Automated image-based phenotypic analysis in zebrafish embryos

PubMed Central

Vogt, Andreas; Cholewinski, Andrzej; Shen, Xiaoqiang; Nelson, Scott; Lazo, John S.; Tsang, Michael; Hukriede, Neil A.

2009-01-01

Presently, the zebrafish is the only vertebrate model compatible with contemporary paradigms of drug discovery. Zebrafish embryos are amenable to automation necessary for high-throughput chemical screens, and optical transparency makes them potentially suited for image-based screening. However, the lack of tools for automated analysis of complex images presents an obstacle to utilizing the zebrafish as a high-throughput screening model. We have developed an automated system for imaging and analyzing zebrafish embryos in multi-well plates regardless of embryo orientation and without user intervention. Images of fluorescent embryos were acquired on a high-content reader and analyzed using an artificial intelligence-based image analysis method termed Cognition Network Technology (CNT). CNT reliably detected transgenic fluorescent embryos (Tg(fli1:EGFP)y1) arrayed in 96-well plates and quantified intersegmental blood vessel development in embryos treated with small molecule inhibitors of anigiogenesis. The results demonstrate it is feasible to adapt image-based high-content screening methodology to measure complex whole organism phenotypes. PMID:19235725

Droplet-based magnetic bead immunoassay using microchannel-connected multiwell plates (μCHAMPs) for the detection of amyloid beta oligomers.

PubMed

Park, Min Cheol; Kim, Moojong; Lim, Gun Taek; Kang, Sung Min; An, Seong Soo A; Kim, Tae Song; Kang, Ji Yoon

2016-06-21

Multiwell plates are regularly used in analytical research and clinical diagnosis but often require laborious washing steps and large sample or reagent volumes (typically, 100 μL per well). To overcome such drawbacks in the conventional multiwell plate, we present a novel microchannel-connected multiwell plate (μCHAMP) that can be used for automated disease biomarker detection in a small sample volume by performing droplet-based magnetic bead immunoassay inside the plate. In this μCHAMP-based immunoassay platform, small volumes (30-50 μL) of aqueous-phase working droplets are stably confined within each well by the simple microchannel structure (200-300 μm in height and 0.5-1 mm in width), and magnetic beads are exclusively transported into an adjacent droplet through the oil-filled microchannels assisted by a magnet array aligned beneath and controlled by a XY-motorized stage. Using this μCHAMP-based platform, we were able to perform parallel detection of synthetic amyloid beta (Aβ) oligomers as a model analyte for the early diagnosis of Alzheimer's disease (AD). This platform easily simplified the laborious and consumptive immunoassay procedure by achieving automated parallel immunoassay (32 assays per operation in 3-well connected 96-well plate) within 1 hour and at low sample consumption (less than 10 μL per assay) with no cumbersome manual washing step. Moreover, it could detect synthetic Aβ oligomers even below 10 pg mL(-1) concentration with a calculated detection limit of ∼3 pg mL(-1). Therefore, the μCHAMP and droplet-based magnetic bead immunoassay, with the combination of XY-motorized magnet array, would be a useful platform in the diagnosis of human disease, including AD, which requires low consumption of the patient's body fluid sample and automation of the entire immunoassay procedure for high processing capacity.

Strain Library Imaging Protocol for high-throughput, automated single-cell microscopy of large bacterial collections arrayed on multiwell plates.

PubMed

Shi, Handuo; Colavin, Alexandre; Lee, Timothy K; Huang, Kerwyn Casey

2017-02-01

Single-cell microscopy is a powerful tool for studying gene functions using strain libraries, but it suffers from throughput limitations. Here we describe the Strain Library Imaging Protocol (SLIP), which is a high-throughput, automated microscopy workflow for large strain collections that requires minimal user involvement. SLIP involves transferring arrayed bacterial cultures from multiwell plates onto large agar pads using inexpensive replicator pins and automatically imaging the resulting single cells. The acquired images are subsequently reviewed and analyzed by custom MATLAB scripts that segment single-cell contours and extract quantitative metrics. SLIP yields rich data sets on cell morphology and gene expression that illustrate the function of certain genes and the connections among strains in a library. For a library arrayed on 96-well plates, image acquisition can be completed within 4 min per plate.

Optogenetic stimulation of multiwell MEA plates for neural and cardiac applications

NASA Astrophysics Data System (ADS)

Clements, Isaac P.; Millard, Daniel C.; Nicolini, Anthony M.; Preyer, Amanda J.; Grier, Robert; Heckerling, Andrew; Blum, Richard A.; Tyler, Phillip; McSweeney, K. M.; Lu, Yi-Fan; Hall, Diana; Ross, James D.

2016-03-01

Microelectrode array (MEA) technology enables advanced drug screening and "disease-in-a-dish" modeling by measuring the electrical activity of cultured networks of neural or cardiac cells. Recent developments in human stem cell technologies, advancements in genetic models, and regulatory initiatives for drug screening have increased the demand for MEA-based assays. In response, Axion Biosystems previously developed a multiwell MEA platform, providing up to 96 MEA culture wells arrayed into a standard microplate format. Multiwell MEA-based assays would be further enhanced by optogenetic stimulation, which enables selective excitation and inhibition of targeted cell types. This capability for selective control over cell culture states would allow finer pacing and probing of cell networks for more reliable and complete characterization of complex network dynamics. Here we describe a system for independent optogenetic stimulation of each well of a 48-well MEA plate. The system enables finely graded control of light delivery during simultaneous recording of network activity in each well. Using human induced pluripotent stem cell (hiPSC) derived cardiomyocytes and rodent primary neuronal cultures, we demonstrate high channel-count light-based excitation and suppression in several proof-of-concept experimental models. Our findings demonstrate advantages of combining multiwell optical stimulation and MEA recording for applications including cardiac safety screening, neural toxicity assessment, and advanced characterization of complex neuronal diseases.

Hydrogel-Based Fluorescent Dual pH and Oxygen Sensors Loaded in 96-Well Plates for High-Throughput Cell Metabolism Studies.

PubMed

Wu, Shanshan; Wu, Siying; Yi, Zheyuan; Zeng, Fei; Wu, Weizhen; Qiao, Yuan; Zhao, Xingzhong; Cheng, Xing; Tian, Yanqing

2018-02-13

In this study, we developed fluorescent dual pH and oxygen sensors loaded in multi-well plates for in-situ and high-throughput monitoring of oxygen respiration and extracellular acidification during microbial cell growth for understanding metabolism. Biocompatible PHEMA-co-PAM materials were used as the hydrogel matrix. A polymerizable oxygen probe (OS2) derived from PtTFPP and a polymerizable pH probe (S2) derived from fluorescein were chemically conjugated into the matrix to solve the problem of the probe leaching from the matrix. Gels were allowed to cure directly on the bottom of 96-well plates at room-temperature via redox polymerization. The influence of matrix's composition on the sensing behaviors was investigated to optimize hydrogels with enough robustness for repeatable use with good sensitivity. Responses of the dual sensing hydrogels to dissolved oxygen (DO) and pH were studied. These dual oxygen-pH sensing plates were successfully used for microbial cell-based screening assays, which are based on the measurement of fluorescence intensity changes induced by cellular oxygen consumption and pH changes during microbial growth. This method may provide a real-time monitoring of cellular respiration, acidification, and a rapid kinetic assessment of multiple samples for cell viability as well as high-throughput drug screening. All of these assays can be carried out by a conventional plate reader.

Cellphone-Based Hand-Held Microplate Reader for Point-of-Care Testing of Enzyme-Linked Immunosorbent Assays.

PubMed

Berg, Brandon; Cortazar, Bingen; Tseng, Derek; Ozkan, Haydar; Feng, Steve; Wei, Qingshan; Chan, Raymond Yan-Lok; Burbano, Jordi; Farooqui, Qamar; Lewinski, Michael; Di Carlo, Dino; Garner, Omai B; Ozcan, Aydogan

2015-08-25

Standard microplate based enzyme-linked immunosorbent assays (ELISA) are widely utilized for various nanomedicine, molecular sensing, and disease screening applications, and this multiwell plate batched analysis dramatically reduces diagnosis costs per patient compared to nonbatched or nonstandard tests. However, their use in resource-limited and field-settings is inhibited by the necessity for relatively large and expensive readout instruments. To mitigate this problem, we created a hand-held and cost-effective cellphone-based colorimetric microplate reader, which uses a 3D-printed opto-mechanical attachment to hold and illuminate a 96-well plate using a light-emitting-diode (LED) array. This LED light is transmitted through each well, and is then collected via 96 individual optical fibers. Captured images of this fiber-bundle are transmitted to our servers through a custom-designed app for processing using a machine learning algorithm, yielding diagnostic results, which are delivered to the user within ∼1 min per 96-well plate, and are visualized using the same app. We successfully tested this mobile platform in a clinical microbiology laboratory using FDA-approved mumps IgG, measles IgG, and herpes simplex virus IgG (HSV-1 and HSV-2) ELISA tests using a total of 567 and 571 patient samples for training and blind testing, respectively, and achieved an accuracy of 99.6%, 98.6%, 99.4%, and 99.4% for mumps, measles, HSV-1, and HSV-2 tests, respectively. This cost-effective and hand-held platform could assist health-care professionals to perform high-throughput disease screening or tracking of vaccination campaigns at the point-of-care, even in resource-poor and field-settings. Also, its intrinsic wireless connectivity can serve epidemiological studies, generating spatiotemporal maps of disease prevalence and immunity.

A Student-Made Inexpensive Multichannel Pipet

ERIC Educational Resources Information Center

Dragojlovic, Veljko

2009-01-01

An inexpensive multichannel pipet designed to deliver small volumes of liquid simultaneously to wells in a multiwell plate can be prepared by students in a single laboratory period. The multichannel pipet is made of disposable plastic 1 mL syringes and drilled plastic plates, which are used to make plunger and barrel assemblies. Application of the…

Guidelines for Microplate Selection in High Content Imaging.

PubMed

Trask, Oscar J

2018-01-01

Since the inception of commercialized automated high content screening (HCS) imaging devices in the mid to late 1990s, the adoption of media vessels typically used to house and contain biological specimens for interrogation has transitioned from microscope slides and petri dishes into multi-well microtiter plates called microplates. The early 96- and 384-well microplates commonly used in other high-throughput screening (HTS) technology applications were often not designed for optical imaging. Since then, modifications and the use of next-generation materials with improved optical clarity have enhanced the quality of captured images, reduced autofocusing failures, and empowered the use of higher power magnification objectives to resolve fine detailed measurements at the subcellular pixel level. The plethora of microplates and their applications requires practitioners of high content imaging (HCI) to be especially diligent in the selection and adoption of the best plates for running longitudinal studies or larger screening campaigns. While the highest priority in experimental design is the selection of the biological model, the choice of microplate can alter the biological response and ultimately may change the experimental outcome. This chapter will provide readers with background, troubleshooting guidelines, and considerations for choosing an appropriate microplate.

The RABiT: a rapid automated biodosimetry tool for radiological triage. II. Technological developments.

PubMed

Garty, Guy; Chen, Youhua; Turner, Helen C; Zhang, Jian; Lyulko, Oleksandra V; Bertucci, Antonella; Xu, Yanping; Wang, Hongliang; Simaan, Nabil; Randers-Pehrson, Gerhard; Lawrence Yao, Y; Brenner, David J

2011-08-01

Over the past five years the Center for Minimally Invasive Radiation Biodosimetry at Columbia University has developed the Rapid Automated Biodosimetry Tool (RABiT), a completely automated, ultra-high throughput biodosimetry workstation. This paper describes recent upgrades and reliability testing of the RABiT. The RABiT analyses fingerstick-derived blood samples to estimate past radiation exposure or to identify individuals exposed above or below a cut-off dose. Through automated robotics, lymphocytes are extracted from fingerstick blood samples into filter-bottomed multi-well plates. Depending on the time since exposure, the RABiT scores either micronuclei or phosphorylation of the histone H2AX, in an automated robotic system, using filter-bottomed multi-well plates. Following lymphocyte culturing, fixation and staining, the filter bottoms are removed from the multi-well plates and sealed prior to automated high-speed imaging. Image analysis is performed online using dedicated image processing hardware. Both the sealed filters and the images are archived. We have developed a new robotic system for lymphocyte processing, making use of an upgraded laser power and parallel processing of four capillaries at once. This system has allowed acceleration of lymphocyte isolation, the main bottleneck of the RABiT operation, from 12 to 2 sec/sample. Reliability tests have been performed on all robotic subsystems. Parallel handling of multiple samples through the use of dedicated, purpose-built, robotics and high speed imaging allows analysis of up to 30,000 samples per day.

The RABiT: A Rapid Automated Biodosimetry Tool For Radiological Triage. II. Technological Developments

PubMed Central

Garty, Guy; Chen, Youhua; Turner, Helen; Zhang, Jian; Lyulko, Oleksandra; Bertucci, Antonella; Xu, Yanping; Wang, Hongliang; Simaan, Nabil; Randers-Pehrson, Gerhard; Yao, Y. Lawrence; Brenner, David J.

2011-01-01

Purpose Over the past five years the Center for Minimally Invasive Radiation Biodosimetry at Columbia University has developed the Rapid Automated Biodosimetry Tool (RABiT), a completely automated, ultra-high throughput biodosimetry workstation. This paper describes recent upgrades and reliability testing of the RABiT. Materials and methods The RABiT analyzes fingerstick-derived blood samples to estimate past radiation exposure or to identify individuals exposed above or below a cutoff dose. Through automated robotics, lymphocytes are extracted from fingerstick blood samples into filter-bottomed multi-well plates. Depending on the time since exposure, the RABiT scores either micronuclei or phosphorylation of the histone H2AX, in an automated robotic system, using filter-bottomed multi-well plates. Following lymphocyte culturing, fixation and staining, the filter bottoms are removed from the multi-well plates and sealed prior to automated high-speed imaging. Image analysis is performed online using dedicated image processing hardware. Both the sealed filters and the images are archived. Results We have developed a new robotic system for lymphocyte processing, making use of an upgraded laser power and parallel processing of four capillaries at once. This system has allowed acceleration of lymphocyte isolation, the main bottleneck of the RABiT operation, from 12 to 2 sec/sample. Reliability tests have been performed on all robotic subsystems. Conclusions Parallel handling of multiple samples through the use of dedicated, purpose-built, robotics and high speed imaging allows analysis of up to 30,000 samples per day. PMID:21557703

The Summer Apprentice Program, 1992

DTIC Science & Technology

1992-01-01

AD-A266 726 LABORATORY NOTE NO. 81 The Summer Apprentice Program 1992 Katherine Ellen Renn D TL C Thornton Samuel Mu JLE E DDavid M. Dahle JUL 0 6...Webster mice at 15-17 days gestation. The neocortex was removed, minced, and placed in media containing 0.08% acetylated trypsin at 370 C for one hour...plated on pure glial cultures. The cells were plated in 15 mm multiwell plates (2.5 x 10 -5 cells/well). The cultures were maintained at 370 C in a 5

Efficient method for the detection of microbially-produced antibacterial substances from food systems.

PubMed

Morgan, S M; Hickey, R; Ross, R P; Hill, C

2000-07-01

A novel method for the isolation of microbially-derived inhibitory substances from food sources was developed. The method involves an enrichment step coupled to a killing assay which is initially carried out in multiwell plates. The technique has advantages in that large numbers of samples can be tested in parallel. The assay can be completed in less than 60 h and is more sensitive than direct plating due to the enrichment step. This novel screening approach was compared with the standard direct plating approach in an effort to identify the antimicrobial potential of a number of Kefir grains. Kefir grains were incubated in 10% reconstituted skim milk for 20 h at 32 degrees C to enable production of any potential biopreservatives. Following overnight incubation, fermentates were aliquoted into multi-well plates and a known number of indicator cells was added to each well. The fermentates were incubated for a further 20 h and counts were carried out to determine whether a reduction in indicator cell numbers had occurred. A reduction in cell-forming units indicated the presence of an inhibitory substance and these inhibitory fermentates were selected for further investigation. Using the protocol outlined, Kefir fermentates capable of inhibiting Listeria innocua DPC1770 and Escherichia coli O157:H45 were identified.

Uniform electric field generation in circular multi-well culture plates using polymeric inserts

NASA Astrophysics Data System (ADS)

Tsai, Hsieh-Fu; Cheng, Ji-Yen; Chang, Hui-Fang; Yamamoto, Tadashi; Shen, Amy Q.

2016-05-01

Applying uniform electric field (EF) in vitro in the physiological range has been achieved in rectangular shaped microchannels. However, in a circular-shaped device, it is difficult to create uniform EF from two electric potentials due to different electrical resistances originated from the length difference between the diameter of the circle and the length of any parallel chord of the bottom circular chamber where cells are cultured. To address this challenge, we develop a three-dimensional (3D) computer-aided designed (CAD) polymeric insert to create uniform EF in circular shaped multi-well culture plates. A uniform EF with a coefficient of variation (CV) of 1.2% in the 6-well plate can be generated with an effective stimulation area percentage of 69.5%. In particular, NIH/3T3 mouse embryonic fibroblast cells are used to validate the performance of the 3D designed Poly(methyl methacrylate) (PMMA) inserts in a circular-shaped 6-well plate. The CAD based inserts can be easily scaled up (i.e., 100 mm dishes) to further increase effective stimulation area percentages, and also be implemented in commercially available cultureware for a wide variety of EF-related research such as EF-cell interaction and tissue regeneration studies.

Surface adsorption considerations when working with amyloid fibrils in multiwell plates and Eppendorf tubes

PubMed Central

Murray, Amber N; Palhano, Fernando L; Bieschke, Jan; Kelly, Jeffery W

2013-01-01

The accumulation of cross-β-sheet amyloid fibrils is the hallmark of amyloid diseases. Recently, we reported the discovery of amyloid disaggregase activities in extracts from mammalian cells and Caenorhabditis elegans. However, we have discovered a problem with the interpretation of our previous results as Aβ disaggregation in vitro. Here, we show that Aβ fibrils adsorb to the plastic surface of multiwell plates and Eppendorf tubes. This adsorption is markedly increased in the presence of complex biological mixtures subjected to a denaturing air-water interface. The time-dependent loss of thioflavin T fluorescence that we interpreted previously as disaggregation is due to increased adsorption of Aβ amyloid to the surfaces of multiwell plates and Eppendorf tubes in the presence of biological extracts. As the proteins in biological extracts denature over time at the air-water interface due to agitation/shaking, their adsorption increases, in turn promoting adsorption of amyloid fibrils. We delineate important control experiments that quantify the extent of amyloid adsorption to the surface of plastic and quartz containers. Based on the results described in this article, we conclude that our interpretation of the kinetic fibril disaggregation assay data previously reported in Bieschke et al., Protein Sci 2009;18:2231–2241 and Murray et al., Protein Sci 2010;19:836–846 is invalid when used as evidence for a disaggregase activity. Thus, we correct the two prior publications reporting that worm or mammalian cell extracts disaggregate Aβ amyloid fibrils in vitro at 37°C (see Corrigenda in this issue of Protein Science). We apologize for misinterpreting our previous data and for any confounding experimental efforts this may have caused. PMID:23963844

Surface adsorption considerations when working with amyloid fibrils in multiwell plates and Eppendorf tubes.

PubMed

Murray, Amber N; Palhano, Fernando L; Bieschke, Jan; Kelly, Jeffery W

2013-11-01

The accumulation of cross-β-sheet amyloid fibrils is the hallmark of amyloid diseases. Recently, we reported the discovery of amyloid disaggregase activities in extracts from mammalian cells and Caenorhabditis elegans. However, we have discovered a problem with the interpretation of our previous results as Aβ disaggregation in vitro. Here, we show that Aβ fibrils adsorb to the plastic surface of multiwell plates and Eppendorf tubes. This adsorption is markedly increased in the presence of complex biological mixtures subjected to a denaturing air-water interface. The time-dependent loss of thioflavin T fluorescence that we interpreted previously as disaggregation is due to increased adsorption of Aβ amyloid to the surfaces of multiwell plates and Eppendorf tubes in the presence of biological extracts. As the proteins in biological extracts denature over time at the air-water interface due to agitation/shaking, their adsorption increases, in turn promoting adsorption of amyloid fibrils. We delineate important control experiments that quantify the extent of amyloid adsorption to the surface of plastic and quartz containers. Based on the results described in this article, we conclude that our interpretation of the kinetic fibril disaggregation assay data previously reported in Bieschke et al., Protein Sci 2009;18:2231-2241 and Murray et al., Protein Sci 2010;19:836-846 is invalid when used as evidence for a disaggregase activity. Thus, we correct the two prior publications reporting that worm or mammalian cell extracts disaggregate Aβ amyloid fibrils in vitro at 37°C (see Corrigenda in this issue of Protein Science). We apologize for misinterpreting our previous data and for any confounding experimental efforts this may have caused. © 2013 The Protein Society.

A facile method to screen inhibitors of protein-protein interactions including MDM2-p53 displayed on T7 phage.

PubMed

Ishi, Kazutomo; Sugawara, Fumio

2008-05-01

Protein-protein interactions are essential in many biological processes including cell cycle and apoptosis. It is currently of great medical interest to inhibit specific protein-protein interactions in order to treat a variety of disease states. Here, we describe a facile multiwell plate assay method using T7 phage display to screen for candidate inhibitors of protein-protein interactions. Because T7 phage display is an effective method for detecting protein-protein interactions, we aimed to utilize this technique to screen for small-molecule inhibitors that disrupt these types of interaction. We used the well-characterized interaction between p53 and MDM2 and an inhibitor of this interaction, nutlin 3, as a model system to establish a new screening method. Phage particles displaying p53 interacted with GST-MDM2 immobilized on 96-well plates, and the interaction was inhibited by nutlin 3. Multiwell plate assay was then performed using a natural product library, which identified dehydroaltenusin as a candidate inhibitor of the p53-MDM2 interaction. We discuss the potential applications of this novel T7 phage display methodology, which we propose to call 'reverse phage display'.

High Throughput Microplate Respiratory Measurements Using Minimal Quantities Of Isolated Mitochondria

PubMed Central

Rogers, George W.; Brand, Martin D.; Petrosyan, Susanna; Ashok, Deepthi; Elorza, Alvaro A.; Ferrick, David A.; Murphy, Anne N.

2011-01-01

Recently developed technologies have enabled multi-well measurement of O2 consumption, facilitating the rate of mitochondrial research, particularly regarding the mechanism of action of drugs and proteins that modulate metabolism. Among these technologies, the Seahorse XF24 Analyzer was designed for use with intact cells attached in a monolayer to a multi-well tissue culture plate. In order to have a high throughput assay system in which both energy demand and substrate availability can be tightly controlled, we have developed a protocol to expand the application of the XF24 Analyzer to include isolated mitochondria. Acquisition of optimal rates requires assay conditions that are unexpectedly distinct from those of conventional polarography. The optimized conditions, derived from experiments with isolated mouse liver mitochondria, allow multi-well assessment of rates of respiration and proton production by mitochondria attached to the bottom of the XF assay plate, and require extremely small quantities of material (1–10 µg of mitochondrial protein per well). Sequential measurement of basal, State 3, State 4, and uncoupler-stimulated respiration can be made in each well through additions of reagents from the injection ports. We describe optimization and validation of this technique using isolated mouse liver and rat heart mitochondria, and apply the approach to discover that inclusion of phosphatase inhibitors in the preparation of the heart mitochondria results in a specific decrease in rates of Complex I-dependent respiration. We believe this new technique will be particularly useful for drug screening and for generating previously unobtainable respiratory data on small mitochondrial samples. PMID:21799747

Electroacoustics: A New Mechanism for Driving Individually Addressable Jetting and Other Microfluidic Manipulations in Multiwell Arrays

NASA Astrophysics Data System (ADS)

Yeo, Leslie; Rezk, Amgad

2017-11-01

The low take-up of microfluidic technology at the laboratory bench despite 25 years of advances can be attributed to the reluctance of practitioners to adopt new and sophisticated technology, which requires substantial retraining, as well as the large investments that have already been made in the vast array of existing laboratory equipment. A way to circumvent this is to design microfluidic technology to retrofit existing laboratory technology such as microscope stages, microplate readers, etc. This is however not without challenge as existing microfluidic devices themselves often require large ancillary equipment to drive fluidic actuation/detection, which are not always amenable to integration into these existing laboratory formats. We have developed a low-cost and scalable modular plug-and-play microplatform that facilitates individual addressability of each well in a microarray plate for sample dispensing, mixing and preconcentration, as well as its ejection via jetting/nebulisation for subsequent analysis. As this cannot be achieved using standard acoustofluidics, we have developed a new electroacoustic mechanism that allows the transmission of high frequency sound waves into each well while uniquely confining the electric field off the piezoelectric chip.

Open-top selective plane illumination microscope for conventionally mounted specimens.

PubMed

McGorty, Ryan; Liu, Harrison; Kamiyama, Daichi; Dong, Zhiqiang; Guo, Su; Huang, Bo

2015-06-15

We have developed a new open-top selective plane illumination microscope (SPIM) compatible with microfluidic devices, multi-well plates, and other sample formats used in conventional inverted microscopy. Its key element is a water prism that compensates for the aberrations introduced when imaging at 45 degrees through a coverglass. We have demonstrated its unique high-content imaging capability by recording Drosophila embryo development in environmentally-controlled microfluidic channels and imaging zebrafish embryos in 96-well plates. We have also shown the imaging of C. elegans and moving Drosophila larvae on coverslips.

The high throughput biomedicine unit at the institute for molecular medicine Finland: high throughput screening meets precision medicine.

PubMed

Pietiainen, Vilja; Saarela, Jani; von Schantz, Carina; Turunen, Laura; Ostling, Paivi; Wennerberg, Krister

2014-05-01

The High Throughput Biomedicine (HTB) unit at the Institute for Molecular Medicine Finland FIMM was established in 2010 to serve as a national and international academic screening unit providing access to state of the art instrumentation for chemical and RNAi-based high throughput screening. The initial focus of the unit was multiwell plate based chemical screening and high content microarray-based siRNA screening. However, over the first four years of operation, the unit has moved to a more flexible service platform where both chemical and siRNA screening is performed at different scales primarily in multiwell plate-based assays with a wide range of readout possibilities with a focus on ultraminiaturization to allow for affordable screening for the academic users. In addition to high throughput screening, the equipment of the unit is also used to support miniaturized, multiplexed and high throughput applications for other types of research such as genomics, sequencing and biobanking operations. Importantly, with the translational research goals at FIMM, an increasing part of the operations at the HTB unit is being focused on high throughput systems biological platforms for functional profiling of patient cells in personalized and precision medicine projects.

An Automated Method for High-Throughput Screening of Arabidopsis Rosette Growth in Multi-Well Plates and Its Validation in Stress Conditions.

PubMed

De Diego, Nuria; Fürst, Tomáš; Humplík, Jan F; Ugena, Lydia; Podlešáková, Kateřina; Spíchal, Lukáš

2017-01-01

High-throughput plant phenotyping platforms provide new possibilities for automated, fast scoring of several plant growth and development traits, followed over time using non-invasive sensors. Using Arabidops is as a model offers important advantages for high-throughput screening with the opportunity to extrapolate the results obtained to other crops of commercial interest. In this study we describe the development of a highly reproducible high-throughput Arabidopsis in vitro bioassay established using our OloPhen platform, suitable for analysis of rosette growth in multi-well plates. This method was successfully validated on example of multivariate analysis of Arabidopsis rosette growth in different salt concentrations and the interaction with varying nutritional composition of the growth medium. Several traits such as changes in the rosette area, relative growth rate, survival rate and homogeneity of the population are scored using fully automated RGB imaging and subsequent image analysis. The assay can be used for fast screening of the biological activity of chemical libraries, phenotypes of transgenic or recombinant inbred lines, or to search for potential quantitative trait loci. It is especially valuable for selecting genotypes or growth conditions that improve plant stress tolerance.

Next generation platforms for high-throughput biodosimetry

PubMed Central

Repin, Mikhail; Turner, Helen C.; Garty, Guy; Brenner, David J.

2014-01-01

Here the general concept of the combined use of plates and tubes in racks compatible with the American National Standards Institute/the Society for Laboratory Automation and Screening microplate formats as the next generation platforms for increasing the throughput of biodosimetry assays was described. These platforms can be used at different stages of biodosimetry assays starting from blood collection into microtubes organised in standardised racks and ending with the cytogenetic analysis of samples in standardised multiwell and multichannel plates. Robotically friendly platforms can be used for different biodosimetry assays in minimally equipped laboratories and on cost-effective automated universal biotech systems. PMID:24837249

High-throughput measurements of biochemical responses using the plate::vision multimode 96 minilens array reader.

PubMed

Huang, Kuo-Sen; Mark, David; Gandenberger, Frank Ulrich

2006-01-01

The plate::vision is a high-throughput multimode reader capable of reading absorbance, fluorescence, fluorescence polarization, time-resolved fluorescence, and luminescence. Its performance has been shown to be quite comparable with other readers. When the reader is integrated into the plate::explorer, an ultrahigh-throughput screening system with event-driven software and parallel plate-handling devices, it becomes possible to run complicated assays with kinetic readouts in high-density microtiter plate formats for high-throughput screening. For the past 5 years, we have used the plate::vision and the plate::explorer to run screens and have generated more than 30 million data points. Their throughput, performance, and robustness have speeded up our drug discovery process greatly.

A Multiwell Platform for Studying Stiffness-Dependent Cell Biology

PubMed Central

Mih, Justin D.; Sharif, Asma S.; Liu, Fei; Marinkovic, Aleksandar; Symer, Matthew M.; Tschumperlin, Daniel J.

2011-01-01

Adherent cells are typically cultured on rigid substrates that are orders of magnitude stiffer than their tissue of origin. Here, we describe a method to rapidly fabricate 96 and 384 well platforms for routine screening of cells in tissue-relevant stiffness contexts. Briefly, polyacrylamide (PA) hydrogels are cast in glass-bottom plates, functionalized with collagen, and sterilized for cell culture. The Young's modulus of each substrate can be specified from 0.3 to 55 kPa, with collagen surface density held constant over the stiffness range. Using automated fluorescence microscopy, we captured the morphological variations of 7 cell types cultured across a physiological range of stiffness within a 384 well plate. We performed assays of cell number, proliferation, and apoptosis in 96 wells and resolved distinct profiles of cell growth as a function of stiffness among primary and immortalized cell lines. We found that the stiffness-dependent growth of normal human lung fibroblasts is largely invariant with collagen density, and that differences in their accumulation are amplified by increasing serum concentration. Further, we performed a screen of 18 bioactive small molecules and identified compounds with enhanced or reduced effects on soft versus rigid substrates, including blebbistatin, which abolished the suppression of lung fibroblast growth at 1 kPa. The ability to deploy PA gels in multiwell plates for high throughput analysis of cells in tissue-relevant environments opens new opportunities for the discovery of cellular responses that operate in specific stiffness regimes. PMID:21637769

A multiwell platform for studying stiffness-dependent cell biology.

PubMed

Mih, Justin D; Sharif, Asma S; Liu, Fei; Marinkovic, Aleksandar; Symer, Matthew M; Tschumperlin, Daniel J

2011-01-01

Adherent cells are typically cultured on rigid substrates that are orders of magnitude stiffer than their tissue of origin. Here, we describe a method to rapidly fabricate 96 and 384 well platforms for routine screening of cells in tissue-relevant stiffness contexts. Briefly, polyacrylamide (PA) hydrogels are cast in glass-bottom plates, functionalized with collagen, and sterilized for cell culture. The Young's modulus of each substrate can be specified from 0.3 to 55 kPa, with collagen surface density held constant over the stiffness range. Using automated fluorescence microscopy, we captured the morphological variations of 7 cell types cultured across a physiological range of stiffness within a 384 well plate. We performed assays of cell number, proliferation, and apoptosis in 96 wells and resolved distinct profiles of cell growth as a function of stiffness among primary and immortalized cell lines. We found that the stiffness-dependent growth of normal human lung fibroblasts is largely invariant with collagen density, and that differences in their accumulation are amplified by increasing serum concentration. Further, we performed a screen of 18 bioactive small molecules and identified compounds with enhanced or reduced effects on soft versus rigid substrates, including blebbistatin, which abolished the suppression of lung fibroblast growth at 1 kPa. The ability to deploy PA gels in multiwell plates for high throughput analysis of cells in tissue-relevant environments opens new opportunities for the discovery of cellular responses that operate in specific stiffness regimes.

76 FR 60531 - National Institute of Justice Interview Room Recording Systems and License Plate Readers Workshop

Federal Register 2010, 2011, 2012, 2013, 2014

2011-09-29

... Justice Interview Room Recording Systems and License Plate Readers Workshop AGENCY: National Institute of Justice. ACTION: Notice of the Interview Room Recording Systems and License Plate Readers Workshops.... The focus of the workshops is the development of NIJ performance standards for Interview Room...

Membrane-on-a-chip: microstructured silicon/silicon-dioxide chips for high-throughput screening of membrane transport and viral membrane fusion.

PubMed

Kusters, Ilja; van Oijen, Antoine M; Driessen, Arnold J M

2014-04-22

Screening of transport processes across biological membranes is hindered by the challenge to establish fragile supported lipid bilayers and the difficulty to determine at which side of the membrane reactants reside. Here, we present a method for the generation of suspended lipid bilayers with physiological relevant lipid compositions on microstructured Si/SiO2 chips that allow for high-throughput screening of both membrane transport and viral membrane fusion. Simultaneous observation of hundreds of single-membrane channels yields statistical information revealing population heterogeneities of the pore assembly and conductance of the bacterial toxin α-hemolysin (αHL). The influence of lipid composition and ionic strength on αHL pore formation was investigated at the single-channel level, resolving features of the pore-assembly pathway. Pore formation is inhibited by a specific antibody, demonstrating the applicability of the platform for drug screening of bacterial toxins and cell-penetrating agents. Furthermore, fusion of H3N2 influenza viruses with suspended lipid bilayers can be observed directly using a specialized chip architecture. The presented micropore arrays are compatible with fluorescence readout from below using an air objective, thus allowing high-throughput screening of membrane transport in multiwell formats in analogy to plate readers.

Multi Spectral Fluorescence Imager (MSFI)

NASA Technical Reports Server (NTRS)

Caron, Allison

2016-01-01

Genetic transformation with in vivo reporter genes for fluorescent proteins can be performed on a variety of organisms to address fundamental biological questions. Model organisms that may utilize an ISS imager include unicellular organisms (Saccharomyces cerevisiae), plants (Arabidopsis thaliana), and invertebrates (Caenorhabditis elegans). The multispectral fluorescence imager (MSFI) will have the capability to accommodate 10 cm x 10 cm Petri plates, various sized multi-well culture plates, and other custom culture containers. Features will include programmable temperature and light cycles, ethylene scrubbing (less than 25 ppb), CO2 control (between 400 ppm and ISS-ambient levels in units of 100 ppm) and sufficient airflow to prevent condensation that would interfere with imaging.

An Automated Microfluidic Multiplexer for Fast Delivery of C. elegans Populations from Multiwells

PubMed Central

Ghorashian, Navid; Gökçe, Sertan Kutal; Guo, Sam Xun; Everett, William Neil; Ben-Yakar, Adela

2013-01-01

Automated biosorter platforms, including recently developed microfluidic devices, enable and accelerate high-throughput and/or high-resolution bioassays on small animal models. However, time-consuming delivery of different organism populations to these systems introduces a major bottleneck to executing large-scale screens. Current population delivery strategies rely on suction from conventional well plates through tubing periodically exposed to air, leading to certain disadvantages: 1) bubble introduction to the sample, interfering with analysis in the downstream system, 2) substantial time drain from added bubble-cleaning steps, and 3) the need for complex mechanical systems to manipulate well plate position. To address these concerns, we developed a multiwell-format microfluidic platform that can deliver multiple distinct animal populations from on-chip wells using multiplexed valve control. This Population Delivery Chip could operate autonomously as part of a relatively simple setup that did not require any of the major mechanical moving parts typical of plate-handling systems to address a given well. We demonstrated automatic serial delivery of 16 distinct C. elegans worm populations to a single outlet without introducing any bubbles to the samples, causing cross-contamination, or damaging the animals. The device achieved delivery of more than 90% of the population preloaded into a given well in 4.7 seconds; an order of magnitude faster than delivery modalities in current use. This platform could potentially handle other similarly sized model organisms, such as zebrafish and drosophila larvae or cellular micro-colonies. The device’s architecture and microchannel dimensions allow simple expansion for processing larger numbers of populations. PMID:24069313

Magnetic manipulation device for the optimization of cell processing conditions.

PubMed

Ito, Hiroshi; Kato, Ryuji; Ino, Kosuke; Honda, Hiroyuki

2010-02-01

Variability in human cell phenotypes make it's advancements in optimized cell processing necessary for personalized cell therapy. Here we propose a strategy of palm-top sized device to assist physically manipulating cells for optimizing cell preparations. For the design of such a device, we combined two conventional approaches: multi-well plate formatting and magnetic cell handling using magnetite cationic liposomes (MCLs). From our previous works, we showed the labeling applications of MCL on adhesive cells for various tissue engineering approaches. To feasibly transfer cells in multi-well plate, we here evaluated the magnetic response of MCL-labeled suspension type cells. The cell handling performance of Jurkat cells proved to be faster and more robust compared to MACS (Magnetic Cell Sorting) bead methods. To further confirm our strategy, prototype palm-top sized device "magnetic manipulation device (MMD)" was designed. In the device, the actual cell transportation efficacy of Jurkat cells was satisfying. Moreover, as a model of the most distributed clinical cell processing, primary peripheral blood mononuclear cells (PBMCs) from different volunteers were evaluated. By MMD, individual PBMCs indicated to have optimum Interleukin-2 (IL-2) concentrations for the expansion. Such huge differences of individual cells indicated that MMD, our proposing efficient and self-contained support tool, could assist the feasible and cost-effective optimization of cell processing in clinical facilities. Copyright (c) 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

High-throughput cocrystal slurry screening by use of in situ Raman microscopy and multi-well plate.

