSE significantly increased PTEN activity and decreased AKT phosphorylation in A549 cells. Transfection of miR-19a and -19b mimics reversed the up-regulations of IGF2R and PTEN gene expression and abrogated the GSE induced anti-proliferative response. Additionally, oral administration of leucoselect phytosome, comprised of standardized grape seed oligomeric procyanidins complexed with soy phospholipids, to athymic nude mice via gavage, significantly down-regulated miR-19a, -19b and the miR-17-92 cluster host gene (MIR17HG) expressions, increased IGF-2R, PTEN, decreased phosphorylated-AKT in A549 xenograft tumors, and markedly inhibited tumor growth. To confirm the absorption of orally administered GSE, plasma procyanidin B1 levels, between 60 and 90 min after gavage of leucoselect phytosome (400 mg/kg), were measured by LC/MS at week 2 and 8 of treatment; the estimated concentration that was associated with 50% growth inhibition (IC50) (1.3 μg/mL) in vitro was much higher than the IC50 (0.032-0.13 μg/ml) observed in vivo. Our findings reveal novel antineoplastic mechanisms by GSE and support the clinical translation of leucoselect phytosome as an anti-neoplastic and chemopreventive agent for lung cancer. PMID:27289489

  6. Automated protein NMR resonance assignments.

    PubMed

    Wan, Xiang; Xu, Dong; Slupsky, Carolyn M; Lin, Guohui

    2003-01-01

    NMR resonance peak assignment is one of the key steps in solving an NMR protein structure. The assignment process links resonance peaks to individual residues of the target protein sequence, providing the prerequisite for establishing intra- and inter-residue spatial relationships between atoms. The assignment process is tedious and time-consuming, which could take many weeks. Though there exist a number of computer programs to assist the assignment process, many NMR labs are still doing the assignments manually to ensure quality. This paper presents (1) a new scoring system for mapping spin systems to residues, (2) an automated adjacency information extraction procedure from NMR spectra, and (3) a very fast assignment algorithm based on our previous proposed greedy filtering method and a maximum matching algorithm to automate the assignment process. The computational tests on 70 instances of (pseudo) experimental NMR data of 14 proteins demonstrate that the new score scheme has much better discerning power with the aid of adjacency information between spin systems simulated across various NMR spectra. Typically, with automated extraction of adjacency information, our method achieves nearly complete assignments for most of the proteins. The experiment shows very promising perspective that the fast automated assignment algorithm together with the new score scheme and automated adjacency extraction may be ready for practical use. PMID:16452794

  7. Automating Finance

    ERIC Educational Resources Information Center

    Moore, John

    2007-01-01

    In past years, higher education's financial management side has been riddled with manual processes and aging mainframe applications. This article discusses schools which had taken advantage of an array of technologies that automate billing, payment processing, and refund processing in the case of overpayment. The investments are well worth it:…

  8. Automated dispenser

    SciTech Connect

    Hollen, R.M.; Stalnaker, N.D.

    1989-04-06

    An automated dispenser having a conventional pipette attached to an actuating cylinder through a flexible cable for delivering precise quantities of a liquid through commands from remotely located computer software. The travel of the flexible cable is controlled by adjustable stops and a locking shaft. The pipette can be positioned manually or by the hands of a robot. 1 fig.

  9. Influence of DNA extraction method, 16S rRNA targeted hypervariable regions, and sample origin on microbial diversity detected by 454 pyrosequencing in marine chemosynthetic ecosystems.

    PubMed

    Cruaud, Perrine; Vigneron, Adrien; Lucchetti-Miganeh, Céline; Ciron, Pierre Emmanuel; Godfroy, Anne; Cambon-Bonavita, Marie-Anne

    2014-08-01

    Next-generation sequencing (NGS) opens up exciting possibilities for improving our knowledge of environmental microbial diversity, allowing rapid and cost-effective identification of both cultivated and uncultivated microorganisms. However, library preparation, sequencing, and analysis of the results can provide inaccurate representations of the studied community compositions. Therefore, all these steps need to be taken into account carefully. Here we evaluated the effects of DNA extraction methods, targeted 16S rRNA hypervariable regions, and sample origins on the diverse microbes detected by 454 pyrosequencing in marine cold seep and hydrothermal vent sediments. To assign the reads with enough taxonomic precision, we built a database with about 2,500 sequences from Archaea and Bacteria from deep-sea marine sediments, affiliated according to reference publications in the field. Thanks to statistical and diversity analyses as well as inference of operational taxonomic unit (OTU) networks, we show that (i) while DNA extraction methods do not seem to affect the results for some samples, they can lead to dramatic changes for others; and (ii) the choice of amplification and sequencing primers also considerably affects the microbial community detected in the samples. Thereby, very different proportions of pyrosequencing reads were obtained for some microbial lineages, such as the archaeal ANME-1, ANME-2c, and MBG-D and deltaproteobacterial subgroups. This work clearly indicates that the results from sequencing-based analyses, such as pyrosequencing, should be interpreted very carefully. Therefore, the combination of NGS with complementary approaches, such as fluorescence in situ hybridization (FISH)/catalyzed reporter deposition (CARD)-FISH or quantitative PCR (Q-PCR), would be desirable to gain a more comprehensive picture of environmental microbial communities. PMID:24837380

  10. Influence of DNA Extraction Method, 16S rRNA Targeted Hypervariable Regions, and Sample Origin on Microbial Diversity Detected by 454 Pyrosequencing in Marine Chemosynthetic Ecosystems

    PubMed Central

    Cruaud, Perrine; Vigneron, Adrien; Lucchetti-Miganeh, Céline; Ciron, Pierre Emmanuel; Godfroy, Anne

    2014-01-01

    Next-generation sequencing (NGS) opens up exciting possibilities for improving our knowledge of environmental microbial diversity, allowing rapid and cost-effective identification of both cultivated and uncultivated microorganisms. However, library preparation, sequencing, and analysis of the results can provide inaccurate representations of the studied community compositions. Therefore, all these steps need to be taken into account carefully. Here we evaluated the effects of DNA extraction methods, targeted 16S rRNA hypervariable regions, and sample origins on the diverse microbes detected by 454 pyrosequencing in marine cold seep and hydrothermal vent sediments. To assign the reads with enough taxonomic precision, we built a database with about 2,500 sequences from Archaea and Bacteria from deep-sea marine sediments, affiliated according to reference publications in the field. Thanks to statistical and diversity analyses as well as inference of operational taxonomic unit (OTU) networks, we show that (i) while DNA extraction methods do not seem to affect the results for some samples, they can lead to dramatic changes for others; and (ii) the choice of amplification and sequencing primers also considerably affects the microbial community detected in the samples. Thereby, very different proportions of pyrosequencing reads were obtained for some microbial lineages, such as the archaeal ANME-1, ANME-2c, and MBG-D and deltaproteobacterial subgroups. This work clearly indicates that the results from sequencing-based analyses, such as pyrosequencing, should be interpreted very carefully. Therefore, the combination of NGS with complementary approaches, such as fluorescence in situ hybridization (FISH)/catalyzed reporter deposition (CARD)-FISH or quantitative PCR (Q-PCR), would be desirable to gain a more comprehensive picture of environmental microbial communities. PMID:24837380

  11. Automation in biological crystallization

    PubMed Central

    Shaw Stewart, Patrick; Mueller-Dieckmann, Jochen

    2014-01-01

    Crystallization remains the bottleneck in the crystallographic process leading from a gene to a three-dimensional model of the encoded protein or RNA. Automation of the individual steps of a crystallization experiment, from the preparation of crystallization cocktails for initial or optimization screens to the imaging of the experiments, has been the response to address this issue. Today, large high-throughput crystallization facilities, many of them open to the general user community, are capable of setting up thousands of crystallization trials per day. It is thus possible to test multiple constructs of each target for their ability to form crystals on a production-line basis. This has improved success rates and made crystallization much more convenient. High-throughput crystallization, however, cannot relieve users of the task of producing samples of high quality. Moreover, the time gained from eliminating manual preparations must now be invested in the careful evaluation of the increased number of experiments. The latter requires a sophisticated data and laboratory information-management system. A review of the current state of automation at the individual steps of crystallization with specific attention to the automation of optimization is given. PMID:24915074

  12. Automating the analytical laboratory via the Chemical Analysis Automation paradigm

    SciTech Connect

    Hollen, R.; Rzeszutko, C.

    1997-10-01

    To address the need for standardization within the analytical chemistry laboratories of the nation, the Chemical Analysis Automation (CAA) program within the US Department of Energy, Office of Science and Technology`s Robotic Technology Development Program is developing laboratory sample analysis systems that will automate the environmental chemical laboratories. The current laboratory automation paradigm consists of islands-of-automation that do not integrate into a system architecture. Thus, today the chemist must perform most aspects of environmental analysis manually using instrumentation that generally cannot communicate with other devices in the laboratory. CAA is working towards a standardized and modular approach to laboratory automation based upon the Standard Analysis Method (SAM) architecture. Each SAM system automates a complete chemical method. The building block of a SAM is known as the Standard Laboratory Module (SLM). The SLM, either hardware or software, automates a subprotocol of an analysis method and can operate as a standalone or as a unit within a SAM. The CAA concept allows the chemist to easily assemble an automated analysis system, from sample extraction through data interpretation, using standardized SLMs without the worry of hardware or software incompatibility or the necessity of generating complicated control programs. A Task Sequence Controller (TSC) software program schedules and monitors the individual tasks to be performed by each SLM configured within a SAM. The chemist interfaces with the operation of the TSC through the Human Computer Interface (HCI), a logical, icon-driven graphical user interface. The CAA paradigm has successfully been applied in automating EPA SW-846 Methods 3541/3620/8081 for the analysis of PCBs in a soil matrix utilizing commercially available equipment in tandem with SLMs constructed by CAA.

  13. Ribosomal RNA-based panbacterial polymerase chain reaction for rapid diagnosis of septicaemia in Intensive Care Unit patients.

    PubMed

    Gupta, Mahua Das; Kaur, Harsimran; Ray, Pallab; Gautam, Vikas; Puri, G D

    2016-01-01

    Early diagnosis and treatment of sepsis by appropriate antibiotics is of utmost importance. Therefore, we evaluated 16S rRNA panbacterial polymerase chain reaction (PCR) for rapid diagnosis of sepsis in 49 adult patients in Intensive Care Units (ICUs) and compared it with an automated blood culture. 8 ml of 10 ml blood collected was inoculated into BACTEC® aerobic bottle and the remaining 2 ml was used for DNA extraction and PCR. 109 of 115 (93%) episodes of suspected sepsis showed concordant results between automated culture and PCR. Six episodes were positive by PCR only. Panbacterial PCR reduces turnaround time with rapid differentiation between systemic inflammatory response syndrome and sepsis. PMID:27080778

  14. Quantitation of efletirizine in human plasma and urine using automated solid-phase extraction and column-switching high-performance liquid chromatography.

    PubMed

    Coe, R A; DeCesare, L S; Lee, J W

    1999-07-01

    A heart-cut column-switching, ion-pair, reversed-phase HPLC system was used for the quantitation of efletirizine (EFZ) in biological fluids. The analyte and an internal standard (I.S.) were extracted from human EDTA plasma by C18 solid-phase extraction (SPE) using a RapidTrace workstation. The eluent from the SPE was evaporated, reconstituted and injected onto the HPLC column. Urine samples were diluted and injected directly without the need of extraction. The compounds of interest were separated from most of the extraneous matrix materials by the first C18 column, and switched onto a second C18 column for further separation using a mobile phase of stronger eluting capability. Linearity range was 10-2000 ng ml(-1) for plasma and 0.05-10 microg ml(-1) for urine. The lower limit of quantitation (LOQ) was 10 ng from 1 ml of plasma, with a signal-to-noise ratio of 15:1. Inter-day precision and bias of quality control samples (QCs) were <5% for plasma and <7% for urine. Selectivity was established against six other antihistamines, three analogs of efletirizine, and on 12 control plasma lots and nine control urine lots. Recovery was 90.0% for EFZ and 89.5% for I.S. from plasma. One hundred samples can be processed in every 2.75 h on a 10-module RapidTrace workstation with minimal human attention. Method ruggedness were tested on three brands of SPE and six different lots of one SPE brand. Performance ruggedness was demonstrated by different analysts on multiple HPLC systems. Analyte stability through sample storage, extraction process (benchtop, freeze-thaw, refrigeration after extraction) and chromatography (on-system, reinjection) was established. PMID:10448959

  15. Rapid analysis of three β-agonist residues in food of animal origin by automated on-line solid-phase extraction coupled to liquid chromatography and tandem mass spectrometry.