PubMed

Kojima, Takashi; Tsutsumi, Shunichirou; Yamamoto, Katsuhiko; Ikeda, Yukihiro; Moriwaki, Toshiya

2010-10-31

Cocrystal has attracted much attention in order to improve poor physicochemical properties, since cocrystal former crystallize with the ionic drugs as well as nonionic drugs. Cocrystal screening was usually conducted by crystallization, slurry and co-grinding techniques, however sensitivity, cost and time for screening were limited because of issues such as dissociation of cocrystal during crystallization and cost and time required for slurry and co-grinding methods. To overcome these issues, novel high-throughput cocrystal slurry screening was developed by using in situ Raman microscope and a multi-well plate. Cocrystal screening of indomethacin was conducted with 46 cocrystal formers and potential cocrystals were prepared on a large scale for the characterization with powder X-ray diffractometry, thermal analysis, and Raman microscopy and (1)H NMR spectroscopy. Compared with the characterization of scale-up cocrystals, the cocrystal screening indicated that indomethacin structured novel cocrystals with D/L-mandelic acid, nicotinamide, lactamide and benzamide which was not obtained in the screening with crystallization technique previously reported. In addition, the screening provided not only information of cocrystal formation within a day but also information of equilibrium of cocrystal formation and polymorphic transformation in one screening. Information obtained in this screening allows effective solid form selection by saving cost and time for the development. Copyright © 2010 Elsevier B.V. All rights reserved.

Development of LEDs-based microplate reader for bioanalytical assay measurements

NASA Astrophysics Data System (ADS)

Alaruri, Sami D.; Katzlinger, Michael; Schinwald, Bernhard; Kronberger, Georg; Atzler, Joseph

2013-10-01

The optical design for an LEDs-based microplate reader that can perform fluorescence intensity (top and bottom), absorbance, luminescence and time-resolved fluorescence measurements is described. The microplate reader is the first microplate reader in the marketplace that incorporates LEDs as excitation light sources. Absorbance measurements over the 0-3.5 optical density range for caffeine solution are presented. Additionally, fluorescence intensity readings collected at 535 and 625 nm from a green and a red RediPlateTM are reported. Furthermore, fluorescence decay lifetime measurements obtained for Eu (europium) and Sm (samarium) standard solutions using 370 nm excitation are presented. The microplate reader detection limits for the fluorescence intensity top, fluorescence intensity bottom, fluorescence polarization and time-resolved fluorescence modes are 1.5 fmol 100 µL-1 fluorescein (384-well plate), 25 fmol 100 µL-1 fluorescein (384-well plate), 5 mP at 10 nM fluorescein (black 384-well plate) and 30 amol 100 µL-1 europium solution (white 384-well plate), respectively.

Quantifying cell mono-layer cultures by video imaging.

PubMed

Miller, K S; Hook, L A

1996-04-01

A method is described in which the relative number of adherent cells in multi-well tissue-culture plates is assayed by staining the cells with Giemsa and capturing the image of the stained cells with a video camera and charged-coupled device. The resultant image is quantified using the associated video imaging software. The method is shown to be sensitive and reproducible and should be useful for studies where quantifying relative cell numbers and/or proliferation in vitro is required.

Next generation platforms for high-throughput biodosimetry.

PubMed

Repin, Mikhail; Turner, Helen C; Garty, Guy; Brenner, David J

2014-06-01

Here the general concept of the combined use of plates and tubes in racks compatible with the American National Standards Institute/the Society for Laboratory Automation and Screening microplate formats as the next generation platforms for increasing the throughput of biodosimetry assays was described. These platforms can be used at different stages of biodosimetry assays starting from blood collection into microtubes organised in standardised racks and ending with the cytogenetic analysis of samples in standardised multiwell and multichannel plates. Robotically friendly platforms can be used for different biodosimetry assays in minimally equipped laboratories and on cost-effective automated universal biotech systems. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The impact of cell culture equipment on energy loss.

PubMed

Davies, Lleucu B; Kiernan, Michael N; Bishop, Joanna C; Thornton, Catherine A; Morgan, Gareth

2014-01-01

Light energy of discrete wavelengths supplied via lasers and broadband intense pulsed light have been used therapeutically for many years. In vitro models complement clinical studies, especially for the elucidation of underlying mechanisms of action. Clarification that light energy reaches the cells is necessary when developing protocols for the treatment of cells using in vitro models. Few studies report on energy loss in cell culture equipment. The ability of energy from light with therapeutic potential to reach cells in culture needs to be determined; this includes determining the proportion of light energy lost within standard cell culture media and cell culture vessels. The energy absorption of cell culture media, with/without the pH indicator dye phenol red, and the loss of energy within different plastics and glassware used typically for in vitro cell culture were investigated using intense pulsed light and a yellow pulsed dye laser. Media containing phenol red have a distinctive absorption peak (560 nm) absent in phenol red-free media and restored by the addition of phenol red. For both light sources, energy loss was lowest in standard polystyrene tissue culture flasks or multi-well plates and highest in polypropylene vessels or glass tubes. The effects of phenol red-free media on the absorption of energy varied with the light source used. Phenol red-free media are the media of choice; polystyrene vessels with flat surfaces such as culture flasks or multi-well plates should be used in preference to polypropylene or glass vessels.

Miniaturized microscope for high throughput screening of tumor spheroids in microfluidic devices

NASA Astrophysics Data System (ADS)

Uranga, Javier; Rodríguez-Pena, Alejandro; Gahigiro, Desiré; Ortiz-de-Solorzano, Carlos

2018-02-01

High-throughput in vitro screening of highly physiological three-dimensional cell cultures (3D-HTS) is rapidly gaining importance in preclinical studies, to study the effect of the microenvironment in tumor development, and to evaluate the efficacy of new anticancer drugs. Furthermore, it could also be envisioned the use of 3D-HTS systems in personalized anti-cancer treatment planning, based on tumor organoids or spheroids grown from tumor biopsies or isolated tumor circulating cells. Most commercial, multi-well plate based 3D-HTS systems are large, expensive, and are based on the use of multi-well plates that hardly provide a physiological environment and require the use of large amounts of biological material and reagents. In this paper we present a novel, miniaturized inverted microscope (hereinafter miniscospe), made up of low-cost, mass producible parts, that can be used to monitor the growth of living tumor cell spheroids within customized three-dimensional microfluidic platforms. Our 3D-HTS miniscope combines phase contrast imaging based on oblique back illumination technique with traditional widefield epi-fluorescence imaging, implemented using miniaturized electro-optical parts and gradient-index refraction lenses. This small (3x6x2cm), lightweight device can effectively image overtime the growth of (>200) tumor spheroids in a controlled and reproducible environment. Our miniscope can be used to acquire time-lapse images of cellular living spheroids over the course of several hours and captures their growth before and after drug treatment, to evaluate the effectiveness of the drug.

Development of an automated MODS plate reader to detect early growth of Mycobacterium tuberculosis.

PubMed

Comina, G; Mendoza, D; Velazco, A; Coronel, J; Sheen, P; Gilman, R H; Moore, D A J; Zimic, M

2011-06-01

In this work, an automated microscopic observation drug susceptibility (MODS) plate reader has been developed. The reader automatically handles MODS plates and after autofocussing digital images are acquired of the characteristic microscopic cording structures of Mycobacterium tuberculosis, which are the identification method utilized in the MODS technique to detect tuberculosis and multidrug resistant tuberculosis. In conventional MODS, trained technicians manually move the MODS plate on the stage of an inverted microscope while trying to locate and focus upon the characteristic microscopic cording colonies. In centres with high tuberculosis diagnostic demand, sufficient time may not be available to adequately examine all cultures. An automated reader would reduce labour time and the handling of M. tuberculosis cultures by laboratory personnel. Two hundred MODS culture images (100 from tuberculosis positive and 100 from tuberculosis negative sputum samples confirmed by a standard MODS reading using a commercial microscope) were acquired randomly using the automated MODS plate reader. A specialist analysed these digital images with the help of a personal computer and designated them as M. tuberculosis present or absent. The specialist considered four images insufficiently clear to permit a definitive reading. The readings from the 196 valid images resulted in a 100% agreement with the conventional nonautomated standard reading. The automated MODS plate reader combined with open-source MODS pattern recognition software provides a novel platform for high throughput automated tuberculosis diagnosis. © 2011 The Authors Journal of Microscopy © 2011 Royal Microscopical Society.

77 FR 58580 - Interview Room Recording System Standard and License Plate Reader Standard Workshops

Federal Register 2010, 2011, 2012, 2013, 2014

2012-09-21

... DEPARTMENT OF JUSTICE Office of Justice Programs [OJP (NIJ) Docket No. 1603] Interview Room Recording System Standard and License Plate Reader Standard Workshops AGENCY: National Institute of Justice, DOJ. [[Page 58581

Fabrication of multi-well chips for spheroid cultures and implantable constructs through rapid prototyping techniques.

PubMed

Lopa, Silvia; Piraino, Francesco; Kemp, Raymond J; Di Caro, Clelia; Lovati, Arianna B; Di Giancamillo, Alessia; Moroni, Lorenzo; Peretti, Giuseppe M; Rasponi, Marco; Moretti, Matteo

2015-07-01

Three-dimensional (3D) culture models are widely used in basic and translational research. In this study, to generate and culture multiple 3D cell spheroids, we exploited laser ablation and replica molding for the fabrication of polydimethylsiloxane (PDMS) multi-well chips, which were validated using articular chondrocytes (ACs). Multi-well ACs spheroids were comparable or superior to standard spheroids, as revealed by glycosaminoglycan and type-II collagen deposition. Moreover, the use of our multi-well chips significantly reduced the operation time for cell seeding and medium refresh. Exploiting a similar approach, we used clinical-grade fibrin to generate implantable multi-well constructs allowing for the precise distribution of multiple cell types. Multi-well fibrin constructs were seeded with ACs generating high cell density regions, as shown by histology and cell fluorescent staining. Multi-well constructs were compared to standard constructs with homogeneously distributed ACs. After 7 days in vitro, expression of SOX9, ACAN, COL2A1, and COMP was increased in both constructs, with multi-well constructs expressing significantly higher levels of chondrogenic genes than standard constructs. After 5 weeks in vivo, we found that despite a dramatic size reduction, the cell distribution pattern was maintained and glycosaminoglycan content per wet weight was significantly increased respect to pre-implantation samples. In conclusion, multi-well chips for the generation and culture of multiple cell spheroids can be fabricated by low-cost rapid prototyping techniques. Furthermore, these techniques can be used to generate implantable constructs with defined architecture and controlled cell distribution, allowing for in vitro and in vivo investigation of cell interactions in a 3D environment. © 2015 Wiley Periodicals, Inc.

Using PATIMDB to Create Bacterial Transposon Insertion Mutant Libraries

PubMed Central

Urbach, Jonathan M.; Wei, Tao; Liberati, Nicole; Grenfell-Lee, Daniel; Villanueva, Jacinto; Wu, Gang; Ausubel, Frederick M.

2015-01-01

PATIMDB is a software package for facilitating the generation of transposon mutant insertion libraries. The software has two main functions: process tracking and automated sequence analysis. The process tracking function specifically includes recording the status and fates of multiwell plates and samples in various stages of library construction. Automated sequence analysis refers specifically to the pipeline of sequence analysis starting with ABI files from a sequencing facility and ending with insertion location identifications. The protocols in this unit describe installation and use of PATIMDB software. PMID:19343706

A novel cell-based assay for inhibitory anti-muscarinic type 3 receptor antibodies in primary Sjögren's syndrome.

PubMed

Bastian, Isabell; Gordon, Tom P; Jackson, Michael W

2015-12-01

Inhibitory autoantibodies acting at the muscarinic acetylcholine receptor type 3 (M3R) are postulated to mediate autonomic dysfunction, including decreased salivary and lacrimal gland output and extra-glandular manifestations, in patients with primary Sjögren's syndrome. However, the contention that anti-M3R antibodies are pathogenic in patients remains untested, due to a lack of assays both sophisticated enough to detect inhibitory anti-M3R antibodies yet suitable for screening large patient cohorts. In the current study, we have established a cell-based bioassay of M3R activity, based on dual transfection of the M3R and a luciferase reporter gene. The bioassay is capable of capturing real-time agonist-mediated signalling of the M3R, which is inhibited specifically by patient IgG that have previously been demonstrated to have anti-M3R activity. The assay can be run in multi-well culture plates, and analysed using simple luminescence readers. As such, the new bioassay incorporating M3R-mediated luciferase transduction is the first assay adaptable to common diagnostic platforms that is capable of determining the presence in patient serum of functionally active anti-M3R autoantibodies. The new bioassay should prove useful for large cohort screening studies aiming to correlate the presence in patients of inhibitory anti-M3R antibodies with symptoms of both glandular and extra-glandular autonomic dysfunction. Copyright © 2015 Elsevier B.V. All rights reserved.

A novel high-throughput imaging system for automated analyses of avoidance behavior in zebrafish larvae

PubMed Central

Pelkowski, Sean D.; Kapoor, Mrinal; Richendrfer, Holly A.; Wang, Xingyue; Colwill, Ruth M.; Creton, Robbert

2011-01-01

Early brain development can be influenced by numerous genetic and environmental factors, with long-lasting effects on brain function and behavior. The identification of these factors is facilitated by recent innovations in high-throughput screening. However, large-scale screening in whole organisms remains challenging, in particular when studying changes in brain function or behavior in vertebrate model systems. In this study, we present a novel imaging system for high-throughput analyses of behavior in zebrafish larvae. The three-camera system can image twelve multiwell plates simultaneously and is unique in its ability to provide local visual stimuli in the wells of a multiwell plate. The acquired images are converted into a series of coordinates, which characterize the location and orientation of the larvae. The developed imaging techniques were tested by measuring avoidance behaviors in seven-day-old zebrafish larvae. The system effectively quantified larval avoidance and revealed an increased edge preference in response to a blue or red ‘bouncing ball’ stimulus. Larvae also avoid a bouncing ball stimulus when it is counter-balanced with a stationary ball, but do not avoid blinking balls counter-balanced with a stationary ball. These results indicate that the seven-day-old larvae respond specifically to movement, rather than color, size, or local changes in light intensity. The imaging system and assays for measuring avoidance behavior may be used to screen for genetic and environmental factors that cause developmental brain disorders and for novel drugs that could prevent or treat these disorders. PMID:21549762

Development of a novel cell sorting method that samples population diversity in flow cytometry.

PubMed

Osborne, Geoffrey W; Andersen, Stacey B; Battye, Francis L

2015-11-01

Flow cytometry based electrostatic cell sorting is an important tool in the separation of cell populations. Existing instruments can sort single cells into multi-well collection plates, and keep track of cell of origin and sorted well location. However currently single sorted cell results reflect the population distribution and fail to capture the population diversity. Software was designed that implements a novel sorting approach, "Slice and Dice Sorting," that links a graphical representation of a multi-well plate to logic that ensures that single cells are sampled and sorted from all areas defined by the sort region/s. Therefore the diversity of the total population is captured, and the more frequently occurring or rarer cell types are all sampled. The sorting approach was tested computationally, and using functional cell based assays. Computationally we demonstrate that conventional single cell sorting can sample as little as 50% of the population diversity dependant on the population distribution, and that Slice and Dice sorting samples much more of the variety present within a cell population. We then show by sorting single cells into wells using the Slice and Dice sorting method that there are cells sorted using this method that would be either rarely sorted, or not sorted at all using conventional single cell sorting approaches. The present study demonstrates a novel single cell sorting method that samples much more of the population diversity than current methods. It has implications in clonal selection, stem cell sorting, single cell sequencing and any areas where population heterogeneity is of importance. © 2015 International Society for Advancement of Cytometry.

A novel high-throughput imaging system for automated analyses of avoidance behavior in zebrafish larvae.

PubMed

Pelkowski, Sean D; Kapoor, Mrinal; Richendrfer, Holly A; Wang, Xingyue; Colwill, Ruth M; Creton, Robbert

2011-09-30

Early brain development can be influenced by numerous genetic and environmental factors, with long-lasting effects on brain function and behavior. The identification of these factors is facilitated by recent innovations in high-throughput screening. However, large-scale screening in whole organisms remains challenging, in particular when studying changes in brain function or behavior in vertebrate model systems. In this study, we present a novel imaging system for high-throughput analyses of behavior in zebrafish larvae. The three-camera system can image 12 multiwell plates simultaneously and is unique in its ability to provide local visual stimuli in the wells of a multiwell plate. The acquired images are converted into a series of coordinates, which characterize the location and orientation of the larvae. The developed imaging techniques were tested by measuring avoidance behaviors in seven-day-old zebrafish larvae. The system effectively quantified larval avoidance and revealed an increased edge preference in response to a blue or red 'bouncing ball' stimulus. Larvae also avoid a bouncing ball stimulus when it is counter-balanced with a stationary ball, but do not avoid blinking balls counter-balanced with a stationary ball. These results indicate that the seven-day-old larvae respond specifically to movement, rather than color, size, or local changes in light intensity. The imaging system and assays for measuring avoidance behavior may be used to screen for genetic and environmental factors that cause developmental brain disorders and for novel drugs that could prevent or treat these disorders. Copyright © 2011 Elsevier B.V. All rights reserved.

pH measurement and a rational and practical pH control strategy for high throughput cell culture system.

PubMed

Zhou, Haiying; Purdie, Jennifer; Wang, Tongtong; Ouyang, Anli

2010-01-01

The number of therapeutic proteins produced by cell culture in the pharmaceutical industry continues to increase. During the early stages of manufacturing process development, hundreds of clones and various cell culture conditions are evaluated to develop a robust process to identify and select cell lines with high productivity. It is highly desirable to establish a high throughput system to accelerate process development and reduce cost. Multiwell plates and shake flasks are widely used in the industry as the scale down model for large-scale bioreactors. However, one of the limitations of these two systems is the inability to measure and control pH in a high throughput manner. As pH is an important process parameter for cell culture, this could limit the applications of these scale down model vessels. An economical, rapid, and robust pH measurement method was developed at Eli Lilly and Company by employing SNARF-4F 5-(-and 6)-carboxylic acid. The method demonstrated the ability to measure the pH values of cell culture samples in a high throughput manner. Based upon the chemical equilibrium of CO(2), HCO(3)(-), and the buffer system, i.e., HEPES, we established a mathematical model to regulate pH in multiwell plates and shake flasks. The model calculates the required %CO(2) from the incubator and the amount of sodium bicarbonate to be added to adjust pH to a preset value. The model was validated by experimental data, and pH was accurately regulated by this method. The feasibility of studying the pH effect on cell culture in 96-well plates and shake flasks was also demonstrated in this study. This work shed light on mini-bioreactor scale down model construction and paved the way for cell culture process development to improve productivity or product quality using high throughput systems. Copyright 2009 American Institute of Chemical Engineers

A comparative analysis of standard microtiter plate reading versus imaging in cellular assays.

PubMed

Bushway, Paul J; Mercola, Mark; Price, Jeffrey H

2008-08-01

We evaluated the performance of two plate readers (the Beckman Coulter [Fullerton, CA] DTX and the PerkinElmer [Wellesley, MA] EnVision) and a plate imager (the General Electric [Fairfield, CT] IN Cell 1000 Analyzer) in a primary fluorescent cellular screen of 10,000 Molecular Libraries Screening Center Network library compounds for up- and down-regulation of vascular cell adhesion molecule (VCAM)-1, which has been shown to be up-regulated in atherothrombotic vascular disease and is a general indicator of chronic inflammatory disease. Prior to screening, imaging of a twofold, six-step titration of fluorescent cells in a 384-well test plate showed greater consistency, sensitivity, and dynamic range of signal detection curves throughout the detection range, as compared to the plate readers. With the same 384-well test plate, the detection limits for fluorescent protein-labeled cells on the DTX and EnVision instruments were 2,250 and 560 fluorescent cells per well, respectively, as compared to 280 on the IN Cell 1000. During VCAM screening, sensitivity was critical for detection of antagonists, which reduced brightness of the primary immunofluorescence readout; inhibitor controls yielded Z' values of 0.41 and 0.16 for the IN Cell 1000 and EnVision instruments, respectively. The best 1% of small molecule inhibitors from all platforms were visually confirmed using images from the IN Cell 1000. The EnVision and DTX plate readers mutually identified approximately 57% and 21%, respectively, of the VCAM-1 inhibitors visually confirmed in the IN Cell best 1% of inhibitors. Furthermore, the plate reader hits were largely exclusive, with only 6% agreement across all platforms (three hits out of 47). Taken together, the imager outperformed the plate readers at hit detection in this bimodal assay because of superior sensitivity and had the advantage of speeding hit confirmation during post-acquisition analysis.

Automated measurement of zebrafish larval movement

PubMed Central

Cario, Clinton L; Farrell, Thomas C; Milanese, Chiara; Burton, Edward A

2011-01-01

Abstract The zebrafish is a powerful vertebrate model that is readily amenable to genetic, pharmacological and environmental manipulations to elucidate the molecular and cellular basis of movement and behaviour. We report software enabling automated analysis of zebrafish movement from video recordings captured with cameras ranging from a basic camcorder to more specialized equipment. The software, which is provided as open-source MATLAB functions, can be freely modified and distributed, and is compatible with multiwell plates under a wide range of experimental conditions. Automated measurement of zebrafish movement using this technique will be useful for multiple applications in neuroscience, pharmacology and neuropsychiatry. PMID:21646414

Automated measurement of zebrafish larval movement.

PubMed

Cario, Clinton L; Farrell, Thomas C; Milanese, Chiara; Burton, Edward A

2011-08-01

The zebrafish is a powerful vertebrate model that is readily amenable to genetic, pharmacological and environmental manipulations to elucidate the molecular and cellular basis of movement and behaviour. We report software enabling automated analysis of zebrafish movement from video recordings captured with cameras ranging from a basic camcorder to more specialized equipment. The software, which is provided as open-source MATLAB functions, can be freely modified and distributed, and is compatible with multiwell plates under a wide range of experimental conditions. Automated measurement of zebrafish movement using this technique will be useful for multiple applications in neuroscience, pharmacology and neuropsychiatry.

Piezo- and solenoid valve-based liquid dispensing for miniaturized assays.

PubMed

Niles, Walter D; Coassin, Peter J

2005-04-01

Miniaturization of biological assays requires dispensing liquids in the submicroliter range of volumes. Accuracy and reproducibility of dispensing this range depend on both the dispenser and the receptacle in which the assay is constructed. Miniaturization technologies developed by Aurora Discovery, Inc. (San Diego, CA) include high-density multiwell plates for assay samples and reagent storage, as well as piezo-based and solenoid valve-based liquid dispensers. Some basic principles of small-volume dispensing by jetting are described to provide context for dispenser design and function. Performance of the latest instruments incorporating these dispensing devices is presented.

A versatile and low-cost open source pipetting robot for automation of toxicological and ecotoxicological bioassays

PubMed Central

Seiler, Thomas-Benjamin; Ruchter, Nadine; Schumann, Mark; Döring, Ricarda; Cofalla, Catrina; Ostfeld, Avi; Salomons, Elad; Schüttrumpf, Holger; Hollert, Henner

2017-01-01

In the past decades, bioassays and whole-organism bioassay have become important tools not only in compliance testing of industrial chemicals and plant protection products, but also in the monitoring of environmental quality. With few exceptions, such test systems are discontinuous. They require exposure of the biological test material in small units, such as multiwell plates, during prolonged incubation periods, and do not allow online read-outs. It is mostly due to these shortcomings that applications in continuous monitoring of, e.g., drinking or surface water quality are largely missing. We propose the use of pipetting robots that can be used to automatically exchange samples in multiwell plates with fresh samples in a semi-static manner, as a potential solution to overcome these limitations. In this study, we developed a simple and low-cost, versatile pipetting robot constructed partly using open-source hardware that has a small footprint and can be used for online monitoring of water quality by means of an automated whole-organism bioassay. We tested its precision in automated 2-fold dilution series and used it for exposure of zebrafish embryos (Danio rerio)–a common model species in ecotoxicology—to cadmium chloride and permethrin. We found that, compared to conventional static or semi-static exposure scenarios, effects of the two chemicals in zebrafish embryos generally occurred at lower concentrations, and analytically verified that the increased frequency of media exchange resulted in a greater availability of the chemical. In combination with advanced detection systems this custom-made pipetting robot has the potential to become a valuable tool in future monitoring strategies for drinking and surface water. PMID:28622373

A versatile and low-cost open source pipetting robot for automation of toxicological and ecotoxicological bioassays.

PubMed

Steffens, Sebastian; Nüßer, Leonie; Seiler, Thomas-Benjamin; Ruchter, Nadine; Schumann, Mark; Döring, Ricarda; Cofalla, Catrina; Ostfeld, Avi; Salomons, Elad; Schüttrumpf, Holger; Hollert, Henner; Brinkmann, Markus

2017-01-01

In the past decades, bioassays and whole-organism bioassay have become important tools not only in compliance testing of industrial chemicals and plant protection products, but also in the monitoring of environmental quality. With few exceptions, such test systems are discontinuous. They require exposure of the biological test material in small units, such as multiwell plates, during prolonged incubation periods, and do not allow online read-outs. It is mostly due to these shortcomings that applications in continuous monitoring of, e.g., drinking or surface water quality are largely missing. We propose the use of pipetting robots that can be used to automatically exchange samples in multiwell plates with fresh samples in a semi-static manner, as a potential solution to overcome these limitations. In this study, we developed a simple and low-cost, versatile pipetting robot constructed partly using open-source hardware that has a small footprint and can be used for online monitoring of water quality by means of an automated whole-organism bioassay. We tested its precision in automated 2-fold dilution series and used it for exposure of zebrafish embryos (Danio rerio)-a common model species in ecotoxicology-to cadmium chloride and permethrin. We found that, compared to conventional static or semi-static exposure scenarios, effects of the two chemicals in zebrafish embryos generally occurred at lower concentrations, and analytically verified that the increased frequency of media exchange resulted in a greater availability of the chemical. In combination with advanced detection systems this custom-made pipetting robot has the potential to become a valuable tool in future monitoring strategies for drinking and surface water.

Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates

NASA Astrophysics Data System (ADS)

Maioli, Vincent; Chennell, George; Sparks, Hugh; Lana, Tobia; Kumar, Sunil; Carling, David; Sardini, Alessandro; Dunsby, Chris

2016-11-01

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format.

Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates.

PubMed

Maioli, Vincent; Chennell, George; Sparks, Hugh; Lana, Tobia; Kumar, Sunil; Carling, David; Sardini, Alessandro; Dunsby, Chris

2016-11-25

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format.

Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates

PubMed Central

Maioli, Vincent; Chennell, George; Sparks, Hugh; Lana, Tobia; Kumar, Sunil; Carling, David; Sardini, Alessandro; Dunsby, Chris

2016-01-01

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format. PMID:27886235

Next generation 1536-well oligonucleotide synthesizer with on-the-fly dispense.

PubMed

Jensen, Michael; Roberts, Lester; Johnson, Andrew; Fukushima, Marilyn; Davis, Ronald

2014-02-10

Here we report the development of our Next Generation Automated Multiplexed Oligonucleotide Synthesizer (NG-1536-AMOS), capable of producing 1536 samples in a single run using a multi-well filtered titer plate. With the potential to synthesize up to 3456 samples per plate, we converted the BioRAPTR Flying Reagent Dispenser into an open-well system where spent reagents are drained to waste under vacuum. During synthesis, reagents are delivered on-the-fly to each micro-titer well at volumes ≤5 μl with plate speeds up to 150 mm/s. Using gas-phase cleavage and deprotection, a full plate of 1536 60 mers may be processed with same-day turnaround with an average yield per well at 3.5 nmol. Final product at only $0.00277/base is eluted into a low-volume collection plate for immediate use in downstream application (e.g. Biomek FX for versatile sample handling). Also, crude oligonucleotide quality is comparable to that of commercial synthesis instrumentation, with an error rate on the NG-1536-AMOS platform of 1.53/717 bases. Furthermore, mass spectral analysis on strands synthesized up to 80 bases showed high purity with an average coupling efficiency of 99.5%. Copyright © 2013 Elsevier B.V. All rights reserved.

Automatic license plate reader: a solution to avoiding vehicle pursuit

NASA Astrophysics Data System (ADS)

Jordan, Stanley K.

1997-01-01

The Massachusetts Governor's Auto Theft Strike Force has tested an automatic license plate reader (LPR) to recover stolen cars and catch car thieves, without vehicle pursuit. Experiments were conducted at the Sumner Tunnel in Boston, and proved the feasibility of a LPR for identifying stolen cars instantly. The same technology can be applied to other law-enforcement objectives.

A high throughput respirometric assay for mitochondrial biogenesis and toxicity

PubMed Central

Beeson, Craig C.; Beeson, Gyda C.; Schnellmann, Rick G.

2010-01-01

Mitochondria are a common target of toxicity for drugs and other chemicals, and results in decreased aerobic metabolism and cell death. In contrast, mitochondrial biogenesis restores cell vitality and there is a need for new agents to induce biogenesis. Current cell-based models of mitochondrial biogenesis or toxicity are inadequate because cultured cell lines are highly glycolytic with minimal aerobic metabolism and altered mitochondrial physiology. In addition, there are no high-throughput, real-time assays that assess mitochondrial function. We adapted primary cultures of renal proximal tubular cells (RPTC) that exhibit in vivo levels of aerobic metabolism, are not glycolytic, and retain higher levels of differentiated functions and used the Seahorse Biosciences analyzer to measure mitochondrial function in real time in multi-well plates. Using uncoupled respiration as a marker of electron transport chain (ETC) integrity, the nephrotoxicants cisplatin, HgCl2 and gentamicin exhibited mitochondrial toxicity prior to decreases in basal respiration and cell death. Conversely, using FCCP-uncoupled respiration as a marker of maximal ETC activity, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), SRT1720, resveratrol, daidzein, and metformin produced mitochondrial biogenesis in RPTC. The merger of the RPTC model and multi-well respirometry results in a single high throughput assay to measure mitochondrial biogenesis and toxicity, and nephrotoxic potential. PMID:20465991

High-throughput SRCD using multi-well plates and its applications

NASA Astrophysics Data System (ADS)

Hussain, Rohanah; Jávorfi, Tamás; Rudd, Timothy R.; Siligardi, Giuliano

2016-12-01

The sample compartment for high-throughput synchrotron radiation circular dichroism (HT-SRCD) has been developed to satisfy an increased demand of protein characterisation in terms of folding and binding interaction properties not only in the traditional field of structural biology but also in the growing research area of material science with the potential to save time by 80%. As the understanding of protein behaviour in different solvent environments has increased dramatically the development of novel functions such as recombinant proteins modified to have different functions from harvesting solar energy to metabolonics for cleaning heavy and metal and organic molecule pollutions, there is a need to characterise speedily these system.

Plug-and-actuate on demand: multimodal individual addressability of microarray plates using modular hybrid acoustic wave technology.

PubMed

Rezk, Amgad R; Ramesan, Shwathy; Yeo, Leslie Y

2018-01-30

The microarray titre plate remains a fundamental workhorse in genomic, proteomic and cellomic analyses that underpin the drug discovery process. Nevertheless, liquid handling technologies for sample dispensing, processing and transfer have not progressed significantly beyond conventional robotic micropipetting techniques, which are not only at their fundamental sample size limit, but are also prone to mechanical failure and contamination. This is because alternative technologies to date suffer from a number of constraints, mainly their limitation to carry out only a single liquid operation such as dispensing or mixing at a given time, and their inability to address individual wells, particularly at high throughput. Here, we demonstrate the possibility for true sequential or simultaneous single- and multi-well addressability in a 96-well plate using a reconfigurable modular platform from which MHz-order hybrid surface and bulk acoustic waves can be coupled to drive a variety of microfluidic modes including mixing, sample preconcentration and droplet jetting/ejection in individual or multiple wells on demand, thus constituting a highly versatile yet simple setup capable of improving the functionality of existing laboratory protocols and processes.

Artefacts found in computed radiography.

PubMed

Cesar, L J; Schueler, B A; Zink, F E; Daly, T R; Taubel, J P; Jorgenson, L L

2001-02-01

Artefacts on radiographic images are distracting and may compromise accurate diagnosis. Although most artefacts that occur in conventional radiography have become familiar, computed radiography (CR) systems produce artefacts that differ from those found in conventional radiography. We have encountered a variety of artefacts in CR images that were produced from four different models plate reader. These artefacts have been identified and traced to the imaging plate, plate reader, image processing software or laser printer or to operator error. Understanding the potential sources of CR artefacts will aid in identifying and resolving problems quickly and help prevent future occurrences.

Regulation of Glucose Utilization by Estradiol in Breast Cancer

DTIC Science & Technology

2014-10-01

counts/min) weremeasured using 350 l of lysate. Protein concentration was determined using the BCA assay according to the manufacturer’s...instructions and measured on a Powerwave XS plate reader (Biotek). Counts were normalized to protein concentration. Glycolysis Assay—MCF-7 cells growing in 6...determined using the BCA assay according to the manufacturer’s instructions and mea- sured on a Powerwave XS plate reader. Counts were normal- ized to

Bioactive contaminants leach from disposable laboratory plasticware.

PubMed

McDonald, G Reid; Hudson, Alan L; Dunn, Susan M J; You, Haitao; Baker, Glen B; Whittal, Randy M; Martin, Jonathan W; Jha, Amitabh; Edmondson, Dale E; Holt, Andrew

2008-11-07

Disposable plasticware such as test tubes, pipette tips, and multiwell assay or culture plates are used routinely in most biological research laboratories. Manufacturing of plastics requires the inclusion of numerous chemicals to enhance stability, durability, and performance. Some lubricating (slip) agents, exemplified by oleamide, also occur endogenously in humans and are biologically active, and cationic biocides are included to prevent bacterial colonization of the plastic surface. We demonstrate that these manufacturing agents leach from laboratory plasticware into a standard aqueous buffer, dimethyl sulfoxide, and methanol and can have profound effects on proteins and thus on results from bioassays of protein function. These findings have far-reaching implications for the use of disposable plasticware in biological research.

Optimizing multi-dimensional high throughput screening using zebrafish

PubMed Central

Truong, Lisa; Bugel, Sean M.; Chlebowski, Anna; Usenko, Crystal Y.; Simonich, Michael T.; Massey Simonich, Staci L.; Tanguay, Robert L.

2016-01-01

The use of zebrafish for high throughput screening (HTS) for chemical bioactivity assessments is becoming routine in the fields of drug discovery and toxicology. Here we report current recommendations from our experiences in zebrafish HTS. We compared the effects of different high throughput chemical delivery methods on nominal water concentration, chemical sorption to multi-well polystyrene plates, transcription responses, and resulting whole animal responses. We demonstrate that digital dispensing consistently yields higher data quality and reproducibility compared to standard plastic tip-based liquid handling. Additionally, we illustrate the challenges in using this sensitive model for chemical assessment when test chemicals have trace impurities. Adaptation of these better practices for zebrafish HTS should increase reproducibility across laboratories. PMID:27453428

15 years of zebrafish chemical screening

PubMed Central

Rennekamp, Andrew J.; Peterson, Randall T.

2015-01-01

In 2000, the first chemical screen using living zebrafish in a multi-well plate was reported. Since then, more than 60 additional screens have been published describing whole-organism drug and pathway discovery projects in zebrafish. To investigate the scope of the work reported in the last 14 years and to identify trends in the field, we analyzed the discovery strategies of 64 primary research articles from the literature. We found that zebrafish screens have expanded beyond the use of developmental phenotypes to include behavioral, cardiac, metabolic, proliferative and regenerative endpoints. Additionally, many creative strategies have been used to uncover the mechanisms of action of new small molecules including chemical phenocopy, genetic phenocopy, mutant rescue, and spatial localization strategies. PMID:25461724

Migration of phthalates on culture plates - an important challenge to consider for in vitro studies.

PubMed

Frohnert Hansen, Juliana; Boas, Malene; Møller Brorson, Marianne; Frederiksen, Hanne; Hartoft-Nielsen, Marie-Louise; Krogh Rasmussen, Åse; Main, Katharina M; Feldt-Rasmussen, Ulla

2016-01-01

Phthalates are endocrine disruptors of the reproductive system and suspected to influence many other organ and hormone systems. They are also semi-volatile organic compounds present in the gas phase in the environment. Their mode of action has been investigated in numerous in vitro studies. Multi-well culture plates are typically used to study phthalates in cell cultures. In a pilot study, we observed evidence of phthalate migration in 24-well culture plates. As this has not previously been described, we investigated the phenomenon in more detail. Primary human thyroid epithelial cell cultures (n = 8 cultures) were exposed to either di-ethyl phthalate (DEP), di-n-butyl phthalate (DnBP), mono-n-butyl phthalate (MnBP) or di-(2-ethylhexyl) phthalate (DEHP). Measurement of phthalate metabolites by mass spectrometry demonstrated that the short-branched DEP was able to migrate to adjacent wells when added to cell culture plates. DnBP also seemed to be able to migrate, unlike the long-branched DEHP or the monoester MnBP which did not seem to have this ability. High background levels of phthalate metabolites were also observed, which might compromise results from low dose phthalate studies. In conclusion, the migration of phthalates which is probably caused by their volatile properties might lead to false interpretation of study results.

Usage of DNA Fingerprinting Technology for Quality Control in Molecular Lab Bench Work.

PubMed

McIntosh, Linda Y; Lal, Janella E; Qin, Dahui

2018-01-01

One of the major quality assurance (QA) goals in many molecular laboratories is to avoid sample pipetting errors on the lab bench; especially when pipetting into multiwell plates. A pipetting error can cause a switch in patient samples, which can lead to recording the wrong results for the patient samples involved. Such pipetting errors are difficult to identify when it happens in lab bench work. DNA fingerprinting is a powerful tool in determining sample identities. Our laboratory has explored the usage of this technology in our QA process and successfully established that DNA fingerprinting can be used to monitor possible sample switch in gene rearrangement lab bench work. We use florescent light to quench the florescence in the gene rearrangement polymerase chain reaction products. After that, DNA fingerprinting technology is used to identify the sample DNA in the gene rearrangement polymerase chain reaction plate. The result is compared with the corresponding patient's blood sample DNA to determine whether there is a sample switch during the lab bench work.

Chapter 17 Sterile Plate-Based Vitrification of Adherent Human Pluripotent Stem Cells and Their Derivatives Using the TWIST Method.

PubMed

Neubauer, Julia C; Stracke, Frank; Zimmermann, Heiko

2017-01-01

Due to their high biological complexity, e.g., their close cell-to-cell contacts, cryopreservation of human pluripotent stem cells with standard slow-rate protocols often is inefficient and can hardly be standardized. Vitrification that means ultrafast freezing already showed very good viability and recovery rates for this sensitive cell system, but is only applicable for low cell numbers, bears a high risk of contamination, and can hardly be implemented under GxP regulations. In this chapter, a sterile plate-based vitrification method for adherent pluripotent stem cells and their derivatives is presented based on a procedure and device for human embryonic stem cells developed by Beier et al. (Cryobiology 66:8-16, 2013). This protocol overcomes the limitations of conventional vitrification procedures resulting in the highly efficient preservation of ready-to-use adherent pluripotent stem cells with the possibility of vitrifying cells in multi-well formats for direct application in high-throughput screenings.

Use of a Fluorometric Imaging Plate Reader in high-throughput screening

NASA Astrophysics Data System (ADS)

Groebe, Duncan R.; Gopalakrishnan, Sujatha; Hahn, Holly; Warrior, Usha; Traphagen, Linda; Burns, David J.

1999-04-01

High-throughput screening (HTS) efforts at Abbott Laboratories have been greatly facilitated by the use of a Fluorometric Imaging Plate Reader. The FLIPR consists of an incubated cabinet with integrated 96-channel pipettor and fluorometer. An argon laser is used to excite fluorophores in a 96-well microtiter plate and the emitted fluorometer. An argon laser is used to excite fluorophores in a 96-well microtiter plate and the emitted fluorescence is imaged by a cooled CCD camera. The image data is downloaded from the camera and processed to average the signal form each well of the microtiter pate for each time point. The data is presented in real time on the computer screen, facilitating interpretation and trouble-shooting. In addition to fluorescence, the camera can also detect luminescence form firefly luciferase.

GryphSens: A Smartphone-Based Portable Diagnostic Reader for the Rapid Detection of Progesterone in Milk

PubMed Central

Jang, Hyunwook; Ahmed, Syed Rahin; Neethirajan, Suresh

2017-01-01

Enzyme-linked immunosorbent assay (ELISA) is a popular assay technique for the detection and quantification of various biological substances due its high sensitivity and specificity. More often, it requires large and expensive laboratory instruments, which makes it difficult to conduct when the tests must be performed quickly at the point-of-care (POC). To increase portability and ease of use, we propose a portable diagnostic system based on a Raspberry Pi imaging sensor for the rapid detection of progesterone in milk samples. We designed, assembled, and tested a standalone portable diagnostic reader and validated it for progesterone detection against a standard ELISA assay using a commercial plate reader. The portable POC device yielded consistent results, regardless of differences in the cameras and flashlights between various smartphone devices. An Android application was built to provide front-end access to users, control the diagnostic reader, and display and store the progesterone measurement on the smartphone. The diagnostic reader takes images of the samples, reads the pixel values, processes the results, and presents the results on the handheld device. The proposed POC reader can perform to superior levels of performance as a plate reader, while adding the desirable qualities of portability and ease of use. PMID:28489036

GryphSens: A Smartphone-Based Portable Diagnostic Reader for the Rapid Detection of Progesterone in Milk.

PubMed

Jang, Hyunwook; Ahmed, Syed Rahin; Neethirajan, Suresh

2017-05-10

Enzyme-linked immunosorbent assay (ELISA) is a popular assay technique for the detection and quantification of various biological substances due its high sensitivity and specificity. More often, it requires large and expensive laboratory instruments, which makes it difficult to conduct when the tests must be performed quickly at the point-of-care (POC). To increase portability and ease of use, we propose a portable diagnostic system based on a Raspberry Pi imaging sensor for the rapid detection of progesterone in milk samples. We designed, assembled, and tested a standalone portable diagnostic reader and validated it for progesterone detection against a standard ELISA assay using a commercial plate reader. The portable POC device yielded consistent results, regardless of differences in the cameras and flashlights between various smartphone devices. An Android application was built to provide front-end access to users, control the diagnostic reader, and display and store the progesterone measurement on the smartphone. The diagnostic reader takes images of the samples, reads the pixel values, processes the results, and presents the results on the handheld device. The proposed POC reader can perform to superior levels of performance as a plate reader, while adding the desirable qualities of portability and ease of use.

Optimizing multi-dimensional high throughput screening using zebrafish.

PubMed

Truong, Lisa; Bugel, Sean M; Chlebowski, Anna; Usenko, Crystal Y; Simonich, Michael T; Simonich, Staci L Massey; Tanguay, Robert L

2016-10-01

The use of zebrafish for high throughput screening (HTS) for chemical bioactivity assessments is becoming routine in the fields of drug discovery and toxicology. Here we report current recommendations from our experiences in zebrafish HTS. We compared the effects of different high throughput chemical delivery methods on nominal water concentration, chemical sorption to multi-well polystyrene plates, transcription responses, and resulting whole animal responses. We demonstrate that digital dispensing consistently yields higher data quality and reproducibility compared to standard plastic tip-based liquid handling. Additionally, we illustrate the challenges in using this sensitive model for chemical assessment when test chemicals have trace impurities. Adaptation of these better practices for zebrafish HTS should increase reproducibility across laboratories. Copyright © 2016 Elsevier Inc. All rights reserved.

Development of carbon plasma-coated multiwell plates for high-throughput mass spectrometric analysis of highly lipophilic fermentation products.

PubMed

Heinig, Uwe; Scholz, Susanne; Dahm, Pia; Grabowy, Udo; Jennewein, Stefan

2010-08-01

Classical approaches to strain improvement and metabolic engineering rely on rapid qualitative and quantitative analyses of the metabolites of interest. As an analytical tool, mass spectrometry (MS) has proven to be efficient and nearly universally applicable for timely screening of metabolites. Furthermore, gas chromatography (GC)/MS- and liquid chromatography (LC)/MS-based metabolite screens can often be adapted to high-throughput formats. We recently engineered a Saccharomyces cerevisiae strain to produce taxa-4(5),11(12)-diene, the first pathway-committing biosynthetic intermediate for the anticancer drug Taxol, through the heterologous and homologous expression of several genes related to isoprenoid biosynthesis. To date, GC/MS- and LC/MS-based high-throughput methods have been inherently difficult to adapt to the screening of isoprenoid-producing microbial strains due to the need for extensive sample preparation of these often highly lipophilic compounds. In the current work, we examined different approaches to the high-throughput analysis of taxa-4(5),11(12)-diene biosynthesizing yeast strains in a 96-deep-well format. Carbon plasma coating of standard 96-deep-well polypropylene plates allowed us to circumvent the inherent solvent instability of commonly used deep-well plates. In addition, efficient adsorption of the target isoprenoid product by the coated plates allowed rapid and simple qualitative and quantitative analyses of the individual cultures. Copyright 2010 Elsevier Inc. All rights reserved.

Characterization of Early Cortical Neural Network ...

EPA Pesticide Factsheets

We examined the development of neural network activity using microelectrode array (MEA) recordings made in multi-well MEA plates (mwMEAs) over the first 12 days in vitro (DIV). In primary cortical cultures made from postnatal rats, action potential spiking activity was essentially absent on DIV 2 and developed rapidly between DIV 5 and 12. Spiking activity was primarily sporadic and unorganized at early DIV, and became progressively more organized with time in culture, with bursting parameters, synchrony and network bursting increasing between DIV 5 and 12. We selected 12 features to describe network activity and principal components analysis using these features demonstrated a general segregation of data by age at both the well and plate levels. Using a combination of random forest classifiers and Support Vector Machines, we demonstrated that 4 features (CV of within burst ISI, CV of IBI, network spike rate and burst rate) were sufficient to predict the age (either DIV 5, 7, 9 or 12) of each well recording with >65% accuracy. When restricting the classification problem to a binary decision, we found that classification improved dramatically, e.g. 95% accuracy for discriminating DIV 5 vs DIV 12 wells. Further, we present a novel resampling approach to determine the number of wells that might be needed for conducting comparisons of different treatments using mwMEA plates. Overall, these results demonstrate that network development on mwMEA plates is similar to

Further Evaluation of DNT Hazard Screening using Neural Networks from Rat Cortical Neurons on Multi-well Microelectrode Arrays

EPA Science Inventory

Thousands of chemicals have not been characterized for their DNT potential. Due to the need for DNT hazard identification, efforts to develop screening assays for DNT potential is a high priority. Multi-well microelectrode arrays (MEA) measure the spontaneous activity of electr...

High-throughput and automated diagnosis of antimicrobial resistance using a cost-effective cellphone-based micro-plate reader

NASA Astrophysics Data System (ADS)

Feng, Steve; Tseng, Derek; di Carlo, Dino; Garner, Omai B.; Ozcan, Aydogan

2016-12-01

Routine antimicrobial susceptibility testing (AST) can prevent deaths due to bacteria and reduce the spread of multi-drug-resistance, but cannot be regularly performed in resource-limited-settings due to technological challenges, high-costs, and lack of trained professionals. We demonstrate an automated and cost-effective cellphone-based 96-well microtiter-plate (MTP) reader, capable of performing AST without the need for trained diagnosticians. Our system includes a 3D-printed smartphone attachment that holds and illuminates the MTP using a light-emitting-diode array. An inexpensive optical fiber-array enables the capture of the transmitted light of each well through the smartphone camera. A custom-designed application sends the captured image to a server to automatically determine well-turbidity, with results returned to the smartphone in ~1 minute. We tested this mobile-reader using MTPs prepared with 17 antibiotics targeting Gram-negative bacteria on clinical isolates of Klebsiella pneumoniae, containing highly-resistant antimicrobial profiles. Using 78 patient isolate test-plates, we demonstrated that our mobile-reader meets the FDA-defined AST criteria, with a well-turbidity detection accuracy of 98.21%, minimum-inhibitory-concentration accuracy of 95.12%, and a drug-susceptibility interpretation accuracy of 99.23%, with no very major errors. This mobile-reader could eliminate the need for trained diagnosticians to perform AST, reduce the cost-barrier for routine testing, and assist in spatio-temporal tracking of bacterial resistance.

Technologies That Enable Accurate and Precise Nano- to Milliliter-Scale Liquid Dispensing of Aqueous Reagents Using Acoustic Droplet Ejection.

PubMed

Sackmann, Eric K; Majlof, Lars; Hahn-Windgassen, Annett; Eaton, Brent; Bandzava, Temo; Daulton, Jay; Vandenbroucke, Arne; Mock, Matthew; Stearns, Richard G; Hinkson, Stephen; Datwani, Sammy S

2016-02-01

Acoustic liquid handling uses high-frequency acoustic signals that are focused on the surface of a fluid to eject droplets with high accuracy and precision for various life science applications. Here we present a multiwell source plate, the Echo Qualified Reservoir (ER), which can acoustically transfer over 2.5 mL of fluid per well in 25-nL increments using an Echo 525 liquid handler. We demonstrate two Labcyte technologies-Dynamic Fluid Analysis (DFA) methods and a high-voltage (HV) grid-that are required to maintain accurate and precise fluid transfers from the ER at this volume scale. DFA methods were employed to dynamically assess the energy requirements of the fluid and adjust the acoustic ejection parameters to maintain a constant velocity droplet. Furthermore, we demonstrate that the HV grid enhances droplet velocity and coalescence at the destination plate. These technologies enabled 5-µL per destination well transfers to a 384-well plate, with accuracy and precision values better than 4%. Last, we used the ER and Echo 525 liquid handler to perform a quantitative polymerase chain reaction (qPCR) assay to demonstrate an application that benefits from the flexibility and larger volume capabilities of the ER. © 2015 Society for Laboratory Automation and Screening.

Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes

PubMed Central

Bray, Mark-Anthony; Singh, Shantanu; Han, Han; Davis, Chadwick T.; Borgeson, Blake; Hartland, Cathy; Kost-Alimova, Maria; Gustafsdottir, Sigrun M.; Gibson, Christopher C.; Carpenter, Anne E.

2016-01-01

In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profiles. This protocol describes the design and execution of experiments using Cell Painting, a morphological profiling assay multiplexing six fluorescent dyes imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Cells are plated in multi-well plates, perturbed with the treatments to be tested, stained, fixed, and imaged on a high-throughput microscope. Then, automated image analysis software identifies individual cells and measures ~1,500 morphological features (various measures of size, shape, texture, intensity, etc.) to produce a rich profile suitable for detecting subtle phenotypes. Profiles of cell populations treated with different experimental perturbations can be compared to suit many goals, such as identifying the phenotypic impact of chemical or genetic perturbations, grouping compounds and/or genes into functional pathways, and identifying signatures of disease. Cell culture and image acquisition takes two weeks; feature extraction and data analysis take an additional 1-2 weeks. PMID:27560178

Automated sample area definition for high-throughput microscopy.

PubMed

Zeder, M; Ellrott, A; Amann, R

2011-04-01

High-throughput screening platforms based on epifluorescence microscopy are powerful tools in a variety of scientific fields. Although some applications are based on imaging geometrically defined samples such as microtiter plates, multiwell slides, or spotted gene arrays, others need to cope with inhomogeneously located samples on glass slides. The analysis of microbial communities in aquatic systems by sample filtration on membrane filters followed by multiple fluorescent staining, or the investigation of tissue sections are examples. Therefore, we developed a strategy for flexible and fast definition of sample locations by the acquisition of whole slide overview images and automated sample recognition by image analysis. Our approach was tested on different microscopes and the computer programs are freely available (http://www.technobiology.ch). Copyright © 2011 International Society for Advancement of Cytometry.

Quantum Effects at a Proton Relaxation at Low Temperatures

NASA Astrophysics Data System (ADS)

Kalytka, V. A.; Korovkin, M. V.

2016-11-01

Quantum effects during migratory polarization in multi-well crystals (including multi-well silicates and crystalline hydrates) are investigated in a variable electric field at low temperatures by direct quantum-mechanical calculations. Based on analytical solution of the quantum Liouville kinetic equation in the linear approximation for the polarizing field, the non-stationary density matrix is calculated for an ensemble of non-interacting protons moving in the field of one-dimensional multi-well crystal potential relief of rectangular shape. An expression for the complex dielectric constant convenient for a comparison with experiment and calculation of relaxer parameters is derived using the nonequilibrium polarization density matrix. The density matrix apparatus can be used for analytical investigation of the quantum mechanism of spontaneous polarization of a ferroelectric material (KDP and DKDP).

Evaluation of the Neuroactivity of ToxCast Compounds Using Multi-well Microelectrode Array Recordings in Primary Cortical Neurons

EPA Science Inventory

Evaluation of the Neuroactivity of ToxCast Compounds Using Multi-well Microelectrode Array Recordings in Primary Cortical Neurons P Valdivia1, M Martin2, WR LeFew3, D Hall3, J Ross1, K Houck2 and TJ Shafer3 1Axion Biosystems, Atlanta GA and 2NCCT, 3ISTD, NHEERL, ORD, US EPA, RT...

Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation.

PubMed

John, Gernot T; Klimant, Ingo; Wittmann, Christoph; Heinzle, Elmar

2003-03-30

Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96-well polystyrene microtiter plates. The oxygen-sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an internal reference. The sensor measured dissolved oxygen accurately in optically well-defined media. Oxygen transfer coefficients, k(L)a, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker. For this purpose, the dissolved oxygen was initially depleted by the addition of sodium dithionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite. k(L)a values in one commercial reader were about 10 to 40 h(-1). k(L)a values were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor. Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation in the microtiter plate were used to estimate k(L)a values in a 96-well microtiter plate. The resulting values were about 130 h(-1), which is in the lower range of typical stirred fermentors. The resulting maximum oxygen transfer rate was 26 mM h(-1). Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 829-836, 2003.

Evaluation of the Minnesota Easy Culture System II Bi-Plate and Tri-Plate for identification of common mastitis pathogens in milk.

PubMed

Royster, E; Godden, S; Goulart, D; Dahlke, A; Rapnicki, P; Timmerman, J

2014-01-01

The objective of this study was to validate use of the Minnesota Easy Culture System II Bi-Plate and Tri-Plate (University of Minnesota Laboratory for Udder Health, St. Paul) to identify common mastitis pathogens in milk. A total of 283 quarter and composite milk samples submitted to the University of Minnesota Laboratory for Udder Health during the spring of 2010 were cultured simultaneously using 3 methods: standard laboratory culture (reference method) and the Minnesota Easy Culture System II Bi-Plate and Tri-Plate methods. Bi-Plate and Tri-Plate cultures were incubated for 18 to 24h and interpreted by 2 independent, untrained readers within 5h of each other. An experienced technician completed the standard laboratory culture. For each sample, all 3 study personnel recorded the culture result (yes/no) for each of the following diagnostic categories: no bacterial growth (NG), mixed (2 organisms), contaminated (3 or more organisms), gram-positive (GP), gram-negative (GN), Staphylococcus spp., Streptococcus spp., Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Enterococcus spp., Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Klebsiella spp., and other. For each category, the prevalence, sensitivity, specificity, accuracy, and predictive values of a positive and negative test were calculated, and the agreement between readers and between each reader and the laboratory was assessed. Specificity, overall accuracy, and negative predictive values were generally high (>80%) for the Bi-Plate and Tri-Plate for each category. Sensitivity and positive predictive values were intermediate (>60%) or high (>80%) for the broad categories of NG, GP, GN, Staphylococcus spp. and Streptococcus spp., and for Staph. aureus, but were generally lower (<60%) for other more specific categories. Similarly, interreader agreement (kappa value) was moderate to substantial (40-80%) for the broad categories of NG, GP, GN, Staphylococcus spp. and Streptococcus spp., and for Staph. aureus and E. coli, but was lower for other categories. The Tri-Plate had a higher sensitivity, accuracy, and negative predictive value for Streptococcus spp., and higher interreader agreement for some of the more specific categories. Our conclusion was that Bi-Plate and Tri-Plate results will be most reliable when used to classify infections in broad diagnostic categories such NG, GP, or GN. The Bi-Plate and Tri-Plate will have intermediate ability to identify infections as being caused by Staphylococcus spp., Streptococcus spp., or Staph. aureus. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The loss and recovery of vertebrate vision examined in microplates.

PubMed

Thorn, Robert J; Clift, Danielle E; Ojo, Oladele; Colwill, Ruth M; Creton, Robbert

2017-01-01

Regenerative medicine offers potentially ground-breaking treatments of blindness and low vision. However, as new methodologies are developed, a critical question will need to be addressed: how do we monitor in vivo for functional success? In the present study, we developed novel behavioral assays to examine vision in a vertebrate model system. In the assays, zebrafish larvae are imaged in multiwell or multilane plates while various red, green, blue, yellow or cyan objects are presented to the larvae on a computer screen. The assays were used to examine a loss of vision at 4 or 5 days post-fertilization and a gradual recovery of vision in subsequent days. The developed assays are the first to measure the loss and recovery of vertebrate vision in microplates and provide an efficient platform to evaluate novel treatments of visual impairment.

Multiwell fracturing experiments. [Nitrogen foam fracture treatment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warpinski, N.

The objective of the Multiwell fracturing experiments is to test and develop the technology for the efficient stimulation of tight, lenticular gas sands. This requires basic understanding of: (1) fracture behavior and geometry in this complex lithologic environment, and (2) subsequent production into the created fracture. The intricate interplay of the hydraulic fracture with the lens geometry, the internal reservoir characteristics (fractures, reservoir breaks, etc.), the in situ stresses, and the mechanical defects (fracture, bedding, etc.) need to be defined in order to develop a successful stimulation program. The stimulation phase of the Multiwell Experiment is concerned with: (1) determiningmore » important rock/reservoir properties that influence or control fracture geometry and behavior, (2) designing fracture treatments to achieve a desired size and objectives, and (3) conducting post-treatment analyses to evaluate the effectiveness of the treatment. Background statement, project description, results and evaluation of future plans are presented. 5 refs., 2 figs., 2 tabs.« less

Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates.

PubMed

Gehring, Andrew G; Brewster, Jeffrey D; He, Yiping; Irwin, Peter L; Paoli, George C; Simons, Tawana; Tu, Shu-I; Uknalis, Joseph

2015-12-04

Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 10⁵ cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.

The use of BAS-TR imaging plates calibration in determining the resolving power of Fuji BAS-1800II image plate reader

NASA Astrophysics Data System (ADS)

Alnaimi, R.

2018-01-01

The importance of this work lies in assuring the reliability of the results obtained from both imaging plates type BAS-TR and Fuji Image Reader BAS-1800II as they are widely used in calculating essential x-ray sources parameters such as the source size, x-ray flux and brilliance, hence, the calibration presented in this work. For such quantitative analysis, a common practice used by many researchers, where Gold resolution meshes are utilised for such purpose, however not quite successful due to the transmission effect of high energy photons at their edges as well as the pixeling effect while magnifying the scanned image to secure the edge spread function (ESF) data. In contrast, the use of resolution test target (RTT) and wire mesh grid together with a set of test samples i.e. Stanley blades, Ta, Ti and Si wafer of 100, 300, 15, and 490 micron thickness respectively appeared to be efficient in determining IP pixel size and the resolution of the reader. Two different experiments were conducted using two different targets and lasers of very different performance. The first, was a 15 μm VHS video tape composed of Mylar as carrier film with Fe2O3 and CrO2 powder. Nd:YAG laser of long pulse 800 ps, 50 Hz repetition rate and single shot were utilised. Whereas, the second experiment were conducted on a 9μm C wire and a short pulse 500fs Cerberus single shot laser was used. The results obtained from both experiments were pretty much similar. The imaging plate spatial resolution was measured to be: 3.4 ± 0.2 pixels and a pixel size of 41.26 ± 1.4 μm, whereas the smallest resolvable object visible to the reader (1:1 imaging with magnification factor) was of order 140.3 ± 0.3 microns. This appeared to be worse by a factor of three which indicates the importance of the reader's calibration on a regular basis, and at the same time one has to reconsider any related work and calculation based upon the previous nominal values.

High-Throughput Mechanobiology Screening Platform Using Micro- and Nanotopography.

PubMed

Hu, Junqiang; Gondarenko, Alexander A; Dang, Alex P; Bashour, Keenan T; O'Connor, Roddy S; Lee, Sunwoo; Liapis, Anastasia; Ghassemi, Saba; Milone, Michael C; Sheetz, Michael P; Dustin, Michael L; Kam, Lance C; Hone, James C

2016-04-13

We herein demonstrate the first 96-well plate platform to screen effects of micro- and nanotopographies on cell growth and proliferation. Existing high-throughput platforms test a limited number of factors and are not fully compatible with multiple types of testing and assays. This platform is compatible with high-throughput liquid handling, high-resolution imaging, and all multiwell plate-based instrumentation. We use the platform to screen for topographies and drug-topography combinations that have short- and long-term effects on T cell activation and proliferation. We coated nanofabricated "trench-grid" surfaces with anti-CD3 and anti-CD28 antibodies to activate T cells and assayed for interleukin 2 (IL-2) cytokine production. IL-2 secretion was enhanced at 200 nm trench width and >2.3 μm grating pitch; however, the secretion was suppressed at 100 nm width and <0.5 μm pitch. The enhancement on 200 nm grid trench was further amplified with the addition of blebbistatin to reduce contractility. The 200 nm grid pattern was found to triple the number of T cells in long-term expansion, a result with direct clinical applicability in adoptive immunotherapy.

THE RABIT: A RAPID AUTOMATED BIODOSIMETRY TOOL FOR RADIOLOGICAL TRIAGE

PubMed Central

Garty, Guy; Chen, Youhua; Salerno, Alessio; Turner, Helen; Zhang, Jian; Lyulko, Oleksandra; Bertucci, Antonella; Xu, Yanping; Wang, Hongliang; Simaan, Nabil; Randers-Pehrson, Gerhard; Yao, Y. Lawrence; Amundson, Sally A.; Brenner, David J.

2010-01-01

In response to the recognized need for high throughput biodosimetry methods for use after large scale radiological events, a logical approach is complete automation of standard biodosimetric assays that are currently performed manually. We describe progress to date on the RABIT (Rapid Automated BIodosimetry Tool), designed to score micronuclei or γ-H2AX fluorescence in lymphocytes derived from a single drop of blood from a fingerstick. The RABIT system is designed to be completely automated, from the input of the capillary blood sample into the machine, to the output of a dose estimate. Improvements in throughput are achieved through use of a single drop of blood, optimization of the biological protocols for in-situ analysis in multi-well plates, implementation of robotic plate and liquid handling, and new developments in high-speed imaging. Automating well-established bioassays represents a promising approach to high-throughput radiation biodosimetry, both because high throughputs can be achieved, but also because the time to deployment is potentially much shorter than for a new biological assay. Here we describe the development of each of the individual modules of the RABIT system, and show preliminary data from key modules. Ongoing is system integration, followed by calibration and validation. PMID:20065685

Understanding Interactions between Hydrogeologic Factors, Design Variables, and System Operations for Multi-Well Aquifer Storage and Recovery Systems

NASA Astrophysics Data System (ADS)

Majumdar, S.; Miller, G. R.; Smith, B.; Sheng, Z.

2017-12-01

Aquifer Storage and Recovery (ASR) system is a powerful tool for managing our present and future freshwater supplies. It involves injection of excess water into an aquifer, storing and later recovering it when needed, such as in a drought or during peak demand periods. Multi-well ASR systems, such as the Twin Oaks Facility in San Antonio, consist of a group of wells that are used for simultaneous injection and extraction of stored water. While significant research has gone into examining the effects of hydraulic and operational factors on recovery efficiency for single ASR well, little is known about how multi-well systems respond to these factors and how energy uses may vary. In this study, we created a synthetic ASR model in MODFLOW to test a range of multi-well scenarios. We altered design parameters (well spacing, pumping capacity, well configuration), hydrogeologic factors (regional hydraulic gradient, hydraulic conductivity, dispersivity), and operational variables (injection and withdrawal durations; pumping rates) to determine the response of the system across a realistic range of interrelated parameters. We then computed energy use for each simulation, based on the hydraulic head in each well and standard pump factors, as well as recovery efficiency, based on tracer concentration in recovered water from the wells. The tracer concentration in the groundwater was determined using MT3DMS. We observed that the recovery and energy efficiencies for the Multi-well ASR system decrease with the increase in well spacing and hydraulic gradient. When longitudinal dispersivity was doubled, the recovery and energy efficiencies were nearly halved. Another finding from our study suggests that we can recover nearly 90% of the water after two successive cycles of operation. The results will be used to develop generalized operational guidelines for meeting freshwater demands and also optimise the energy consumed during pumping.

A novel 384-multiwell microelectrode array for the impedimetric monitoring of Tau protein induced neurodegenerative processes.

PubMed

Jahnke, Heinz-Georg; Krinke, Dana; Seidel, Diana; Lilienthal, Katharina; Schmidt, Sabine; Azendorf, Ronny; Fischer, Michael; Mack, Till; Striggow, Frank; Althaus, Holger; Schober, Andreas; Robitzki, Andrea A

2017-02-15

Over the last decades, countless bioelectronic monitoring systems were developed for the analysis of cells as well as complex tissues. Most studies addressed the sensitivity and specificity of the bioelectronic detection method in comparison to classical molecular biological assays. In contrast, the up scaling as a prerequisite for the practical application of these novel bioelectronic monitoring systems is mostly only discussed theoretically. In this context, we developed a novel 384-multiwell microelectrode array (MMEA) based measurement system for the sensitive label-free real-time monitoring of neurodegenerative processes by impedance spectroscopy. With respect to the needs of productive screening systems for robust and reproducible measurements on high numbers of plates, we focused on reducing the critical contacting of more than 400 electrodes for a 384-MMEA. Therefore, we introduced an on top array of immersive counter electrodes that are individually addressed by a multiplexer and connected all measurement electrodes on the 384-MMEA to a single contact point. More strikingly, our novel approach provided a comparable signal stability and sensitivity similar to an array with integrated counter electrodes. Next, we optimized a SH-SY5Y cell based tauopathy model by introducing a novel 5-fold Tau mutation eliminating the need of artificial tauopathy induction. In combination with our novel 384-MMEA based measurement system, the concentration and time dependent neuroregenerative effect of the kinase inhibitor SRN-003-556 could be quantitatively monitored. Thus, our novel screening system could be a useful tool to identify and develop potential novel therapeutics in the field of Tau-related neurodegenerative diseases. Copyright © 2016. Published by Elsevier B.V.

Incubation under fluid dynamic conditions markedly improves the structural preservation in vitro of explanted skeletal muscles.

PubMed

Carton, Flavia; Calderan, Laura; Malatesta, Manuela

2017-11-28

Explanted organs and tissues represent suitable experimental systems mimicking the functional and structural complexity of the living organism, with positive ethical and economic impact on research activities. However, their preservation in culture is generally limited, thus hindering their application as experimental models for biomedical research. In the present study, we investigated the potential of an innovative fluid dynamic culture system to improve the structural preservation in vitro of explanted mouse skeletal muscles (soleus). We used light and transmission electron microscopy to compare the morphological features of muscles maintained either in multiwell plates under conventional conditions or in a bioreactor mimicking the flow of physiological fluids. Our results demonstrate that fluid dynamic conditions markedly slowed the progressive structural deterioration of the muscle tissue occurring during the permanence in the culture medium, prolonging the preservation of some organelles such as mitochondria up to 48 h.

Incubation under fluid dynamic conditions markedly improves the structural preservation in vitro of explanted skeletal muscles

PubMed Central

Carton, Flavia; Calderan, Laura; Malatesta, Manuela

2017-01-01

Explanted organs and tissues represent suitable experimental systems mimicking the functional and structural complexity of the living organism, with positive ethical and economic impact on research activities. However, their preservation in culture is generally limited, thus hindering their application as experimental models for biomedical research. In the present study, we investigated the potential of an innovative fluid dynamic culture system to improve the structural preservation in vitro of explanted mouse skeletal muscles (soleus). We used light and transmission electron microscopy to compare the morphological features of muscles maintained either in multiwell plates under conventional conditions or in a bioreactor mimicking the flow of physiological fluids. Our results demonstrate that fluid dynamic conditions markedly slowed the progressive structural deterioration of the muscle tissue occurring during the permanence in the culture medium, prolonging the preservation of some organelles such as mitochondria up to 48 h. PMID:29313601

Localization-based super-resolution imaging meets high-content screening.

PubMed

Beghin, Anne; Kechkar, Adel; Butler, Corey; Levet, Florian; Cabillic, Marine; Rossier, Olivier; Giannone, Gregory; Galland, Rémi; Choquet, Daniel; Sibarita, Jean-Baptiste

2017-12-01

Single-molecule localization microscopy techniques have proven to be essential tools for quantitatively monitoring biological processes at unprecedented spatial resolution. However, these techniques are very low throughput and are not yet compatible with fully automated, multiparametric cellular assays. This shortcoming is primarily due to the huge amount of data generated during imaging and the lack of software for automation and dedicated data mining. We describe an automated quantitative single-molecule-based super-resolution methodology that operates in standard multiwell plates and uses analysis based on high-content screening and data-mining software. The workflow is compatible with fixed- and live-cell imaging and allows extraction of quantitative data like fluorophore photophysics, protein clustering or dynamic behavior of biomolecules. We demonstrate that the method is compatible with high-content screening using 3D dSTORM and DNA-PAINT based super-resolution microscopy as well as single-particle tracking.

Growthcurver: an R package for obtaining interpretable metrics from microbial growth curves.

PubMed

Sprouffske, Kathleen; Wagner, Andreas

2016-04-19

Plate readers can measure the growth curves of many microbial strains in a high-throughput fashion. The hundreds of absorbance readings collected simultaneously for hundreds of samples create technical hurdles for data analysis. Growthcurver summarizes the growth characteristics of microbial growth curve experiments conducted in a plate reader. The data are fitted to a standard form of the logistic equation, and the parameters have clear interpretations on population-level characteristics, like doubling time, carrying capacity, and growth rate. Growthcurver is an easy-to-use R package available for installation from the Comprehensive R Archive Network (CRAN). The source code is available under the GNU General Public License and can be obtained from Github (Sprouffske K, Growthcurver sourcecode, 2016).

Comparative stain removal properties of four commercially available denture cleaning products: an in vitro study.

PubMed

Alam, M; Jagger, R; Vowles, R; Moran, J

2011-02-01

Formulations of commercially available denture cleaners vary widely. Unfortunately, comparative data to suggest which products are the most effective can become invalid as newer products are introduced or formulations are changed. The aim of this in vitro study was to measure the stain removal properties of four currently available denture cleaners. Stain was deposited on multi-well polystyrene saliva coated microplates using multiple chlorhexidine and tea solutions. Following drying, each stained well was exposed to a solution of denture cleaner, dried again and the amount of stain remaining measured using a microplate reader. The cleaning procedure was repeated with further multiple exposures of the wells to solutions of the denture cleaners. All denture cleaners removed stain better than water used as a control. At five cleaning cycles only one of the cleaners (Superdrug Cleaning Powder) had removed 100% of the stain. At 30 cycles three of the cleaners had removed 100% of the stain. All the commercial denture cleaners removed stain. Superdrug Cleaning Powder, which contains sodium percarbonate and sodium lauryl sulphate, was particularly effective. © 2010 John Wiley & Sons A/S.

Fabrication and optimisation of a fused filament 3D-printed microfluidic platform

NASA Astrophysics Data System (ADS)

Tothill, A. M.; Partridge, M.; James, S. W.; Tatam, R. P.

2017-03-01

A 3D-printed microfluidic device was designed and manufactured using a low cost (2000) consumer grade fusion deposition modelling (FDM) 3D printer. FDM printers are not typically used, or are capable, of producing the fine detailed structures required for microfluidic fabrication. However, in this work, the optical transparency of the device was improved through manufacture optimisation to such a point that optical colorimetric assays can be performed in a 50 µl device. A colorimetric enzymatic cascade assay was optimised using glucose oxidase and horseradish peroxidase for the oxidative coupling of aminoantipyrine and chromotropic acid to produce a blue quinoneimine dye with a broad absorbance peaking at 590 nm for the quantification of glucose in solution. For comparison the assay was run in standard 96 well plates with a commercial plate reader. The results show the accurate and reproducible quantification of 0-10 mM glucose solution using a 3D-printed microfluidic optical device with performance comparable to that of a plate reader assay.

Fluorescence lifetime plate reader: Resolution and precision meet high-throughput

PubMed Central

Petersen, Karl J.; Peterson, Kurt C.; Muretta, Joseph M.; Higgins, Sutton E.; Gillispie, Gregory D.; Thomas, David D.

2014-01-01

We describe a nanosecond time-resolved fluorescence spectrometer that acquires fluorescence decay waveforms from each well of a 384-well microplate in 3 min with signal-to-noise exceeding 400 using direct waveform recording. The instrument combines high-energy pulsed laser sources (5–10 kHz repetition rate) with a photomultiplier and high-speed digitizer (1 GHz) to record a fluorescence decay waveform after each pulse. Waveforms acquired from rhodamine or 5-((2-aminoethyl)amino) naphthalene-1-sulfonic acid dyes in a 384-well plate gave lifetime measurements 5- to 25-fold more precise than the simultaneous intensity measurements. Lifetimes as short as 0.04 ns were acquired by interleaving with an effective sample rate of 5 GHz. Lifetime measurements resolved mixtures of single-exponential dyes with better than 1% accuracy. The fluorescence lifetime plate reader enables multiple-well fluorescence lifetime measurements with an acquisition time of 0.5 s per well, suitable for high-throughput fluorescence lifetime screening applications. PMID:25430092

[Establishment of an in vitro screening model for steroid 5 alpha-reductase inhibitors with the microplate reader].