    PubMed

    Mi, Jiebo; Li, Shujing; Xu, Hong; Liang, Wei; Sun, Tao

    2014-09-01

    An automated online solid-phase extraction with liquid chromatography and tandem mass spectrometry method was developed and validated for the detection of clenbuterol, salbutamol, and ractopamine in food of animal origin. The samples from the food matrix were pretreated with an online solid-phase extraction cartridge by Oasis MCX for <5 min after acid hydrolysis for 30 min. The peak focusing mode was used to elute the target compounds directly onto a C18 column. Chromatographic separation was achieved under gradient conditions using a mobile phase composed of acetonitrile/0.1% formic acid in aqueous solution. Each analyte was detected in two multiple reaction monitoring transitions via an electrospray ionization source in a positive mode. The relative standard deviations ranged from 2.6 to 10.5%, and recovery was between 76.7 and 107.2% at all quality control levels. The limits of quantification of three β-agonists were in the range of 0.024-0.29 μg/kg in pork, sausage, and milk powder, respectively. This newly developed method offers high sensitivity and minimum sample pretreatment for the high-throughput analysis of β-agonist residues. PMID:24916570

  16. Semi-automated building extraction from airborne laser scanning data. (Polish Title: Półautomatyczne modelowanie brył budynków na podstawie danych z lotniczego skaningu laserowego)

    NASA Astrophysics Data System (ADS)

    Marjasiewicz, M.; Malej, T.

    2014-12-01

    The main idea of this project is to introduce a conception of semi - automated method for building model extraction from Airborne Laser Scanning data. The presented method is based on the RANSAC algorithm, which provides automatic collection planes for roofs model creation. In the case of Airborne Laser Scanning, the algorithm can process point clouds influenced with noise and erroneous measurement (gross errors). The RANSAC algorithm is based on the iterative processing of a set of points in order to estimate the geometric model. Research of u sing algorithm for ALS data was performed in available Cloud Compare and SketchUP software. An important aspect in this research was algorithm parameters selection, which was made on the basis of characteristics of point cloud and scanned objects. Analysis showed that the accuracy of plane extraction with RANSAC algorithm does not exceed 20 centimeters for point clouds of density 4 pts . /m 2 . RANSAC can be successfully used in buildings modelling based on ALS data. Roofs created by the presented method could be used in visualizations on a much better level than Level of Detail 2 by CityGML standard. If model is textured it can represent LoD3 standard.

  17. Multiresidue trace analysis of pharmaceuticals, their human metabolites and transformation products by fully automated on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry.

    PubMed

    García-Galán, María Jesús; Petrovic, Mira; Rodríguez-Mozaz, Sara; Barceló, Damià

    2016-09-01

    A novel, fully automated analytical methodology based on dual column liquid chromatography coupled to tandem mass spectrometry (LC-LC-MS(2)) has been developed and validated for the analysis of 12 pharmaceuticals and 20 metabolites and transformation products in different types of water (influent and effluent wastewaters and surface water). Two LC columns were used - one for pre-concentration of the sample and the second for separation and analysis - so that water samples were injected directly in the chromatographic system. Besides the many advantages of the methodology, such as minimization of the sample volume required and its manipulation, both compounds ionized in positive and negative mode could be analyzed simultaneously without compromising the sensitivity. A comparative study of different mobile phases, gradients and LC pre-concentration columns was carried out to obtain the best analytical performance. Limits of detection (MLODs) achieved were in the low ngL(-1) range for all the compounds. The method was successfully applied to study the presence of the target analytes in different wastewater and surface water samples collected near the city of Girona (Catalonia, Spain). Data on the environmental presence and fate of pharmaceutical metabolites and TPs is still scarce, highlighting the relevance of the developed methodology. PMID:27343613

  18. An Integrative Analysis of microRNA and mRNA Profiling in CML Stem Cells.

    PubMed

    Nassar, Farah J; El Eit, Rabab; Nasr, Rihab

    2016-01-01

    Integrative analysis of microRNA (miRNA) and messenger RNA (mRNA) in Chronic Myeloid leukemia (CML) stem cells is an important technique to study the involvement of miRNA and their targets in CML stem cells self-renewal, maintenance, and therapeutic resistance. Here, we describe a simplified integrative analysis using Ingenuity Pathway Analysis software after performing proper RNA extraction, miRNA and mRNA microarray and data analysis. PMID:27581151

  19. A semi-automated system for quantifying the oxidative potential of ambient particles in aqueous extracts using the dithiothreitol (DTT) assay: results from the Southeastern Center for Air Pollution and Epidemiology (SCAPE)

    NASA Astrophysics Data System (ADS)

    Fang, T.; Verma, V.; Guo, H.; King, L. E.; Edgerton, E. S.; Weber, R. J.

    2014-07-01

    A variety of methods are used to measure the capability of particulate matter (PM) to catalytically generate reactive oxygen species (ROS) in vivo, also defined as the aerosol oxidative potential. A widely used measure of aerosol oxidative potential is the dithiothreitol (DTT) assay, which monitors the depletion of DTT (a surrogate for cellular antioxidants) as catalyzed by the redox-active species in PM. However, a major constraint in the routine use of the DTT assay for integrating it with the large-scale health studies is its labor-intensive and time-consuming protocol. To specifically address this concern, we have developed a semi-automated system for quantifying the oxidative potential of aerosol liquid extracts using the DTT assay. The system, capable of unattended analysis at one sample per hour, has a high analytical precision (Coefficient of Variation of 12% for standards, 4% for ambient samples), and reasonably low limit of detection (0.31 nmol min-1). Comparison of the automated approach with the manual method conducted on ambient samples yielded good agreement (slope = 1.08 ± 0.12, r2 = 0.92, N = 9). The system was utilized for the Southeastern Center for Air Pollution and Epidemiology (SCAPE) to generate an extensive data set on DTT activity of ambient particles collected from contrasting environments (urban, road-side, and rural) in the southeastern US. We find that water-soluble PM2.5 DTT activity on a per air volume basis was spatially uniform and often well correlated with PM2.5 mass (r = 0.49 to 0.88), suggesting regional sources contributing to the PM oxidative potential in southeast US. However, the greater heterogeneity in the intrinsic DTT activity (per PM mass basis) across seasons indicates variability in the DTT activity associated with aerosols from sources that vary with season. Although developed for the DTT assay, the instrument can also be used to determine oxidative potential with other acellular assays.

  20. Automated lithocell

    NASA Astrophysics Data System (ADS)

    Englisch, Andreas; Deuter, Armin

    1990-06-01

    Integration and automation have gained more and more ground in modern IC-manufacturing. It is difficult to make a direct calculation of the profit these investments yield. On the other hand, the demands to man, machine and technology have increased enormously of late; it is not difficult to see that only by means of integration and automation can these demands be coped with. Here are some salient points: U the complexity and costs incurred by the equipment and processes have got significantly higher . owing to the reduction of all dimensions, the tolerances within which the various process steps have to be carried out have got smaller and smaller and the adherence to these tolerances more and more difficult U the cycle time has become more and more important both for the development and control of new processes and, to a great extent, for a rapid and reliable supply to the customer. In order that the products be competitive under these conditions, all sort of costs have to be reduced and the yield has to be maximized. Therefore, the computer-aided control of the equipment and the process combined with an automatic data collection and a real-time SPC (statistical process control) has become absolutely necessary for successful IC-manufacturing. Human errors must be eliminated from the execution of the various process steps by automation. The work time set free in this way makes it possible for the human creativity to be employed on a larger scale in stabilizing the processes. Besides, a computer-aided equipment control can ensure the optimal utilization of the equipment round the clock.

  1. Rapid and automated analysis of aflatoxin M1 in milk and dairy products by online solid phase extraction coupled to ultra-high-pressure-liquid-chromatography tandem mass spectrometry.

    PubMed

    Campone, Luca; Piccinelli, Anna Lisa; Celano, Rita; Pagano, Imma; Russo, Mariateresa; Rastrelli, Luca

    2016-01-01

    This study reports a fast and automated analytical procedure for the analysis of aflatoxin M1 (AFM1) in milk and dairy products. The method is based on the simultaneous protein precipitation and AFM1 extraction, by salt-induced liquid-liquid extraction (SI-LLE), followed by an online solid-phase extraction (online SPE) coupled to ultra-high-pressure-liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis to the automatic pre-concentration, clean up and sensitive and selective determination of AFM1. The main parameters affecting the extraction efficiency and accuracy of the analytical method were studied in detail. In the optimal conditions, acetonitrile and NaCl were used as extraction/denaturant solvent and salting-out agent in SI-LLE, respectively. After centrifugation, the organic phase (acetonitrile) was diluted with water (1:9 v/v) and purified (1mL) by online C18 cartridge coupled with an UHPLC column. Finally, selected reaction monitoring (SRM) acquisition mode was applied to the detection of AFM1. Validation studies were carried out on different dairy products (whole and skimmed cow milk, yogurt, goat milk, and powder infant formula), providing method quantification limits about 25 times lower than AFM1 maximum levels permitted by EU regulation 1881/2006 in milk and dairy products for direct human consumption. Recoveries (86-102%) and repeatability (RSD<3, n=6) meet the performance criteria required by EU regulation N. 401/2006 for the determination of the levels of mycotoxins in foodstuffs. Moreover, no matrix effects were observed in the different milk and dairy products studied. The proposed method improves the performance of AFM1 analysis in milk samples as AFM1 determination is performed with a degree of accuracy higher than the conventional methods. Other advantages are the reduction of sample preparation procedure, time and cost of the analysis, enabling high sample throughput that meet the current concerns of food safety and the public

  2. RNA Interference

    MedlinePlus

    ... NIGMS Home > Science Education > RNA Interference Fact Sheet RNA Interference Fact Sheet Tagline (Optional) Middle/Main Content Area What is RNA interference? RNA interference (RNAi) is a natural process ...

  3. RNA topology

    PubMed Central

    2013-01-01

    A new variety on non-coding RNA has been discovered by several groups: circular RNA (circRNA). This discovery raises intriguing questions about the possibility of the existence of knotted RNA molecules and the existence of a new class of enzymes changing RNA topology, RNA topoisomerases. PMID:23603781

  4. Epigenetic silencing of miR-218 by the lncRNA CCAT1, acting via BMI1, promotes an altered cell cycle transition in the malignant transformation of HBE cells induced by cigarette smoke extract.

    PubMed

    Lu, Lu; Xu, Hui; Luo, Fei; Liu, Xinlu; Lu, Xiaolin; Yang, Qianlei; Xue, Junchao; Chen, Chao; Shi, Le; Liu, Qizhan

    2016-08-01

    Cigarette smoking is the strongest risk factor for the development of lung cancer, the leading cause of cancer-related deaths. However, the molecular mechanisms leading to lung cancer are largely unknown. A long-noncoding RNA (lncRNA), CCAT1, regarded as cancer-associated, has been investigated extensively. Moreover, the molecular mechanisms of lncRNAs in regulation of microRNAs (miRNAs) induced by cigarette smoke remain unclear. In the present investigation, cigarette smoke extract (CSE) caused an altered cell cycle and increased CCAT1 levels and decreased miR-218 levels in human bronchial epithelial (HBE) cells. Depletion of CCAT1 attenuated the CSE-induced decreases of miR-218 levels, suggesting that miR-218 is negatively regulated by CCAT1 in HBE cells exposed to CSE. The CSE-induced increases of BMI1 levels and blocked by CCAT1 siRNA were attenuated by an miR-218 inhibitor. Moreover, in CSE-transformed HBE cells, the CSE-induced cell cycle changes and elevated neoplastic capacity were reversed by CCAT1 siRNA or BMI1 siRNA. This epigenetic silencing of miR-218 by CCAT1 induces an altered cell cycle transition through BMI1 and provides a new mechanism for CSE-induced lung carcinogenesis. PMID:27212446

  5. Automated microdialysis-based system for in situ microsampling and investigation of lead bioavailability in terrestrial environments under physiologically based extraction conditions.

    PubMed

    Rosende, María; Magalhães, Luis M; Segundo, Marcela A; Miró, Manuel

    2013-10-15

    In situ automatic microdialysis sampling under batch-flow conditions is herein proposed for the first time for expedient assessment of the kinetics of lead bioaccessibility/bioavailability in contaminated and agricultural soils exploiting the harmonized physiologically based extraction test (UBM). Capitalized upon a concentric microdialysis probe immersed in synthetic gut fluids, the miniaturized flow system is harnessed for continuous monitoring of lead transfer across the permselective microdialysis membrane to mimic the diffusive transport of metal species through the epithelium of the stomach and of the small intestine. Besides, the addition of the UBM gastrointestinal fluid surrogates at a specified time frame is fully mechanized. Distinct microdialysis probe configurations and membranes types were investigated in detail to ensure passive sampling under steady-state dialytic conditions for lead. Using a 3-cm-long polysulfone membrane with averaged molecular weight cutoff of 30 kDa in a concentric probe and a perfusate flow rate of 2.0 μL min(-1), microdialysis relative recoveries in the gastric phase were close to 100%, thereby omitting the need for probe calibration. The automatic leaching method was validated in terms of bias in the analysis of four soils with different physicochemical properties and containing a wide range of lead content (16 ± 3 to 1216 ± 42 mg kg(-1)) using mass balance assessment as a quality control tool. No significant differences between the mass balance and the total lead concentration in the suite of analyzed soils were encountered (α = 0.05). Our finding that the extraction of soil-borne lead for merely one hour in the GI phase suffices for assessment of the bioavailable fraction as a result of the fast immobilization of lead species at near-neutral conditions would assist in providing risk assessment data from the UBM test on a short notice. PMID:24016003

  6. Validation of a sensitive and automated 96-well solid-phase extraction liquid chromatography-tandem mass spectrometry method for the determination of desloratadine and 3-hydroxydesloratadine in human plasma.

    PubMed

    Yang, Liyu; Clement, Robert P; Kantesaria, Bhavna; Reyderman, Larisa; Beaudry, Francis; Grandmaison, Charles; Di Donato, Lorella; Masse, Robert; Rudewicz, Patrick J

    2003-07-25

    To support clinical development, a liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method was developed and validated for the determination of desloratadine (descarboethoxyloratadine) and 3-OH desloratadine (3-hydroxydescarboethoxyloratadine) concentrations in human plasma. The method consisted of automated 96-well solid-phase extraction for sample preparation and liquid chromatography/turbo ionspray tandem mass spectrometry for analysis. [2H(4)]Desloratadine and [2H(4)]3-OH desloratadine were used as internal standards (I.S.). A quadratic regression (weighted 1/concentration(2)) gave the best fit for calibration curves over the concentration range of 25-10000 pg/ml for both desloratadine and 3-OH desloratadine. There was no interference from endogenous components in the blank plasma tested. The accuracy (%bias) at the lower limit of quantitation (LLOQ) was -12.8 and +3.4% for desloratadine and 3-OH desloratadine, respectively. The precision (%CV) for samples at the LLOQ was 15.1 and 10.9% for desloratadine and 3-OH desloratadine, respectively. For quality control samples at 75, 1000 and 7500 pg/ml, the between run %CV was extracts (up to 185 h at 5 degrees C). This LC-MS-MS method for the determination of desloratadine and 3-OH desloratadine in human plasma met regulatory requirements for selectivity, sensitivity, goodness of fit, precision, accuracy and stability. PMID:12860030

  7. Standard operation protocol for analysis of lipid hydroperoxides in human serum using a fully automated method based on solid-phase extraction and liquid chromatography-mass spectrometry in selected reaction monitoring.

    PubMed

    Ferreiro-Vera, C; Ribeiro, Joana P N; Mata-Granados, J M; Priego-Capote, F; Luque de Castro, M D

    2011-09-23

    Standard operating procedures (SOPs) are of paramount importance in the analytical field to ensure the reproducibility of the results obtained among laboratories. SOPs gain special interest when the aim is the analysis of potentially unstable compounds. An SOP for analysis of lipid hydroperoxides (HpETEs) is here reported after optimization of the critical steps to be considered in their analysis in human serum from sampling to final analysis. The method is based on automated hyphenation between solid-phase extraction (SPE) and liquid chromatography-mass spectrometry (LC-MS). The developed research involves: (i) optimization of the SPE and LC-MS steps with a proper synchronization; (ii) validation of the method-viz. accuracy study (estimated as 86.4% as minimum value), evaluation of sensitivity and precision, which ranged from 2.5 to 7.0 ng/mL (0.25-0.70 ng on column) as quantification limit and precision below 13.2%), and robustness study (reusability of the cartridge for 5 times without affecting the accuracy and precision of the method); (iii) stability study, involving freeze-thaw stability, short-term and long-term stability and stock solution stability tests. The results thus obtained allow minimizing both random and systematic variation of the metabolic profiles of the target compounds by correct application of the established protocol. PMID:21851945

  8. Determination of nine benzotriazole UV stabilizers in environmental water samples by automated on-line solid phase extraction coupled with high-performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Liu, Runzeng; Ruan, Ting; Wang, Thanh; Song, Shanjun; Guo, Feng; Jiang, Guibin

    2014-03-01

    A method using automated on-line solid phase extraction coupled with a high-performance liquid chromatography-tandem mass spectrometry system was developed for the determination of emerging benzotriazole UV stabilizers (BZTs) in different environmental water matrices including river water, sewage influent and effluent. Water sample was injected directly and the analytes were preconcentrated on a Polar Advantage II on-line SPE cartridge. After cleanup step the target BZTs were eluted in back flush mode and then separated on a liquid chromatography column. Experimental parameters such as sample loading flow rate, SPE cartridge, pH value and methanol ratio in the sample were optimized in detail. The method detection limits ranged from 0.21 to 2.17 ng/L. Recoveries of the target BZTs at 50 ng/L spiking level ranged from 76% to 114% and the inter-day RSDs ranged from 1% to 15%. The optimized method was successfully applied to analyze twelve water samples collected from different wastewater treatment plants and rivers, and five BZTs (UV-P, UV-329, UV-350, UV-234 and UV-328) were detected with concentrations up to 37.1 ng/L. The proposed method is simple, sensitive and suitable for simultaneous analysis and monitoring of BZTs in water samples. PMID:24468355

  9. Automated Desalting Apparatus

    NASA Technical Reports Server (NTRS)

    Spencer, Maegan K.; Liu, De-Ling; Kanik, Isik; Beegle, Luther

    2010-01-01

    Because salt and metals can mask the signature of a variety of organic molecules (like amino acids) in any given sample, an automated system to purify complex field samples has been created for the analytical techniques of electrospray ionization/ mass spectroscopy (ESI/MS), capillary electrophoresis (CE), and biological assays where unique identification requires at least some processing of complex samples. This development allows for automated sample preparation in the laboratory and analysis of complex samples in the field with multiple types of analytical instruments. Rather than using tedious, exacting protocols for desalting samples by hand, this innovation, called the Automated Sample Processing System (ASPS), takes analytes that have been extracted through high-temperature solvent extraction and introduces them into the desalting column. After 20 minutes, the eluent is produced. This clear liquid can then be directly analyzed by the techniques listed above. The current apparatus including the computer and power supplies is sturdy, has an approximate mass of 10 kg, and a volume of about 20 20 20 cm, and is undergoing further miniaturization. This system currently targets amino acids. For these molecules, a slurry of 1 g cation exchange resin in deionized water is packed into a column of the apparatus. Initial generation of the resin is done by flowing sequentially 2.3 bed volumes of 2N NaOH and 2N HCl (1 mL each) to rinse the resin, followed by .5 mL of deionized water. This makes the pH of the resin near neutral, and eliminates cross sample contamination. Afterward, 2.3 mL of extracted sample is then loaded into the column onto the top of the resin bed. Because the column is packed tightly, the sample can be applied without disturbing the resin bed. This is a vital step needed to ensure that the analytes adhere to the resin. After the sample is drained, oxalic acid (1 mL, pH 1.6-1.8, adjusted with NH4OH) is pumped into the column. Oxalic acid works as a

  10. Pressure-driven mesofluidic platform integrating automated on-chip renewable micro-solid-phase extraction for ultrasensitive determination of waterborne inorganic mercury.

    PubMed

    Portugal, Lindomar A; Laglera, Luis M; Anthemidis, Aristidis N; Ferreira, Sérgio L C; Miró, Manuel

    2013-06-15

    A dedicated pressure-driven mesofluidic platform incorporating on-chip sample clean-up and analyte preconcentration is herein reported for expedient determination of trace level concentrations of waterborne inorganic mercury. Capitalizing upon the Lab-on-a-Valve (LOV) concept, the mesofluidic device integrates on-chip micro-solid phase extraction (μSPE) in automatic disposable mode followed by chemical vapor generation and gas-liquid separation prior to in-line atomic fluorescence spectrometric detection. In contrast to prevailing chelating sorbents for Hg(II), bare poly(divinylbenzene-N-vinylpyrrolidone) copolymer sorptive beads were resorted to efficient uptake of Hg(II) in hydrochloric acid milieu (pH=2.3) without the need for metal derivatization nor pH adjustment of prior acidified water samples for preservation to near-neutral conditions. Experimental variables influencing the sorptive uptake and retrieval of target species and the evolvement of elemental mercury within the miniaturized integrated reaction chamber/gas-liquid separator were investigated in detail. Using merely <10 mg of sorbent, the limits of detection and quantification at the 3s(blank) and 10s(blank) levels, respectively, for a sample volume of 3 mL were 12 and 42 ng L(-1) Hg(II) with a dynamic range extending up to 5.0 μg L(-1). The proposed mesofluidic platform copes with the requirements of regulatory bodies (US-EPA, WHO, EU-Commission) for drinking water quality and surface waters that endorse maximum allowed concentrations of mercury spanning from 0.07 to 6.0 μg L(-1). Demonstrated with the analysis of aqueous samples of varying matrix complexity, the LOV approach afforded reliable results with relative recoveries of 86-107% and intermediate precision down to 9% in the renewable μSPE format. PMID:23618176

  11. Algorithms Could Automate Cancer Diagnosis

    NASA Technical Reports Server (NTRS)

    Baky, A. A.; Winkler, D. G.

    1982-01-01

    Five new algorithms are a complete statistical procedure for quantifying cell abnormalities from digitized images. Procedure could be basis for automated detection and diagnosis of cancer. Objective of procedure is to assign each cell an atypia status index (ASI), which quantifies level of abnormality. It is possible that ASI values will be accurate and economical enough to allow diagnoses to be made quickly and accurately by computer processing of laboratory specimens extracted from patients.

  12. Expression ratio of histone demethylase KDM3A to protamine-1 mRNA is predictive of successful testicular sperm extraction in men with obstructive and non-obstructive azoospermia.