PubMed

Wu, Jian-Hui; Sun, Zu-Yue

2013-06-01

To establish an in vitro screening model for steroid 5 alpha-reductase inhibitors using the microplate reader. Steroid 5 alpha-reductase was obtained from the liver of female rats, an in vitro screening model for steroid 5 alpha-reductase inhibitors established using the 96-well plate and microplate reader after determination of the enzymatic activity, and the reliability of the model verified with the known 5 alpha-reductase inhibitors epristeride and finasteride. Added to the 96-well plate were the final concentrations of testosterone (0-40 micromol/L), NADPH (22 micromol/L), epristeride (0-60 nmol/L) or finasteride (0-60 nmol/ L) and steroid 5 alpha-reductase (20 microl), the total volume of each well adjusted to 200 microl with Tris-Hcl buffer. The 96-well plate was placed in the microplate reader, mixed and incubated at 37 degrees C, followed by detection of the A340nm value at 0 and 10 min and analysis of the data. The Km value of steroid 5 alpha-reductase was 3.794 micromol/L, with a Vmax of 0.271 micromol/(L. min). The Ki of epristeride was 148.2 nmol/L, with an IC50 of 31.5 nmol/L, and the enzymatic reaction kinetic curve suggested that epristeride was an uncompetitive enzyme inhibitor. The Ki of finasteride was 158. 8 nmol/L, with an IC50 of 13.6 nmol/L. The enzymatic reaction kinetic curve showed that both epristeride and finasteride were competitive enzyme inhibitors, similar to those reported in the published literature. A screening model was successfully established, which could rapidly and effectively screen steroid 5 alpha-reductase inhibitors in vitro.

Optimization of a multi-well array SERS chip

NASA Astrophysics Data System (ADS)

Abell, J. L.; Driskell, J. D.; Dluhy, R. A.; Tripp, R. A.; Zhao, Y.-P.

2009-05-01

SERS-active substrates are fabricated by oblique angle deposition and patterned by a polymer-molding technique to provide a uniform array for high throughput biosensing and multiplexing. Using a conventional SERS-active molecule, 1,2-Bis(4-pyridyl)ethylene (BPE), we show that this device provides a uniform Raman signal enhancement from well to well. The patterning technique employed in this study demonstrates a flexibility allowing for patterning control and customization, and performance optimization of the substrate. Avian influenza is analyzed to demonstrate the ability of this multi-well patterned SERS substrate for biosensing.

Matrix Rigidity Regulates Cancer Cell Growth and Cellular Phenotype

PubMed Central

Tilghman, Robert W.; Cowan, Catharine R.; Mih, Justin D.; Koryakina, Yulia; Gioeli, Daniel; Slack-Davis, Jill K.; Blackman, Brett R.; Tschumperlin, Daniel J.; Parsons, J. Thomas

2010-01-01

Background The mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness) of the microenvironment and how this response varies among cancer cell lines. Methodology/Principal Findings In this study we used a recently developed 96-well plate system that arrays extracellular matrix-conjugated polyacrylamide gels that increase in stiffness by at least 50-fold across the plate. This plate was used to determine how changes in the rigidity of the extracellular matrix modulate the biological properties of tumor cells. The cell lines tested fall into one of two categories based on their proliferation on substrates of differing stiffness: “rigidity dependent” (those which show an increase in cell growth as extracellular rigidity is increased), and “rigidity independent” (those which grow equally on both soft and stiff substrates). Cells which grew poorly on soft gels also showed decreased spreading and migration under these conditions. More importantly, seeding the cell lines into the lungs of nude mice revealed that the ability of cells to grow on soft gels in vitro correlated with their ability to grow in a soft tissue environment in vivo. The lung carcinoma line A549 responded to culture on soft gels by expressing the differentiated epithelial marker E-cadherin and decreasing the expression of the mesenchymal transcription factor Slug. Conclusions/Significance These observations suggest that the mechanical properties of the matrix environment play a significant role in regulating the proliferation and the morphological properties of cancer cells. Further, the multiwell format of the soft-plate assay is a useful and effective adjunct to established 3-dimensional cell culture models. PMID:20886123

Protocols and programs for high-throughput growth and aging phenotyping in yeast.

PubMed

Jung, Paul P; Christian, Nils; Kay, Daniel P; Skupin, Alexander; Linster, Carole L

2015-01-01

In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting "Colony Forming Units". To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens.

Acoustic auditing as a real-time, non-invasive quality control process for both source and assay plates.

PubMed

Olechno, Joseph; Ellson, Richard; Browning, Brent; Stearns, Richard; Mutz, Mitchell; Travis, Michael; Qureshi, Shehrzad; Shieh, Jean

2005-08-01

Acoustic auditing is a non-destructive, non-invasive technique to monitor the composition and volume of fluids in open or sealed microplates and storage tubes. When acoustic energy encounters an interface between two materials, some of the energy passes through the interface, while the remainder is reflected. Acoustic energy applied to the bottom of a multi-well plate or a storage tube is reflected by the fluid contents of the microplate or tube. The amplitude of these reflections or echoes correlates directly with properties of the fluid, including the speed of sound and the concentration of water in the fluid. Once the speed of sound in the solution is known from the analysis of these echoes, it is easy to determine the depth of liquid and, thereby, the volume by monitoring how long it takes for sound energy to reflect off the fluid meniscus. This technique is rapid (>100,000 samples per day), precise (<1% coefficient of variation for hydration measurements, <4% coefficient of variation for volume measurements), and robust. It does not require uncapping tubes or unsealing or unlidding microplates. The sound energy is extremely gentle and has no deleterious impact upon the fluid or compounds dissolved in it.

Automated Lab-on-a-Chip Technology for Fish Embryo Toxicity Tests Performed under Continuous Microperfusion (μFET).

PubMed

Zhu, Feng; Wigh, Adriana; Friedrich, Timo; Devaux, Alain; Bony, Sylvie; Nugegoda, Dayanthi; Kaslin, Jan; Wlodkowic, Donald

2015-12-15

The fish embryo toxicity (FET) biotest has gained popularity as one of the alternative approaches to acute fish toxicity tests in chemical hazard and risk assessment. Despite the importance and common acceptance of FET, it is still performed in multiwell plates and requires laborious and time-consuming manual manipulation of specimens and solutions. This work describes the design and validation of a microfluidic Lab-on-a-Chip technology for automation of the zebrafish embryo toxicity test common in aquatic ecotoxicology. The innovative device supports rapid loading and immobilization of large numbers of zebrafish embryos suspended in a continuous microfluidic perfusion as a means of toxicant delivery. Furthermore, we also present development of a customized mechatronic automation interface that includes a high-resolution USB microscope, LED cold light illumination, and miniaturized 3D printed pumping manifolds that were integrated to enable time-resolved in situ analysis of developing fish embryos. To investigate the applicability of the microfluidic FET (μFET) in toxicity testing, copper sulfate, phenol, ethanol, caffeine, nicotine, and dimethyl sulfoxide were tested as model chemical stressors. Results obtained on a chip-based system were compared with static protocols performed in microtiter plates. This work provides evidence that FET analysis performed under microperfusion opens a brand new alternative for inexpensive automation in aquatic ecotoxicology.

Robotic multi-well planar patch-clamp for native and primary mammalian cells

PubMed Central

Milligan, Carol J; Li, Jing; Sukumar, Piruthivi; Majeed, Yasser; Dallas, Mark L; English, Anne; Emery, Paul; Porter, Karen E; Smith, Andrew M; McFadzean, Ian; Beccano-Kelly, Dayne; Bahnasi, Yahya; Cheong, Alex; Naylor, Jacqueline; Zeng, Fanning; Liu, Xing; Gamper, Nikita; Jiang, Lin-Hua; Pearson, Hugh A; Peers, Chris; Robertson, Brian; Beech, David J

2009-01-01

Multi-well robotic planar patch-clamp has become common in drug development and safety programmes because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favoured method. Here we show the wider potential of the multi-well approach with the capability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints, and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by pre-programmed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 hr depending on the experimental design and yields 16-33 cell recordings. PMID:19197268

Reasonable Policies on Automated License Plate Readers Act

THOMAS, 113th Congress

Rep. Capuano, Michael E. [D-MA-7

2013-07-10

House - 09/13/2013 Referred to the Subcommittee on Crime, Terrorism, Homeland Security, and Investigations. (All Actions) Tracker: This bill has the status IntroducedHere are the steps for Status of Legislation:

7 CFR 353.9 - Standards for accreditation of non-government facilities to perform laboratory seed health...

Code of Federal Regulations, 2013 CFR

2013-01-01

... seed requires a stereo microscope. Visual examination of tissue requires a compound light microscope... equipment; fluorescent microscopes; plate readers; spectrophotometers; and the appropriate assay materials...

7 CFR 353.9 - Standards for accreditation of non-government facilities to perform laboratory seed health...

Code of Federal Regulations, 2011 CFR

2011-01-01

... seed requires a stereo microscope. Visual examination of tissue requires a compound light microscope... equipment; fluorescent microscopes; plate readers; spectrophotometers; and the appropriate assay materials...

7 CFR 353.9 - Standards for accreditation of non-government facilities to perform laboratory seed health...

Code of Federal Regulations, 2010 CFR

2010-01-01

... seed requires a stereo microscope. Visual examination of tissue requires a compound light microscope... equipment; fluorescent microscopes; plate readers; spectrophotometers; and the appropriate assay materials...

7 CFR 353.9 - Standards for accreditation of non-government facilities to perform laboratory seed health...

Code of Federal Regulations, 2014 CFR

2014-01-01

... seed requires a stereo microscope. Visual examination of tissue requires a compound light microscope... equipment; fluorescent microscopes; plate readers; spectrophotometers; and the appropriate assay materials...

Antibacterial and antifungal activities of different parts of Tribulus terrestris L. growing in Iraq

PubMed Central

Al-Bayati, Firas A.; Al-Mola, Hassan F.

2008-01-01

Antimicrobial activity of organic and aqueous extracts from fruits, leaves and roots of Tribulus terrestris L., an Iraqi medicinal plant used as urinary anti-infective in folk medicine, was examined against 11 species of pathogenic and non-pathogenic microorganisms: Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Corynebacterium diphtheriae, Escherichia coli, Proteus vulgaris, Serratia marcescens, Salmonella typhimurium, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida albicans using microdilution method in 96 multiwell microtiter plates. All the extracts from the different parts of the plant showed antimicrobial activity against most tested microorganisms. The most active extract against both Gram-negative and Gram-positive bacteria was ethanol extract from the fruits with a minimal inhibitory concentration (MIC) value of 0.15 mg/ml against B. subtilis, B. cereus, P. vulgaris and C. diphtheriae. In addition, the same extract from the same plant part demonstrated the strongest antifungal activity against C. albicans with an MIC value of 0.15 mg/ml. PMID:18257138

Liquid-handling Lego robots and experiments for STEM education and research

PubMed Central

Gerber, Lukas C.; Calasanz-Kaiser, Agnes; Hyman, Luke; Voitiuk, Kateryna; Patil, Uday

2017-01-01

Liquid-handling robots have many applications for biotechnology and the life sciences, with increasing impact on everyday life. While playful robotics such as Lego Mindstorms significantly support education initiatives in mechatronics and programming, equivalent connections to the life sciences do not currently exist. To close this gap, we developed Lego-based pipetting robots that reliably handle liquid volumes from 1 ml down to the sub-μl range and that operate on standard laboratory plasticware, such as cuvettes and multiwell plates. These robots can support a range of science and chemistry experiments for education and even research. Using standard, low-cost household consumables, programming pipetting routines, and modifying robot designs, we enabled a rich activity space. We successfully tested these activities in afterschool settings with elementary, middle, and high school students. The simplest robot can be directly built from the widely used Lego Education EV3 core set alone, and this publication includes building and experiment instructions to set the stage for dissemination and further development in education and research. PMID:28323828

Liquid-handling Lego robots and experiments for STEM education and research.

PubMed

Gerber, Lukas C; Calasanz-Kaiser, Agnes; Hyman, Luke; Voitiuk, Kateryna; Patil, Uday; Riedel-Kruse, Ingmar H

2017-03-01

Liquid-handling robots have many applications for biotechnology and the life sciences, with increasing impact on everyday life. While playful robotics such as Lego Mindstorms significantly support education initiatives in mechatronics and programming, equivalent connections to the life sciences do not currently exist. To close this gap, we developed Lego-based pipetting robots that reliably handle liquid volumes from 1 ml down to the sub-μl range and that operate on standard laboratory plasticware, such as cuvettes and multiwell plates. These robots can support a range of science and chemistry experiments for education and even research. Using standard, low-cost household consumables, programming pipetting routines, and modifying robot designs, we enabled a rich activity space. We successfully tested these activities in afterschool settings with elementary, middle, and high school students. The simplest robot can be directly built from the widely used Lego Education EV3 core set alone, and this publication includes building and experiment instructions to set the stage for dissemination and further development in education and research.

A model for predicting field-directed particle transport in the magnetofection process.

PubMed

Furlani, Edward P; Xue, Xiaozheng

2012-05-01

To analyze the magnetofection process in which magnetic carrier particles with surface-bound gene vectors are attracted to target cells for transfection using an external magnetic field and to obtain a fundamental understanding of the impact of key factors such as particle size and field strength on the gene delivery process. A numerical model is used to study the field-directed transport of the carrier particle-gene vector complex to target cells in a conventional multiwell culture plate system. The model predicts the transport dynamics and the distribution of particle accumulation at the target cells. The impact of several factors that strongly influence gene vector delivery is assessed including the properties of the carrier particles, the strength of the field source, and its extent and proximity relative to the target cells. The study demonstrates that modeling can be used to predict and optimize gene vector delivery in the magnetofection process for novel and conventional in vitro systems.

High-throughput investigation of single and binary protein adsorption isotherms in anion exchange chromatography employing multivariate analysis.

PubMed

Field, Nicholas; Konstantinidis, Spyridon; Velayudhan, Ajoy

2017-08-11

The combination of multi-well plates and automated liquid handling is well suited to the rapid measurement of the adsorption isotherms of proteins. Here, single and binary adsorption isotherms are reported for BSA, ovalbumin and conalbumin on a strong anion exchanger over a range of pH and salt levels. The impact of the main experimental factors at play on the accuracy and precision of the adsorbed protein concentrations is quantified theoretically and experimentally. In addition to the standard measurement of liquid concentrations before and after adsorption, the amounts eluted from the wells are measured directly. This additional measurement corroborates the calculation based on liquid concentration data, and improves precision especially under conditions of weak or moderate interaction strength. The traditional measurement of multicomponent isotherms is limited by the speed of HPLC analysis; this analytical bottleneck is alleviated by careful multivariate analysis of UV spectra. Copyright © 2017. Published by Elsevier B.V.

High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells.

PubMed

Carlson-Stevermer, Jared; Goedland, Madelyn; Steyer, Benjamin; Movaghar, Arezoo; Lou, Meng; Kohlenberg, Lucille; Prestil, Ryan; Saha, Krishanu

2016-01-12

CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Development and Application of a High Throughput Protein Unfolding Kinetic Assay

PubMed Central

Wang, Qiang; Waterhouse, Nicklas; Feyijinmi, Olusegun; Dominguez, Matthew J.; Martinez, Lisa M.; Sharp, Zoey; Service, Rachel; Bothe, Jameson R.; Stollar, Elliott J.

2016-01-01

The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses. PMID:26745729

Unmixing of fluorescence spectra to resolve quantitative time-series measurements of gene expression in plate readers.

PubMed

Lichten, Catherine A; White, Rachel; Clark, Ivan B N; Swain, Peter S

2014-02-03

To connect gene expression with cellular physiology, we need to follow levels of proteins over time. Experiments typically use variants of Green Fluorescent Protein (GFP), and time-series measurements require specialist expertise if single cells are to be followed. Fluorescence plate readers, however, a standard in many laboratories, can in principle provide similar data, albeit at a mean, population level. Nevertheless, extracting the average fluorescence per cell is challenging because autofluorescence can be substantial. Here we propose a general method for correcting plate reader measurements of fluorescent proteins that uses spectral unmixing and determines both the fluorescence per cell and the errors on that fluorescence. Combined with strain collections, such as the GFP fusion collection for budding yeast, our methodology allows quantitative measurements of protein levels of up to hundreds of genes and therefore provides complementary data to high throughput studies of transcription. We illustrate the method by following the induction of the GAL genes in Saccharomyces cerevisiae for over 20 hours in different sugars and argue that the order of appearance of the Leloir enzymes may be to reduce build-up of the toxic intermediate galactose-1-phosphate. Further, we quantify protein levels of over 40 genes, again over 20 hours, after cells experience a change in carbon source (from glycerol to glucose). Our methodology is sensitive, scalable, and should be applicable to other organisms. By allowing quantitative measurements on a per cell basis over tens of hours and over hundreds of genes, it should increase our understanding of the dynamic changes that drive cellular behaviour.

Unmixing of fluorescence spectra to resolve quantitative time-series measurements of gene expression in plate readers

PubMed Central

2014-01-01

Background To connect gene expression with cellular physiology, we need to follow levels of proteins over time. Experiments typically use variants of Green Fluorescent Protein (GFP), and time-series measurements require specialist expertise if single cells are to be followed. Fluorescence plate readers, however, a standard in many laboratories, can in principle provide similar data, albeit at a mean, population level. Nevertheless, extracting the average fluorescence per cell is challenging because autofluorescence can be substantial. Results Here we propose a general method for correcting plate reader measurements of fluorescent proteins that uses spectral unmixing and determines both the fluorescence per cell and the errors on that fluorescence. Combined with strain collections, such as the GFP fusion collection for budding yeast, our methodology allows quantitative measurements of protein levels of up to hundreds of genes and therefore provides complementary data to high throughput studies of transcription. We illustrate the method by following the induction of the GAL genes in Saccharomyces cerevisiae for over 20 hours in different sugars and argue that the order of appearance of the Leloir enzymes may be to reduce build-up of the toxic intermediate galactose-1-phosphate. Further, we quantify protein levels of over 40 genes, again over 20 hours, after cells experience a change in carbon source (from glycerol to glucose). Conclusions Our methodology is sensitive, scalable, and should be applicable to other organisms. By allowing quantitative measurements on a per cell basis over tens of hours and over hundreds of genes, it should increase our understanding of the dynamic changes that drive cellular behaviour. PMID:24495318

A multiwell format assay for heparanase.

PubMed

Behzad, Farhad; Brenchley, Paul E C

2003-09-15

This assay employs a biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate that is covalently linked to the surface of 96-well immunoassay plates. The ratio of biotin:HSGAG and the coating concentration of substrate bound to the wells have been optimized and allow removal of biotin HSGAG within 60 min of incubation at 37 degrees C in assay buffer with a standard dilution of bacterial heparitinase or platelet heparanase. Loss of biotin signal from the well surface is detected on incubation with peroxidase-streptavidin followed by color development using 3,3',5,5'-tetramethylbenzidine as the peroxidase substrate. The new assay allows specific detection of heparanase activity in multiple samples in a total time of 3 h including a 1-h substrate digestion step and is a significant improvement with regard to sensitivity, specificity, and ease of handling of multiple samples compared to other described assays. Heparanase specifically degrades the biotinylated HSGAG substrate, when used with an optimized assay buffer. A range of enzymes including collagenase, trypsin, plasmin, pepsin, chondroitinases, hyaluronidase, and neuraminidase show no effect on the substrate under optimized assay conditions. The covalent linkage of the substrate to the well prevents leaching of substrate and allows preparation and long-term storage of substrate-coated plates. The assay can be used to detect heparanase levels in clinical samples and cell culture supernatants and is ideal as a screening method for antagonists of enzyme activity.

Characterization of ToxCast Phase II compounds disruption of ...

EPA Pesticide Factsheets

The development of multi-well microelectrode array (mwMEA) systems has increased in vitro screening throughput making them an effective method to screen and prioritize large sets of compounds for potential neurotoxicity. In the present experiments, a multiplexed approach was used to determine compound effects on both neural function and cell health in primary cortical networks grown on mwMEA plates following exposure to ~1100 compounds from EPA’s Phase II ToxCast libraries. On DIV 13, baseline activity (40 min) was recorded prior to exposure to each compound at 40 µM. DMSO and the GABAA antagonist bicuculline (BIC) were included as controls on each mwMEA plate. Changes in spontaneous network activity (mean firing rate; MFR) and cell viability (lactate dehydrogenase; LDH and CellTiter Blue; CTB) were assessed within the same well following compound exposure. Activity calls (“hits”) were established using the 90th and 20th percentiles of the compound-induced change in MFR (medians of triplicates) across all tested compounds; compounds above (top 10% of compounds increasing MFR), and below (bottom 20% of compounds decreasing MFR) these thresholds, respectively were considered hits. MFR was altered beyond one of these thresholds by 322 compounds. Four compound categories accounted for 66% of the hits, including: insecticides (e.g. abamectin, lindane, prallethrin), pharmaceuticals (e.g. haloperidol, reserpine), fungicides (e.g. hexaconazole, fenamidone), and h

High-throughput microfluidic mixing and multiparametric cell sorting for bioactive compound screening.

PubMed

Young, Susan M; Curry, Mark S; Ransom, John T; Ballesteros, Juan A; Prossnitz, Eric R; Sklar, Larry A; Edwards, Bruce S

2004-03-01

HyperCyt, an automated sample handling system for flow cytometry that uses air bubbles to separate samples sequentially introduced from multiwell plates by an autosampler. In a previously documented HyperCyt configuration, air bubble separated compounds in one sample line and a continuous stream of cells in another are mixed in-line for serial flow cytometric cell response analysis. To expand capabilities for high-throughput bioactive compound screening, the authors investigated using this system configuration in combination with automated cell sorting. Peptide ligands were sampled from a 96-well plate, mixed in-line with fluo-4-loaded, formyl peptide receptor-transfected U937 cells, and screened at a rate of 3 peptide reactions per minute with approximately 10,000 cells analyzed per reaction. Cell Ca(2+) responses were detected to as little as 10(-11) M peptide with no detectable carryover between samples at up to 10(-7) M peptide. After expansion in culture, cells sort-purified from the 10% highest responders exhibited enhanced sensitivity and more sustained responses to peptide. Thus, a highly responsive cell subset was isolated under high-throughput mixing and sorting conditions in which response detection capability spanned a 1000-fold range of peptide concentration. With single-cell readout systems for protein expression libraries, this technology offers the promise of screening millions of discrete compound interactions per day.

The use of multiwell culture plates in the duckweed toxicity test-a case study on Zn nanoparticles.

PubMed

Kalčíková, Gabriela; Marolt, Gregor; Kokalj, Anita Jemec; Gotvajn, Andreja Žgajnar

2018-06-11

Extensive production of nanomaterials of various properties needs to be coupled with rapid toxicity testing in order to provide information about their potential risks to the environment and human health. Miniaturization of toxicity tests may accelerate economical testing of nanomaterials, but is not a common practice. We describe a case study to miniaturize a commonly used toxicity test with plant duckweed Lemna minor. 6-well, 12-well and 24-well culture plates were used to assess their potential use for the duckweed toxicity test with potassium chloride as reference material. The results were compared to the standard test design using 100 mL glass beakers. The comparison showed that the best agreement was with the 6-well vessels. This set-up was further used for toxicity testing of zinc oxide nanoparticles (ZnO NP) and zinc chlorides. Zinc was not adsorbed onto either glass or plastic walls of the miniaturized system. We assume that in both vessels a fast agglomeration and settling of ZnO NP took place. Linear regression and statistical testing indicated a good correlation between the toxicity results obtained in the standard test and miniaturized 6-well vessels. The miniaturization of the test system for assessing the biological effect of nanomaterials on Lemna minor could become an appropriate alternative to the traditionally used high volume vessels. Copyright © 2018. Published by Elsevier B.V.

Thousand-fold fluorescent signal amplification for mHealth diagnostics

PubMed Central

Balsam, Joshua; Rasooly, Reuven; Bruck, Hugh Alan; Rasooly, Avraham

2013-01-01

The low sensitivity of Mobile Health (mHealth) optical detectors, such as those found on mobile phones, is a limiting factor for many mHealth clinical applications. To improve sensitivity, we have combined two approaches for optical signal amplification: (1) a computational approach based on an image stacking algorithm to decrease the image noise and enhance weak signals, and (2) an optical signal amplifier utilizing a capillary tube array. These approaches were used in a detection system which includes a multi-wavelength LEDs capable of exciting many fluorophores in multiple wavelengths, a mobile phone or a webcam as a detector, and capillary tube array configured with 36 capillary tubes for signal enhancement. The capillary array enables a ~100X increase in signal sensitivity for fluorescein, reducing the limit of detection (LOD) for mobile phones and webcams from 1000 nM to 10 nM. Computational image stacking enables another ~10X increase in signal sensitivity, further reducing the LOD for webcam from 10 nM to 1 nM. To demonstrate the feasibility of the device for the detection of disease-related biomarkers, Adenovirus DNA labeled with SYBR Green or fluorescein was analyzed by both our capillary array and a commercial plate reader. The LOD for the capillary array was 5ug/mL, and that of the plate reader was 1 ug/mL. Similar results were obtained using DNA stained with fluorescein. The combination of the two signal amplification approaches enables a ~1000X increase in LOD for the webcam platform. This brings it into the range of a conventional plate reader while using a smaller sample volume (10ul) than the plate reader requires (100 ul). This suggests that such a device could be suitable for biosensing applications where up to 10 fold smaller sample sizes are needed. The simple optical configuration for mHealth described in this paper employing the combined capillary and image processing signal amplification is capable of measuring weak fluorescent signals without the need of dedicated laboratories. It has the potential to be used to increase sensitivity of other optically based mHealth technologies, and may increase mHealth’s clinical utility, especially for telemedicine and for resource-poor settings and global health applications. PMID:23928092

Thousand-fold fluorescent signal amplification for mHealth diagnostics.

PubMed

Balsam, Joshua; Rasooly, Reuven; Bruck, Hugh Alan; Rasooly, Avraham

2014-01-15

The low sensitivity of Mobile Health (mHealth) optical detectors, such as those found on mobile phones, is a limiting factor for many mHealth clinical applications. To improve sensitivity, we have combined two approaches for optical signal amplification: (1) a computational approach based on an image stacking algorithm to decrease the image noise and enhance weak signals, and (2) an optical signal amplifier utilizing a capillary tube array. These approaches were used in a detection system which includes multi-wavelength LEDs capable of exciting many fluorophores in multiple wavelengths, a mobile phone or a webcam as a detector, and capillary tube array configured with 36 capillary tubes for signal enhancement. The capillary array enables a ~100× increase in signal sensitivity for fluorescein, reducing the limit of detection (LOD) for mobile phones and webcams from 1000 nM to 10nM. Computational image stacking enables another ~10× increase in signal sensitivity, further reducing the LOD for webcam from 10nM to 1 nM. To demonstrate the feasibility of the device for the detection of disease-related biomarkers, adenovirus DNA labeled with SYBR green or fluorescein was analyzed by both our capillary array and a commercial plate reader. The LOD for the capillary array was 5 ug/mL, and that of the plate reader was 1 ug/mL. Similar results were obtained using DNA stained with fluorescein. The combination of the two signal amplification approaches enables a ~1000× increase in LOD for the webcam platform. This brings it into the range of a conventional plate reader while using a smaller sample volume (10 ul) than the plate reader requires (100 ul). This suggests that such a device could be suitable for biosensing applications where up to 10 fold smaller sample sizes are needed. The simple optical configuration for mHealth described in this paper employing the combined capillary and image processing signal amplification is capable of measuring weak fluorescent signals without the need of dedicated laboratories. It has the potential to be used to increase sensitivity of other optically based mHealth technologies, and may increase mHealth's clinical utility, especially for telemedicine and for resource-poor settings and global health applications. Published by Elsevier B.V.

Evaluation of the light scattering and the turbidity microtiter plate-based methods for the detection of the excipient-mediated drug precipitation inhibition.

PubMed

Petruševska, Marija; Urleb, Uroš; Peternel, Luka

2013-11-01

The excipient-mediated precipitation inhibition is classically determined by the quantification of the dissolved compound in the solution. In this study, two alternative approaches were evaluated, one is the light scattering (nephelometer) and other is the turbidity (plate reader) microtiter plate-based methods which are based on the quantification of the compound precipitate. Following the optimization of the nephelometer settings (beam focus, laser gain) and the experimental conditions, the screening of 23 excipients on the precipitation inhibition of poorly soluble fenofibrate and dipyridamole was performed. The light scattering method resulted in excellent correlation (r>0.91) between the calculated precipitation inhibitor parameters (PIPs) and the precipitation inhibition index (PI(classical)) obtained by the classical approach for fenofibrate and dipyridamole. Among the evaluated PIPs AUC100 (nephelometer) resulted in only four false positives and lack of false negatives. In the case of the turbidity-based method a good correlation of the PI(classical) was obtained for the PIP maximal optical density (OD(max), r=0.91), however, only for fenofibrate. In the case of the OD(max) (plate reader) five false positives and two false negatives were identified. In conclusion, the light scattering-based method outperformed the turbidity-based one and could be reliably used for identification of novel precipitation inhibitors. Copyright © 2013 Elsevier B.V. All rights reserved.

New design of a passive type RADFET reader for enhanced sensitivity

NASA Astrophysics Data System (ADS)

Lee, Dae-Hee

2016-07-01

We present a new design of a passive type RADFET reader with enhanced radiation sensitivity. Using a electostatic plate, we have applied a static electric field to the gate voltage, which impacts a positive biasing on the p-type MOSFET. The resultant effect shows that 1.8 times of radiation sensitivity increased when we measured the threshold voltage shift of the RADFET exposed to 30 krad irradiation. We discuss further about the characteristic changes of a RADFET according to the positive biasing on the gate voltage.

A Comprehensive Numerical Model for Simulating Fluid Transport in Nanopores

PubMed Central

Zhang, Yuan; Yu, Wei; Sepehrnoori, Kamy; Di, Yuan

2017-01-01

Since a large amount of nanopores exist in tight oil reservoirs, fluid transport in nanopores is complex due to large capillary pressure. Recent studies only focus on the effect of nanopore confinement on single-well performance with simple planar fractures in tight oil reservoirs. Its impacts on multi-well performance with complex fracture geometries have not been reported. In this study, a numerical model was developed to investigate the effect of confined phase behavior on cumulative oil and gas production of four horizontal wells with different fracture geometries. Its pore sizes were divided into five regions based on nanopore size distribution. Then, fluid properties were evaluated under different levels of capillary pressure using Peng-Robinson equation of state. Afterwards, an efficient approach of Embedded Discrete Fracture Model (EDFM) was applied to explicitly model hydraulic and natural fractures in the reservoirs. Finally, three fracture geometries, i.e. non-planar hydraulic fractures, non-planar hydraulic fractures with one set natural fractures, and non-planar hydraulic fractures with two sets natural fractures, are evaluated. The multi-well performance with confined phase behavior is analyzed with permeabilities of 0.01 md and 0.1 md. This work improves the analysis of capillarity effect on multi-well performance with complex fracture geometries in tight oil reservoirs. PMID:28091599

A Comprehensive Numerical Model for Simulating Fluid Transport in Nanopores

NASA Astrophysics Data System (ADS)

Zhang, Yuan; Yu, Wei; Sepehrnoori, Kamy; di, Yuan

2017-01-01

Since a large amount of nanopores exist in tight oil reservoirs, fluid transport in nanopores is complex due to large capillary pressure. Recent studies only focus on the effect of nanopore confinement on single-well performance with simple planar fractures in tight oil reservoirs. Its impacts on multi-well performance with complex fracture geometries have not been reported. In this study, a numerical model was developed to investigate the effect of confined phase behavior on cumulative oil and gas production of four horizontal wells with different fracture geometries. Its pore sizes were divided into five regions based on nanopore size distribution. Then, fluid properties were evaluated under different levels of capillary pressure using Peng-Robinson equation of state. Afterwards, an efficient approach of Embedded Discrete Fracture Model (EDFM) was applied to explicitly model hydraulic and natural fractures in the reservoirs. Finally, three fracture geometries, i.e. non-planar hydraulic fractures, non-planar hydraulic fractures with one set natural fractures, and non-planar hydraulic fractures with two sets natural fractures, are evaluated. The multi-well performance with confined phase behavior is analyzed with permeabilities of 0.01 md and 0.1 md. This work improves the analysis of capillarity effect on multi-well performance with complex fracture geometries in tight oil reservoirs.

Characterizing Microbe-Environment Interactions Through Experimental Evolution: The Autonomous Adaptive Directed Evolution Chamber

NASA Astrophysics Data System (ADS)

Ibanez, C. R.; Blaich, J.; Owyang, S.; Storrs, A.; Moffet, A.; Wong, N.; Zhou, J.; Gentry, D.

2015-12-01

We are developing a laboratory system for studying micro- to meso-scale interactions between microorganisms and their physicochemical environments. The Autonomous Adaptive Directed Evolution Chamber (AADEC) cultures microorganisms in controlled,small-scale geochemical environments. It observes corresponding microbial interactions to these environments and has the ability to adjust thermal, chemical, and other parameters in real time in response to these interactions. In addition to the sensed data, the system allows the generation of time-resolved ecological, genomic, etc. samples on the order of microbial generations. The AADEC currently houses cultures in liquid media and controls UVC radiation, heat exposure, and nutrient supply. In a proof-of-concept experimental evolution application, it can increase UVC radiation resistance of Escherichia coli cultures by iteratively exposing them to UVC and allowing the surviving cells to regrow. A baseline characterization generated a million fold resistance increase. This demonstration uses a single-well growth chamber prototype, but it was limited by scalability. We have expanded upon this system by implementing a microwell plate compatible fluidics system and sensor housing. This microwell plate system increases the diversity of microbial interactions seen in response to the geochemical environments generated by the system, allowing greater control over individual cultures' environments and detection of rarer events. The custom microfluidic card matches the footprint of a standard microwell plate. This card enables controllable fluid flow between wells and introduces multiple separate exposure and sensor chambers, increasing the variety of sensors compatible with the system. This gives the device control over scale and the interconnectedness of environments within the system. The increased controllability of the multiwell system provides a platform for implementing machine learning algorithms that will autonomously adjust geochemical environmental parameters.

Poster — Thur Eve — 02: Measurement of CT radiation profile width using Fuji CR imaging plate raw data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bjarnason, T A; Department of Radiology, University of British Columbia, Vancouver; Yang, C J

2014-08-15

Measuring the CT collimation width and assessing the shape of the overall profile is a relatively straightforward quality control (QC) measure that impacts both image quality and patient dose, and is often required at acceptance and routine testing. Most CT facilities have access to computed radiography (CR) systems, so performing CT collimation profile assessments using CR plates requires no additional equipment. Previous studies have shown how to effectively use CR plates to measure the radiation profile width. However, a major limitation of the previous work is that the full dynamic range of CR detector plates are not used, since themore » CR processing technology reduces the dynamic range of the DICOM output to 2{sup 10}, requiring the sensitivity and latitude settings of CR reader to be adjusted to prevent clipping of the CT profile data. Such adjustments to CR readers unnecessarily complicate the QC procedure. These clipping artefacts hinder the ability to accurately assess CT collimation width because the full-width at half maximum value of the penumbras are not properly determined if the maximum dose of the profile is not available. Furthermore, any inconsistencies in the radiation profile shape are lost if the profile plateau is clipped off. In this work we developed an opensource Matlab script for straightforward CT profile width measurements using raw CR data that also allows assessment of the profile shape without clipping, and applied this approach during CT QC.« less

Simultaneous and iterative weighted regression analysis of toxicity tests using a microplate reader.

PubMed

Galgani, F; Cadiou, Y; Gilbert, F

1992-04-01

A system is described for determination of LC50 or IC50 by an iterative process based on data obtained from a plate reader using a marine unicellular alga as a target species. The esterase activity of Tetraselmis suesica on fluorescein diacetate as a substrate was measured using a fluorescence titerplate. Simultaneous analysis of results was performed using an iterative process adopting the sigmoid function Y = y/1 (dose of toxicant/IC50)slope for dose-response relationships. IC50 (+/- SEM) was estimated (P less than 0.05). An application with phosalone as a toxicant is presented.

Material for surface-enhanced Raman spectroscopy, and SER sensors and method for preparing same

NASA Technical Reports Server (NTRS)

Farquharson, Stuart (Inventor); Nelson, Chad (Inventor); Lee, Yuan-Hsiang (Inventor)

2003-01-01

Metal-doped sol-gel materials, suitable for use as sensors for surface-enhanced Raman spectroscopic analysis for trace chemical detection, are produced by effecting gelation and solvent removal of a doped sol-gel under mild temperature conditions. At least in certain instances reaction and drying will desirably be effected in an oxygen-starved environment. The metal of the sol-gel material functions, when irradiated, to produce a plasmon field for interaction with molecules of an analyte in contact therewith, increasing by orders of magnitude Raman photons that are generate by excitation radiation, and the method allows matching of the metal and metal particle size to a wavelength of light (or incident radiation, e.g., laser radiation) to generate surface plasmons. The porosity of the sol-gel material dramatically increases the surface area, and thereby the amount of metal exposed for analyte interaction. The sensors provided may be in the form of glass vials, fiber optics, multi-well micro-sample plates, etc., having surface coatings of the doped sol-gel material, to provide sampling systems for use in a Raman instrument.

Radiation dosimetry in cell biology: comparison of calculated and measured absorbed dose for a range of culture vessels and clinical beam qualities.

PubMed

Claridge Mackonis, Elizabeth; Hammond, Lauren; Esteves, Ana I S; Suchowerska, Natalka

2018-02-01

Cell culture studies are frequently used to evaluate the effects of cancer treatments such as radiotherapy, hormone therapy, chemotherapy, nanoparticle enhancement, and to determine any synergies between the treatments. To achieve valid results, the absorbed dose of each therapy needs to be well known and controlled. In this study, we aim to determine the uncertainty associated with radiation exposure in different experimental conditions. We have performed an in-depth evaluation of the absorbed dose and dose distribution that would be delivered to a cell sample when cultivated in a number of the more popular designs of culture vessels. We focus on exposure to two beam types: a kilovoltage x-ray beam and a megavoltage photon beam, both of which are routinely used to treat cancer patients in the clinical environment. Our results identify large variations of up to 16% in the absorbed dose across multi-well culture plates, which if ignored in radiobiological experiments, have the potential to lead to erroneous conclusions.