    PubMed

    Javadirad, S M; Hojati, Z; Ghaedi, K; Nasr-Esfahani, M H

    2016-05-01

    To evaluate the predictive value of histone demethylase KDM3A to protamine 1 (PRM1) mRNA expression ratio as a reliable marker of sperm retrieval in men with obstructive and non-obstructive azoospermia (NOA). Fifty eight azoospermic men, including 44 with NOA and 14 with obstructive azoospermia (OA). Testis tissue samples were collected from azoospermic men who have been referred for testicular sperm extraction (TESE) and micro-TESE. Relative expression ratio of KDM3A to PRM1 was analyzed after selection of approved reference genes. Histological classification of testis biopsies was performed. Sperm retrieval following TESE and micro-TESE was evaluated. A sperm retrieval prediction sensitivity of 95% was established when the Cq of PRM1 became smaller than the Cq of both KDM3A and GAPDH genes. However, azoospermic men with down-regulated KDM3A and decreased expression of PRM1 mRNA showed very low success for sperm retrieval (<25%), even after micro-TESE surgery. The KDM3A to PRM1 mRNA expression ratio can be used as a reliable marker of successful testicular sperm extraction in men with obstructive and non-obstructive azoospermia with 95% sensitivity. PMID:27027467

  13. Maneuver Automation Software

    NASA Technical Reports Server (NTRS)

    Uffelman, Hal; Goodson, Troy; Pellegrin, Michael; Stavert, Lynn; Burk, Thomas; Beach, David; Signorelli, Joel; Jones, Jeremy; Hahn, Yungsun; Attiyah, Ahlam; Illsley, Jeannette

    2009-01-01

    The Maneuver Automation Software (MAS) automates the process of generating commands for maneuvers to keep the spacecraft of the Cassini-Huygens mission on a predetermined prime mission trajectory. Before MAS became available, a team of approximately 10 members had to work about two weeks to design, test, and implement each maneuver in a process that involved running many maneuver-related application programs and then serially handing off data products to other parts of the team. MAS enables a three-member team to design, test, and implement a maneuver in about one-half hour after Navigation has process-tracking data. MAS accepts more than 60 parameters and 22 files as input directly from users. MAS consists of Practical Extraction and Reporting Language (PERL) scripts that link, sequence, and execute the maneuver- related application programs: "Pushing a single button" on a graphical user interface causes MAS to run navigation programs that design a maneuver; programs that create sequences of commands to execute the maneuver on the spacecraft; and a program that generates predictions about maneuver performance and generates reports and other files that enable users to quickly review and verify the maneuver design. MAS can also generate presentation materials, initiate electronic command request forms, and archive all data products for future reference.

  14. Both Automation and Paper.

    ERIC Educational Resources Information Center

    Purcell, Royal

    1988-01-01

    Discusses the concept of a paperless society and the current situation in library automation. Various applications of automation and telecommunications are addressed, and future library automation is considered. Automation at the Monroe County Public Library in Bloomington, Indiana, is described as an example. (MES)

  15. A semi-automated system for quantifying the oxidative potential of ambient particles in aqueous extracts using the dithiothreitol (DTT) assay: results from the Southeastern Center for Air Pollution and Epidemiology (SCAPE)

    NASA Astrophysics Data System (ADS)

    Fang, T.; Verma, V.; Guo, H.; King, L. E.; Edgerton, E. S.; Weber, R. J.

    2015-01-01

    A variety of methods are used to measure the capability of particulate matter (PM) to catalytically generate reactive oxygen species (ROS) in vivo, also defined as the aerosol oxidative potential. A widely used measure of aerosol oxidative potential is the dithiothreitol (DTT) assay, which monitors the depletion of DTT (a surrogate for cellular antioxidants) as catalyzed by the redox-active species in PM. However, a major constraint in the routine use of the DTT assay for integrating it with large-scale health studies is its labor-intensive and time-consuming protocol. To specifically address this concern, we have developed a semi-automated system for quantifying the oxidative potential of aerosol liquid extracts using the DTT assay. The system, capable of unattended analysis at one sample per hour, has a high analytical precision (coefficient of variation of 15% for positive control, 4% for ambient samples) and reasonably low limit of detection (0.31 nmol min-1). Comparison of the automated approach with the manual method conducted on ambient samples yielded good agreement (slope = 1.08 ± 0.12, r2 = 0.92, N = 9). The system was utilized for the Southeastern Center for Air Pollution & Epidemiology (SCAPE) to generate an extensive data set on DTT activity of ambient particles collected from contrasting environments (urban, roadside, and rural) in the southeastern US. We find that water-soluble PM2.5 DTT activity on a per-air-volume basis was spatially uniform and often well correlated with PM2.5 mass (r = 0.49 to 0.88), suggesting regional sources contributing to the PM oxidative potential in the southeastern US. The correlation may also suggest a mechanistic explanation (oxidative stress) for observed PM2.5 mass-health associations. The heterogeneity in the intrinsic DTT activity (per-PM-mass basis) across seasons indicates variability in the DTT activity associated with aerosols from sources that vary with season. Although developed for the DTT assay, the

  16. Automated Defect Classification (ADC)

    Energy Science and Technology Software Center (ESTSC)

    1998-01-01

    The ADC Software System is designed to provide semiconductor defect feature analysis and defect classification capabilities. Defect classification is an important software method used by semiconductor wafer manufacturers to automate the analysis of defect data collected by a wide range of microscopy techniques in semiconductor wafer manufacturing today. These microscopies (e.g., optical bright and dark field, scanning electron microscopy, atomic force microscopy, etc.) generate images of anomalies that are induced or otherwise appear on wafermore » surfaces as a result of errant manufacturing processes or simple atmospheric contamination (e.g., airborne particles). This software provides methods for analyzing these images, extracting statistical features from the anomalous regions, and applying supervised classifiers to label the anomalies into user-defined categories.« less

  17. Automated synthetic scene generation

    NASA Astrophysics Data System (ADS)

    Givens, Ryan N.

    Physics-based simulations generate synthetic imagery to help organizations anticipate system performance of proposed remote sensing systems. However, manually constructing synthetic scenes which are sophisticated enough to capture the complexity of real-world sites can take days to months depending on the size of the site and desired fidelity of the scene. This research, sponsored by the Air Force Research Laboratory's Sensors Directorate, successfully developed an automated approach to fuse high-resolution RGB imagery, lidar data, and hyperspectral imagery and then extract the necessary scene components. The method greatly reduces the time and money required to generate realistic synthetic scenes and developed new approaches to improve material identification using information from all three of the input datasets.

  18. Automating Frame Analysis

    SciTech Connect

    Sanfilippo, Antonio P.; Franklin, Lyndsey; Tratz, Stephen C.; Danielson, Gary R.; Mileson, Nicholas D.; Riensche, Roderick M.; McGrath, Liam

    2008-04-01

    Frame Analysis has come to play an increasingly stronger role in the study of social movements in Sociology and Political Science. While significant steps have been made in providing a theory of frames and framing, a systematic characterization of the frame concept is still largely lacking and there are no rec-ognized criteria and methods that can be used to identify and marshal frame evi-dence reliably and in a time and cost effective manner. Consequently, current Frame Analysis work is still too reliant on manual annotation and subjective inter-pretation. The goal of this paper is to present an approach to the representation, acquisition and analysis of frame evidence which leverages Content Analysis, In-formation Extraction and Semantic Search methods to provide a systematic treat-ment of a Frame Analysis and automate frame annotation.

  19. RNA genetics

    SciTech Connect

    Domingo, E. ); Holland, J.J. . Dept. of Biology); Ahlquist, P. . Dept. of Plant Pathology)

    1988-01-01

    This book contains the proceedings on RNA genetics: Retroviruses, Viroids, and RNA recombination, Volume 2. Topics covered include: Replication of retrovirus genomes, Hepatitis B virus replication, and Evolution of RNA viruses.

  20. Automated data entry system: performance issues

    NASA Astrophysics Data System (ADS)

    Thoma, George R.; Ford, Glenn

    2001-12-01

    This paper discusses the performance of a system for extracting bibliographic fields from scanned pages in biomedical journals to populate MEDLINE, the flagship database of the national Library of Medicine (NLM), and heavily used worldwide. This system consists of automated processes to extract the article title, author names, affiliations and abstract, and manual workstations for the entry of other required fields such as pagination, grant support information, databank accession numbers and others needed for a completed bibliographic record in MEDLINE. Labor and time data are given for (1) a wholly manual keyboarding process to create the records, (2) an OCR-based system that requires all fields except the abstract to be manually input, and (3) a more automated system that relies on document image analysis and understanding techniques for the extraction of several fields. It is shown that this last, most automated, approach requires less than 25% of the labor effort in the first, manual, process.

  1. Method and Apparatus for Automated Isolation of Nucleic Acids from Small Cell Samples

    NASA Technical Reports Server (NTRS)

    Sundaram, Shivshankar; Prabhakarpandian, Balabhaskar; Pant, Kapil; Wang, Yi

    2014-01-01

    RNA isolation is a ubiquitous need, driven by current emphasis on microarrays and miniaturization. With commercial systems requiring 100,000 to 1,000,000 cells for successful isolation, there is a growing need for a small-footprint, easy-to-use device that can harvest nucleic acids from much smaller cell samples (1,000 to 10,000 cells). The process of extraction of RNA from cell cultures is a complex, multi-step one, and requires timed, asynchronous operations with multiple reagents/buffers. An added complexity is the fragility of RNA (subject to degradation) and its reactivity to surface. A novel, microfluidics-based, integrated cartridge has been developed that can fully automate the complex process of RNA isolation (lyse, capture, and elute RNA) from small cell culture samples. On-cartridge cell lysis is achieved using either reagents or high-strength electric fields made possible by the miniaturized format. Traditionally, silica-based, porous-membrane formats have been used for RNA capture, requiring slow perfusion for effective capture. In this design, high efficiency capture/elution are achieved using a microsphere-based "microfluidized" format. Electrokinetic phenomena are harnessed to actively mix microspheres with the cell lysate and capture/elution buffer, providing important advantages in extraction efficiency, processing time, and operational flexibility. Successful RNA isolation was demonstrated using both suspension (HL-60) and adherent (BHK-21) cells. Novel features associated with this development are twofold. First, novel designs that execute needed processes with improved speed and efficiency were developed. These primarily encompass electric-field-driven lysis of cells. The configurations include electrode-containing constructs, or an "electrode-less" chip design, which is easy to fabricate and mitigates fouling at the electrode surface; and the "fluidized" extraction format based on electrokinetically assisted mixing and contacting of microbeads

  2. A method of extracting disease-related microRNAs through the propagation algorithm using the environmental factor based global miRNA network.

    PubMed

    Ha, Jihwan; Kim, Hyunjin; Yoon, Youngmi; Park, Sanghyun

    2015-01-01

    MicroRNAs (miRNA) are known to be involved in the development of various diseases. Hence various scientists in the field have been utilized computational analyses to determine the relationship between miRNA and diseases. However, the knowledge of miRNA and disease is still very limited. Therefore, we combined Environmental Factor (EF) data to a miRNA global network. Increasing research has shown that relationship between miRNAs and EFs play a significant role in classifying types of diseases. Environmental Factors consist of radiation, drugs, viruses, alcohol, cigarettes, and stress. Our global network considered all the interactions between every pair of miRNAs, which has led to precise analyses in comparison to local networks. As a result, our approaches' performance demonstrated its effectiveness in identifying disease-related miRNA and this is the area under the ROC curve (AUC) of 74.46%. Furthermore, comparative experiment has shown that our approach performs comparable to other existing methods with an accuracy of 94%, 90% and 96% for breast cancer, colonic cancer, and lung cancer respectively. In conclusion, these results support that our research has broadened new biological insights on identifying disease-related miRNAs. PMID:26405945

  3. Posttranscriptional silencing of the lncRNA MALAT1 by miR-217 inhibits the epithelial-mesenchymal transition via enhancer of zeste homolog 2 in the malignant transformation of HBE cells induced by cigarette smoke extract.

    PubMed

    Lu, Lu; Luo, Fei; Liu, Yi; Liu, Xinlu; Shi, Le; Lu, Xiaolin; Liu, Qizhan

    2015-12-01

    Lung cancer is regarded as the leading cause of cancer-related deaths, and cigarette smoking is one of the strongest risk factors for the development of lung cancer. However, the mechanisms for cigarette smoke-induced lung carcinogenesis remain unclear. The present study investigated the effects of an miRNA (miR-217) on levels of an lncRNA (MALAT1) and examined the role of these factors in the epithelial-mesenchymal transition (EMT) induced by cigarette smoke extract (CSE) in human bronchial epithelial (HBE) cells. In these cells, CSE caused decreases of miR-217 levels and increases in lncRNA MALAT1 levels. Over-expression of miR-217 with a mimic attenuated the CSE-induced increase of MALAT1 levels, and reduction of miR-217 levels by an inhibitor enhanced expression of MALAT1. Moreover, the CSE-induced increase of MALAT1 expression was blocked by an miR-217 mimic, indicating that miR-217 negatively regulates MALAT1 expression. Knockdown of MALAT1 reversed CSE-induced increases of EZH2 (enhancer of zeste homolog 2) and H3K27me3 levels. In addition to the alteration from epithelial to spindle-like mesenchymal morphology, chronic exposure of HBE cells to CSE increased the levels of EZH2, H3K27me3, vimentin, and N-cadherin and decreased E-cadherin levels, effects that were reversed by MALAT1 siRNA or EZH2 siRNA. The results indicate that miR-217 regulation of EZH2/H3K27me3 via MALAT1 is involved in CSE-induced EMT and malignant transformation of HBE cells. The posttranscriptional silencing of MALAT1 by miR-217 provides a link, through EZH2, between ncRNAs and the EMT and establishes a mechanism for CSE-induced lung carcinogenesis. PMID:26415832

  4. An Automated System of Knickpoint Definition and Extraction from a Digital Elevation Model (DEM): Implications for Efficient Large-Scale Mapping and Statistical Analyses of Knickpoint Distributions in Fluvial Networks

    NASA Astrophysics Data System (ADS)

    Neely, A. B.; Bookhagen, B.; Burbank, D. W.

    2014-12-01

    Knickpoints, or convexities in a stream's longitudinal profile, often delineate boundaries in stream networks that separate reaches eroding at different rates resulting from sharp temporal or spatial changes in uplift rate, contributing drainage area, precipitation, or bedrock lithology. We explicitly defined the geometry of a knickpoint in a manner that can be identified using an algorithm which operates in accordance with the stream power incision model, using a chi-plot analysis approach. This method allows for comparison between the real stream profile extracted from a DEM, and a linear best-fit line profile in chi-elevation space, representing a steady state theoretical stream functioning in accordance to uniform temporal and spatial conditions listed above. Assessing where the stream of interest is "under-steepened" and "over-steepened" with respect to a theoretical linear profile reveals knickpoints as certain points of slope inflection, extractable by our algorithm. We tested our algorithm on a 1m resolution LiDAR DEM of Santa Cruz Island (SCI), a tectonically active island 25km south of Santa Barbara, CA with an estimated uplift rate between 0.5 and 1.2mm/yr calculated from uplifted paleoshorelines. We have identified 1025 knickpoints using our algorithm and compared the position of these knickpoints to a similarly-sized dataset of knickpoints manually selected from distance-elevation longitudinal stream profiles for the same region. Our algorithm reduced mapping time by 99.3% and agreed with knickpoint positions from the manually selected knickpoint map for 85% of the 1025 knickpoints. Discrepancies can arise from inconsistencies in manual knickpoint selection that are not present in an automated computation. Additionally, the algorithm measures useful characteristics for each knickpoint allowing for quick statistical analyses. Histograms of knickpoint elevation and chi coordinate have a 3 peaked distribution, possibly expressing 3 levels of uplifted

  5. A Rapid Protocol of Crude RNA/DNA Extraction for RT-qPCR Detection and Quantification of 'Candidatus Phytoplasma prunorum'

    PubMed Central

    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

  6. A Rapid Protocol of Crude RNA/DNA Extraction for RT-qPCR Detection and Quantification of 'Candidatus Phytoplasma prunorum'.

    PubMed

    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

  7. RNA genetics

    SciTech Connect

    Domingo, E.; Holland, J.J.; Ahlquist, P.

    1988-01-01

    These three volumes comprise reference on RNA genomes. The replication, mutation, recombination-assortment, and extreme evolutionary variability of RNA viruses and related RNA replicons is emphasized. The replication mechanisms of positive, negative, and double-stranded RNA viruses of animals and plants are featured.

  8. Automated Methods for Multiplexed Pathogen Detection

    SciTech Connect

    Straub, Tim M.; Dockendorff, Brian P.; Quinonez-Diaz, Maria D.; Valdez, Catherine O.; Shutthanandan, Janani I.; Tarasevich, Barbara J.; Grate, Jay W.; Bruckner-Lea, Cindy J.

    2005-09-01

    Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides ''live vs. dead'' capabilities. However

  9. RNA Crystallization

    NASA Technical Reports Server (NTRS)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  10. RNA helicases

    PubMed Central

    Owttrim, George W.

    2013-01-01

    Similar to proteins, RNA molecules must fold into the correct conformation and associate with protein complexes in order to be functional within a cell. RNA helicases rearrange RNA secondary structure and RNA-protein interactions in an ATP-dependent reaction, performing crucial functions in all aspects of RNA metabolism. In prokaryotes, RNA helicase activity is associated with roles in housekeeping functions including RNA turnover, ribosome biogenesis, translation and small RNA metabolism. In addition, RNA helicase expression and/or activity are frequently altered during cellular response to abiotic stress, implying they perform defined roles during cellular adaptation to changes in the growth environment. Specifically, RNA helicases contribute to the formation of cold-adapted ribosomes and RNA degradosomes, implying a role in alleviation of RNA secondary structure stabilization at low temperature. A common emerging theme involves RNA helicases acting as scaffolds for protein-protein interaction and functioning as molecular clamps, holding RNA-protein complexes in specific conformations. This review highlights recent advances in DEAD-box RNA helicase association with cellular response to abiotic stress in prokaryotes. PMID:23093803

  11. Components for automated microfluidics sample preparation and analysis

    NASA Astrophysics Data System (ADS)

    Archer, M.; Erickson, J. S.; Hilliard, L. R.; Howell, P. B., Jr.; Stenger, D. A.; Ligler, F. S.; Lin, B.

    2008-02-01

    The increasing demand for portable devices to detect and identify pathogens represents an interdisciplinary effort between engineering, materials science, and molecular biology. Automation of both sample preparation and analysis is critical for performing multiplexed analyses on real world samples. This paper selects two possible components for such automated portable analyzers: modified silicon structures for use in the isolation of nucleic acids and a sheath flow system suitable for automated microflow cytometry. Any detection platform that relies on the genetic content (RNA and DNA) present in complex matrices requires careful extraction and isolation of the nucleic acids in order to ensure their integrity throughout the process. This sample pre-treatment step is commonly performed using commercially available solid phases along with various molecular biology techniques that require multiple manual steps and dedicated laboratory space. Regardless of the detection scheme, a major challenge in the integration of total analysis systems is the development of platforms compatible with current isolation techniques that will ensure the same quality of nucleic acids. Silicon is an ideal candidate for solid phase separations since it can be tailored structurally and chemically to mimic the conditions used in the laboratory. For analytical purposes, we have developed passive structures that can be used to fully ensheath one flow stream with another. As opposed to traditional flow focusing methods, our sheath flow profile is truly two dimensional, making it an ideal candidate for integration into a microfluidic flow cytometer. Such a microflow cytometer could be used to measure targets captured on either antibody- or DNA-coated beads.

  12. Automated External Defibrillator

    MedlinePlus

    ... from the NHLBI on Twitter. What Is an Automated External Defibrillator? An automated external defibrillator (AED) is a portable device that ... Institutes of Health Department of Health and Human Services USA.gov

  13. Automated labeling in document images

    NASA Astrophysics Data System (ADS)

    Kim, Jongwoo; Le, Daniel X.; Thoma, George R.

    2000-12-01

    The National Library of Medicine (NLM) is developing an automated system to produce bibliographic records for its MEDLINER database. This system, named Medical Article Record System (MARS), employs document image analysis and understanding techniques and optical character recognition (OCR). This paper describes a key module in MARS called the Automated Labeling (AL) module, which labels all zones of interest (title, author, affiliation, and abstract) automatically. The AL algorithm is based on 120 rules that are derived from an analysis of journal page layouts and features extracted from OCR output. Experiments carried out on more than 11,000 articles in over 1,000 biomedical journals show the accuracy of this rule-based algorithm to exceed 96%.

  14. Sensors and Automated Analyzers for Radionuclides

    SciTech Connect

    Grate, Jay W.; Egorov, Oleg B.

    2003-03-27

    The production of nuclear weapons materials has generated large quantities of nuclear waste and significant environmental contamination. We have developed new, rapid, automated methods for determination of radionuclides using sequential injection methodologies to automate extraction chromatographic separations, with on-line flow-through scintillation counting for real time detection. This work has progressed in two main areas: radionuclide sensors for water monitoring and automated radiochemical analyzers for monitoring nuclear waste processing operations. Radionuclide sensors have been developed that collect and concentrate radionuclides in preconcentrating minicolumns with dual functionality: chemical selectivity for radionuclide capture and scintillation for signal output. These sensors can detect pertechnetate to below regulatory levels and have been engineered into a prototype for field testing. A fully automated process monitor has been developed for total technetium in nuclear waste streams. This instrument performs sample acidification, speciation adjustment, separation and detection in fifteen minutes or less.

  15. Image segmentation for automated dental identification

    NASA Astrophysics Data System (ADS)

    Haj Said, Eyad; Nassar, Diaa Eldin M.; Ammar, Hany H.

    2006-02-01

    Dental features are one of few biometric identifiers that qualify for postmortem identification; therefore, creation of an Automated Dental Identification System (ADIS) with goals and objectives similar to the Automated Fingerprint Identification System (AFIS) has received increased attention. As a part of ADIS, teeth segmentation from dental radiographs films is an essential step in the identification process. In this paper, we introduce a fully automated approach for teeth segmentation with goal to extract at least one tooth from the dental radiograph film. We evaluate our approach based on theoretical and empirical basis, and we compare its performance with the performance of other approaches introduced in the literature. The results show that our approach exhibits the lowest failure rate and the highest optimality among all full automated approaches introduced in the literature.

  16. Workflow automation architecture standard

    SciTech Connect

    Moshofsky, R.P.; Rohen, W.T.

    1994-11-14

    This document presents an architectural standard for application of workflow automation technology. The standard includes a functional architecture, process for developing an automated workflow system for a work group, functional and collateral specifications for workflow automation, and results of a proof of concept prototype.

  17. Automated Characterization Of Vibrations Of A Structure

    NASA Technical Reports Server (NTRS)

    Bayard, David S.; Yam, Yeung; Mettler, Edward; Hadaegh, Fred Y.; Milman, Mark H.; Scheid, Robert E.

    1992-01-01

    Automated method of characterizing dynamical properties of large flexible structure yields estimates of modal parameters used by robust control system to stabilize structure and minimize undesired motions. Based on extraction of desired modal and control-design data from responses of structure to known vibrational excitations. Applicable to terrestrial structures where vibrations are important - aircraft, buildings, bridges, cranes, and drill strings.

  18. Automation in Clinical Microbiology

    PubMed Central

    Ledeboer, Nathan A.