Non-contact method for directing electrotaxis

NASA Astrophysics Data System (ADS)

Ahirwar, Dinesh K.; Nasser, Mohd W.; Jones, Travis H.; Sequin, Emily K.; West, Joseph D.; Henthorne, Timothy L.; Javor, Joshua; Kaushik, Aniruddha M.; Ganju, Ramesh K.; Subramaniam, Vish V.

2015-06-01

We present a method to induce electric fields and drive electrotaxis (galvanotaxis) without the need for electrodes to be in contact with the media containing the cell cultures. We report experimental results using a modification of the transmembrane assay, demonstrating the hindrance of migration of breast cancer cells (SCP2) when an induced a.c. electric field is present in the appropriate direction (i.e. in the direction of migration). Of significance is that migration of these cells is hindered at electric field strengths many orders of magnitude (5 to 6) below those previously reported for d.c. electrotaxis, and even in the presence of a chemokine (SDF-1α) or a growth factor (EGF). Induced a.c. electric fields applied in the direction of migration are also shown to hinder motility of non-transformed human mammary epithelial cells (MCF10A) in the presence of the growth factor EGF. In addition, we also show how our method can be applied to other cell migration assays (scratch assay), and by changing the coil design and holder, that it is also compatible with commercially available multi-well culture plates.

Control of proliferation rate of N27 dopaminergic neurons using Transcranial Magnetic Stimulation orientation

NASA Astrophysics Data System (ADS)

Meng, Yiwen; Hadimani, Ravi; Anantharam, Vellareddy; Kanthasamy, Anumantha; Jiles, David

2015-03-01

Transcranial magnetic stimulation (TMS) has been used to investigate possible treatments for a variety of neurological disorders. However, the effect that magnetic fields have on neurons has not been well documented in the literature. We have investigated the effect of different orientation of magnetic field generated by TMS coils with a monophasic stimulator on the proliferation rate of N27 neuronal cells cultured in flasks and multi-well plates. The proliferation rate of neurons would increase by exposed horizontally adherent N27 cells to a magnetic field pointing upward through the neuronal proliferation layer compared with the control group. On the other hand, proliferation rate would decrease in cells exposed to a magnetic field pointing downward through the neuronal growth layer compared with the control group. We confirmed results obtained from the Trypan-blue and automatic cell counting methods with those from the CyQuant and MTS cell viability assays. Our findings could have important implications for the preclinical development of TMS treatments of neurological disorders and represents a new method to control the proliferation rate of neuronal cells.

Near-infrared fluorescence image quality test methods for standardized performance evaluation

NASA Astrophysics Data System (ADS)

Kanniyappan, Udayakumar; Wang, Bohan; Yang, Charles; Ghassemi, Pejhman; Wang, Quanzeng; Chen, Yu; Pfefer, Joshua

2017-03-01

Near-infrared fluorescence (NIRF) imaging has gained much attention as a clinical method for enhancing visualization of cancers, perfusion and biological structures in surgical applications where a fluorescent dye is monitored by an imaging system. In order to address the emerging need for standardization of this innovative technology, it is necessary to develop and validate test methods suitable for objective, quantitative assessment of device performance. Towards this goal, we develop target-based test methods and investigate best practices for key NIRF imaging system performance characteristics including spatial resolution, depth of field and sensitivity. Characterization of fluorescence properties was performed by generating excitation-emission matrix properties of indocyanine green and quantum dots in biological solutions and matrix materials. A turbid, fluorophore-doped target was used, along with a resolution target for assessing image sharpness. Multi-well plates filled with either liquid or solid targets were generated to explore best practices for evaluating detection sensitivity. Overall, our results demonstrate the utility of objective, quantitative, target-based testing approaches as well as the need to consider a wide range of factors in establishing standardized approaches for NIRF imaging system performance.

The effects of laser immunotherapy on cancer cell migration

NASA Astrophysics Data System (ADS)

Bahavar, Cody F.; Zhou, Feifan; Hasanjee, Aamr M.; Layton, Elivia; Lam, Anh; Chen, Wei R.; Vaughan, Melville B.

2016-03-01

Laser immunotherapy (LIT) uses laser irradiation and immunological stimulation to target all types of metastases and creates a long-term tumor resistance. Glycated chitosan (GC) is the immunological stimulant used in LIT. Interestingly, GC can act as a surfactant for single-walled carbon nanotubes (SWNTs) to immunologically modify SWNTs. SWNT-GC retains the optical properties of SWNTs and the immunological functions of GC to help increase the selectivity of the laser and create a more optimal immune response. One essential aspect of understanding this immune response is knowing how laser irradiation affects cancer cells' ability to metastasize. In this experiment, a cell migration assay was performed. A 2mm circular elastomer plugs were placed at the bottom of multi-well dishes. Pre-cancerous keratinocytes, different tumor cells, and fibroblasts were then plated separately in treated wells. Once the cells reached 100% confluence, they were irradiated by either a 980nm or 805nm wavelength laser. The goal was to determine the effects of laser irradiation and immunological stimulation on cancer cell migration in vitro, paying the way to understand the mechanism of LIT in treating metastatic tumors in cancer patients.

Development of a spontaneously active dorsal root ganglia assay using multiwell multielectrode arrays

PubMed Central

Newberry, Kim; Wang, Shuya; Hoque, Nina; Kiss, Laszlo; Ahlijanian, Michael K.; Herrington, James

2016-01-01

In vitro phenotypic assays of sensory neuron activity are important tools for identifying potential analgesic compounds. These assays are typically characterized by hyperexcitable and/or abnormally, spontaneously active cells. Whereas manual electrophysiology experiments provide high-resolution biophysical data to characterize both in vitro models and potential therapeutic modalities (e.g., action potential characteristics, the role of specific ion channels, and receptors), these techniques are hampered by their low throughput. We have established a spontaneously active dorsal root ganglia (DRG) platform using multiwell multielectrode arrays (MEAs) that greatly increase the ability to evaluate the effects of multiple compounds and conditions on DRG excitability within the context of a cellular network. We show that spontaneous DRG firing can be attenuated with selective Na+ and Ca2+ channel blockers, as well as enhanced with K+ channel blockers. In addition, spontaneous activity can be augmented with both the transient receptor potential cation channel subfamily V member 1 agonist capsaicin and the peptide bradykinin and completely blocked with neurokinin receptor antagonists. Finally, we validated the use of this assay by demonstrating that commonly used neuropathic pain therapeutics suppress DRG spontaneous activity. Overall, we have optimized primary rat DRG cells on a multiwell MEA platform to generate and characterize spontaneously active cultures that have the potential to be used as an in vitro phenotypic assay to evaluate potential therapeutics in rodent models of pain. PMID:27052585

Inter-operator variation in ELISPOT analysis of measles virus-specific IFN-gamma-secreting T cells.

PubMed

Ryan, J E; Ovsyannikova, I G; Dhiman, N; Pinsky, N A; Vierkant, R A; Jacobson, R M; Poland, G A

2005-01-01

The ELISPOT assay is a highly sensitive technique used for the detection of individual cytokine releasing cells. We have developed an IFN-gamma ELISPOT assay utilizing unfractionated frozen peripheral blood mononuclear cells (PBMC) to quantify the frequency of measles virus (MV)-specific IFN-gamma-secreting T cells in 117 healthy children who had been previously immunized with two doses of the measles-mumps-rubella vaccine. We have also estimated the variability associated with the quantification of ELISPOT plates and compared the number of MV-specific IFN-gamma-secreting T cells for each subject as determined by two different operators of an ELISPOT reader. The median frequency of MV-specific IFN-gamma-producing memory T cells detected by this assay was 0.005 % and 0.01 % as determined by an in-house and commercial operator, respectively. Although we found a significant correlation (r = 0.83, p<0.0001) between the number of spots counted by the commercial and in-house operators of an ELISPOT reader, the median number of spots counted by the commercial operator was twice the number of spots counted by an in-house operator (p<0.001). This demonstrates the importance of using a common ELISPOT reader and operator, among other parameters, to quantify the number of spots when a large volume of plates are being scanned and analyzed.

Ultra-sensitive detection using integrated waveguide technologies

USDA-ARS?s Scientific Manuscript database

There is a pressing need to detect analytes at very low concentrations, such as food- and water-borne pathogens (e.g. E. coli O157:H7) and biothreat agents (e.g., anthrax, toxins). Common fluorescence detection methods, such as 96 well plate readers, are not sufficiently sensitive for low concentra...

A Non-imaging High Throughput Approach to Chemical Library Screening at the Unmodified Adenosine-A3 Receptor in Living Cells.

PubMed

Arruda, Maria Augusta; Stoddart, Leigh A; Gherbi, Karolina; Briddon, Stephen J; Kellam, Barrie; Hill, Stephen J

2017-01-01

Recent advances in fluorescent ligand technology have enabled the study of G protein-coupled receptors in their native environment without the need for genetic modification such as addition of N-terminal fluorescent or bioluminescent tags. Here, we have used a non-imaging plate reader (PHERAstar FS) to monitor the binding of fluorescent ligands to the human adenosine-A 3 receptor (A 3 AR; CA200645 and AV039), stably expressed in CHO-K1 cells. To verify that this method was suitable for the study of other GPCRs, assays at the human adenosine-A 1 receptor, and β 1 and β 2 adrenoceptors (β 1 AR and β 2 AR; BODIPY-TMR-CGP-12177) were also carried out. Affinity values determined for the binding of the fluorescent ligands CA200645 and AV039 to A 3 AR for a range of classical adenosine receptor antagonists were consistent with A 3 AR pharmacology and correlated well ( R 2 = 0.94) with equivalent data obtained using a confocal imaging plate reader (ImageXpress Ultra). The binding of BODIPY-TMR-CGP-12177 to the β 1 AR was potently inhibited by low concentrations of the β 1 -selective antagonist CGP 20712A (pK i 9.68) but not by the β 2 -selective antagonist ICI 118551(pK i 7.40). Furthermore, in experiments conducted in CHO K1 cells expressing the β 2 AR this affinity order was reversed with ICI 118551 showing the highest affinity (pK i 8.73) and CGP20712A (pK i 5.68) the lowest affinity. To determine whether the faster data acquisition of the non-imaging plate reader (~3 min per 96-well plate) was suitable for high throughput screening (HTS), we screened the LOPAC library for inhibitors of the binding of CA200645 to the A 3 AR. From the initial 1,263 compounds evaluated, 67 hits (defined as those that inhibited the total binding of 25 nM CA200645 by ≥40%) were identified. All compounds within the library that had medium to high affinity for the A 3 AR (pK i ≥6) were successfully identified. We found three novel compounds in the library that displayed unexpected sub-micromolar affinity for the A 3 AR. These were K114 (pK i 6.43), retinoic acid p -hydroxyanilide (pK i 6.13) and SU 6556 (pK i 6.17). Molecular docking of these latter three LOPAC library members provided a plausible set of binding poses within the vicinity of the established orthosteric A 3 AR binding pocket. A plate reader based library screening using an untagged receptor is therefore possible using fluorescent ligand opening the possibility of its use in compound screening at natively expressed receptors.

Phase sensitive spectral domain interferometry for label free biomolecular interaction analysis and biosensing applications

NASA Astrophysics Data System (ADS)

Chirvi, Sajal

Biomolecular interaction analysis (BIA) plays vital role in wide variety of fields, which include biomedical research, pharmaceutical industry, medical diagnostics, and biotechnology industry. Study and quantification of interactions between natural biomolecules (proteins, enzymes, DNA) and artificially synthesized molecules (drugs) is routinely done using various labeled and label-free BIA techniques. Labeled BIA (Chemiluminescence, Fluorescence, Radioactive) techniques suffer from steric hindrance of labels on interaction site, difficulty of attaching labels to molecules, higher cost and time of assay development. Label free techniques with real time detection capabilities have demonstrated advantages over traditional labeled techniques. The gold standard for label free BIA is surface Plasmon resonance (SPR) that detects and quantifies the changes in refractive index of the ligand-analyte complex molecule with high sensitivity. Although SPR is a highly sensitive BIA technique, it requires custom-made sensor chips and is not well suited for highly multiplexed BIA required in high throughput applications. Moreover implementation of SPR on various biosensing platforms is limited. In this research work spectral domain phase sensitive interferometry (SD-PSI) has been developed for label-free BIA and biosensing applications to address limitations of SPR and other label free techniques. One distinct advantage of SD-PSI compared to other label-free techniques is that it does not require use of custom fabricated biosensor substrates. Laboratory grade, off-the-shelf glass or plastic substrates of suitable thickness with proper surface functionalization are used as biosensor chips. SD-PSI is tested on four separate BIA and biosensing platforms, which include multi-well plate, flow cell, fiber probe with integrated optics and fiber tip biosensor. Sensitivity of 33 ng/ml for anti-IgG is achieved using multi-well platform. Principle of coherence multiplexing for multi-channel label-free biosensing applications is introduced. Simultaneous interrogation of multiple biosensors is achievable with a single spectral domain phase sensitive interferometer by coding the individual sensograms in coherence-multiplexed channels. Experimental results demonstrating multiplexed quantitative biomolecular interaction analysis of antibodies binding to antigen coated functionalized biosensor chip surfaces on different platforms are presented.

Laboratory evaluation of 3M Petrifilms and University of Minnesota Bi-plates as potential on-farm tests for clinical mastitis.

PubMed

McCarron, J L; Keefe, G P; McKenna, S L B; Dohoo, I R; Poole, D E

2009-05-01

The objective was to determine test characteristics and compare 2 potential on-farm culture systems for clinical mastitis, the Minnesota Easy Culture System II Bi-plate and Petrifilm. The tests were evaluated using clinically positive mastitic milk samples (n = 282) to determine their ability to differentiate appropriate treatment groups; all cases that had gram-positive growth were considered treatment candidates (n = 161), whereas cases that grew gram-negative organisms only or yielded no bacterial growth were classified as no treatment (n = 121). For Petrifilm, both undiluted and 1:10 diluted milk samples were used. To create treatment categories, 2 types of Petrifilms were used, Aerobic Count (AC) and Coliform Count (CC). Both Bi-plates and Petrifilms were read after 24 h of incubation. Analysis was conducted at various colony count thresholds for the Petrifilm test system. The combination of Petrifilms that had the highest sensitivity classified a case as gram-negative if there were > or =20 colonies present on the CC. If there were <20 colonies present on the CC and >5 colonies present on the AC, a case would be classified as gram-positive. The Bi-plate had a sensitivity of 97.9% and a specificity of 68.6%. The Petrifilm test system had a sensitivity of 93.8% and a specificity of 70.1%. There was no significant difference in the sensitivities between the tests. All Bi-plates and Petrifilms were read by a laboratory technician and a group of masked readers with limited microbiology training. Kappa values for the masked readers were 0.75 for Bi-plates and 0.84 and 0.86 for AC and CC Petrifilms, respectively. The Bi-plate and Petrifilm were able to successfully categorize clinical cases of mastitis into 2 treatments based on their ability to detect the presence of a gram-positive organism. Neither method had the ability to determine if a sample was contaminated. The results of this study indicate that both tests were able to appropriately categorize cases, which could potentially result in a reduction in the quantity of antibiotics used to treat clinical cases of mastitis.

TGF-β1-Mediated Differentiation of Fibroblasts Is Associated with Increased Mitochondrial Content and Cellular Respiration

PubMed Central

Negmadjanov, Ulugbek; Godic, Zarko; Rizvi, Farhan; Emelyanova, Larisa; Ross, Gracious; Richards, John; Holmuhamedov, Ekhson L.; Jahangir, Arshad

2015-01-01

Objectivs Cytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts. Methods Cultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers) and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit. Results Treatment with TGF-β1 (5 ng/mL) induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU) compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU). TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU) and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU), as well as mitochondrial DNA content (530 ± 12 μg DNA/106 cells vs. 307 ± 9 μg DNA/106 cells in control). TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O2/min/106 cells) and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O2/min/106 cells). Conclusions TGF-β1 induced differentiation of fibroblasts is accompanied by energetic remodeling of myofibroblasts with an increase in mitochondrial respiration and mitochondrial content. PMID:25849590

Probe colorimeter for quantitating enzyme-linked immunosorbent assays and other colorimetric assays performed with microplates.

PubMed Central

Ackerman, S B; Kelley, E A

1983-01-01

The performance of a fiberoptic probe colorimeter (model PC800; Brinkmann Instruments, Inc., Westbury, N.Y.) for quantitating enzymatic or colorimetric assays in 96-well microtiter plates was compared with the performances of a spectrophotometer (model 240; Gilford Instrument Laboratories, Inc., Oberlin, Ohio) and a commercially available enzyme immunoassay reader (model MR590; Dynatech Laboratories, Inc., Alexandria, Va.). Alkaline phosphatase-p-nitrophenyl phosphate in 3 M NaOH was used as the chromophore source. Six types of plates were evaluated for use with the probe colorimeter; they generated reproducibility values (100% coefficient of variation) ranging from 91 to 98% when one individual made 24 independent measurements on the same dilution of chromophore on each plate. Eleven individuals each performed 24 measurements with the colorimeter on either a visually light (absorbance of 0.10 at 420 nm) or a dark (absorbance of 0.80 at 420 nm) dilution of chromophore; reproducibilities averaged 87% for the light dilution and 97% for the dark dilution. When one individual measured the same chromophore sample at least 20 times in the colorimeter, in the spectrophotometer or in the enzyme immunoassay reader, reproducibility for each instrument was greater than 99%. Measurements of a dilution series of chromophore in a fixed volume indicated that the optical responses of each instrument were linear in a range of 0.05 to 1.10 absorbance units. Images PMID:6341399

Probe colorimeter for quantitating enzyme-linked immunosorbent assays and other colorimetric assays performed with microplates.

PubMed

Ackerman, S B; Kelley, E A

1983-03-01

The performance of a fiberoptic probe colorimeter (model PC800; Brinkmann Instruments, Inc., Westbury, N.Y.) for quantitating enzymatic or colorimetric assays in 96-well microtiter plates was compared with the performances of a spectrophotometer (model 240; Gilford Instrument Laboratories, Inc., Oberlin, Ohio) and a commercially available enzyme immunoassay reader (model MR590; Dynatech Laboratories, Inc., Alexandria, Va.). Alkaline phosphatase-p-nitrophenyl phosphate in 3 M NaOH was used as the chromophore source. Six types of plates were evaluated for use with the probe colorimeter; they generated reproducibility values (100% coefficient of variation) ranging from 91 to 98% when one individual made 24 independent measurements on the same dilution of chromophore on each plate. Eleven individuals each performed 24 measurements with the colorimeter on either a visually light (absorbance of 0.10 at 420 nm) or a dark (absorbance of 0.80 at 420 nm) dilution of chromophore; reproducibilities averaged 87% for the light dilution and 97% for the dark dilution. When one individual measured the same chromophore sample at least 20 times in the colorimeter, in the spectrophotometer or in the enzyme immunoassay reader, reproducibility for each instrument was greater than 99%. Measurements of a dilution series of chromophore in a fixed volume indicated that the optical responses of each instrument were linear in a range of 0.05 to 1.10 absorbance units.

Interfering with DNA Damage Signals: Radiosensitizing Prostate Cancer Using Small Peptides

DTIC Science & Technology

2009-05-01

25-l sample of the supernatant was analyzed for free phosphate in the malachite green assay by dilution with 100 l of a developing solution... malachite green). After incubation for 15 min, the release of phosphate was quantified by measuring the absorbance at 650 nm in a microtiter plate reader

Comparison of the three optical platforms for measurement of cellular respiration.

PubMed

Kondrashina, Alina V; Ogurtsov, Vladimir I; Papkovsky, Dmitri B

2015-01-01

We compared three optical platforms for measurement of cellular respiration: absolute oxygen consumption rates (OCRs) in hermetically sealed microcuvettes, relative OCRs measured in a 96-well plate with oil seal, and steady-state oxygenation of cells in an open 96-well plate. Using mouse embryonic fibroblasts cell line, the phosphorescent intracellular O2 probe MitoXpress-Intra, and time-resolved fluorescence reader, we determined algorithms for conversion of relative OCRs and cell oxygenation into absolute OCRs, thereby allowing simple high-throughput measurement of absolute OCR values. Copyright © 2014 Elsevier Inc. All rights reserved.

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen dye.

PubMed

Moreno, Luis A; Cox, Kendra L

2010-11-05

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit. The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 μL sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode. Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

PubMed Central

Moreno, Luis A.; Cox, Kendra L.

2010-01-01

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit. The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 μL sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode. Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program. PMID:21189464

Development of Multiwell-Plate Methods Using Pure Cultures of Methanogens To Identify New Inhibitors for Suppressing Ruminant Methane Emissions.

PubMed

Weimar, M R; Cheung, J; Dey, D; McSweeney, C; Morrison, M; Kobayashi, Y; Whitman, W B; Carbone, V; Schofield, L R; Ronimus, R S; Cook, G M

2017-08-01

Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H 2 and CO 2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against M. maripaludis and M. AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions. IMPORTANCE Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a high-throughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions. Copyright © 2017 American Society for Microbiology.

Tissue Engineering Platforms to Replicate the Tumor Microenvironment of Multiple Myeloma.

PubMed

Zhang, Wenting; Lee, Woo Y; Zilberberg, Jenny

2017-01-01

We described here the manufacturing and implementation of two prototype perfusion culture devices designed primarily for the cultivation of difficult-to-preserve primary patient-derived multiple myeloma cells (MMC). The first device consists of an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment. The second platform is a 96-well plate-modified perfusion culture device that can be utilized to reconstruct several tissue and tumor microenvironments utilizing both primary human and murine cells. This culture device was designed and fabricated specifically to: (1) enable the preservation of primary MMC for downstream use in biological studies and chemosensitivity analyses and, (2) provide a high-throughput format that is compatible with plate readers specifically seeing that this system is built on an industry standard 96-well tissue culture plate.

Fluorescence-PCR Assays and Isolation of Luminescent Bacterial Clones Using an Automated Plate Reader

ERIC Educational Resources Information Center

Crowley, Thomas E.

2011-01-01

The genes responsible for luminescence in various species of the marine microorganism "Photobacterium", have been used for many years as a tool by researchers and instructors. In particular, the "lux" operon of "Photobacterium fischeri" has been used by many instructors to teach recombinant DNA techniques. Two methods using an automated plate…

78 FR 59065 - Interview Room Recording System Standard and License Plate Reader Standard Workshops

Federal Register 2010, 2011, 2012, 2013, 2014

2013-09-25

.... Space is limited at each workshop, and as a result, only 50 participants will be allowed to register for... organization. Exceptions to this limit may occur, should space allow. Participants planning to attend are responsible for their own travel arrangements. DATES: Both workshops will be held on Saturday, October 19...

Longitudinal measurement of extracellular matrix rigidity in 3D tumor models using particle-tracking microrheology.

PubMed

Jones, Dustin P; Hanna, William; El-Hamidi, Hamid; Celli, Jonathan P

2014-06-10

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(ω). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Automated Patch-Clamp Methods for the hERG Cardiac Potassium Channel.

PubMed

Houtmann, Sylvie; Schombert, Brigitte; Sanson, Camille; Partiseti, Michel; Bohme, G Andrees

2017-01-01

The human Ether-a-go-go Related Gene (hERG) product has been identified as a central ion channel underlying both familial forms of elongated QT interval on the electrocardiogram and drug-induced elongation of the same QT segment. Indeed, reduced function of this potassium channel involved in the repolarization of the cardiac action potential can produce a type of life-threatening cardiac ventricular arrhythmias called Torsades de Pointes (TdP). Therefore, hERG inhibitory activity of newly synthetized molecules is a relevant structure-activity metric for compound prioritization and optimization in medicinal chemistry phases of drug discovery. Electrophysiology remains the gold standard for the functional assessment of ion channel pharmacology. The recent years have witnessed automatization and parallelization of the manual patch-clamp technique, allowing higher throughput screening on recombinant hERG channels. However, the multi-well plate format of automatized patch-clamp does not allow visual detection of potential micro-precipitation of poorly soluble compounds. In this chapter we describe bench procedures for the culture and preparation of hERG-expressing CHO cells for recording on an automated patch-clamp workstation. We also show that the sensitivity of the assay can be improved by adding a surfactant to the extracellular medium.

Integrative Bioengineering Institute

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eddington, David; Magin,L,Richard; Hetling, John

2009-01-09

Microfabrication enables many exciting experimental possibilities for medicine and biology that are not attainable through traditional methods. However, in order for microfabricated devices to have an impact they must not only provide a robust solution to a current unmet need, but also be simple enough to seamlessly integrate into standard protocols. Broad dissemination of bioMEMS has been stymied by the common aim of replacing established and well accepted protocols with equally or more complex devices, methods, or materials. The marriage of a complex, difficult to fabricate bioMEMS device with a highly variable biological system is rarely successful. Instead, the designmore » philosophy of my lab aims to leverage a beneficial microscale phenomena (e.g. fast diffusion at the microscale) within a bioMEMS device and adapt to established methods (e.g. multiwell plate cell culture) and demonstrate a new paradigm for the field (adapt instead of replace). In order for the field of bioMEMS to mature beyond novel proof-of-concept demonstrations, researchers must focus on developing systems leveraging these phenomena and integrating into standard labs, which have largely been ignored. Towards this aim, the Integrative Bioengineering Institute has been established.« less

Principles of using Cold Atmospheric Plasma Stimulated Media for Cancer Treatment

PubMed Central

Yan, Dayun; Talbot, Annie; Nourmohammadi, Niki; Cheng, Xiaoqian; Canady, Jerome; Sherman, Jonathan; Keidar, Michael

2015-01-01

To date, the significant anti-cancer capacity of cold atmospheric plasma (CAP) on dozens of cancer cell lines has been demonstrated in vitro and in mice models. Conventionally, CAP was directly applied to irradiate cancer cells or tumor tissue. Over past three years, the CAP irradiated media was also found to kill cancer cells as effectively as the direct CAP treatment. As a novel strategy, using the CAP stimulated (CAPs) media has become a promising anti-cancer tool. In this study, we demonstrated several principles to optimize the anti-cancer capacity of the CAPs media on glioblastoma cells and breast cancer cells. Specifically, using larger wells on a multi-well plate, smaller gaps between the plasma source and the media, and smaller media volume enabled us to obtain a stronger anti-cancer CAPs media composition without increasing the treatment time. Furthermore, cysteine was the main target of effective reactive species in the CAPs media. Glioblastoma cells were more resistant to the CAPs media than breast cancer cells. Glioblastoma cells consumed the effective reactive species faster than breast cancer cells did. In contrast to nitric oxide, hydrogen peroxide was more likely to be the effective reactive species. PMID:26677750

Identification and Correction of Additive and Multiplicative Spatial Biases in Experimental High-Throughput Screening.

PubMed

Mazoure, Bogdan; Caraus, Iurie; Nadon, Robert; Makarenkov, Vladimir

2018-06-01

Data generated by high-throughput screening (HTS) technologies are prone to spatial bias. Traditionally, bias correction methods used in HTS assume either a simple additive or, more recently, a simple multiplicative spatial bias model. These models do not, however, always provide an accurate correction of measurements in wells located at the intersection of rows and columns affected by spatial bias. The measurements in these wells depend on the nature of interaction between the involved biases. Here, we propose two novel additive and two novel multiplicative spatial bias models accounting for different types of bias interactions. We describe a statistical procedure that allows for detecting and removing different types of additive and multiplicative spatial biases from multiwell plates. We show how this procedure can be applied by analyzing data generated by the four HTS technologies (homogeneous, microorganism, cell-based, and gene expression HTS), the three high-content screening (HCS) technologies (area, intensity, and cell-count HCS), and the only small-molecule microarray technology available in the ChemBank small-molecule screening database. The proposed methods are included in the AssayCorrector program, implemented in R, and available on CRAN.

Antifungal efficacy of thymol, carvacrol, eugenol and menthol as alternative agents to control the growth of food-relevant fungi.

PubMed

Abbaszadeh, S; Sharifzadeh, A; Shokri, H; Khosravi, A R; Abbaszadeh, A

2014-06-01

This work is an attempt to examine the antifungal activity of thymol, carvacrol, eugenol and menthol against 11 food-decaying fungi. The susceptibility test for the compounds was carried out in terms of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) using microdilution method in 96 multi-well microtiter plates. Results indicated that all compounds were effective to varying extents against various fungal isolates, with the highest efficacy displayed by carvacrol (mean MIC value: 154.5 μg/mL) (P<0.05). The incorporation of increased concentrations of all compounds to the media led to progressive and significant reduction in growth for all fungi. The most potent inhibitory activity of thymol, carvacrol, eugenol and menthol was found for Cladosporium spp. (MIC: 100 μg/mL), Aspergillus spp. (MIC: 100 μg/mL), Cladosporium spp. (MIC: 350 μg/mL), and Aspergillus spp. and Cladosporium spp. (MIC: 125 μg/mL), respectively. Thus, the application of these herbal components could be considered as a good alternatives to inhibit fungal growth and to reduce the use of synthetic fungicides. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Developing a novel fiber optic fluorescence device for multiplexed high-throughput cytotoxic screening.

PubMed

Lee, Dennis; Barnes, Stephen

2010-01-01

The need for new pharmacological agents is unending. Yet the drug discovery process has changed substantially over the past decade and continues to evolve in response to new technologies. There is presently a high demand to reduce discovery time by improving specific lab disciplines and developing new technology platforms in the area of cell-based assay screening. Here we present the developmental concept and early stage testing of the Ab-Sniffer, a novel fiber optic fluorescence device for high-throughput cytotoxicity screening using an immobilized whole cell approach. The fused silica fibers are chemically functionalized with biotin to provide interaction with fluorescently labeled, streptavidin functionalized alginate-chitosan microspheres. The microspheres are also functionalized with Concanavalin A to facilitate binding to living cells. By using lymphoma cells and rituximab in an adaptation of a well-known cytotoxicity protocol we demonstrate the utility of the Ab-Sniffer for functional screening of potential drug compounds rather than indirect, non-functional screening via binding assay. The platform can be extended to any assay capable of being tied to a fluorescence response including multiple target cells in each well of a multi-well plate for high-throughput screening.

Image plates as x-ray detectors in plasma physics experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gales, S.G.; Bentley, C.D.

2004-10-01

The performance of image plates based on the photostimulable phosphor BaF(Br,l):Eu{sup 2+} has been investigated and compared with x-ray film. Evaluation of detective quantum efficiency (DQE), sensitivity, dynamic range, and linearity was carried out for several types of commercially available image plate, using the Excalibur soft x-ray calibration facility at AWE. Image plate response was found to be linear over a dynamic range of 5 orders of magnitude. One type of image plate was found to have a number of advantages for soft x-ray detection, with a measured sensitivity 1 order of magnitude greater than that of Kodak Industrex CXmore » and DEF-5 x-ray film. The DQE of this plate was found to be superior to that of film at low [less than 10{sup 3} photons/(50 {mu}m){sup 2}] and high fluxes [greater than 10{sup 4} photons/(50 {mu}m){sup 2}]. The spatial resolution of image plates, scanned with several models of commercial image plate readers, has been evaluated using a USAF resolution test target. The highest spatial resolution measured is 35 {mu}m. Though this is significantly lower than the resolution possible with film, it is sufficient for many applications. Image plates were fielded in a refractive x-ray lens imaging diagnostic on the 1 TW Helen laser and these results are discussed.« less

Measurements of 4 Atmospheric Trace Gases Outside Homes Adjacent to a Multiwell Pad During Drilling, Hydraulic Fracturing, and Production Phases, Using Low-Cost Sensors and Artificial Neural Network Quantification Techniques

NASA Astrophysics Data System (ADS)

Casey, J. G.; Ilie, A. M. C.; Coffey, E.; Collier-Oxandale, A. M.; Hannigan, M.; Vaccaro, C.

2017-12-01

In Colorado and elsewhere in North America, the oil and gas production industry has been growing alongside and in the midst of increasing urban and rural populations. These coinciding trends have resulted in a growing number of people living in close proximity to petroleum production and processing activities, leading to potential public health impacts. Combustion-related emissions from heavy-duty diesel vehicle traffic, generators, compressors, and production stream flaring can potentially lead to locally enhanced levels of nitrogen oxides (NOx), carbon monoxide (CO), and carbon dioxide (CO2). Venting and fugitive emissions of production stream constituents can potentially lead to locally enhanced levels of methane (CH4) and volatile organic compounds (VOCs), some of which (like benzene) are known carcinogens. NOx and VOC emissions can also potentially increase local ozone (O3) production. After learning of a large new multiwell pad on the outskirts of Greeley, Colorado, we were able to quickly mobilize portable air quality monitors outfitted with low-cost gas sensors that respond to CH4, CO2, CO, and O3. The air quality monitors were installed outside homes adjacent to the new multiwell pad several weeks prior to the first spud date. An anemometer was also installed outside one of the homes in order to monitor wind speed and direction. Measurements continued during drilling, hydraulic fracturing, and production phases. The sensors were periodically collocated with reference instruments at a nearby regulatory air quality monitoring site towards calibration via field normalization and validation. Artificial Neural Networks were employed to map sensor signals to trace gas mole fractions during collocation periods. We present measurements of CH4, CO2, CO, and O3 in context with wellpad activities and local meteorology. CO and O3 observations are presented in context with regional measurements and National Ambient Air Quality Standards for each. Wind speed and direction measurements were used to indicate when air masses originated from the direction of the multiwell pad. CO2 mole fractions were used to estimate planetary boundary layer height and CH4 mole fractions were used to identify periods conducive to the pooling and accumulation of production stream venting and fugitive emissions.

Development of a new microtiter plate format for clinically relevant assays.

PubMed

Piletska, Elena V; Piletsky, Stanislav S; Whitcombe, Michael J; Chianella, Iva; Piletsky, Sergey A

2012-02-21

A new format for the microtiter plate-based assays was proposed. The novelty involves the use of disk-shaped inserts for immobilization of biological and chemical reagents. The internal opening of the disks allows measurements of the reactions by standard microtiter plate readers without any additional steps involving liquid handling. Ideally the plate end-users just have to add the sample and take the measurement without any need of multiple reagent additions or transfer of the liquid to a different plate. The novel assay format also allows handling of reagents which are not soluble in an aqueous environment. As a proof of concept we describe here several model reactions which are compatible with microtiter plate format, such as monitoring enzymatic reactions catalyzed by glucose oxidase (GOx) and urease, measurements of proteins by BCA assay, analysis of pH, and concentration of antioxidants. The "mix and match" approach in the disk-shape format allows multiplexing and could be particularly useful for high throughput screening. One of the potential application areas for this novel assay format could be in a multianalyte system for measurement of clinically relevant analytes in primary care.

Performance evaluation of three computed radiography systems using methods recommended in American Association of Physicists in Medicine Report 93.

PubMed

Muhogora, Wilbroad; Padovani, Renato; Bonutti, Faustino; Msaki, Peter; Kazema, R

2011-07-01

The performances of three clinical computed radiography (CR) systems, (Agfa CR 75 (with CRMD 4.0 image plates), Kodak CR 850 (with Kodak GP plates) and Kodak CR 850A (with Kodak GP plates)) were evaluated using six tests recommended in American Association of Physicists in Medicine Report 93. The results indicated variable performances with majority being within acceptable limits. The variations were mainly attributed to differences in detector formulations, plate readers' characteristics, and aging effects. The differences of the mean low contrast scores between the imaging systems for three observers were statistically significant for Agfa and Kodak CR 850A (P=0.009) and for Kodak CR systems (P=0.006) probably because of the differences in ages. However, the differences were not statistically significant between Agfa and Kodak CR 850 (P=0.284) suggesting similar perceived image quality. The study demonstrates the need to implement quality control program regularly.

75 FR 75186 - License Plate Reader Standard Special Technical Committee Request for Proposals for Certification...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-02

... members will participate in six 2-day meetings over a 12-month time period with the goal of completing... begin in January 2011. Travel expenses and per diem will be reimbursed for all STC meetings; however, participation time will not be funded. NIJ is seeking representatives from (1) certification bodies and (2) test...

Microfluidic Flow Injection Analysis with Thermal Lens Microscopic Detection for Determination of NGAL

NASA Astrophysics Data System (ADS)

Radovanović, Tatjana; Liu, Mingqiang; Likar, Polona; Klemenc, Matjaž; Franko, Mladen

2015-06-01

A combined microfluidic flow injection analysis-thermal lens microscopy (FIA-TLM) system was applied for determination of neutrophil gelatinase-associated lipocalin (NGAL)—a biomarker of acute kidney injury. NGAL was determined following a commercial ELISA assay and transfer of the resulting solution into the FIA-TLM system with a 100 m deep microchannel. At an excitation power of 100 mW, the FIA-TLM provided about seven times lower limits of detection (1.5 pg as compared to a conventional ELISA test, and a sample throughput of six samples per minute, which compares favorably with sample throughput of the microtiter plate reader, which reads 96 wells in about 30 min. Comparison of results for NGAL in plasma samples from healthy individuals and for NGAL dynamics in patients undergoing coronary angiography measured with transmission mode spectrometry on a microtiter plate reader and with FIA-TLM showed good agreement. In addition to improved LOD, the high sensitivity of FIA-TLM offers possibilities of a further reduction of the total reaction time of the NGAL ELISA test by sacrificing some of the sensitivity while reducing the duration of individual incubation steps.