    2013-01-01

    Historically, the trend toward automation in clinical pathology laboratories has largely bypassed the clinical microbiology laboratory. In this article, we review the historical impediments to automation in the microbiology laboratory and offer insight into the reasons why we believe that we are on the cusp of a dramatic change that will sweep a wave of automation into clinical microbiology laboratories. We review the currently available specimen-processing instruments as well as the total laboratory automation solutions. Lastly, we outline the types of studies that will need to be performed to fully assess the benefits of automation in microbiology laboratories. PMID:23515547

  19. Automation of industrial bioprocesses.

    PubMed

    Beyeler, W; DaPra, E; Schneider, K

    2000-01-01

    The dramatic development of new electronic devices within the last 25 years has had a substantial influence on the control and automation of industrial bioprocesses. Within this short period of time the method of controlling industrial bioprocesses has changed completely. In this paper, the authors will use a practical approach focusing on the industrial applications of automation systems. From the early attempts to use computers for the automation of biotechnological processes up to the modern process automation systems some milestones are highlighted. Special attention is given to the influence of Standards and Guidelines on the development of automation systems. PMID:11092132

  20. Moving RNA moves RNA forward.

    PubMed

    Peng, Lina; Li, Yujiao; Zhang, Lan; Yu, Wenqiang

    2013-10-01

    Cell communication affects all aspects of cell structure and behavior, such as cell proliferation, differentiation, division, and coordination of various physiological functions. The moving RNA in plants and mammalian cells indicates that nucleic acid could be one of the various types of messengers for cell communication. The microvesicle is a critical pathway that mediates RNA moving and keeps moving RNA stable in body fluids. When moving miRNA enters the target cell, it functions by altering the gene expression profile and significantly inhibiting mRNA translation in recipient cells. Thus, moving RNA may act as a long-range modulator during development, organogenesis, and tumor metastasis. PMID:24008386

  1. Protein-RNA networks revealed through covalent RNA marks.

    PubMed

    Lapointe, Christopher P; Wilinski, Daniel; Saunders, Harriet A J; Wickens, Marvin

    2015-12-01

    Protein-RNA networks are ubiquitous and central in biological control. We present an approach termed RNA Tagging that enables the user to identify protein-RNA interactions in vivo by analyzing purified cellular RNA, without protein purification or cross-linking. An RNA-binding protein of interest is fused to an enzyme that adds uridines to the end of RNA. RNA targets bound by the chimeric protein in vivo are covalently marked with uridines and subsequently identified from extracted RNA via high-throughput sequencing. We used this approach to identify hundreds of RNAs bound by a Saccharomyces cerevisiae PUF protein, Puf3p. The results showed that although RNA-binding proteins productively bind specific RNAs to control their function, they also 'sample' RNAs without exerting a regulatory effect. We used the method to uncover hundreds of new and likely regulated targets for a protein without canonical RNA-binding domains, Bfr1p. RNA Tagging is well suited to detect and analyze protein-RNA networks in vivo. PMID:26524240

  2. Protein-RNA networks revealed through covalent RNA marks

    PubMed Central

    Lapointe, Christopher P.; Wilinski, Daniel; Saunders, Harriet A. J.; Wickens, Marvin

    2015-01-01

    Protein-RNA networks are ubiquitous and central in biological control. We present an approach, termed “RNA Tagging,” that identifies protein-RNA interactions in vivo by analyzing purified cellular RNA, without protein purification or crosslinking. An RNA-binding protein of interest is fused to an enzyme that adds uridines to the end of RNA. RNA targets bound by the chimeric protein in vivo are covalently marked with uridines and subsequently identified from extracted RNA using high-throughput sequencing. We used this approach to identify hundreds of RNAs bound by a Saccharomyces cerevisiae PUF protein, Puf3p. The method revealed that while RNA-binding proteins productively bind specific RNAs to control their function, they also “sample” RNAs without exerting a regulatory effect. We exploited the method to uncover hundreds of new and likely regulated targets for a protein without canonical RNA-binding domains, Bfr1p. The RNA Tagging approach is well-suited to detect and analyze protein-RNA networks in vivo. PMID:26524240

  3. Optimization of Droplet Digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR: Clinical implications for quantification of Oseltamivir-resistant subpopulations.

    PubMed

    Taylor, Sean C; Carbonneau, Julie; Shelton, Dawne N; Boivin, Guy

    2015-11-01

    The recent introduction of Droplet Digital PCR (ddPCR) has provided researchers with a tool that permits direct quantification of nucleic acids from a wide range of samples with increased precision and sensitivity versus RT-qPCR. The sample interdependence of RT-qPCR stemming from the measurement of Cq and ΔCq values is eliminated with ddPCR which provides an independent measure of the absolute nucleic acid concentration for each sample without standard curves thereby reducing inter-well and inter-plate variability. Well-characterized RNA purified from H275-wild type (WT) and H275Y-point mutated (MUT) neuraminidase of influenza A (H1N1) pandemic 2009 virus was used to demonstrate a ddPCR optimization workflow to assure robust data for downstream analysis. The ddPCR reaction mix was also tested with RT-qPCR and gave excellent reaction efficiency (between 90% and 100%) with the optimized MUT/WT duplexed assay thus enabling the direct comparison of the two platforms from the same reaction mix and thermal cycling protocol. ddPCR gave a marked improvement in sensitivity (>30-fold) for mutation abundance using a mixture of purified MUT and WT RNA and increased precision (>10 fold, p<0.05 for both inter- and intra-assay variability) versus RT-qPCR from patient samples to accurately identify residual mutant viral population during recovery. PMID:26315318

  4. Determining RNA quality for NextGen sequencing: some exceptions to the gold standard rule of 23S to 16S rRNA ratio

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using next-generation-sequencing technology to assess entire transcriptomes requires high quality starting RNA. Currently, RNA quality is routinely judged using automated microfluidic gel electrophoresis platforms and associated algorithms. Here we report that such automated methods generate false-n...

  5. Effect of RNA Integrity Determined With the Agilent 2100 Bioanalyzer on Bacterial RNA Quantification with RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA integrity is critical for successful RNA quantification. The level of integrity required differs among sources and extraction procedures and has not been determined for bacterial RNA. Three RNA isolation methods were evaluated for their ability to produce high quality RNA from D. dadantii. The i...

  6. ANDSystem: an Associative Network Discovery System for automated literature mining in the field of biology

    PubMed Central

    2015-01-01

    Background Sufficient knowledge of molecular and genetic interactions, which comprise the entire basis of the functioning of living systems, is one of the necessary requirements for successfully answering almost any research question in the field of biology and medicine. To date, more than 24 million scientific papers can be found in PubMed, with many of them containing descriptions of a wide range of biological processes. The analysis of such tremendous amounts of data requires the use of automated text-mining approaches. Although a handful of tools have recently been developed to meet this need, none of them provide error-free extraction of highly detailed information. Results The ANDSystem package was developed for the reconstruction and analysis of molecular genetic networks based on an automated text-mining technique. It provides a detailed description of the various types of interactions between genes, proteins, microRNA's, metabolites, cellular components, pathways and diseases, taking into account the specificity of cell lines and organisms. Although the accuracy of ANDSystem is comparable to other well known text-mining tools, such as Pathway Studio and STRING, it outperforms them in having the ability to identify an increased number of interaction types. Conclusion The use of ANDSystem, in combination with Pathway Studio and STRING, can improve the quality of the automated reconstruction of molecular and genetic networks. ANDSystem should provide a useful tool for researchers working in a number of different fields, including biology, biotechnology, pharmacology and medicine. PMID:25881313

  7. RNA epigenetics

    PubMed Central

    Liu, Nian; Pan, Tao

    2014-01-01

    Summary Mammalian messenger and long non-coding RNA contain tens of thousands of post-transcriptional chemical modifications. Among these, the N6-methyl-adenosine (m6A) modification is the most abundant and can be removed by specific mammalian enzymes. M6A modification is recognized by families of RNA binding proteins that affect many aspects of mRNA function. mRNA/lncRNA modification represents another layer of epigenetic regulation of gene expression, analogous to DNA methylation and histone modification. PMID:24768686

  8. Automated imaging system for single molecules

    DOEpatents

    Schwartz, David Charles; Runnheim, Rodney; Forrest, Daniel

    2012-09-18

    There is provided a high throughput automated single molecule image collection and processing system that requires minimal initial user input. The unique features embodied in the present disclosure allow automated collection and initial processing of optical images of single molecules and their assemblies. Correct focus may be automatically maintained while images are collected. Uneven illumination in fluorescence microscopy is accounted for, and an overall robust imaging operation is provided yielding individual images prepared for further processing in external systems. Embodiments described herein are useful in studies of any macromolecules such as DNA, RNA, peptides and proteins. The automated image collection and processing system and method of same may be implemented and deployed over a computer network, and may be ergonomically optimized to facilitate user interaction.

  9. Automated DNA Sequencing System

    SciTech Connect

    Armstrong, G.A.; Ekkebus, C.P.; Hauser, L.J.; Kress, R.L.; Mural, R.J.

    1999-04-25

    Oak Ridge National Laboratory (ORNL) is developing a core DNA sequencing facility to support biological research endeavors at ORNL and to conduct basic sequencing automation research. This facility is novel because its development is based on existing standard biology laboratory equipment; thus, the development process is of interest to the many small laboratories trying to use automation to control costs and increase throughput. Before automation, biology Laboratory personnel purified DNA, completed cycle sequencing, and prepared 96-well sample plates with commercially available hardware designed specifically for each step in the process. Following purification and thermal cycling, an automated sequencing machine was used for the sequencing. A technician handled all movement of the 96-well sample plates between machines. To automate the process, ORNL is adding a CRS Robotics A- 465 arm, ABI 377 sequencing machine, automated centrifuge, automated refrigerator, and possibly an automated SpeedVac. The entire system will be integrated with one central controller that will direct each machine and the robot. The goal of this system is to completely automate the sequencing procedure from bacterial cell samples through ready-to-be-sequenced DNA and ultimately to completed sequence. The system will be flexible and will accommodate different chemistries than existing automated sequencing lines. The system will be expanded in the future to include colony picking and/or actual sequencing. This discrete event, DNA sequencing system will demonstrate that smaller sequencing labs can achieve cost-effective the laboratory grow.

  10. Enhancing Seismic Calibration Research Through Software Automation

    SciTech Connect

    Ruppert, S; Dodge, D; Elliott, A; Ganzberger, M; Hauk, T; Matzel, E; Ryall, F

    2004-07-09

    observations. Even partial automation of this second tier, through development of prototype tools to extract observations and make many thousands of scientific measurements, has significantly increased the efficiency of the scientists who construct and validate integrated calibration surfaces. This achieved gain in efficiency and quality control is likely to continue and even accelerate through continued application of information science and scientific automation. Data volume and calibration research requirements have increased by several orders of magnitude over the past decade. Whereas it was possible for individual researchers to download individual waveforms and make time-consuming measurements event by event in the past, with the Terabytes of data available today, a software automation framework must exist to efficiently populate and deliver quality data to the researcher. This framework must also simultaneously provide the researcher with robust measurement and analysis tools that can handle and extract groups of events effectively and isolate the researcher from the now onerous task of database management and metadata collection necessary for validation and error analysis. We have succeeded in automating many of the collection, parsing, reconciliation and extraction tasks, individually. Several software automation prototypes have been produced and have resulted in demonstrated gains in efficiency of producing scientific data products. Future software automation tasks will continue to leverage database and information management technologies in addressing additional scientific calibration research tasks.

  11. Automation for System Safety Analysis

    NASA Technical Reports Server (NTRS)

    Malin, Jane T.; Fleming, Land; Throop, David; Thronesbery, Carroll; Flores, Joshua; Bennett, Ted; Wennberg, Paul

    2009-01-01

    This presentation describes work to integrate a set of tools to support early model-based analysis of failures and hazards due to system-software interactions. The tools perform and assist analysts in the following tasks: 1) extract model parts from text for architecture and safety/hazard models; 2) combine the parts with library information to develop the models for visualization and analysis; 3) perform graph analysis and simulation to identify and evaluate possible paths from hazard sources to vulnerable entities and functions, in nominal and anomalous system-software configurations and scenarios; and 4) identify resulting candidate scenarios for software integration testing. There has been significant technical progress in model extraction from Orion program text sources, architecture model derivation (components and connections) and documentation of extraction sources. Models have been derived from Internal Interface Requirements Documents (IIRDs) and FMEA documents. Linguistic text processing is used to extract model parts and relationships, and the Aerospace Ontology also aids automated model development from the extracted information. Visualizations of these models assist analysts in requirements overview and in checking consistency and completeness.