Design and installation of a subsea multiwell drilling template at Garden Banks Block 189

DOE Office of Scientific and Technical Information (OSTI.GOV)

Domangue, R.J.

1991-09-01

This paper describes the economics, design, and installation of a simple drilling template that was used to retain or save subsea exploratory and delineation wells in Garden Banks Block 189 (GB 189) in the Gulf of Mexico.

A review of the multiwell experiment in tight gas sandstones of the Mesaverde Group, Piceance Basin, Colorado

USGS Publications Warehouse

Nelson, P.H.

2002-01-01

The Cretaceous Iles and Williams Fork Formations of the Mesaverde Group contain important reservoir and source rocks for basin-centered gas accumulations in the Piceance Basin of northwestern Colorado. The sandstones in these formations have very low permeability, so low that successful production of gas requires the presence of fractures. To increase gas production, the natural fracture system of these "tight gas sandstones" must be augmented by inducing artificial fractures, while minimizing the amount of formation damage due to introduced fluids. The Multiwell Experiment was undertaken to provide geological characterization, obtain physical property data, and perform stimulation experiments in the Iles and Williams Fork Formations. Three vertical wells and one follow-up slant well were drilled, logged, partially cored, tested for gas production, stimulated in various manners, and tested again. Drawing from published reports and papers, this review paper presents well log, core, and test data from the Multiwell Experiment while emphasizing the geological controls on gas production at the site. Gas production is controlled primarily by a set of regional fractures trending west-northwest. The fractures are vertical, terminating at lithologic boundaries within and at the upper and lower boundaries of sandstone beds. Fractures formed preferentially in sandstones where in situ stress and fracture gradients are lower than in shales and mudstones. The fractures cannot be identified adequately in vertical wellbores; horizontal wells are required. Because present-day maximum horizontal stress is aligned with the regional fractures, artificial fractures induced by pressuring the wellbore form parallel to the regional fractures rather than linking them, with consequent limitations upon enhancement of gas production.

Statistical Analysis of Zebrafish Locomotor Response.

PubMed

Liu, Yiwen; Carmer, Robert; Zhang, Gaonan; Venkatraman, Prahatha; Brown, Skye Ashton; Pang, Chi-Pui; Zhang, Mingzhi; Ma, Ping; Leung, Yuk Fai

2015-01-01

Zebrafish larvae display rich locomotor behaviour upon external stimulation. The movement can be simultaneously tracked from many larvae arranged in multi-well plates. The resulting time-series locomotor data have been used to reveal new insights into neurobiology and pharmacology. However, the data are of large scale, and the corresponding locomotor behavior is affected by multiple factors. These issues pose a statistical challenge for comparing larval activities. To address this gap, this study has analyzed a visually-driven locomotor behaviour named the visual motor response (VMR) by the Hotelling's T-squared test. This test is congruent with comparing locomotor profiles from a time period. Different wild-type (WT) strains were compared using the test, which shows that they responded differently to light change at different developmental stages. The performance of this test was evaluated by a power analysis, which shows that the test was sensitive for detecting differences between experimental groups with sample numbers that were commonly used in various studies. In addition, this study investigated the effects of various factors that might affect the VMR by multivariate analysis of variance (MANOVA). The results indicate that the larval activity was generally affected by stage, light stimulus, their interaction, and location in the plate. Nonetheless, different factors affected larval activity differently over time, as indicated by a dynamical analysis of the activity at each second. Intriguingly, this analysis also shows that biological and technical repeats had negligible effect on larval activity. This finding is consistent with that from the Hotelling's T-squared test, and suggests that experimental repeats can be combined to enhance statistical power. Together, these investigations have established a statistical framework for analyzing VMR data, a framework that should be generally applicable to other locomotor data with similar structure.

Statistical Analysis of Zebrafish Locomotor Response

PubMed Central

Zhang, Gaonan; Venkatraman, Prahatha; Brown, Skye Ashton; Pang, Chi-Pui; Zhang, Mingzhi; Ma, Ping; Leung, Yuk Fai

2015-01-01

Zebrafish larvae display rich locomotor behaviour upon external stimulation. The movement can be simultaneously tracked from many larvae arranged in multi-well plates. The resulting time-series locomotor data have been used to reveal new insights into neurobiology and pharmacology. However, the data are of large scale, and the corresponding locomotor behavior is affected by multiple factors. These issues pose a statistical challenge for comparing larval activities. To address this gap, this study has analyzed a visually-driven locomotor behaviour named the visual motor response (VMR) by the Hotelling’s T-squared test. This test is congruent with comparing locomotor profiles from a time period. Different wild-type (WT) strains were compared using the test, which shows that they responded differently to light change at different developmental stages. The performance of this test was evaluated by a power analysis, which shows that the test was sensitive for detecting differences between experimental groups with sample numbers that were commonly used in various studies. In addition, this study investigated the effects of various factors that might affect the VMR by multivariate analysis of variance (MANOVA). The results indicate that the larval activity was generally affected by stage, light stimulus, their interaction, and location in the plate. Nonetheless, different factors affected larval activity differently over time, as indicated by a dynamical analysis of the activity at each second. Intriguingly, this analysis also shows that biological and technical repeats had negligible effect on larval activity. This finding is consistent with that from the Hotelling’s T-squared test, and suggests that experimental repeats can be combined to enhance statistical power. Together, these investigations have established a statistical framework for analyzing VMR data, a framework that should be generally applicable to other locomotor data with similar structure. PMID:26437184

Evaluation of Multi-Well Microelectrode Arrays for Neurotoxicity Screening Using a Chemical Training Set

EPA Science Inventory

Microelectrode array (MEA) approaches have been proposed as a tool for detecting functional changes in electrically-excitable cells, including neurons, exposed to drugs, chemical or particles. However, conventional single well-MEA systems lack the throughput necessary for screen...

Results of the multiwell experiment in situ stresses, natural fractures, and other geological controls on reservoirs

NASA Astrophysics Data System (ADS)

Lorenz, John C.; Warpinski, Norman R.; Teufel, Lawrence W.; Branagan, Paul T.; Sattler, Allan R.; Northrop, David A.

Hundreds of millions of cubic meters of natural gas are locked up in low-permeability, natural gas reservoirs. The Multiwell Experiment (MWX) was designed to characterize such reservoirs, typical of much of the western United States, and to assess and develop a technology for the production of this unconventional resource. Flow-rate tests of the MWX reservoirs indicate a system permeability that is several orders of magnitude higher than laboratory permeability measurements made on matrix-rock sandstones. This enhanced permeability is caused by natural fractures. The single set of fractures present in the reservoirs provides a significant permeability anisotropy that is aligned with the maximum in situ horizontal stress. Hydraulic fractures therefore form parallel to the natural fractures and are consequently an inefficient mechanism for stimulation. Successful stimulation may be possible by perturbing the local stress field with a large hydraulic fracture in one well so that a second hydraulic fracture in an offset well propagates transverse to the natural fracture permeability trend.

A field-shaping multi-well avalanche detector for direct conversion amorphous selenium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goldan, A. H.; Zhao, W.

2013-01-15

Purpose: A practical detector structure is proposed to achieve stable avalanche multiplication gain in direct-conversion amorphous selenium radiation detectors. Methods: The detector structure is referred to as a field-shaping multi-well avalanche detector. Stable avalanche multiplication gain is achieved by eliminating field hot spots using high-density avalanche wells with insulated walls and field-shaping inside each well. Results: The authors demonstrate the impact of high-density insulated wells and field-shaping to eliminate the formation of both field hot spots in the avalanche region and high fields at the metal-semiconductor interface. Results show a semi-Gaussian field distribution inside each well using the field-shaping electrodes,more » and the electric field at the metal-semiconductor interface can be one order-of-magnitude lower than the peak value where avalanche occurs. Conclusions: This is the first attempt to design a practical direct-conversion amorphous selenium detector with avalanche gain.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warpinski, N.R.; Branagan, P.T.; Sattler, A.R.

This paper is a case study of the stimulation and testing of tight, lenticular sands in the paludal interval of the Mesaverde group in the Piceance basin at DOE's Multiwell Experiment (MWX) site in Colorado. Topics discussed include geologic data, stress test results, well testing, laboratory core studies, stimulation and stimulation analyses, and postfracture operations.

Evaluation of the Sequential Spot Sampler (S3) for time-resolved measurement of PM2.5 sulfate and nitrate through lab and field measurements

NASA Astrophysics Data System (ADS)

Hecobian, A.; Evanoski-Cole, A.; Eiguren-Fernandez, A.; Sullivan, A. P.; Lewis, G. S.; Hering, S. V.; Collett, J. L., Jr.

2016-02-01

The Sequential Spot Sampler (S3), a newly developed instrument to collect aerosols for time-resolved chemical composition measurements, was evaluated in the laboratory and field for the measurement of particulate sulfate and nitrate. The S3 uses a multi-temperature condensation growth tube to grow individual aerosols to droplets which are then deposited as a ˜ 1 mm diameter dry spot at the end of the growth tube in a 100 µL well of a multi-well plate. The well plate advances automatically to provide a sequence of time-resolved samples. The collected aerosols are subsequently analyzed in the laboratory. The sample is concentrated during the collection process, and the laboratory extraction and analysis steps can be automated. The well plate, as received from the field, is placed onto a needle-based autosampler that adds liquid for sample extraction and injects sample extract from each well onto an ion chromatograph for analysis. Laboratory evaluation for sulfate and nitrate ions showed that poly ether ether ketone (PEEK) used as well plate material does not contribute any artifacts; a 60 min extraction procedure leads to the recovery of sulfate and nitrate from the dry spots at above 95 % extraction efficiency; and samples stored frozen and analyzed up to 23 months later show less than a 10 % change in sulfate and nitrate concentrations. The limit of detection was 0.5 µg m-3 for sulfate and 0.2 µg m-3 for nitrate for a 1 h sampling period. In a month-long field study conducted in southern California, two S3s were deployed alongside a URG denuder-filter-pack and a Particle-Into-Liquid Sampler combined with an Ion Chromatograph (PILS-IC). Collocated S3 sampler concentrations compared by linear regression show good agreement, with r2 = 0.99 and slope = 0.99 (±0.004) µg m-3 for sulfate and r2 = 0.99 and slope = 1.0 (±0.006) µg m-3 for nitrate. When compared to the URG denuder-filter-pack and the PILS-IC, the S3 sulfate and nitrate concentrations yielded correlations above 0.84 for the square of the correlation coefficient and regression slopes close to 1.

Secure Integration of Radio Frequency Identification (RFID) Technology into a Supply Chain

DTIC Science & Technology

2005-09-01

serves as the rough equivalent of a license plate on an automobile . Figure 1 (below) illustrates the typical construction of an RFID tag. An antenna...writable passive tags (RW) Reprogrammable Class 3 Semi-active tags Reprogrammable Class 4 Active tags Reprogrammable Class 5 Readers... Reprogrammable Table 1. EPC Tag Classes[3]. Table 2 summarizes the advantages, disadvantages and applications of each type of tag. Tag Type Advantages

Optimization of the HyPer sensor for robust real-time detection of hydrogen peroxide in the rice blast fungus.

PubMed

Huang, Kun; Caplan, Jeff; Sweigard, James A; Czymmek, Kirk J; Donofrio, Nicole M

2017-02-01

Reactive oxygen species (ROS) production and breakdown have been studied in detail in plant-pathogenic fungi, including the rice blast fungus, Magnaporthe oryzae; however, the examination of the dynamic process of ROS production in real time has proven to be challenging. We resynthesized an existing ROS sensor, called HyPer, to exhibit optimized codon bias for fungi, specifically Neurospora crassa, and used a combination of microscopy and plate reader assays to determine whether this construct could detect changes in fungal ROS during the plant infection process. Using confocal microscopy, we were able to visualize fluctuating ROS levels during the formation of an appressorium on an artificial hydrophobic surface, as well as during infection on host leaves. Using the plate reader, we were able to ascertain measurements of hydrogen peroxide (H 2 O 2 ) levels in conidia as detected by the MoHyPer sensor. Overall, by the optimization of codon usage for N. crassa and related fungal genomes, the MoHyPer sensor can be used as a robust, dynamic and powerful tool to both monitor and quantify H 2 O 2 dynamics in real time during important stages of the plant infection process. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Comparison of fluorescence-based methods to determine nanoparticle uptake by phagocytes and non-phagocytic cells in vitro

PubMed Central

Claudia, Meindl; Kristin, Öhlinger; Jennifer, Ober; Eva, Roblegg; Eleonore, Fröhlich

2017-01-01

At many portals of entry the relative uptake by phagocytes and non-phagocytic cells has a prominent effect on availability and biological action of nanoparticles (NPs). Cellular uptake can be determined for fluorescence-labeled NPs. The present study compares three methods (plate reader, flow cytometry and image analysis) in order to investigate the influence of particle size and functionalization and medium content on cellular uptake of fluorescence–labeled polystyrene particles and to study the respective method’s suitability for uptake studies. For comparison between the techniques, ratios of macrophage to alveolar epithelial cell uptakes were used. Presence of serum protein in the exposure solution decreased uptake of carboxyl-functionalized and non-functionalized particles; there was no clear effect for the amine-functionalized particles. The 200 nm non- or carboxyl-functionalized NPs were taken up preferentially by phagocytes while for amine-functionalized particles preference was lowest. The presence of the serum slightly increased the preference for these particles. In conclusion, due to the possibility of calibration, plate reader measurements might present a better option than the other techniques to (semi)quantify differences between phagocytes and non-phagocytic cells for particles with different fluorescence. In order to obtain unbiased data the fluorescent labeling has to fulfill certain requirements. PMID:28065592

Antitrichomonal activity of Peganum harmala alkaloid extract against trichomoniasis in pigeon (Columba livia domestica).

PubMed

Tabari, M A; Youssefi, M R; Moghadamnia, A A

2017-06-01

1. This study was designed to evaluate the antitrichomonal effects of P. harmala alkaloid extract against T. gallinae, both in vitro and in vivo, as well as comparing it to that of metronidazole, conventional antitrichomonal medication and harmine and harmaline, the two alkaloids present in P. harmala. 2. T. gallinae were collected by the wet mount method from infected free-living pigeons. The in vitro assay was performed using multi-well plates containing test compounds in final concentrations of 5, 10, 15, 20, 30, 50 or 100 μg/ml. The in vivo assay was done on 60 experimentally infected pigeons dosed with metronidazole at 50 mg/kg body weight (BW) or alkaloids at 25 mg/kg BW. 3. The 24 h minimum inhibitory concentration (MIC) of alkaloid extract was 15 µg/ml while that of metronidazole was 50 µg/ml. Harmine and harmaline revealed 24 h MIC of 30 and 100 µg/ml, respectively. Treatment of infected pigeons with alkaloids led to a full recovery after 3 d but with metronidazole total eradication of trophozoites was not achieved. 4. In conclusion, data of the present study suggested P. harmala is a potent natural anti-trichomonal agent, effective against T. gallinae.

A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells.

PubMed

Nakamura, Mikiko; Suzuki, Ayako; Akada, Junko; Yarimizu, Tohru; Iwakiri, Ryo; Hoshida, Hisashi; Akada, Rinji

2015-08-01

Escherichia coli plasmids are commonly used for gene expression experiments in mammalian cells, while PCR-amplified DNAs are rarely used even though PCR is a much faster and easier method to construct recombinant DNAs. One difficulty may be the limited amount of DNA produced by PCR. For direct utilization of PCR-amplified DNA in transfection experiments, efficient transfection with a smaller amount of DNA should be attained. For this purpose, we investigated two enhancer reagents, polyethylene glycol and tRNA, for a chemical transfection method. The addition of the enhancers to a commercial transfection reagent individually and synergistically exhibited higher transfection efficiency applicable for several mammalian cell culture lines in a 96-well plate. By taking advantage of a simple transfection procedure using PCR-amplified DNA, SV40 and rabbit β-globin terminator lengths were minimized. The terminator length is short enough to design in oligonucleotides; thus, terminator primers can be used for the construction and analysis of numerous mutations, deletions, insertions, and tag-fusions at the 3'-terminus of any gene. The PCR-mediated gene manipulation with the terminator primers will transform gene expression by allowing for extremely simple and high-throughput experiments with small-scale, multi-well, and mammalian cell cultures.

Microengineering methods for cell-based microarrays and high-throughput drug-screening applications.

PubMed

Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

2011-09-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time consuming, and often faces concerns due to the extensive use of animals. To improve cost effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems has facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell-based drug-screening models which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell-based drug-screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds great potential to provide repeatable 3D cell-based constructs with high temporal, spatial control and versatility.

Unstirred Water Layers and the Kinetics of Organic Cation Transport

PubMed Central

Shibayama, Takahiro; Morales, Mark; Zhang, Xiaohong; Martinez, Lucy; Berteloot, Alfred; Secomb, Timothy W.; Wright, Stephen H.

2015-01-01

Purpose Unstirred water layers (UWLs) present an unavoidable complication in the measurement of transport kinetics in cultured cells and the high rates of transport achieved by overexpressing heterologous transporters exacerbate the UWL effect. This study examined the correlation between measured Jmax and Kt values and the effect of manipulating UWL thickness or transport Jmax on the accuracy of experimentally determined kinetics of the multidrug transporters, OCT2 and MATE1. Methods Transport of TEA and MPP was measured in CHO cells that stably expressed human OCT2 or MATE1. UWL thickness was manipulated by vigorous reciprocal shaking. Several methods were used to manipulate maximal transport rates. Results Vigorous stirring stimulated uptake of OCT2-mediated transport by decreasing apparent Kt (Ktapp) values. Systematic reduction in transport rates was correlated with reduction in Ktapp values. The slope of these relationships indicated a 1500 µm UWL in multiwell plates. Reducing the influence of UWLs (by decreasing either their thickness or the Jmax of substrate transport) reduced Ktapp by 2-fold to >10-fold. Conclusions Failure to take into account the presence of UWLs in experiments using cultured cells to measure transport kinetics can result in significant underestimates of the affinity of multidrug transporters for substrates. PMID:25791216

Fluoro-luminometric real-time measurement of bacterial viability and killing.

PubMed

Lehtinen, Janne; Virta, Marko; Lilius, Esa Matti

2003-10-01

The viability and killing of Escherichia coli was measured on a real-time basis using a fluoro-luminometric device, which allows successive measurements of fluorescence and bioluminescence without user intervention. Bacteria were made fluorescent and bioluminescent by expression of gfp and insect luciferase (lucFF) genes. The green fluorescent protein (GFP) is a highly fluorescent, extremely stable protein, which accumulates in cells during growth, and therefore the measured fluorescence signal was proportional to the total number of cells. The luciferase reaction is dependent of ATP produced by living cells, so that the bioluminescence level was a direct measure of the viable cells. In contrast to the bacterial luciferase, the insect luciferase uses a water-soluble and nonvolatile substrate, which makes automated multi-well microplate assay possible. For the validation of the assay, the proportion of living and dead cell populations was experimentally modified by incubating E. coli cells in the presence of various ethanol concentrations. Bacterial viability and killing measured by a fluoro-luminometric assay correlated fairly well with the reference methods: conventional plate counting, optical density measurement and various flow cytometric analyses. The real-time assay described here allows following the changes in bacterial cultures and assessing the bactericidal and other effects of various chemical, immunological and physical agents simultaneously in large numbers of samples.

Microengineering Methods for Cell Based Microarrays and High-Throughput Drug Screening Applications

PubMed Central

Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

2011-01-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time-consuming and often face ethical concerns due to extensive use of animals. To improve cost-effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems have facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell based drug-screening models, which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell based drug screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds a great potential to provide repeatable 3D cell based constructs with high temporal, spatial control and versatility. PMID:21725152

Solutions to decrease a systematic error related to AAPH addition in the fluorescence-based ORAC assay.

PubMed

Mellado-Ortega, Elena; Zabalgogeazcoa, Iñigo; Vázquez de Aldana, Beatriz R; Arellano, Juan B

2017-02-15

Oxygen radical absorbance capacity (ORAC) assay in 96-well multi-detection plate readers is a rapid method to determine total antioxidant capacity (TAC) in biological samples. A disadvantage of this method is that the antioxidant inhibition reaction does not start in all of the 96 wells at the same time due to technical limitations when dispensing the free radical-generating azo initiator 2,2'-azobis (2-methyl-propanimidamide) dihydrochloride (AAPH). The time delay between wells yields a systematic error that causes statistically significant differences in TAC determination of antioxidant solutions depending on their plate position. We propose two alternative solutions to avoid this AAPH-dependent error in ORAC assays. Copyright © 2016 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

James, S St.; Argento, D; Stewart, R

Purpose: The University of Washington Medical Center offers neutron therapy for the palliative and definitive treatment of selected cancers. In vivo field verification has the potential to improve the safe and effective delivery of neutron therapy. We propose a portal imaging method that relies on the creation of positron emitting isotopes (11C and 15O) through (n, 2n) reactions with a PMMA plate placed below the patient. After field delivery, the plate is retrieved from the vault and imaged using a reader that detects annihilation photons. The spatial pattern of activity produced in the PMMA plate provides information to reconstruct themore » neutron fluence map needed to confirm treatment delivery. Methods: We used MCNP to simulate the accumulation of 11C activity in a slab of PMMA 2 mm thick, and GATE was used to simulate the sensitivity and spatial resolution of a prototype imaging system. BGO crystal thicknesses of 1 cm, 2 cm and 3 cm were simulated with detector separations of 2 cm. Crystal pitches of 2 mm and 4 mm were evaluated. Back-projection of the events was used to create a planar image. The spatial resolution was taken to be the FWHM of the reconstructed point source image. Results: The system sensitivity for a point source in the center of the field of view was found to range from 58% for 1 cm thick BGO with 2 mm crystal pitch to 74% for the 3 cm thick BGO crystals with 4 mm crystal pitch. The spatial resolution at the center of the field of view was found to be 1.5 mm for the system with 2 mm crystal pitch and 2.8 mm for the system with the 4 mm crystal pitch. Conclusion: BGO crystals with 4 mm crystal pitch and 3 cm length would offer the best sensitivity reader.« less

A cost-effective smartphone-based antimicrobial susceptibility test reader for drug resistance testing (Conference Presentation)

NASA Astrophysics Data System (ADS)

Feng, Steve W.; Tseng, Derek; Di Carlo, Dino; Garner, Omai B.; Ozcan, Aydogan

2017-03-01

Antimicrobial susceptibility testing (AST) is commonly used for determining microbial drug resistance, but routine testing, which can significantly reduce the spread of multi-drug resistant organisms, is not regularly performed in resource-limited and field-settings due to technological challenges and lack of trained diagnosticians. We developed a portable cost-effective smartphone-based colorimetric 96-well microtiter plate (MTP) reader capable of automated AST without the need for a trained diagnostician. This system is composed of a smartphone used in conjunction with a 3D-printed opto-mechanical attachment, which holds a set of inexpensive light-emitting-diodes and fiber-optic cables coupled to the 96-well MTP for enabling the capture of the transmitted light through each well by the smartphone camera. Images of the MTP plate are captured at multiple exposures and uploaded to a local or remote server (e.g., a laptop) for automated processing/analysis of the results using a custom-designed smartphone application. Each set of images are combined to generate a high dynamic-range image and analyzed for well turbidity (indicative of bacterial growth), followed by interpretative analysis per plate to determine minimum inhibitory concentration (MIC) and drug susceptibility for the specific bacterium. Results are returned to the originating device within 1 minute and shown to the user in tabular form. We demonstrated the capability of this platform using MTPs prepared with 17 antibiotic drugs targeting Gram-negative bacteria and tested 82 patient isolate MTPs of Klebsiella pneumoniae, achieving well turbidity accuracy of 98.19%, MIC accuracy of 95.15%, and drug susceptibility interpretation accuracy of 99.06%, meeting the FDA defined criteria for AST.

Comparison of imaging characteristics of multiple-beam equalization and storage phosphor direct digitizer radiographic systems

NASA Astrophysics Data System (ADS)

Sankaran, A.; Chuang, Keh-Shih; Yonekawa, Hisashi; Huang, H. K.

1992-06-01

The imaging characteristics of two chest radiographic equipment, Advanced Multiple Beam Equalization Radiography (AMBER) and Konica Direct Digitizer [using a storage phosphor (SP) plate] systems have been compared. The variables affecting image quality and the computer display/reading systems used are detailed. Utilizing specially designed wedge, geometric, and anthropomorphic phantoms, studies were conducted on: exposure and energy response of detectors; nodule detectability; different exposure techniques; various look- up tables (LUTs), gray scale displays and laser printers. Methods for scatter estimation and reduction were investigated. It is concluded that AMBER with screen-film and equalization techniques provides better nodule detectability than SP plates. However, SP plates have other advantages such as flexibility in the selection of exposure techniques, image processing features, and excellent sensitivity when combined with optimum reader operating modes. The equalization feature of AMBER provides better nodule detectability under the denser regions of the chest. Results of diagnostic accuracy are demonstrated with nodule detectability plots and analysis of images obtained with phantoms.

Radiology image orientation processing for workstation display

NASA Astrophysics Data System (ADS)

Chang, Chung-Fu; Hu, Kermit; Wilson, Dennis L.

1998-06-01

Radiology images are acquired electronically using phosphor plates that are read in Computed Radiology (CR) readers. An automated radiology image orientation processor (RIOP) for determining the orientation for chest images and for abdomen images has been devised. In addition, the chest images are differentiated as front (AP or PA) or side (Lateral). Using the processing scheme outlined, hospitals will improve the efficiency of quality assurance (QA) technicians who orient images and prepare the images for presentation to the radiologists.

Medically relevant assays with a simple smartphone and tablet based fluorescence detection system.

PubMed

Wargocki, Piotr; Deng, Wei; Anwer, Ayad G; Goldys, Ewa M

2015-05-20

Cell phones and smart phones can be reconfigured as biomedical sensor devices but this requires specialized add-ons. In this paper we present a simple cell phone-based portable bioassay platform, which can be used with fluorescent assays in solution. The system consists of a tablet, a polarizer, a smart phone (camera) and a box that provides dark readout conditions. The assay in a well plate is placed on the tablet screen acting as an excitation source. A polarizer on top of the well plate separates excitation light from assay fluorescence emission enabling assay readout with a smartphone camera. The assay result is obtained by analysing the intensity of image pixels in an appropriate colour channel. With this device we carried out two assays, for collagenase and trypsin using fluorescein as the detected fluorophore. The results of collagenase assay with the lowest measured concentration of 3.75 µg/mL and 0.938 µg in total in the sample were comparable to those obtained by a microplate reader. The lowest measured amount of trypsin was 930 pg, which is comparable to the low detection limit of 400 pg for this assay obtained in a microplate reader. The device is sensitive enough to be used in point-of-care medical diagnostics of clinically relevant conditions, including arthritis, cystic fibrosis and acute pancreatitis.

Comparison of fluorescence-based methods to determine nanoparticle uptake by phagocytes and non-phagocytic cells in vitro.

PubMed

Claudia, Meindl; Kristin, Öhlinger; Jennifer, Ober; Eva, Roblegg; Eleonore, Fröhlich

2017-03-01

At many portals of entry the relative uptake by phagocytes and non-phagocytic cells has a prominent effect on availability and biological action of nanoparticles (NPs). Cellular uptake can be determined for fluorescence-labeled NPs. The present study compares three methods (plate reader, flow cytometry and image analysis) in order to investigate the influence of particle size and functionalization and medium content on cellular uptake of fluorescence-labeled polystyrene particles and to study the respective method́s suitability for uptake studies. For comparison between the techniques, ratios of macrophage to alveolar epithelial cell uptakes were used. Presence of serum protein in the exposure solution decreased uptake of carboxyl-functionalized and non-functionalized particles; there was no clear effect for the amine-functionalized particles. The 200nm non- or carboxyl-functionalized NPs were taken up preferentially by phagocytes while for amine-functionalized particles preference was lowest. The presence of the serum slightly increased the preference for these particles. In conclusion, due to the possibility of calibration, plate reader measurements might present a better option than the other techniques to (semi)quantify differences between phagocytes and non-phagocytic cells for particles with different fluorescence. In order to obtain unbiased data the fluorescent labeling has to fulfill certain requirements. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Clinical evaluation of CR versus plain film for neonatal ICU applications

NASA Astrophysics Data System (ADS)

Andriole, Katherine P.; Brasch, Robert C.; Gooding, Charles A.; Gould, Robert G.; Huang, H. K.

1995-05-01

The clinical utility of computed radiography (CR) versus screen-film for neonatal intensive care unit (ICU) applications is investigated. The latest versions of standard ST-V and high- resolution HR-V CR imaging plates were compared via measurements of image contrast, spatial resolution and signal-to-noise. The ST-V imaging plate was found to have equivalent spatial resolution and object detectability at a lower required dose than the HR-V, and was therefore chosen as the CR plate to use in clinical trials in which a modified film cassette containing the CR imaging plate, a conventional screen and film was utilized. For 50 portable neonatal chest examinations, plain film was subjectively compared to the perfectly matched, simultaneously obtained CR hardcopy and softcopy images. Grading of overall image quality was on a scale of one (poor) to five (excellent). Readers rated the visualization of various structures in the chest (i.e., lung parenchyma, pulmonary vasculature, tubes/lines) as well as the visualization of pathologic findings. Preliminary results indicate that the image quality of both CR soft and hardcopy are comparable to plain film and that CR may be a suitable alternative to screen-film imaging for portable neonatal chest x rays.

Guidelines for the selection of functional assays to evaluate the hallmarks of cancer.

PubMed

Menyhárt, Otília; Harami-Papp, Hajnalka; Sukumar, Saraswati; Schäfer, Reinhold; Magnani, Luca; de Barrios, Oriol; Győrffy, Balázs

2016-12-01

The hallmarks of cancer capture the most essential phenotypic characteristics of malignant transformation and progression. Although numerous factors involved in this multi-step process are still unknown to date, an ever-increasing number of mutated/altered candidate genes are being identified within large-scale cancer genomic projects. Therefore, investigators need to be aware of available and appropriate techniques capable of determining characteristic features of each hallmark. We review the methods tailored to experimental cancer researchers to evaluate cell proliferation, programmed cell death, replicative immortality, induction of angiogenesis, invasion and metastasis, genome instability, and reprogramming of energy metabolism. Selecting the ideal method is based on the investigator's goals, available equipment and also on financial constraints. Multiplexing strategies enable a more in-depth data collection from a single experiment - obtaining several results from a single procedure reduces variability and saves time and relative cost, leading to more robust conclusions compared to a single end point measurement. Each hallmark possesses characteristics that can be analyzed by immunoblot, RT-PCR, immunocytochemistry, immunoprecipitation, RNA microarray or RNA-seq. In general, flow cytometry, fluorescence microscopy, and multiwell readers are extremely versatile tools and, with proper sample preparation, allow the detection of a vast number of hallmark features. Finally, we also provide a list of hallmark-specific genes to be measured in transcriptome-level studies. Although our list is not exhaustive, we provide a snapshot of the most widely used methods, with an emphasis on methods enabling the simultaneous evaluation of multiple hallmark features. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Combining Facial Recognition, Automatic License Plate Readers and Closed Circuit Television to Create an Interstate Identification System for Wanted Subjects

DTIC Science & Technology

2015-12-01

IPSFRP search request. The candidate list will contain the agency’s requested number (minimum of2) of candidates or a default number of 20 candidates if...INTERSTATE IDENTIFICATION SYSTEM FOR WANTED SUBJECTS 5. FUNDING NUMBERS 6. AUTHOR(S) Michael J. Thomas 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES...Naval Postgraduate School Monterey, CA 93943-5000 8. PERFORMING ORGANIZATION REPORT NUMBER 9. SPONSORING /MONITORING AGENCY NAME(S) AND

Boundary Layer Flow of Air Over Water on a Flat Plate

DTIC Science & Technology

1993-08-01

similar (or coupled self -similar) solution appears to be a global attractor for all initial conditions. 2 Governing Equations A water film of height y...assumptions are self -consistent. The reader may verify that the solution (13) with c(x) given by (16) is self -similar (satisfies (24) without the the...attractor for all solutions of this non-similar family. Self similar boundary layers depend only on q and not on 4. The ý derivatives of u, v and y* may

Low cost, microcontroller based heating device for multi-wavelength differential scanning fluorimetry.

PubMed

Hoeser, Jo; Gnandt, Emmanuel; Friedrich, Thorsten

2018-01-23

Differential scanning fluorimetry is a popular method to estimate the stability of a protein in distinct buffer conditions by determining its 'melting point'. The method requires a temperature controlled fluorescence spectrometer or a RT-PCR machine. Here, we introduce a low-budget version of a microcontroller based heating device implemented into a 96-well plate reader that is connected to a standard fluorescence spectrometer. We demonstrate its potential to determine the 'melting point' of soluble and membranous proteins at various buffer conditions.

Coupling liquid chromatography/mass spectrometry detection with microfluidic droplet array for label-free enzyme inhibition assay.

PubMed

Wang, Xiu-Li; Zhu, Ying; Fang, Qun

2014-01-07

In this work, the combination of droplet-based microfluidics with liquid chromatography/mass spectrometry (LC/MS) was achieved, for providing a fast separation and high-information-content detection method for the analysis of nanoliter-scale droplets with complex compositions. A novel interface method was developed using an oil-covered droplet array chip to couple with an LC/MS system via a capillary sampling probe and a 4 nL injection valve without the need of a droplet extraction device. The present system can perform multistep operations including parallel enzyme inhibition reactions in nanoliter droplets, 4 nL sample injection, fast separation with capillary LC, and label-free detection with ESI-MS, and has significant flexibility in the accurate addressing and sampling of droplets of interest on demand. The system performance was evaluated using angiotensin I and angiotensin II as model samples, and the repeatabilities of peak area for angiotensin I and angiotensin II were 2.7% and 7.5% (RSD, n = 4), respectively. The present system was further applied to the screening for inhibitors of cytochrome P450 (CYP1A2) and measurement of the IC50 value of the inhibitor. The sample consumption for each droplet assay was 100 nL, which is reduced 10-100 times compared with conventional 384-multi-well plate systems usually used in high-throughput drug screening.

Testing Silver Nanoparticle Toxicity Using the Ammonia Oxidizing Bacteria Nitrosomonas Europaea and a High-throughput Assay

NASA Astrophysics Data System (ADS)

Semprini, L.; Bartow, S.; Radniecki, T.

2012-04-01

Understanding the toxicity of nanoparticles on ecologically significant wastewater microbiota, specifically ammonia oxidizing bacteria (AOB), is critical due to the exponential increase in commercialization of nanoparticles as well as the sensitivity of AOB to inhibitors. A high-throughput activity assay was developed to rapidly screen for nanoparticle toxicity on AOB, using a multi-well plate method and AOB Nitrosomonas Europaea. This method demonstrated good agreement with previously established batch bottle assays utilizing both silver ions (Ag+) and nanoparticles (Ag-NPs) as nitrification inhibitors. The method was used to study the inhibition of Ag+ and Ag-NPs (20 nm) on the nitrification by N. Europaea cells grown in fill-and-draw reactors compared exponentially grown batch cells. Results indicate longer hydraulic residence times increased some protection against inhibition as measured by the production of nitrite over a three hour assay. The cells were more sensitive to Ag+ than Ag-NP, which is consistent with our past observations. Studies are currently being conducted to determine the effects that the presence of humic acid and cations on the inhibition and toxicity. Our initial results show that the presence of Mg++ provides protect from Ag-NP inhibition, which partly results from the aggregation of the Ag-NP and a decrease in the rate of oxidation of the Ag-NP to Ag+. The presence of humic acid also provides for some protection from Ag-NP inhibition.

Flatworm models in pharmacological research: the importance of compound stability testing.

PubMed

Stalmans, Sofie; Willems, Maxime; Adriaens, Els; Remon, Jean-Paul; D'Hondt, Matthias; De Spiegeleer, Bart

2014-10-01

Flatworms possess adult pluripotent stem cells, which make them extraordinary experimental model organisms to assess in vivo the undesirable effects of substances on stem cells. Currently, quality practices, implying evaluation of the stability of the test compound under the proposed experimental conditions, are uncommon in this research field. Nevertheless, performing a stability study during the rational design of in vivo assay protocols will result in more reliable assay results. To illustrate the influence of the stability of the test substance on the final experimental outcome, we performed a short-term International Conference on Harmonization (ICH)-based stability study of cyclophosphamide in the culture medium, to which a marine flatworm model Macrostomum lignano is exposed. Using a validated U(H)PLC method, it was demonstrated that the cyclophosphamide concentration in the culture medium at 20°C is lowered to 80% of the initial concentration after 21days. The multiwell plates, flatworms and diatoms, as well as light exposure, did not influence significantly the cyclophosphamide concentration in the medium. The results of the stability study have practical implications on the experimental set-up of the carcinogenicity assay like the frequency of medium renewal. This case study demonstrates the benefits of applying appropriate quality guidelines already during fundamental research increasing the credibility of the results. Copyright © 2014 Elsevier Inc. All rights reserved.

A novel continuous toxicity test system using a luminously modified freshwater bacterium.

PubMed

Cho, Jang-Cheon; Park, Kyung-Je; Ihm, Hyuk-Soon; Park, Ji-Eun; Kim, Se-Young; Kang, Ilnam; Lee, Kyu-Ho; Jahng, Deokjin; Lee, Dong-Hun; Kim, Sang-Jong

2004-09-15

An automated continuous toxicity test system was developed using a recombinant bioluminescent freshwater bacterium. The groundwater-borne bacterium, Janthinobacterium lividum YH9-RC, was modified with luxAB and optimized for toxicity tests using different kinds of organic carbon compounds and heavy metals. luxAB-marked YH9-RC cells were much more sensitive (average 7.3-8.6 times) to chemicals used for toxicity detection than marine Vibrio fischeri cells used in the Microtox assay. Toxicity tests for wastewater samples using the YH9-RC-based toxicity assay showed that EC50-5 min values in an untreated raw wastewater sample (23.9 +/- 12.8%) were the lowest, while those in an effluent sample (76.7 +/- 14.9%) were the highest. Lyophilization conditions were optimized in 384-multiwell plates containing bioluminescent bacteria that were pre-incubated for 15 min in 0.16 M of trehalose prior to freeze-drying, increasing the recovery of bioluminescence and viability by 50%. Luminously modified cells exposed to continuous phenol or wastewater stream showed a rapid decrease in bioluminescence, which fell below detectable range within 1 min. An advanced toxicity test system, featuring automated real-time toxicity monitoring and alerting functions, was designed and finely tuned. This novel continuous toxicity test system can be used for real-time biomonitoring of water toxicity, and can potentially be used as a biological early warning system.

Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells.

PubMed

De Sutter, Valerie; Vanderhaeghen, Rudy; Tilleman, Sofie; Lammertyn, Freya; Vanhoutte, Isabelle; Karimi, Mansour; Inzé, Dirk; Goossens, Alain; Hilson, Pierre

2005-12-01

Although sequence information and genome annotation are improving at an impressive pace, functional ontology is still non-existent or rudimentary for most genes. In this regard, transient expression assays are very valuable for identification of short functional segments in particular pathways, because they can be performed rapidly and at a scale unattainable in stably transformed tissues. Vectors were constructed and protocols developed for systematic transient assays in plant protoplasts. To enhance throughput and reproducibility, protoplast treatments were performed entirely by a liquid-handling robot in multiwell plates, including polyethylene glycol/Ca2+ cell transfection with plasmid mixtures, washes and lysis. All transcriptional readouts were measured using a dual firefly/Renilla luciferase assay, in which the former was controlled by a reporter promoter and the latter by the 35S CaMV promoter, which served as internal normalization standard. The automated protocols were suitable for transient assays in protoplasts prepared from cell cultures of Nicotiana tabacum Bright Yellow-2 and Arabidopsis thaliana. They were implemented in a screen to discover potential regulators of genes coding for key enzymes in nicotine biosynthesis. Two novel tobacco transcription factors were found, NtORC1 and NtJAP1, that positively regulate the putrescine N-methyltransferase (PMT) promoter. In addition, combinatorial tests showed that these two factors act synergistically to induce PMT transcriptional activity. The development and use of high-throughput plant transient expression assays are discussed.

Two continuous coupled assays for ornithine-δ-aminotransferase.

PubMed

Juncosa, Jose I; Lee, Hyunbeom; Silverman, Richard B

2013-09-15

We have developed two new continuous coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ(1)-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of L-glutamate, produced during the regeneration of the cofactor pyridoxal 5'-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red L-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of L-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the L-glutamate oxidase (GluOx) reporter enzyme. Copyright © 2013 Elsevier Inc. All rights reserved.

Determination of excipient based solubility increases using the CheqSol method.

PubMed

Etherson, Kelly; Halbert, Gavin; Elliott, Moira

2014-04-25

Aqueous solubility is an essential characteristic assessed during drug development to determine a compound's drug-likeness since solubility plays an important pharmaceutical role. However, nearly half of the drug candidates discovered today display poor water solubility; therefore methods have to be applied to increase solubility. Solubility determination using the CheqSol method is a novel rapid solubility screening technique for ionisable compounds. The aim of this study is to determine if the CheqSol method can be employed to determine solubility increases of four test drugs (ibuprofen, gliclazide, atenolol and propranolol) induced by non-ionising excipients such as hydroxypropyl-β-cyclodextrin and poloxamers 407 and 188. CheqSol assays were performed for the drugs alone or in combination with varying solubiliser concentrations. The measured intrinsic solubility of all four drugs increased with all the excipients tested in an excipient concentration dependent manner providing results consistent with previous literature. The results demonstrate that it may be possible to use this method to determine the solubility increases induced by non-ionic solubilising excipients with results that are comparable to standard equilibrium based solubility techniques. Since the technique is automated and requires only small drug quantities it may serve as a useful solubility or formulation screening tool providing more detailed physicochemical information than multiwell plate or similar visual systems. Copyright © 2014. Published by Elsevier B.V.

Miniaturizing 3D assay for high-throughput drug and genetic screens for small patient-derived tumor samples (Conference Presentation)

NASA Astrophysics Data System (ADS)

Rotem, Asaf; Garraway, Levi; Su, Mei-Ju; Basu, Anindita; Regev, Aviv; Struhl, Kevin

2017-02-01

Three-dimensional growth conditions reflect the natural environment of cancer cells and are crucial to be performed at drug screens. We developed a 3D assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the 50-year old benchmark assay-soft agar. Using GILA, we performed high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. This phenotypic approach is complementary to our genetic approach that utilizes single-cell RNA-sequencing of a patient sample to identify putative oncogenes that confer sensitivity to drugs designed to specifically inhibit the identified oncoprotein. Currently, we are dealing with a big challenge in our field- the limited number of cells that might be extracted from a biopsy. Small patient-derived samples are hard to test in the traditional multiwell plate and it will be helpful to minimize the culture area and the experimental system. We managed to design a suitable microfluidic device for limited number of cells and perform the assay using image analysis. We aim to test drugs on tumor cells, outside of the patient body- and recommend on the ideal treatment that is tailored to the individual. This device will help to minimize biopsy-sampling volumes and minimize interventions in the patient's tumor.

Capture zone of a multi-well system in bounded peninsula-shaped aquifers.

PubMed

Zarei-Doudeji, Somayeh; Samani, Nozar

2014-08-01

In this paper we present the equation of capture zone for multi-well system in peninsula-shaped confined and unconfined aquifers. The aquifer is rectangular in plan view, bounded along three sides, and extends to infinity at the fourth side. The bounding boundaries are either no-flow (impervious) or in-flow (constant head) so that aquifers with six possible boundary configurations are formed. The well system is consisted of any number of extraction or injection wells or combination of both with any flow rates. The complex velocity potential equations for such a well-aquifer system are derived to delineate the capture envelop. Solutions are provided for the aquifers with and without a uniform regional flow of any directions. The presented equations are of general character and have no limitations in terms of well numbers, positions and types, extraction/injection rate, and regional flow rate and direction. These solutions are presented in form of capture type curves which are useful tools in hands of practitioners to design in-situ groundwater remediation systems, to contain contaminant plumes, to evaluate the surface-subsurface water interaction and to verify numerical models. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry.

PubMed

Shmueli, Ron B; Bhise, Nupura S; Green, Jordan J

2013-03-01

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases(1) and as a technology for regenerative medicine(2). Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery(1,3) . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers(4) that are hydrolytically degradable(5,6) and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)(7), mouse mammary epithelial cells(8), human brain cancer cells(9) and macrovascular (human umbilical vein, HUVECs) endothelial cells(10). A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained(11). In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A sensitive gold nanoparticle-based colorimetric aptasensor for Staphylococcus aureus.

PubMed

Yuan, Jinglei; Wu, Shijia; Duan, Nuo; Ma, Xiaoyuan; Xia, Yu; Chen, Jie; Ding, Zhansheng; Wang, Zhouping

2014-09-01

In this study, a gold nanoparticle-based colorimetric aptasensor for Staphylococcus aureus (S. aureus) using tyramine signal amplification (TSA) technology has been developed. First, the biotinylated aptamer specific for S. aureus was immobilized on the surface of the wells of the microtiter plate via biotin-avidin binding. Then, the target bacteria (S. aureus), biotinylated-aptamer-streptavidin-HRP conjugates, biotinylated tyramine, hydrogen peroxide and avidin-catalase were successively introduced into the wells of the microtiter plate. After that, the existing catalase consumed the hydrogen peroxide. Finally, the freshly prepared gold (III) chloride trihydrate was added, the color of the reaction production would be changed and the absorbance at 550 nm could be measured with a plate reader. Under optimized conditions, there was a linear relationship between the absorbance at 550 nm and the concentration of S. aureus over the range from 10 to 10(6) cfu mL(-1) (with an R² of 0.9947). The limit of the developed method was determined to be 9 cfu mL(-1). Copyright © 2014 Elsevier B.V. All rights reserved.

A Curved Image-Plate Detector System for High-Resolution Synchrotron X-ray Diffraction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sarin, P.; Haggerty, R; Yoon, W

2009-01-01

The developed curved image plate (CIP) is a one-dimensional detector which simultaneously records high-resolution X-ray diffraction (XRD) patterns over a 38.7 2{theta} range. In addition, an on-site reader enables rapid extraction, transfer and storage of X-ray intensity information in {le}30 s, and further qualifies this detector to study kinetic processes in materials science. The CIP detector can detect and store X-ray intensity information linearly proportional to the incident photon flux over a dynamical range of about five orders of magnitude. The linearity and uniformity of the CIP detector response is not compromised in the unsaturated regions of the image plate,more » regardless of saturation in another region. The speed of XRD data acquisition together with excellent resolution afforded by the CIP detector is unique and opens up wide possibilities in materials research accessible through X-ray diffraction. This article presents details of the basic features, operation and performance of the CIP detector along with some examples of applications, including high-temperature XRD.« less

Evaporation-Driven Bioassays in Suspended Droplets.

PubMed

Hernandez-Perez, Ruth; Fan, Z Hugh; Garcia-Cordero, Jose L

2016-07-19

The microtiter plate has been an essential tool for diagnostics, high-throughput screening, and biological assays. We present an alternative platform to perform bioassays in a microplate format that exploits evaporation to drive assay reactions. Our method consists of droplets suspended on plastic pillars; reactions occur in these droplets instead of the wells. The pillars are fabricated by milling, and the rough surface created by this fabrication method pins the droplet to a constant contact line during the assay and also acts as a hydrophobic surface. Upon evaporation, natural convection arising from Marangoni currents mixes solutions in the droplet, which speeds up assay reactions, decreases assay times, and increases limits of detection. As a proof of concept we implemented two colorimetric assays to detect glucose and proteins in only 1.5 μL, without any external devices for mixing and with a digital microscope as a readout mechanism. Our platform is an ideal alternative to the microtiter plate, works with different volumes, is compatible with commercially available reagent dispensers and plate-readers, and could have broad applications in diagnostics and high-throughput screening.

Rapid determination of trace copper in animal feed based on micro-plate colorimetric reaction and statistical partitioning correction.

PubMed

Niu, Yiming; Wang, Jiayi; Zhang, Chi; Chen, Yiqiang

2017-04-15

The objective of this study was to develop a micro-plate based colorimetric assay for rapid and high-throughput detection of copper in animal feed. Copper ion in animal feed was extracted by trichloroacetic acid solution and reduced to cuprous ion by hydroxylamine. The cuprous ion can chelate with 2,2'-bicinchoninic acid to form a Cu-BCA complex which was detected with high sensitivity by micro-plate reader at 354nm. The whole assay procedure can be completed within 20min. To eliminate matrix interference, a statistical partitioning correction approach was proposed, which makes the detection of copper in complex samples possible. The limit of detection was 0.035μg/mL and the detection range was 0.1-10μg/mL of copper in buffer solution. Actual sample analysis indicated that this colorimetric assay produced results consistent with atomic absorption spectrometry analysis. These results demonstrated that the developed assay can be used for rapid determination of copper in animal feed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Sensitive detection of oversulfated chondroitin sulfate in heparin sodium or crude heparin with a colorimetric microplate based assay.

PubMed

Sommers, Cynthia D; Mans, Daniel J; Mecker, Laura C; Keire, David A

2011-05-01

In this work we describe a 96-well microplate assay for oversulfated chondroitin sulfate A (OSCS) in heparin, based on a water-soluble cationic polythiophene polymer (3-(2-(N-(N'-methylimidazole))ethoxy)-4-methylthiophene (LPTP)) and heparinase digestion of heparin. The assay takes advantage of several unique properties of heparin, OSCS, and LPTP, including OSCS inhibition of heparinase I and II activity, the molecular weight dependence of heparin-LPTP spectral shifts, and the distinct association of heparin fragments and OSCS to LPTP. These factors combine to enable detection of the presence of 0.003% w/w spiked OSCS in 10 μg of heparin sodium active pharmaceutical ingredient (API) using a plate reader and with visual detection to 0.1% levels. The same detection limit for OSCS was observed in the presence of 10% levels of dermatan sulfate (DS) or chondroitin sulfate A (CSA) impurities. In addition, we surveyed a selection of crude heparin samples received by the agency in 2008 and 2009 to determine average and extreme DS, CSA, and galactosamine weight percent levels. In the presence of these impurities and the variable heparin content in the crude heparin samples, spiked OSCS was reliably detected to the 0.1% w/w level using a plate reader. Finally, authentically OSCS contaminated heparin sodium API and crude samples were distinguished visually by color from control samples using the LPTP/heparinase test.

Antiproliferative activity of flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the MCF7, KB, and NIH/3T3 cell lines.

PubMed

Nedel, Fernanda; Begnini, Karine; Carvalho, Pedro Henrique de Azambuja; Lund, Rafael Guerra; Beira, Fátima T A; Del Pino, Francisco Augusto B

2012-11-01

This study assessed the antiproliferative effect in vitro of the flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the human breast adenocarcinoma (MCF-7), human mouth epidermal carcinoma (KB), and mouse embryonic fibroblast (NIH 3T3) cell lines, using sulforhodamine B (SRB) assay. A cell density of 2×10(4)/well was seeded in 96-well plates, and samples at different concentrations ranging from 10 to 500 mg/mL were tested. The optical density was determined in an ELISA multiplate reader (Thermo Plate TP-Reader). Results demonstrated that the hexane extract presented antiproliferative activity against both the tumor cell lines KB and MCF-7, presenting a GI(50) (MCF-7=13.09 mg/mL), TGI (KB=37.76 mg/mL), and IL(50) (KB=291.07 mg/mL). Also, the hexane extract presented antiproliferative activity toward NIH 3T3 cells GI(50) (183.65 mg/mL), TGI (280.54 mg/mL), and IL(50) (384.59 mg/mL). The results indicate that the flower hexane extract obtained from M. spicata associated with M. rotundifolia presents an antineoplastic activity against KB and MCF-7, although an antiproliferative effect at a high concentration of the extract was observed toward NIH 3T3.

Rapid High-Throughput Assessment of Aerobic Bacteria in Complex Samples by Fluorescence-Based Oxygen Respirometry

PubMed Central

O'Mahony, Fiach C.; Papkovsky, Dmitri B.

2006-01-01

A simple method has been developed for the analysis of aerobic bacteria in complex samples such as broth and food homogenates. It employs commercial phosphorescent oxygen-sensitive probes to monitor oxygen consumption of samples containing bacteria using standard microtiter plates and fluorescence plate readers. As bacteria grow in aqueous medium, at certain points they begin to deplete dissolved oxygen, which is seen as an increase in probe fluorescence above baseline signal. The time required to reach threshold signal is used to either enumerate bacteria based on a predetermined calibration or to assess the effects of various effectors on the growth of test bacteria by comparison with an untreated control. This method allows for the sensitive (down to a single cell), rapid (0.5 to 12 h) enumeration of aerobic bacteria without the need to conduct lengthy (48 to 72 h) and tedious colony counts on agar plates. It also allows for screening a wide range of chemical and environmental samples for their toxicity. These assays have been validated with different bacteria, including Escherichia coli, Micrococcus luteus, and Pseudomonas fluorescens, with the enumeration of total viable counts in broth and industrial food samples (packaged ham, chicken, and mince meat), and comparison with established agar plating and optical-density-at-600-nm assays has been given. PMID:16461677

Microplate-reader method for the rapid analysis of copper in natural waters with chemiluminescence detection.

PubMed

Durand, Axel; Chase, Zanna; Remenyi, Tomas; Quéroué, Fabien

2012-01-01

We have developed a method for the determination of copper in natural waters at nanomolar levels. The use of a microplate-reader minimizes sample processing time (~25 s per sample), reagent consumption (~120 μL per sample), and sample volume (~700 μL). Copper is detected by chemiluminescence. This technique is based on the formation of a complex between copper and 1,10-phenanthroline and the subsequent emission of light during the oxidation of the complex by hydrogen peroxide. Samples are acidified to pH 1.7 and then introduced directly into a 24-well plate. Reagents are added during data acquisition via two reagent injectors. When trace metal clean protocols are employed, the reproducibility is generally less than 7% on blanks and the detection limit is 0.7 nM for seawater and 0.4 nM for freshwater. More than 100 samples per hour can be analyzed with this technique, which is simple, robust, and amenable to at-sea analysis. Seawater samples from Storm Bay in Tasmania illustrate the utility of the method for environmental science. Indeed other trace metals for which optical detection methods exist (e.g., chemiluminescence, fluorescence, and absorbance) could be adapted to the microplate-reader.

Rapid, convenient method for screening imidazole-containing compounds for heme oxygenase inhibition.

PubMed

Vlahakis, Jason Z; Rahman, Mona N; Roman, Gheorghe; Jia, Zongchao; Nakatsu, Kanji; Szarek, Walter A

2011-01-01

Sensitive assays for measuring heme oxygenase activity have been based on the gas-chromatographic detection of carbon monoxide using elaborate, expensive equipment. The present study describes a rapid and convenient method for screening imidazole-containing candidates for inhibitory activity against heme oxygenase using a plate reader, based on the spectroscopic evaluation of heme degradation. A PowerWave XS plate reader was used to monitor the absorbance (as a function of time) of heme bound to purified truncated human heme oxygenase-1 (hHO-1) in the individual wells of a standard 96-well plate (with or without the addition of a test compound). The degradation of heme by heme oxygenase-1 was initiated using l-ascorbic acid, and the collected relevant absorbance data were analyzed by three different methods to calculate the percent control activity occurring in wells containing test compounds relative to that occurring in control wells with no test compound present. In the cases of wells containing inhibitory compounds, significant shifts in λ(max) from 404 to near 412 nm were observed as well as a decrease in the rate of heme degradation relative to that of the control. Each of the three methods of data processing (overall percent drop in absorbance over 1.5h, initial rate of reaction determined over the first 5 min, and estimated pseudo first-order reaction rate constant determined over 1.5h) gave similar and reproducible results for percent control activity. The fastest and easiest method of data analysis was determined to be that using initial rates, involving data acquisition for only 5 min once reactions have been initiated using l-ascorbic acid. The results of the study demonstrate that this simple assay based on the spectroscopic detection of heme represents a rapid, convenient method to determine the relative inhibitory activity of candidate compounds, and is useful in quickly screening a series or library of compounds for heme oxygenase inhibition. Copyright © 2010 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Evans, R.D.; Lekia, S.D.L.

This paper presents the results of parametric studies of two naturally fractured lenticular tight gas reservoirs, Fluvial E-1 and Puludal Zones 3 and 4, of the U.S. Department of Energy Multi-Well Experiment (MWX) site of Northwestern Colorado. The three-dimensional, two-phase, black oil reservoir simulator that was developed in a previous phase of this research program is also discussed and the capabilities further explored by applying it to several example problems.

High content screening in microfluidic devices

PubMed Central

Cheong, Raymond; Paliwal, Saurabh; Levchenko, Andre

2011-01-01

Importance of the field Miniaturization is key to advancing the state-of-the-art in high content screening (HCS), in order to enable dramatic cost savings through reduced usage of expensive biochemical reagents and to enable large-scale screening on primary cells. Microfluidic technology offers the potential to enable HCS to be performed with an unprecedented degree of miniaturization. Areas covered in this review This perspective highlights a real-world example from the authors’ work of HCS assays implemented in a highly miniaturized microfluidic format. Advantages of this technology are discussed, including cost savings, high throughput screening on primary cells, improved accuracy, the ability to study complex time-varying stimuli, and ease of automation, integration, and scaling. What the reader will gain The reader will understand the capabilities of a new microfluidics-based platform for HCS, and the advantages it provides over conventional plate-based HCS. Take home message Microfluidics technology will drive significant advancements and broader usage and applicability of HCS in drug discovery. PMID:21852997

Photovoltaic Module Encapsulation Design and Materials Selection, Volume 1, Abridged

NASA Technical Reports Server (NTRS)

Cuddihy, E. F.

1982-01-01

A summary version of Volume 1, presenting the basic encapsulation systems, their purposes and requirements, and the characteristics of the most promising candidate systems and materials, as identified and evaluated by the Flat-Plate Solar Array Project is presented. In this summary version considerable detail and much supporting and experimental information has necessarily been omitted. A reader interested in references and literature citations, and in more detailed information on specific topics, should consult Reference 1, JPL Document No. 5101-177, JPL Publication 81-102, DOE/JPL-1012-60 (JPL), June 1, 1982.

Evaluation of Amnion-derived Multipotent Progenitor (AMP) Cells and Amnion-derived Cellular Cytokine Solution (ST266) in Promoting Craniomaxillofacial Regenerative Bone Healing in Critical Size Calvarial Defects

DTIC Science & Technology

2017-10-10

action. MATERIALS AND METHODS Subjects Subjects used in the study include male Fischer 344 (CDF®) rats weighing 280-300 grams obtained from...PBS before being transferred to a flat bottom 96-well plate. Absorbance was then read at 450nm on a Biotek microplate reader. Live/Dead Staining of...incubation, live/dead staining of the scaffolds was imaged on a confocal microscope (Nikon). AMP Cell Differentiation To determine whether AMP

Spatiotemporal Industrial Activity Model for Estimating the Intensity of Oil and Gas Operations in Colorado.

PubMed

Allshouse, William B; Adgate, John L; Blair, Benjamin D; McKenzie, Lisa M

2017-09-05

Oil and gas (O&G) production in the United States has increased in the last 15 years, and operations, which are trending toward large multiwell pads, release hazardous air pollutants. Health studies have relied on proximity to O&G wells as an exposure metric, typically using an inverse distance-weighting (IDW) approach. Because O&G emissions are dependent on multiple factors, a dynamic model is needed to describe the variability in air pollution emissions over space and time. We used information on Colorado O&G activities, production volumes, and air pollutant emission rates from two Colorado basins to create a spatiotemporal industrial activity model to develop an intensity-adjusted IDW well-count metric. The Spearman correlation coefficient between this metric and measured pollutant concentrations was 0.74. We applied our model to households in Greeley, Colorado, which is in the middle of the densely developed Denver-Julesburg basin. Our intensity-adjusted IDW increased the unadjusted IDW dynamic range by a factor of 19 and distinguishes high-intensity events, such as hydraulic fracturing and flowback, from lower-intensity events, such as production at single-well pads. As the frequency of multiwell pads increases, it will become increasingly important to characterize the range of intensities at O&G sites when conducting epidemiological studies.

Effects of Tacrolimus or Sirolimus on the adhesion of vascular wall cells: Controlled in-vitro comparison study.

PubMed

Krüger-Genge, A; Hiebl, B; Franke, R P; Lendlein, A; Jung, F

2017-01-01

In drug eluting stents the cytostatic drugs Sirolimus or Tacrolimus are used to inhibit blood vessel restenosis by limiting the proliferation of smooth muscle cells. However, the cytostatic activity of both drugs was shown to be not cell specific and could also affect the stent endothelialisation, respectively. Currently, only limited in vitro data are available about the impact of Sirolimus and Tacrolimus on endothelial cell proliferation over a broad concentration range. To answer this question the following study was performed.Commercially obtained HUVEC were expanded with DMEM cell culture medium (GIBCO, Germany) supplemented with 5 vol% fetal calf serum on non-coated regular polystyrene-based 24-multiwell plates. For drug testings 2×104 cells/cm2 were seeded and grown for 24 h until 30-40% of the multiwell surfaces were covered and then exposed to Sirolimus (1.0×10-11 - 1.0×10-5 mol/l) or Tacrolimus (2.0×10-8 - 6.2×10-5 mol/l), both dissolved in DMSO. 12, 24 and 48 h after adding the drugs cell numbers per area were quantified by counting the cells in six wells with four fields of view per well, representing 0.6 mm2, using a confocal laser microscope.After 48 h of cell growth in the drug-free cell culture medium, the HUVEC number increased from 2.0×104 to 3.55×104 cells/cm2 (mean cell doubling time: 53.6 h, n = 6). At lower concentrations (≤2.0×10-6 mol/l) Tacrolimus reduced the number of adherent HUVEC significantly less than Sirolimus (p < 0.05). However, at higher concentrations (≥2.07×10-5 mol/l) the effect of Tacrolimus on the number of adherent endothelial cells was significantly greater than that of Sirolimus (p < 0.05). At the highest concentration applied (6.22×10-5 mol/l), Tacrolimus induced detachment of all HUVECs within 12 h after drug application. The number of adherent HUVEC decreased only slightly (about 9%) after Sirolimus application at the highest concentration (1.09×10-5 mol/l).These data show that in a non-flow model the cytostatic drug Tacrolimus reduced the number of adherent endothelial cells less than Sirolimus, as long as the drug concentration did not surpass 10-6 mol/l. At the limits of solubility, Sirolimus (1×10-5 mol/l) reduced the number of adherent endothelial cells less than Tacrolimus (6×10-5 mol/l), which induced detachment of endothelial cells.

A standalone perfusion platform for drug testing and target validation in micro-vessel networks

PubMed Central

Zhang, Boyang; Peticone, Carlotta; Murthy, Shashi K.; Radisic, Milica

2013-01-01

Studying the effects of pharmacological agents on human endothelium includes the routine use of cell monolayers cultivated in multi-well plates. This configuration fails to recapitulate the complex architecture of vascular networks in vivo and does not capture the relationship between shear stress (i.e. flow) experienced by the cells and dose of the applied pharmacological agents. Microfluidic platforms have been applied extensively to create vascular systems in vitro; however, they rely on bulky external hardware to operate, which hinders the wide application of microfluidic chips by non-microfluidic experts. Here, we have developed a standalone perfusion platform where multiple devices were perfused at a time with a single miniaturized peristaltic pump. Using the platform, multiple micro-vessel networks, that contained three levels of branching structures, were created by culturing endothelial cells within circular micro-channel networks mimicking the geometrical configuration of natural blood vessels. To demonstrate the feasibility of our platform for drug testing and validation assays, a drug induced nitric oxide assay was performed on the engineered micro-vessel network using a panel of vaso-active drugs (acetylcholine, phenylephrine, atorvastatin, and sildenafil), showing both flow and drug dose dependent responses. The interactive effects between flow and drug dose for sildenafil could not be captured by a simple straight rectangular channel coated with endothelial cells, but it was captured in a more physiological branching circular network. A monocyte adhesion assay was also demonstrated with and without stimulation by an inflammatory cytokine, tumor necrosis factor-α. PMID:24404058

Micropatterned cell-cell interactions enable functional encapsulation of primary hepatocytes in hydrogel microtissues.

PubMed

Li, Cheri Y; Stevens, Kelly R; Schwartz, Robert E; Alejandro, Brian S; Huang, Joanne H; Bhatia, Sangeeta N

2014-08-01

Drug-induced liver injury is a major cause of drug development failures and postmarket withdrawals. In vitro models that incorporate primary hepatocytes have been shown to be more predictive than model systems which rely on liver microsomes or hepatocellular carcinoma cell lines. Methods to phenotypically stabilize primary hepatocytes ex vivo often rely on mimicry of hepatic microenvironmental cues such as cell-cell interactions and cell-matrix interactions. In this work, we sought to incorporate phenotypically stable hepatocytes into three-dimensional (3D) microtissues, which, in turn, could be deployed in drug-screening platforms such as multiwell plates and diverse organ-on-a-chip devices. We first utilize micropatterning on collagen I to specify cell-cell interactions in two-dimensions, followed by collagenase digestion to produce well-controlled aggregates for 3D encapsulation in polyethylene glycol (PEG) diacrylate. Using this approach, we examined the influence of homotypic hepatocyte interactions and composition of the encapsulating hydrogel, and achieved the maintenance of liver-specific function for over 50 days. Optimally preaggregated structures were subsequently encapsulated using a microfluidic droplet-generator to produce 3D microtissues. Interactions of engineered hepatic microtissues with drugs was characterized by flow cytometry, and yielded both induction of P450 enzymes in response to prototypic small molecules and drug-drug interactions that give rise to hepatotoxicity. Collectively, this study establishes a pipeline for the manufacturing of 3D hepatic microtissues that exhibit stabilized liver-specific functions and can be incorporated into a wide array of emerging drug development platforms.

Cytocompatibility of magnesium and AZ31 alloy with three types of cell lines using a direct in vitro method.

PubMed

Mochizuki, Akira; Yahata, Chie; Takai, Hung

2016-09-01

Magnesium alloys have been investigated by many researchers as a new absorbable biomaterial owing to their excellent degradability with non-maleficence or low-maleficence in living tissues. In the present work, the in vitro cytocompatibility of an Magnesium alloy was investigated by culturing cells directly on it. Investigations were carried out in terms of the cell viability along with the use of scanning electron microscopy to observe its morphology. The cell lines used were derived from fibroblast, endothelial, and smooth muscle cells. Pure magnesium and AZ31 alloy composed of magnesium (96 %), aluminum (3 %), and zinc (1 %) were adopted as models. The viability of cells on the metal samples and on the margin area of a multi-well plate was investigated. For direct culturing on metal, a depression in the viability and morphologically stressed cells were observed. In addition, the cell viability was also depressed for the margin area. To clarify the factors causing the negative effects, the amount of eluted metal ions and pH changes in the medium because of the erosion of the Magnesium samples were investigated, together with the cytotoxicity of sole metal ions corresponding to the composition of the metals. It was found that Mg(2+), Zn(2+), and Al(3+) ions were less toxic at the investigated concentrations, and that these factors will not produce negative effects on cells. Consequently, these factors cannot fully explain the results.

A mini-microscope for in situ monitoring of cells.

PubMed

Kim, Sang Bok; Koo, Kyo-in; Bae, Hojae; Dokmeci, Mehmet R; Hamilton, Geraldine A; Bahinski, Anthony; Kim, Sun Min; Ingber, Donald E; Khademhosseini, Ali

2012-10-21

A mini-microscope was developed for in situ monitoring of cells by modifying off-the-shelf components of a commercial webcam. The mini-microscope consists of a CMOS imaging module, a small plastic lens and a white LED illumination source. The CMOS imaging module was connected to a laptop computer through a USB port for image acquisition and analysis. Due to its compact size, 8 × 10 × 9 cm, the present microscope is portable and can easily fit inside a conventional incubator, and enables real-time monitoring of cellular behaviour. Moreover, the mini-microscope can be used for imaging cells in conventional cell culture flasks, such as Petri dishes and multi-well plates. To demonstrate the operation of the mini-microscope, we monitored the cellular migration of mouse 3T3 fibroblasts in a scratch assay in medium containing three different concentrations of fetal bovine serum (5, 10, and 20%) and demonstrated differential responses depending on serum levels. In addition, we seeded embryonic stem cells inside poly(ethylene glycol) microwells and monitored the formation of stem cell aggregates in real time using the mini-microscope. Furthermore, we also combined a lab-on-a-chip microfluidic device for microdroplet generation and analysis with the mini-microscope and observed the formation of droplets under different flow conditions. Given its cost effectiveness, robust imaging and portability, the presented platform may be useful for a range of applications for real-time cellular imaging using lab-on-a-chip devices at low cost.

A mini-microscope for in situ monitoring of cells†‡

PubMed Central

Kim, Sang Bok; Koo, Kyo-in; Bae, Hojae; Dokmeci, Mehmet R.; Hamilton, Geraldine A.; Bahinski, Anthony; Kim, Sun Min; Ingber, Donald E.

2013-01-01

A mini-microscope was developed for in situ monitoring of cells by modifying off-the-shelf components of a commercial webcam. The mini-microscope consists of a CMOS imaging module, a small plastic lens and a white LED illumination source. The CMOS imaging module was connected to a laptop computer through a USB port for image acquisition and analysis. Due to its compact size, 8 × 10 × 9 cm, the present microscope is portable and can easily fit inside a conventional incubator, and enables real-time monitoring of cellular behaviour. Moreover, the mini-microscope can be used for imaging cells in conventional cell culture flasks, such as Petri dishes and multi-well plates. To demonstrate the operation of the mini-microscope, we monitored the cellular migration of mouse 3T3 fibroblasts in a scratch assay in medium containing three different concentrations of fetal bovine serum (5, 10, and 20%) and demonstrated differential responses depending on serum levels. In addition, we seeded embryonic stem cells inside poly(ethylene glycol) microwells and monitored the formation of stem cell aggregates in real time using the mini-microscope. Furthermore, we also combined a lab-on-a-chip microfluidic device for microdroplet generation and analysis with the mini-microscope and observed the formation of droplets under different flow conditions. Given its cost effectiveness, robust imaging and portability, the presented platform may be useful for a range of applications for real-time cellular imaging using lab-on-a-chip devices at low cost. PMID:22911426

Droplet-based microfluidic flow injection system with large-scale concentration gradient by a single nanoliter-scale injection for enzyme inhibition assay.

PubMed

Cai, Long-Fei; Zhu, Ying; Du, Guan-Sheng; Fang, Qun

2012-01-03

We described a microfluidic chip-based system capable of generating droplet array with a large scale concentration gradient by coupling flow injection gradient technique with droplet-based microfluidics. Multiple modules including sample injection, sample dispersion, gradient generation, droplet formation, mixing of sample and reagents, and online reaction within the droplets were integrated into the microchip. In the system, nanoliter-scale sample solution was automatically injected into the chip under valveless flow injection analysis mode. The sample zone was first dispersed in the microchannel to form a concentration gradient along the axial direction of the microchannel and then segmented into a linear array of droplets by immiscible oil phase. With the segmentation and protection of the oil phase, the concentration gradient profile of the sample was preserved in the droplet array with high fidelity. With a single injection of 16 nL of sample solution, an array of droplets with concentration gradient spanning 3-4 orders of magnitude could be generated. The present system was applied in the enzyme inhibition assay of β-galactosidase to preliminarily demonstrate its potential in high throughput drug screening. With a single injection of 16 nL of inhibitor solution, more than 240 in-droplet enzyme inhibition reactions with different inhibitor concentrations could be performed with an analysis time of 2.5 min. Compared with multiwell plate-based screening systems, the inhibitor consumption was reduced 1000-fold. © 2011 American Chemical Society

Amplified Detection of Prions and Other Amyloids by RT-QuIC in Diagnostics and the Evaluation of Therapeutics and Disinfectants.

PubMed

Caughey, Byron; Orru, Christina D; Groveman, Bradley R; Hughson, Andrew G; Manca, Matteo; Raymond, Lynne D; Raymond, Gregory J; Race, Brent; Saijo, Eri; Kraus, Allison

2017-01-01

Among the most sensitive, specific and practical of methods for detecting prions are the real-time quaking-induced conversion (RT-QuIC) assays. These assays exploit the fundamental self-propagating activity of prions to amplify the presence of prion seeds by as much as a trillion-fold. The reactions can detect most of the known mammalian prion diseases, often with sensitivities greater than those of animal bioassays. RT-QuIC assays are performed in multiwell plates with fluorescence detection and have now reached the sensitivity and practicality required for routine prion disease diagnostics. Some key strains of prions within particular host species, e.g., humans, cattle, and sheep, can be discriminated by comparison of RT-QuIC responses with different recombinant prion protein substrates. The most thoroughly validated diagnostic application of RT-QuIC is in the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) using cerebrospinal fluid. Diagnostic sensitivities as high as 96% can be achieved in less than 24h with specificities of 98%-100%. The ability, if needed, to also test nasal swab samples can increase the RT-QuIC sensitivity for sCJD to virtually 100%. In addition to diagnostic applications, RT-QuIC has also been used in the testing of prion disinfectants and potential therapeutics. Mechanistically related assays are also now being developed for other protein misfolding diseases. © 2017 Elsevier Inc. All rights reserved.

A Novel RFID-Based Sensing Method for Low-Cost Bolt Loosening Monitoring.

PubMed

Wu, Jian; Cui, Xingmei; Xu, Yunpeng

2016-01-28

In coal mines, bolt loosening in the cage guide is affected by the harsh environmental factors and cage hoist vibration, leading to significant threats to work safety. It is crucial, to this effect, to successfully detect the status of multipoint bolts of guide structures. This paper proposes a system to monitor bolt status in harsh environments established based on the RFID technique. A proof-of-concept model was demonstrated consisting of a bolt gearing system, passive UHF RFID tags, a reader, and monitoring software. A tinfoil metal film is fixed on the retaining plate and an RFID tag bonded to a large gear, with the bolt to be detected fixed in the center of a smaller gear. The radio-frequency signal cannot be received by the reader if the tag is completely obscured by the tinfoil, and if the bolt is loose, the tag's antenna is exposed when the gear revolves. A radio-frequency signal that carries corresponding bolt's information is transmitted by the RFID tag to the RFID reader due to coil coupling, identifying loose bolt location and reporting them in the software. Confirmatory test results revealed that the system indeed successfully detects bolt loosening and comparative test results (based on a reed switch multipoint bolt loosening monitor system) provided valuable information regarding the strengths and weaknesses of the proposed system.