  12. Laboratory Automation and Middleware.

    PubMed

    Riben, Michael

    2015-06-01

    The practice of surgical pathology is under constant pressure to deliver the highest quality of service, reduce errors, increase throughput, and decrease turnaround time while at the same time dealing with an aging workforce, increasing financial constraints, and economic uncertainty. Although not able to implement total laboratory automation, great progress continues to be made in workstation automation in all areas of the pathology laboratory. This report highlights the benefits and challenges of pathology automation, reviews middleware and its use to facilitate automation, and reviews the progress so far in the anatomic pathology laboratory. PMID:26065792

  13. Management Planning for Workplace Automation.

    ERIC Educational Resources Information Center

    McDole, Thomas L.

    Several factors must be considered when implementing office automation. Included among these are whether or not to automate at all, the effects of automation on employees, requirements imposed by automation on the physical environment, effects of automation on the total organization, and effects on clientele. The reasons behind the success or…

  14. Automated drilling draws interest

    SciTech Connect

    Not Available

    1985-05-01

    Interest in subsea technology includes recent purchase of both a British yard and Subsea Technology, a Houston-based BOP manufacturer. In France, key personnel from the former Comex Industries have been acquired and a base reinstalled in Marseille. ACB is also investing heavily, with the Norwegians, in automated drilling programs. These automated drilling programs are discussed.

  15. Library Automation Style Guide.

    ERIC Educational Resources Information Center

    Gaylord Bros., Liverpool, NY.

    This library automation style guide lists specific terms and names often used in the library automation industry. The terms and/or acronyms are listed alphabetically and each is followed by a brief definition. The guide refers to the "Chicago Manual of Style" for general rules, and a notes section is included for the convenience of individual…

  16. Automation and Cataloging.

    ERIC Educational Resources Information Center

    Furuta, Kenneth; And Others

    1990-01-01

    These three articles address issues in library cataloging that are affected by automation: (1) the impact of automation and bibliographic utilities on professional catalogers; (2) the effect of the LASS microcomputer software on the cost of authority work in cataloging at the University of Arizona; and (3) online subject heading and classification…

  17. Planning for Office Automation.

    ERIC Educational Resources Information Center

    Sherron, Gene T.

    1982-01-01

    The steps taken toward office automation by the University of Maryland are described. Office automation is defined and some types of word processing systems are described. Policies developed in the writing of a campus plan are listed, followed by a section on procedures adopted to implement the plan. (Author/MLW)

  18. The Automated Office.

    ERIC Educational Resources Information Center

    Naclerio, Nick

    1979-01-01

    Clerical personnel may be able to climb career ladders as a result of office automation and expanded job opportunities in the word processing area. Suggests opportunities in an automated office system and lists books and periodicals on word processing for counselors and teachers. (MF)

  19. Work and Programmable Automation.

    ERIC Educational Resources Information Center

    DeVore, Paul W.

    A new industrial era based on electronics and the microprocessor has arrived, an era that is being called intelligent automation. Intelligent automation, in the form of robots, replaces workers, and the new products, using microelectronic devices, require significantly less labor to produce than the goods they replace. The microprocessor thus…

  20. Automation, Manpower, and Education.

    ERIC Educational Resources Information Center

    Rosenberg, Jerry M.

    Each group in our population will be affected by automation and other forms of technological advancement. This book seeks to identify the needs of these various groups, and to present ways in which educators can best meet them. The author corrects certain prevalent misconceptions concerning manpower utilization and automation. Based on the…

  1. RNA helicases

    PubMed Central

    Ranji, Arnaz

    2010-01-01

    RNA helicases serve multiple roles at the virus-host interface. In some situations, RNA helicases are essential host factors to promote viral replication; however, in other cases they serve as a cellular sensor to trigger the antiviral state in response to viral infection. All family members share the conserved ATP-dependent catalytic core linked to different substrate recognition and protein-protein interaction domains. These flanking domains can be shuffled between different helicases to achieve functional diversity. This review summarizes recent studies, This review summarizes recent studies of RNA helicases in virus biology. First, RNA helicases are catalysts of progressive RNA-protein rearrangements that begin at gene transcription and culminate in release of infectious virus. Second, RNA helicases can act as a scaffold for alternative protein-protein interactions that can defeat the antiviral state. The mounting fundamental understanding of RNA helicases is being used to develop selective and efficacious drugs against human and animal pathogens. The analysis of RNA helicases in virus model systems continues to provide insights into virology, cell biology and immunology and has provided fresh perspective to continue unraveling the complexity of virus-host interactions. PMID:21173576

  2. RNA. Introduction.

    PubMed

    Bao, Marie Z; Kruger, Robert P; Rivas, Fabiola; Smith, Orla; Szewczak, Lara

    2009-02-20

    Two scientists walk into a bar. After a pint and an exchange of pleasantries, one says to the other, "Where do you come from? Scientifically, I mean." The queried scientist responds, "Out of the RNA world." "Don't we all," the asker responds chuckling. Fifteen years ago, the joke would have been made with a nod to the notion that life arose from an RNA-based precursor, the so-called "RNA world." Yet had this conversation happened last week, the scientists would also be grinning in appreciation of the extent to which contemporary cellular biology is steeped in all things RNA. Ours is truly an RNA world.In this year's special review issue, the Cell editorial team has brought together articles focused on RNA in the modern world, providing perspectives on classical and emerging areas of inquiry. We extend our thanks to the many distinguished experts who contributed their time and effort as authors and reviewers to make the issue informative, thought-provoking, and timely. We hope that this collection of articles, written as we stand on the verge of a new wave of RNA biology, edifies and inspires by revealing the inner workings of these versatile molecules and by highlighting the next key questions that need to be addressed as we strive to understand the full functional scope of RNA in cells. PMID:19263588

  3. Conformational readout of RNA by small ligands

    PubMed Central

    Kligun, Efrat; Mandel-Gutfreund, Yael

    2013-01-01

    RNA molecules have highly versatile structures that can fold into myriad conformations, providing many potential pockets for binding small molecules. The increasing number of available RNA structures, in complex with proteins, small ligands and in free form, enables the design of new therapeutically useful RNA-binding ligands. Here we studied RNA ligand complexes from 10 RNA groups extracted from the protein data bank (PDB), including adaptive and non-adaptive complexes. We analyzed the chemical, physical, structural and conformational properties of binding pockets around the ligand. Comparing the properties of ligand-binding pockets to the properties of computed pockets extracted from all available RNA structures and RNA-protein interfaces, revealed that ligand-binding pockets, mainly the adaptive pockets, are characterized by unique properties, specifically enriched in rare conformations of the nucleobase and the sugar pucker. Further, we demonstrate that nucleotides possessing the rare conformations are preferentially involved in direct interactions with the ligand. Overall, based on our comprehensive analysis of RNA-ligand complexes, we suggest that the unique conformations adopted by RNA nucleotides play an important role in RNA recognition by small ligands. We term the recognition of a binding site by a ligand via the unique RNA conformations “RNA conformational readout.” We propose that “conformational readout” is a general way by which RNA binding pockets are recognized and selected from an ensemble of different RNA states. PMID:23618839

  4. Automation in Immunohematology

    PubMed Central

    Bajpai, Meenu; Kaur, Ravneet; Gupta, Ekta

    2012-01-01

    There have been rapid technological advances in blood banking in South Asian region over the past decade with an increasing emphasis on quality and safety of blood products. The conventional test tube technique has given way to newer techniques such as column agglutination technique, solid phase red cell adherence assay, and erythrocyte-magnetized technique. These new technologies are adaptable to automation and major manufacturers in this field have come up with semi and fully automated equipments for immunohematology tests in the blood bank. Automation improves the objectivity and reproducibility of tests. It reduces human errors in patient identification and transcription errors. Documentation and traceability of tests, reagents and processes and archiving of results is another major advantage of automation. Shifting from manual methods to automation is a major undertaking for any transfusion service to provide quality patient care with lesser turnaround time for their ever increasing workload. This article discusses the various issues involved in the process. PMID:22988378

  5. Advances in inspection automation

    NASA Astrophysics Data System (ADS)

    Weber, Walter H.; Mair, H. Douglas; Jansen, Dion; Lombardi, Luciano

    2013-01-01

    This new session at QNDE reflects the growing interest in inspection automation. Our paper describes a newly developed platform that makes the complex NDE automation possible without the need for software programmers. Inspection tasks that are tedious, error-prone or impossible for humans to perform can now be automated using a form of drag and drop visual scripting. Our work attempts to rectify the problem that NDE is not keeping pace with the rest of factory automation. Outside of NDE, robots routinely and autonomously machine parts, assemble components, weld structures and report progress to corporate databases. By contrast, components arriving in the NDT department typically require manual part handling, calibrations and analysis. The automation examples in this paper cover the development of robotic thickness gauging and the use of adaptive contour following on the NRU reactor inspection at Chalk River.

  6. Automated ship image acquisition

    NASA Astrophysics Data System (ADS)

    Hammond, T. R.

    2008-04-01

    The experimental Automated Ship Image Acquisition System (ASIA) collects high-resolution ship photographs at a shore-based laboratory, with minimal human intervention. The system uses Automatic Identification System (AIS) data to direct a high-resolution SLR digital camera to ship targets and to identify the ships in the resulting photographs. The photo database is then searchable using the rich data fields from AIS, which include the name, type, call sign and various vessel identification numbers. The high-resolution images from ASIA are intended to provide information that can corroborate AIS reports (e.g., extract identification from the name on the hull) or provide information that has been omitted from the AIS reports (e.g., missing or incorrect hull dimensions, cargo, etc). Once assembled into a searchable image database, the images can be used for a wide variety of marine safety and security applications. This paper documents the author's experience with the practicality of composing photographs based on AIS reports alone, describing a number of ways in which this can go wrong, from errors in the AIS reports, to fixed and mobile obstructions and multiple ships in the shot. The frequency with which various errors occurred in automatically-composed photographs collected in Halifax harbour in winter time were determined by manual examination of the images. 45% of the images examined were considered of a quality sufficient to read identification markings, numbers and text off the entire ship. One of the main technical challenges for ASIA lies in automatically differentiating good and bad photographs, so that few bad ones would be shown to human users. Initial attempts at automatic photo rating showed 75% agreement with manual assessments.

  7. EXTRACTION AND DETECTION OF ARSENICALS IN SEAWEED VIA ACCELERATED SOLVENT EXTRACTION WITH ION CHROMATOGRAPHIC SEPARATION AND ICP-MS DETECTION

    EPA Science Inventory

    An accelerated solvent extraction (ASE) device was evaluated as a semi-automated means of extracting arsenicals from ribbon kelp. Objective was to investigate effect of experimentally controllable ASE parameters (pressure, temperature, static time and solvent composition) on extr...

  8. First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

    PubMed Central

    Bémer, Pascale; Valentin, Anne Sophie; Héry-Arnaud, Geneviève; Tandé, Didier; Jolivet-Gougeon, Anne; Vincent, Pascal; Kempf, Marie; Lemarié, Carole; Guinard, Jérôme; Bret, Laurent; Cognée, Anne Sophie; Gibaud, Sophie; Burucoa, Christophe; Corvec, Stéphane

    2014-01-01

    The objective of this study was to assess the performance of seven French laboratories for 16S rRNA gene detection by real-time PCR in the diagnosis of bone and joint infection (BJI) to validate a large multicenter study. External quality control (QC) was required owing to the differences in extraction procedures and the molecular equipment used in the different laboratories. Three proficiency sets were organized, including four bacterial DNA extracts and four bead mill-pretreated osteoarticular specimens. Extraction volumes, 16S rRNA gene primers, and sequencing interpretation rules were standardized. In order to assess each laboratory's ability to achieve the best results, scores were assigned, and each QC series was classified as optimal, acceptable, or to be improved. A total of 168 QCs were sent, and 160 responses were analyzed. The expected results were obtained for 93.8%, with the same proportion for extracts (75/80) and clinical specimens (75/80). For the specimens, there was no significant difference between manual and automated extraction. This QC demonstrated the ability to achieve good and homogeneous results using the same 16S rRNA gene PCR with different equipment and validates the possibility of high-quality multicenter studies using molecular diagnosis for BJI. PMID:25411177

  9. First experience of a multicenter external quality assessment of molecular 16S rRNA gene detection in bone and joint infections.

    PubMed

    Plouzeau, Chloé; Bémer, Pascale; Valentin, Anne Sophie; Héry-Arnaud, Geneviève; Tandé, Didier; Jolivet-Gougeon, Anne; Vincent, Pascal; Kempf, Marie; Lemarié, Carole; Guinard, Jérôme; Bret, Laurent; Cognée, Anne Sophie; Gibaud, Sophie; Burucoa, Christophe; Corvec, Stéphane

    2015-02-01

    The objective of this study was to assess the performance of seven French laboratories for 16S rRNA gene detection by real-time PCR in the diagnosis of bone and joint infection (BJI) to validate a large multicenter study. External quality control (QC) was required owing to the differences in extraction procedures and the molecular equipment used in the different laboratories. Three proficiency sets were organized, including four bacterial DNA extracts and four bead mill-pretreated osteoarticular specimens. Extraction volumes, 16S rRNA gene primers, and sequencing interpretation rules were standardized. In order to assess each laboratory's ability to achieve the best results, scores were assigned, and each QC series was classified as optimal, acceptable, or to be improved. A total of 168 QCs were sent, and 160 responses were analyzed. The expected results were obtained for 93.8%, with the same proportion for extracts (75/80) and clinical specimens (75/80). For the specimens, there was no significant difference between manual and automated extraction. This QC demonstrated the ability to achieve good and homogeneous results using the same 16S rRNA gene PCR with different equipment and validates the possibility of high-quality multicenter studies using molecular diagnosis for BJI. PMID:25411177

  10. Automated cognome construction and semi-automated hypothesis generation.

    PubMed

    Voytek, Jessica B; Voytek, Bradley

    2012-06-30

    Modern neuroscientific research stands on the shoulders of countless giants. PubMed alone contains more than 21 million peer-reviewed articles with 40-50,000 more published every month. Understanding the human brain, cognition, and disease will require integrating facts from dozens of scientific fields spread amongst millions of studies locked away in static documents, making any such integration daunting, at best. The future of scientific progress will be aided by bridging the gap between the millions of published research articles and modern databases such as the Allen brain atlas (ABA). To that end, we have analyzed the text of over 3.5 million scientific abstracts to find associations between neuroscientific concepts. From the literature alone, we show that we can blindly and algorithmically extract a "cognome": relationships between brain structure, function, and disease. We demonstrate the potential of data-mining and cross-platform data-integration with the ABA by introducing two methods for semi-automated hypothesis generation. By analyzing statistical "holes" and discrepancies in the literature we can find understudied or overlooked research paths. That is, we have added a layer of semi-automation to a part of the scientific process itself. This is an important step toward fundamentally incorporating data-mining algorithms into the scientific method in a manner that is generalizable to any scientific or medical field. PMID:22584238

  11. RNA Research

    NASA Technical Reports Server (NTRS)

    1998-01-01

    It is generally believed that an RNA World existed at an early stage in the history of life. During this early period, RNA molecules are seen to be potentially involved in both catalysis and the storage of genetic information. It is widely believed that this RNA World was extensive and therefore a sophisticated nucleic acid replication machinery would presumably predate the translation machinery which would not be needed until later stages in the development of life. This view of an extended RNA World is not necessarily correct. From the point of view of exobiology, the difference in these two views mainly affects the significance of studies of the extent of catalysis possible by RNA- In either case, the origin of the translation machinery and the principles of RNA evolution remain central problems in exobiology. Translation presents several interrelated themes of inquiry for exobiology. First, it is essential, for understanding the very origin of life, how peptides and eventually proteins might have come to be made on the early Earth in a template directed manner. Second, it is necessary to understand how a machinery of similar complexity to that found in the ribosomes of modem organisms came to exist by the time of the last common ancestor (as detected by 16S RRNA sequence studies). Third, the RNAs that comprise the ribosome are themselves likely of very early origin and studies of their history may be very informative about the nature of the RNA World. Moreover, studies of these RNAs will contribute to a better understanding of the potential roles of RNA in early evolution.

  12. AXIOME: automated exploration of microbial diversity

    PubMed Central

    2013-01-01

    Background Although high-throughput sequencing of small subunit rRNA genes has revolutionized our understanding of microbial ecosystems, these technologies generate data at depths that benefit from automated analysis. Here we present AXIOME (Automation, eXtension, and Integration Of Microbial Ecology), a highly flexible and extensible management tool for popular microbial ecology analysis packages that promotes reproducibility and customization in microbial research. Findings AXIOME streamlines and manages analysis of small subunit (SSU) rRNA marker data in QIIME and mothur. AXIOME also implements features including the PAired-eND Assembler for Illumina sequences (PANDAseq), non-negative matrix factorization (NMF), multi-response permutation procedures (MRPP), exploring and recovering phylogenetic novelty (SSUnique) and indicator species analysis. AXIOME has a companion graphical user interface (GUI) and is designed to be easily extended to facilitate customized research workflows. Conclusions AXIOME is an actively developed, open source project written in Vala and available from GitHub (http://neufeld.github.com/axiome) and as a Debian package. Axiometic, a GUI companion tool is also freely available (http://neufeld.github.com/axiometic). Given that data analysis has become an important bottleneck for microbial ecology studies, the development of user-friendly computational tools remains a high priority. AXIOME represents an important step in this direction by automating multi-step bioinformatic analyses and enabling the customization of procedures to suit the diverse research needs of the microbial ecology community. PMID:23587322

  13. Automation model of sewerage rehabilitation planning.

    PubMed

    Yang, M D; Su, T C

    2006-01-01

    The major steps of sewerage rehabilitation include inspection of sewerage, assessment of structural conditions, computation of structural condition grades, and determination of rehabilitation methods and materials. Conventionally, sewerage rehabilitation planning relies on experts with professional background that is tedious and time-consuming. This paper proposes an automation model of planning optimal sewerage rehabilitation strategies for the sewer system by integrating image process, clustering technology, optimization, and visualization display. Firstly, image processing techniques, such as wavelet transformation and co-occurrence features extraction, were employed to extract various characteristics of structural failures from CCTV inspection images. Secondly, a classification neural network was established to automatically interpret the structural conditions by comparing the extracted features with the typical failures in a databank. Then, to achieve optimal rehabilitation efficiency, a genetic algorithm was used to determine appropriate rehabilitation methods and substitution materials for the pipe sections with a risk of mal-function and even collapse. Finally, the result from the automation model can be visualized in a geographic information system in which essential information of the sewer system and sewerage rehabilitation plans are graphically displayed. For demonstration, the automation model of optimal sewerage rehabilitation planning was applied to a sewer system in east Taichung, Chinese Taiwan. PMID:17302324

  14. Planning for Office Automation.

    ERIC Educational Resources Information Center

    Mick, Colin K.

    1983-01-01

    Outlines a practical approach to planning for office automation termed the "Focused Process Approach" (the "what" phase, "how" phase, "doing" phase) which is a synthesis of the problem-solving and participatory planning approaches. Thirteen references are provided. (EJS)

  15. Space station automation II

    SciTech Connect

    Chiou, W.C.

    1986-01-01

    This book contains the proceedings of a conference on space station automation. Topics include the following: distributed artificial intelligence for space station energy management systems and computer architecture for tolerobots in earth orbit.

  16. Automated Knowledge Discovery from Simulators

    NASA Technical Reports Server (NTRS)

    Burl, Michael C.; DeCoste, D.; Enke, B. L.; Mazzoni, D.; Merline, W. J.; Scharenbroich, L.

    2006-01-01

    In this paper, we explore one aspect of knowledge discovery from simulators, the landscape characterization problem, where the aim is to identify regions in the input/ parameter/model space that lead to a particular output behavior. Large-scale numerical simulators are in widespread use by scientists and engineers across a range of government agencies, academia, and industry; in many cases, simulators provide the only means to examine processes that are infeasible or impossible to study otherwise. However, the cost of simulation studies can be quite high, both in terms of the time and computational resources required to conduct the trials and the manpower needed to sift through the resulting output. Thus, there is strong motivation to develop automated methods that enable more efficient knowledge extraction.

  17. Automated design of flexible linkers.

    PubMed

    Manion, Charles; Arlitt, Ryan; Campbell, Matthew I; Tumer, Irem; Stone, Rob; Greaney, P Alex

    2016-03-14

    This paper presents a method for the systematic and automated design of flexible organic linkers for construction of metal organic-frameworks (MOFs) in which flexibility, compliance, or other mechanically exotic properties originate at the linker level rather than from the framework kinematics. Our method couples a graph grammar method for systematically generating linker like molecules with molecular dynamics modeling of linkers' mechanical response. Using this approach we have generated a candidate pool of >59,000 hypothetical linkers. We screen linker candidates according to their mechanical behaviors under large deformation, and extract fragments common to the most performant candidate materials. To demonstrate the general approach to MOF design we apply our system to designing linkers for pressure switching MOFs-MOFs that undergo reversible structural collapse after a stress threshold is exceeded. PMID:26687337

  18. Automated Status Notification System

    NASA Technical Reports Server (NTRS)

    2005-01-01

    NASA Lewis Research Center's Automated Status Notification System (ASNS) was born out of need. To prevent "hacker attacks," Lewis' telephone system needed to monitor communications activities 24 hr a day, 7 days a week. With decreasing staff resources, this continuous monitoring had to be automated. By utilizing existing communications hardware, a UNIX workstation, and NAWK (a pattern scanning and processing language), we implemented a continuous monitoring system.

  19. Automated Pilot Advisory System

    NASA Technical Reports Server (NTRS)

    Parks, J. L., Jr.; Haidt, J. G.

    1981-01-01

    An Automated Pilot Advisory System (APAS) was developed and operationally tested to demonstrate the concept that low cost automated systems can provide air traffic and aviation weather advisory information at high density uncontrolled airports. The system was designed to enhance the see and be seen rule of flight, and pilots who used the system preferred it over the self announcement system presently used at uncontrolled airports.

  20. Automated Lattice Perturbation Theory

    SciTech Connect

    Monahan, Christopher

    2014-11-01

    I review recent developments in automated lattice perturbation theory. Starting with an overview of lattice perturbation theory, I focus on the three automation packages currently "on the market": HiPPy/HPsrc, Pastor and PhySyCAl. I highlight some recent applications of these methods, particularly in B physics. In the final section I briefly discuss the related, but distinct, approach of numerical stochastic perturbation theory.

  1. Shielded cells transfer automation

    SciTech Connect

    Fisher, J J

    1984-01-01

    Nuclear waste from shielded cells is removed, packaged, and transferred manually in many nuclear facilities. Radiation exposure is absorbed by operators during these operations and limited only through procedural controls. Technological advances in automation using robotics have allowed a production waste removal operation to be automated to reduce radiation exposure. The robotic system bags waste containers out of glove box and transfers them to a shielded container. Operators control the system outside the system work area via television cameras. 9 figures.

  2. Automated imagery orthorectification pilot

    NASA Astrophysics Data System (ADS)

    Slonecker, E. Terrence; Johnson, Brad; McMahon, Joe

    2009-10-01

    Automated orthorectification of raw image products is now possible based on the comprehensive metadata collected by Global Positioning Systems and Inertial Measurement Unit technology aboard aircraft and satellite digital imaging systems, and based on emerging pattern-matching and automated image-to-image and control point selection capabilities in many advanced image processing systems. Automated orthorectification of standard aerial photography is also possible if a camera calibration report and sufficient metadata is available. Orthorectification of historical imagery, for which only limited metadata was available, was also attempted and found to require some user input, creating a semi-automated process that still has significant potential to reduce processing time and expense for the conversion of archival historical imagery into geospatially enabled, digital f