A Novel RFID-Based Sensing Method for Low-Cost Bolt Loosening Monitoring

PubMed Central

Wu, Jian; Cui, Xingmei; Xu, Yunpeng

2016-01-01

In coal mines, bolt loosening in the cage guide is affected by the harsh environmental factors and cage hoist vibration, leading to significant threats to work safety. It is crucial, to this effect, to successfully detect the status of multipoint bolts of guide structures. This paper proposes a system to monitor bolt status in harsh environments established based on the RFID technique. A proof-of-concept model was demonstrated consisting of a bolt gearing system, passive UHF RFID tags, a reader, and monitoring software. A tinfoil metal film is fixed on the retaining plate and an RFID tag bonded to a large gear, with the bolt to be detected fixed in the center of a smaller gear. The radio-frequency signal cannot be received by the reader if the tag is completely obscured by the tinfoil, and if the bolt is loose, the tag’s antenna is exposed when the gear revolves. A radio-frequency signal that carries corresponding bolt’s information is transmitted by the RFID tag to the RFID reader due to coil coupling, identifying loose bolt location and reporting them in the software. Confirmatory test results revealed that the system indeed successfully detects bolt loosening and comparative test results (based on a reed switch multipoint bolt loosening monitor system) provided valuable information regarding the strengths and weaknesses of the proposed system. PMID:26828498

A novel procedure to assess the non-enzymatic hydrogen-peroxide antioxidant capacity of metabolites with high UV absorption.

PubMed

Csepregi, Kristóf; Hideg, Éva

2016-12-01

Assays assessing non-enzymatic hydrogen peroxide antioxidant capacities are often hampered by the high UV absorption of the sample itself. This is a typical problem in studies using plant extracts with high polyphenol content. Our assay is based on comparing the 405 nm absorption of the product of potassium iodine and hydrogen peroxide in the presence and absence of a putative hydrogen peroxide reactive antioxidant. This method is free of interference with either hydrogen peroxide or antioxidant self-absorption and it is also suitable for high-throughput plate reader applications.

In vitro susceptibility of Prototheca spp. to gentamicin.

PubMed Central

Shahan, T A; Pore, R S

1991-01-01

One hundred strains of Prototheca zopfii, Prototheca wickerhamii, Prototheca moriformis, Prototheca stagnora, and Prototheca ulmnea; five strains of Chlorella protothecoides; and two strains of Candida albicans were obtained from a number of different clinical and environmental sources and were tested for their in vitro susceptibility to the antibacterial agent gentamicin. All Prototheca strains were susceptible to gentamicin at concentrations between 0.3 and 0.9 micrograms/ml. A modified macrobroth dilution MIC assay with a colorimeter and a microbroth dilution assay with a 96-well plate reader were the two methods used to determine the MICs. PMID:1804021

Quantitative digital image analysis of chromogenic assays for high throughput screening of alpha-amylase mutant libraries.

PubMed

Shankar, Manoharan; Priyadharshini, Ramachandran; Gunasekaran, Paramasamy

2009-08-01

An image analysis-based method for high throughput screening of an alpha-amylase mutant library using chromogenic assays was developed. Assays were performed in microplates and high resolution images of the assay plates were read using the Virtual Microplate Reader (VMR) script to quantify the concentration of the chromogen. This method is fast and sensitive in quantifying 0.025-0.3 mg starch/ml as well as 0.05-0.75 mg glucose/ml. It was also an effective screening method for improved alpha-amylase activity with a coefficient of variance of 18%.

What Is the 'Minimum Inhibitory Concentration' (MIC) of Pexiganan Acting on Escherichia coli?-A Cautionary Case Study.

PubMed

Jepson, Alys K; Schwarz-Linek, Jana; Ryan, Lloyd; Ryadnov, Maxim G; Poon, Wilson C K

2016-01-01

We measured the minimum inhibitory concentration (MIC) of the antimicrobial peptide pexiganan acting on Escherichia coli , and found an intrinsic variability in such measurements. These results led to a detailed study of the effect of pexiganan on the growth curve of E. coli, using a plate reader and manual plating (i.e. time-kill curves). The measured growth curves, together with single-cell observations and peptide depletion assays, suggested that addition of a sub-MIC concentration of pexiganan to a population of this bacterium killed a fraction of the cells, reducing peptide activity during the process, while leaving the remaining cells unaffected. This pharmacodynamic hypothesis suggests a considerable inoculum effect, which we quantified. Our results cast doubt on the use of the MIC as 'a measure of the concentration needed for peptide action' and show how 'coarse-grained' studies at the population level give vital information for the correct planning and interpretation of MIC measurements.

Development of Microtiter Plate Culture Method for Rapid Screening of ε-Poly-L-Lysine-Producing Strains.

PubMed

Liu, Yong-Juan; Chen, Xu-Sheng; Zhao, Jun-Jie; Pan, Long; Mao, Zhong-Gui

2017-12-01

ε-Poly-L-lysine (ε-PL) produced by Streptomyces albulus possesses a broad spectrum of antimicrobial activity and is widely used as a food preservative. To extensively screen ε-PL-overproducing strain, we developed an integrated high-throughput screening assay using ribosome engineering technology. The production protocol was scaled down to 24- and 48-deep-well microtiter plates (MTPs). The microplate reader assay was used to monitor ε-PL production. A good correlation was observed between the fermentation results obtained in both 24-(48)-deep-well MTPs and conventional Erlenmeyer flasks. Using this protocol, the production of ε-PL in an entire MTP was determined in <5 min without compromising on accuracy. The high-yielding strain selected through this protocol was also tested in Erlenmeyer flasks. The result showed that the ε-PL production of the high-yielding mutants was nearly 45% higher than that of the parent stain. Thus, development of this protocol is expected to accelerate the selection of ε-PL-overproducing strains.

Disposable luciferase-based microfluidic chip for rapid assay of water pollution.

PubMed

Denisov, Ivan; Lukyanenko, Kirill; Yakimov, Anton; Kukhtevich, Igor; Esimbekova, Elena; Belobrov, Peter

2018-06-21

In the present study, we demonstrate the use of a disposable luciferase-based microfluidic bioassay chip for environmental monitoring and methods for fabrication. The designed microfluidic system includes a chamber with immobilized enzymes of bioluminescent bacteria Photobacterium leiognathi and Vibrio fischeri and their substrates, which dissolve after the introduction of the water sample and thus activate bioluminescent reactions. Limits of detection for copper (II) sulfate, 1,3-dihydroxybenzene and 1,4-benzoquinone for the proposed microfluidic biosensor measured 3 μM, 15 mM, and 2 μM respectively, and these values are higher or close to the level of conventional environmental biosensors based on lyophilized bacteria. Approaches for entrapment of enzymes on poly(methyl methacrylate) (PMMA) plates using a gelatin scaffold and solvent bonding of PMMA chip plates under room temperature were suggested. The proposed microfluidic system may be used with some available luminometers and future portable luminescence readers. © 2018 John Wiley & Sons, Ltd.

Testing a dual-fluorescence assay to monitor the viability of filamentous cyanobacteria.

PubMed

Johnson, Tylor J; Hildreth, Michael B; Gu, Liping; Zhou, Ruanbao; Gibbons, William R

2015-06-01

Filamentous cyanobacteria are currently being engineered to produce long-chain organic compounds, including 3rd generation biofuels. Because of their filamentous morphology, standard methods to quantify viability (e.g., plate counts) are not possible. This study investigated a dual-fluorescence assay based upon the LIVE/DEAD® BacLight™ Bacterial Viability Kit to quantify the percent viability of filamentous cyanobacteria using a microplate reader in a high throughput 96-well plate format. The manufacturer's protocol calls for an optical density normalization step to equalize the numbers of viable and non-viable cells used to generate calibration curves. Unfortunately, the isopropanol treatment used to generate non-viable cells released a blue pigment that altered absorbance readings of the non-viable cell solution, resulting in an inaccurate calibration curve. Thus we omitted this optical density normalization step, and carefully divided cell cultures into two equal fractions before the isopropanol treatment. While the resulting calibration curves had relatively high correlation coefficients, their use in various experiments resulted in viability estimates ranging from below 0% to far above 100%. We traced this to the apparent inaccuracy of the propidium iodide (PI) dye that was to stain only non-viable cells. Through further analysis via microplate reader, as well as confocal and wide-field epi-fluorescence microscopy, we observed non-specific binding of PI in viable filamentous cyanobacteria. While PI will not work for filamentous cyanobacteria, it is possible that other fluorochrome dyes could be used to selectively stain non-viable cells. This will be essential in future studies for screening mutants and optimizing photobioreactor system performance for filamentous cyanobacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.

PubMed

Maxwell, Joshua T; Tsai, Chin-Hsien; Mohiuddin, Tahmina A; Kwong, Jennifer Q

2018-04-27

Ca 2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca 2+ from mitochondria is important for determining the role of mitochondrial Ca 2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca 2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca 2+ uptake using the Ca 2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca 2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca 2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca 2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca 2+ in permeabilized cells using the Ca 2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca 2+ . Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca 2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca 2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

CT radiation profile width measurement using CR imaging plate raw data

PubMed Central

Yang, Chang‐Ying Joseph

2015-01-01

This technical note demonstrates computed tomography (CT) radiation profile measurement using computed radiography (CR) imaging plate raw data showing it is possible to perform the CT collimation width measurement using a single scan without saturating the imaging plate. Previously described methods require careful adjustments to the CR reader settings in order to avoid signal clipping in the CR processed image. CT radiation profile measurements were taken as part of routine quality control on 14 CT scanners from four vendors. CR cassettes were placed on the CT scanner bed, raised to isocenter, and leveled. Axial scans were taken at all available collimations, advancing the cassette for each scan. The CR plates were processed and raw CR data were analyzed using MATLAB scripts to measure collimation widths. The raw data approach was compared with previously established methodology. The quality control analysis scripts are released as open source using creative commons licensing. A log‐linear relationship was found between raw pixel value and air kerma, and raw data collimation width measurements were in agreement with CR‐processed, bit‐reduced data, using previously described methodology. The raw data approach, with intrinsically wider dynamic range, allows improved measurement flexibility and precision. As a result, we demonstrate a methodology for CT collimation width measurements using a single CT scan and without the need for CR scanning parameter adjustments which is more convenient for routine quality control work. PACS numbers: 87.57.Q‐, 87.59.bd, 87.57.uq PMID:26699559

Identification of non-streptococcal organisms from human dental plaque grown on the Streptococcus-selective medium mitis-salivarius agar.

PubMed

Kim, Yeon-Hee; Lee, Si Young

2015-02-01

Mitis-salivarius (MS) agar has been used widely in microbial epidemiological studies because oral viridans streptococci can be selectively grown on this medium. Even though the previous findings reported the limited selecting power of MS agar for streptococcus strains, the identities of non-streptococcal strains from human oral samples which can grow on this medium are not clear yet. In this study, we identified non-streptococcal organisms grown on MS agar plates by polymerase chain reaction (PCR) amplification and sequencing of the 16S ribosomal RNA (rRNA) gene. Eighty bacterial colonies on MS plates were isolated from plaque samples, and bacterial identification was achieved with the rapid ID 32 Strep system and mini API reader. The bacterial colonies identified as non-streptococci by the API system were selected for further identification. The 16S rRNA gene was amplified by PCR and verified using DNA sequencing analysis for identification. Sequences were compared with those of reference organisms in the genome database of the National Center for Biotechnology Information using the Basic Local Alignment Search Tool (BLAST). Among the 11 isolated non-streptococcal strains on MS plates, 3 strains were identified as Actinomyces naeslundii, 7 strains were identified as Actinomyces oris and 1 strain were identified as Actinomyces sp. using Blastn. In this study, we showed that some oral Actinomyces species can grow on Streptococcus-selective MS agar plates. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evaluation and optimization of compound solubilization and delivery methods in a two-tiered ion channel lead optimization triage.

PubMed

Hendricson, Adam W; Gallagher, Liz; Matchett, Michele; Ferrante, Meredith; Spence, Steve; Paiva, Tony; Shou, Wilson; Tertyshnikova, Svetlana; Krambis, Mike; Post-Munson, Deborah; Zhang, Litao; Knox, Ron

2012-04-01

Low-volume dispensing of neat dimethyl sulfoxide (DMSO) into plate-based assays conserves compound, assay reagents, and intermediate dilution plate cost and, as we demonstrate here, significantly improves structure-activity relationship resolution. Acoustic dispensing of DMSO solutions into standard volume 384W plates yielded inconsistent results in studies with 2 cell lines because of apparent effects on the integrity of the cell monolayer (increased intracellular Ca⁺⁺ levels as indicated by elevated basal dye fluorescence after acoustic transfer). PocketTip-mediated transfer was successful at increasing apparent potency on a more consistent basis. Notably, the correlation coefficient among fluorescence imaging plate reader (FLIPR):electrophysiology (EP) across a representative ~125 compound collection was increased ~5× via conversion to a PocketTip direct dispensation, indicating a triage assay more predictive of activity in the decisional patch-clamp assay. Very importantly, the EP-benchmarked false-negative rate as measured by compounds with FLIPR EC₅₀ more than the highest concentration tested fell from >11% to 5% assay-wide, and the relative FLIPR:EP rank-order fidelity increased from 55% to 78%. Elimination of the aqueous intermediate step provided additional benefits, including reduced assay cost, decreased cycle time, and reduced wet compound consumption rate. Direct DMSO dispensing has broad applicability to cell-based functional assays of multiple varieties, especially in cases where limit solubility in assay buffer is a recognized impediment to maximizing interassay connectivity.

A 3D-Printed, Portable, Optical-Sensing Platform for Smartphones Capable of Detecting the Herbicide 2,4-Dichlorophenoxyacetic Acid.

PubMed

Wang, Yijia; Zeinhom, Mohamed M A; Yang, Mingming; Sun, Rongrong; Wang, Shengfu; Smith, Jordan N; Timchalk, Charles; Li, Lei; Lin, Yuehe; Du, Dan

2017-09-05

Onsite rapid detection of herbicides and herbicide residuals in environmental and biological specimens are important for agriculture, environmental concerns, food safety, and health care. The traditional method for herbicide detection requires expensive laboratory equipment and a long turnaround time. In this work, we developed a single-stripe microliter plate smartphone-based colorimetric device for rapid and low-cost in-field tests. This portable smartphone platform is capable of screening eight samples in a single-stripe microplate. The device combined the advantages of small size (50 × 100 × 160 mm 3 ) and low cost ($10). The platform was calibrated by using two different dye solutions, i.e. methyl blue (MB) and rhodamine B, for the red and green channels. The results showed good correlation with results attained from a traditional laboratory reader. We demonstrated the application of this platform for detection of the herbicide 2,4-dichlorophenoxyacetic acid in the range of 1 to 80 ppb. Spiked samples of tap water, rat serum, plasma, and human serum were tested by our device. Recoveries obtained varied from 95.6% to 105.2% for all of the spiked samples using the microplate reader and from 93.7% to 106.9% for all of the samples using the smartphone device. This work validated that the smartphone optical-sensing platform is comparable to the commercial microplate reader; it is eligible for onsite, rapid, and low-cost detection of herbicides for environmental evaluation and biological monitoring.

A 3D-Printed, Portable, Optical-Sensing Platform for Smartphones Capable of Detecting the Herbicide 2,4-Dichlorophenoxyacetic Acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yijia; Zeinhom, Mohamed M. A.; Yang, Mingming

Onsite rapid detection of herbicide and herbicide residuals in environmental and biological specimens is important for agriculture, environment, food safety, and health care. Traditional method for herbicide detection requires expensive laboratory equipment and a long turn-round time. In this work, we developed a single-stripe microliter plate smartphone colorimetric device for rapid and low-cost in-field test. This portable smartphone platform is capable of screening 8 samples in a microplate single-stripe. The device combined the advantages of small size (50×100×160 mm3) and low cost ($10). The platform was calibrated by using two different dye solutions, i.e. methyl blue (MB) and Rhodamine B,more » for green and red channels. The results showed good correlation with results attained from a traditional laboratory reader. We demonstrated the application of this platform for an herbicide, 2,4-Dichlorophenoxyacetic acid detection in the range of 1 ppb to 80 ppb. Spiked samples of tap water, rat serum, plasma and human serum were tested by our device. Recoveries obtained varied from 95.6% to 105.2% for all spiked samples using the microplate reader and from 93.7% to 106.9% using the smartphone device. This work validated that the smartphone optical sensing platform is comparable to the commercial microplate reader, it is eligible for onsite rapid and low-cost detection of herbicide for environmental evaluation and biological monitoring.« less

Teaching And Learning Tectonics With Web-GIS

NASA Astrophysics Data System (ADS)

Anastasio, D. J.; Sahagian, D. L.; Bodzin, A.; Teletzke, A. L.; Rutzmoser, S.; Cirucci, L.; Bressler, D.; Burrows, J. E.

2012-12-01

Tectonics is a new curriculum enhancement consisting of six Web GIS investigations designed to augment a traditional middle school Earth science curriculum. The investigations are aligned to Disciplinary Core Ideas: Earth and Space Science from the National Research Council's (2012) Framework for K-12 Science Education and to tectonics benchmark ideas articulated in the AAAS Project 2061 (2007) Atlas of Science Literacy. The curriculum emphasizes geospatial thinking and scientific inquiry and consists of the following modules: Geohazards, which plate boundary is closest to me? How do we recognize plate boundaries? How does thermal energy move around the Earth? What happens when plates diverge? What happens when plate move sideways past each other? What happens when plates collide? The Web GIS interface uses JavaScript for simplicity, intuition, and convenience for implementation on a variety of platforms making it easier for diverse middle school learners and their teachers to conduct authentic Earth science investigations, including multidisciplinary visualization, analysis, and synthesis of data. Instructional adaptations allow students who are English language learners, have disabilities, or are reluctant readers to perform advanced desktop GIS functions including spatial analysis, map visualization and query. The Web GIS interface integrates graphics, multimedia, and animation in addition to newly developed features, which allow users to explore and discover geospatial patterns that would not be easily visible using typical classroom instructional materials. The Tectonics curriculum uses a spatial learning design model that incorporates a related set of frameworks and design principles. The framework builds on the work of other successful technology-integrated curriculum projects and includes, alignment of materials and assessments with learning goals, casting key ideas in real-world problems, engaging students in scientific practices that foster the use of key ideas, uses geospatial technology, and supports for teachers in adopting and implementing GIS and inquiry-based activities.

Determination of total procyanidins in selected chocolate and confectionery products using DMAC.

PubMed

Payne, Mark J; Hurst, William Jeffrey; Stuart, David A; Ou, Boxin; Fan, Ellen; Ji, Hongping; Kou, Yan

2010-01-01

A simple, specific, high-throughput colorimetric method based on the reaction of 4-dimethylaminocinnamaldehyde (DMAC) with flavan-3-ols was developed to determine total procyanidins in selected cacao-based products. Extracts of defatted samples were dispensed into a 96-well plate and reacted with DMAC. The absorbance of the reaction products was measured at 640 nm and compared to commercially available procyanidin B2 as a standard. The use of the 96-well plates and a plate reader dramatically improved sample throughput. A standard protocol was established and used for further studies. The calibration was found to be linear from 1-100 ppm. The DMAC reagent reacted relatively specifically to (-)-epicatechin, (+)-catechin, epigallocatechin, gallocatechin, the gallates of catechin, epicatechin, gallocatechin, and epigallocatechin, oligomeric procyanidins of cocoa up to n=4, and A-type procyanidins. Little or no reaction occurred with cyanidins and representative compounds of phenolic acids, flavones, flavanones, flavonols, anthocyanidins, isoflavones, and stilbenes. Sample precision studies were carried out on 10 different test materials over several weeks, and yielded RSD values of 4.0 to 9.5%. The method was ring-tested in three laboratories using blinded test materials including cocoa beans, cocoa powder, chocolate liquor, dark chocolate, and milk chocolate. There was excellent agreement of the results between laboratories.

Selectivity verification of cardiac troponin monoclonal antibodies for cardiac troponin detection by using conventional ELISA

NASA Astrophysics Data System (ADS)

Fathil, M. F. M.; Arshad, M. K. Md; Gopinath, Subash C. B.; Adzhri, R.; Ruslinda, A. R.; Hashim, U.

2017-03-01

This paper presents preparation and characterization of conventional enzyme-linked immunosorbent assay (ELISA) for cardiac troponin detection to determine the selectivity of the cardiac troponin monoclonal antibodies. Monoclonal antibodies, used to capture and bind the targets in this experiment, are cTnI monoclonal antibody (MAb-cTnI) and cTnT monoclonal antibody (MAb-cTnT), while both cardiac troponin I (cTnI) and T (cTnT) are used as targets. ELISA is performed inside two microtiter plates for MAb-cTnI and MAb-cTnT. For each plate, monoclonal antibodies are tested by various concentrations of cTnI and cTnT ranging from 0-6400 µg/l. The binding selectivity and level of detection between monoclonal antibodies and antigen are determined through visual observation based on the color change inside each well on the plate. ELISA reader is further used to quantitatively measured the optical density of the color changes, thus produced more accurate reading. The results from this experiment are utilized to justify the use of these monoclonal antibodies as bio-receptors for cardiac troponin detection by using field-effect transistor (FET)-based biosensors coupled with substrate-gate in the future.

Female parthenogenetic apomixis and androsporogenetic parthenogenesis in embryonal cells of Araucaria angustifolia: interpolation of progenesis and asexual heterospory in an artificial sporangium.

PubMed

Durzan, Don J

2012-09-01

Cell fate, development timing and occurrence of reproductive versus apomictic development in gymnosperms are shown to be influenced by culture conditions in vitro. In this study, female parthenogenetic apomixis (fPA), androsporogenetic parthenogenesis (mAP) and progenesis were demonstrated using embryonal initials of Araucaria angustifolia in scaled-up cell suspensions passing through a single-cell bottleneck in darkness and in an artificial sporangium (AS). Expression was based on defined nutrition, hormones and feedforward-adaptive feedback process controls at 23-25 °C and in darkness. In fPA, the nucleus of an embryonal initial undergoes endomitosis and amitosis, forming a diploid egg-equivalent and an apoptotic ventral canal nucleus in a transdifferentiated archegonial tube. Discharge of egg-equivalent cells as parthenospores and their dispersal into the aqueous culture medium were followed by free-nuclear conifer-type proembryogenesis. This replaced the plesiomorphic and central features of proembryogenesis in Araucariaceae. Protoplasmic fusions of embryonal initials were used to reconstruct heterokaryotic expressions of fPA in multiwell plates. In mAP, restitutional meiosis (automixis) was responsible for androsporogenesis and the discharge of monads, dyads, tetrads and polyads. In a display of progenesis, reproductive development was brought to an earlier ontogenetic stage and expressed by embryonal initials. Colchicine increased polyploidy, but androspore formation became aberrant and fragmented. Aberrant automixis led to the formation of chromosomal bouquets, which contributed to genomic silencing in embryonal initials, cytomixis and the formation of pycnotic micronucleated cells. Dispersal of female and male parthenospores displayed heteromorphic asexual heterospory in an aqueous environment.

In situ DMSO hydration measurements of HTS compound libraries.

PubMed

Ellson, R; Stearns, R; Mutz, M; Brown, C; Browning, B; Harris, D; Qureshi, S; Shieh, J; Wold, D

2005-09-01

Compounds used in high throughput screening (HTS) are typically dissolved in DMSO. These solutions are stored automation-friendly racks of wells or tubes. DMSO is hygroscopic and quickly absorbs water from the atmosphere. When present in DMSO compound solutions, water can accelerate degradation and precipitation. Understanding DMSO hydration in an HTS compound library can improve storage and screening methods by managing the impact of water on compound stability. A non-destructive, acoustic method compatible with HTS has been developed to measure water content in DMSO solutions. Performance of this acoustic method was compared with an optical technique and found to be in good agreement. The accuracy and precision of acoustic measurements was shown to be under 3% over the tested range of DMSO solutions (0% to 35% water by volume) and insensitive to the presence of HTS compounds at typical storage concentrations. Time course studies of hydration for wells in 384-well and 1536-well microplates were performed. Well geometry, fluid volume, well position and atmospheric conditions were all factors in hydration rate. High rates of hydration were seen in lower-volume fills, higher-density multi-well plates and when there was a large differential between the humidity of the lab and the water content of the DMSO. For example, a 1536-well microplate filled with 2microL of 100% DMSO exposed for one hour to a laboratory environment with approximately 40% relative humidity will absorb over 6% water by volume. Understanding DMSO hydration rates as well as the ability to reverse library hydration are important steps towards managing stability and availability of compound libraries.

Micropatterned Cell–Cell Interactions Enable Functional Encapsulation of Primary Hepatocytes in Hydrogel Microtissues

PubMed Central

Li, Cheri Y.; Stevens, Kelly R.; Schwartz, Robert E.; Alejandro, Brian S.; Huang, Joanne H.

2014-01-01

Drug-induced liver injury is a major cause of drug development failures and postmarket withdrawals. In vitro models that incorporate primary hepatocytes have been shown to be more predictive than model systems which rely on liver microsomes or hepatocellular carcinoma cell lines. Methods to phenotypically stabilize primary hepatocytes ex vivo often rely on mimicry of hepatic microenvironmental cues such as cell–cell interactions and cell–matrix interactions. In this work, we sought to incorporate phenotypically stable hepatocytes into three-dimensional (3D) microtissues, which, in turn, could be deployed in drug-screening platforms such as multiwell plates and diverse organ-on-a-chip devices. We first utilize micropatterning on collagen I to specify cell–cell interactions in two-dimensions, followed by collagenase digestion to produce well-controlled aggregates for 3D encapsulation in polyethylene glycol (PEG) diacrylate. Using this approach, we examined the influence of homotypic hepatocyte interactions and composition of the encapsulating hydrogel, and achieved the maintenance of liver-specific function for over 50 days. Optimally preaggregated structures were subsequently encapsulated using a microfluidic droplet-generator to produce 3D microtissues. Interactions of engineered hepatic microtissues with drugs was characterized by flow cytometry, and yielded both induction of P450 enzymes in response to prototypic small molecules and drug–drug interactions that give rise to hepatotoxicity. Collectively, this study establishes a pipeline for the manufacturing of 3D hepatic microtissues that exhibit stabilized liver-specific functions and can be incorporated into a wide array of emerging drug development platforms. PMID:24498910

High-throughput on-chip in vivo neural regeneration studies using femtosecond laser nano-surgery and microfluidics

NASA Astrophysics Data System (ADS)

Rohde, Christopher B.; Zeng, Fei; Gilleland, Cody; Samara, Chrysanthi; Yanik, Mehmet F.

2009-02-01

In recent years, the advantages of using small invertebrate animals as model systems for human disease have become increasingly apparent and have resulted in three Nobel Prizes in medicine or chemistry during the last six years for studies conducted on the nematode Caenorhabditis elegans (C. elegans). The availability of a wide array of species-specific genetic techniques, along with the transparency of the worm and its ability to grow in minute volumes make C. elegans an extremely powerful model organism. We present a suite of technologies for complex high-throughput whole-animal genetic and drug screens. We demonstrate a high-speed microfluidic sorter that can isolate and immobilize C. elegans in a well-defined geometry, an integrated chip containing individually addressable screening chambers for incubation and exposure of individual animals to biochemical compounds, and a device for delivery of compound libraries in standard multiwell plates to microfluidic devices. The immobilization stability obtained by these devices is comparable to that of chemical anesthesia and the immobilization process does not affect lifespan, progeny production, or other aspects of animal health. The high-stability enables the use of a variety of key optical techniques. We use this to demonstrate femtosecond-laser nanosurgery and three-dimensional multiphoton microscopy. Used alone or in various combinations these devices facilitate a variety of high-throughput assays using whole animals, including mutagenesis and RNAi and drug screens at subcellular resolution, as well as high-throughput high-precision manipulations such as femtosecond-laser nanosurgery for large-scale in vivo neural degeneration and regeneration studies.

Single-particle imaging for biosensor applications

NASA Astrophysics Data System (ADS)

Yorulmaz, Mustafa; Isil, Cagatay; Seymour, Elif; Yurdakul, Celalettin; Solmaz, Berkan; Koc, Aykut; Ünlü, M. Selim

2017-10-01

Current state-of-the-art technology for in-vitro diagnostics employ laboratory tests such as ELISA that consists of a multi-step test procedure and give results in analog format. Results of these tests are interpreted by the color change in a set of diluted samples in a multi-well plate. However, detection of the minute changes in the color poses challenges and can lead to false interpretations. Instead, a technique that allows individual counting of specific binding events would be useful to overcome such challenges. Digital imaging has been applied recently for diagnostics applications. SPR is one of the techniques allowing quantitative measurements. However, the limit of detection in this technique is on the order of nM. The current required detection limit, which is already achieved with the analog techniques, is around pM. Optical techniques that are simple to implement and can offer better sensitivities have great potential to be used in medical diagnostics. Interference Microscopy is one of the tools that have been investigated over years in optics field. More of the studies have been performed in confocal geometry and each individual nanoparticle was observed separately. Here, we achieve wide-field imaging of individual nanoparticles in a large field-of-view ( 166 μm × 250 μm) on a micro-array based sensor chip in fraction of a second. We tested the sensitivity of our technique on dielectric nanoparticles because they exhibit optical properties similar to viruses and cells. We can detect non-resonant dielectric polystyrene nanoparticles of 100 nm. Moreover, we perform post-processing applications to further enhance visibility.

Adapting the γ-H2AX assay for automated processing in human lymphocytes. 1. Technological aspects.

PubMed

Turner, Helen C; Brenner, David J; Chen, Youhua; Bertucci, Antonella; Zhang, Jian; Wang, Hongliang; Lyulko, Oleksandra V; Xu, Yanping; Shuryak, Igor; Schaefer, Julia; Simaan, Nabil; Randers-Pehrson, Gerhard; Yao, Y Lawrence; Amundson, Sally A; Garty, Guy

2011-03-01

The immunofluorescence-based detection of γ-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this well-established biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated high-throughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure γ-H2AX yields from fingerstick-derived samples of blood. The RABIT workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparin-coated PVC capillaries to the immunolabeling of γ-H2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purpose-built robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of γ-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in γ-H2AX fluorescence in peripheral blood samples irradiated ex vivo with γ rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify γ-H2AX total fluorescence in irradiated peripheral lymphocytes.

Chemical composition, antioxidant and antimicrobial effects of Tunisian Limoniastrum guyonianum Durieu ex Boiss extracts.

PubMed

Bouzidi, Amel; Benzarti, Anissa; Arem, Amira El; Mahfoudhi, Adel; Hammami, Saoussen; Gorcii, Mohamed; Mastouri, Maha; Hellal, Ahmed Noureddine; Mighri, Zine

2016-07-01

In the present investigation, extracts obtained from L. guyonianum Durieu ex Boiss. aerial parts were used to evaluate total phenolic, flavonoid and tannin contents. A study of antioxidant activities of the prepared samples was carried out on the basis of 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2-2'-azino-bis-(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS+.) and ferric reducing antioxidant power (FRAP) assays. Moreover, the efficiency of methanolic, chloroformic and petroleum ether extracts and the deriving fractions from the methanolic extract was tested against human bacterial and fungal pathogenic strains using micro dilution method in 96 multiwell microtiter plate. Furthermore, leaves and stems extracts were subjected to RP-HPLC for phenolic compounds identification. Results showed that polyphenolic contents and antioxidant activities varied considerably as function of solvent polarity. Moreover, antiradical capacities against DPPH, ABTS(+.) and reducing power were maxima in methanol aerial parts extract which showed the highest polyphenol contents (134mg CE/g DW). The antimicrobial activities showed that methanolic, chloroformic and petroleum ether extracts were found to be most potent against Pseudomonas aeruginosa and Staphylococcus aureus with MIC values of 23 and 46μ.mL(-1), respectively. The fractions F(13) and F(16) have a great antifungal potential against Candida glabrata, Candida krusei and Candida parapsilesis (MIC=39μ.mL(-1)). The RP-HPLC analysis lead the identification of gallic, procatechuic and trans-cinnamic acids, methyl-4-hydroxybenzoate, n-propyl-3,4,5-trihydroxybenzoate, epicatechin, naringin and myricetin in L. guyonianum Durieu ex Boiss. leaves and stems extracts.

Improving Cardiac Action Potential Measurements: 2D and 3D Cell Culture.

PubMed

Daily, Neil J; Yin, Yue; Kemanli, Pinar; Ip, Brian; Wakatsuki, Tetsuro

2015-11-01

Progress in the development of assays for measuring cardiac action potential is crucial for the discovery of drugs for treating cardiac disease and assessing cardiotoxicity. Recently, high-throughput methods for assessing action potential using induced pluripotent stem cell (iPSC) derived cardiomyocytes in both two-dimensional monolayer cultures and three-dimensional tissues have been developed. We describe an improved method for assessing cardiac action potential using an ultra-fast cost-effective plate reader with commercially available dyes. Our methods improve dramatically the detection of the fluorescence signal from these dyes and make way for the development of more high-throughput methods for cardiac drug discovery and cardiotoxicity.

In-situ stress measurements at DOE's Multi-Well Experiment site, Mesaverde Group, Rifle, CO

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warpinski, N.R.

Measurements of the vertical distribution of the minimum principal in situ stress in the lower Mesaverde group (7300 to 8100 ft depth) at DOE's Multi-Well Experiment site have been made by conducting small-volume, hydraulic-fracture stress tests through perforations. Accurate, reproducible results were obtained by conducting a number of repeat injections in each zone of interest using a specially designed pump system, modified high-resolution electronic equipment, and a down-hole shut-off tool with a bottom-hole pressure transducer. Stress tests were conducted in marine sandstones and shales as well as a coal, mudstone, and sandstone in a paludal depositional environment; these tests providemore » a detailed stress distribution in this region. The stress magnitudes were found to be dependent on lithology. Marine shales above and below the blanket sands have large horizontal stress - near lithostatic with a frac gradient greater than 1.0 psi/ft (23 kPa/m). This indicates that these rocks do not behave elastically and processes such as creep and possibly fracturing are the dominant mechanisms controlling the stress state. On the other hand, sandstones and siltstones have much lower stresses with a frac gradient of 0.85 to 0.9 psi/ft (19 to 20 kPa/m). Containment of hydraulic fractures would be expected under these conditions. Only three data points were obtained from the paludal interval; no significant stress differences were observed in the different lithologies. 19 references, 8 figures, 1 table.« less

Diagnostic performance of direct traction MR arthrography of the hip: detection of chondral and labral lesions with arthroscopic comparison.

PubMed

Schmaranzer, Florian; Klauser, Andrea; Kogler, Michael; Henninger, Benjamin; Forstner, Thomas; Reichkendler, Markus; Schmaranzer, Ehrenfried

2015-06-01

To assess diagnostic performance of traction MR arthrography of the hip in detection and grading of chondral and labral lesions with arthroscopic comparison. Seventy-five MR arthrograms obtained ± traction of 73 consecutive patients (mean age, 34.5 years; range, 14-54 years) who underwent arthroscopy were included. Traction technique included weight-adapted traction (15-23 kg), a supporting plate for the contralateral leg, and intra-articular injection of 18-27 ml (local anaesthetic and contrast agent). Patients reported on neuropraxia and on pain. Two blinded readers independently assessed femoroacetabular cartilage and labrum lesions which were correlated with arthroscopy. Interobserver agreement was calculated using κ values. Joint distraction ± traction was evaluated in consensus. No procedure had to be stopped. There were no cases of neuropraxia. Accuracy for detection of labral lesions was 92 %/93 %, 91 %/83 % for acetabular lesions, and 92 %/88 % for femoral cartilage lesions for reader 1/reader 2, respectively. Interobserver agreement was moderate (κ = 0.58) for grading of labrum lesions and substantial (κ = 0.7, κ = 0.68) for grading of acetabular and femoral cartilage lesions. Joint distraction was achieved in 72/75 and 14/75 hips with/without traction, respectively. Traction MR arthrography safely enabled accurate detection and grading of labral and chondral lesions. • The used traction technique was well tolerated by most patients. • The used traction technique almost consistently achieved separation of cartilage layers. • Traction MR arthrography enabled accurate detection of chondral and labral lesions.

A fluorescent microplate assay for diarrheic shellfish toxins.

PubMed

Vieytes, M R; Fontal, O I; Leira, F; Baptista de Sousa, J M; Botana, L M

1997-06-01

A fluorescent enzyme inhibition assay for okadaic acid using 4-methylumbelliferyl phosphate and fluorescein diphosphate as substrates for the enzyme phosphatase 2A was developed. In the inhibition assay, performed in a microtiter plate, the PP2A was inhibited by adding okadaic acid and the resulting fluorescence enhancement derived from enzymatic hydrolysis of the substrate was quantified in a fluorescence plate reader. The measurable range of okadaic acid was 3.2 to 3200 pg/ml with an IC50 = 0.1 nM. The detection limit of okadaic acid was 2.56 pg/well in buffer solutions and 12.8 ng/g hepatopancreas in shellfish extracts. The coefficient of variation (CV, n = 22) for each point ranged from 18.80 to 37.90% (mean 28.35%). The proposed method is very convenient, rapid, and sensitive by using the enzyme inhibition assay system and fluorescent reaction as a detection system. This work demonstrates that the fluorescent assay can be used to quantify the amount of okadaic acid in shellfish samples and also is valid for very dilute samples, such as phytoplankton samples.

Parametric study of beam refraction problems across laser anemometer windows

NASA Technical Reports Server (NTRS)

Owen, A. K.

1986-01-01

The experimenter is often required to view flows through a window with a different index of refraction than either the medium being observed or the medium that the laser anemometer is immersed in. The refraction that occurs at the window surfaces may lead to undesirable changes in probe volume position or beam crossing angle and can lead to partial or complete beam uncrossing. This report describes the results of a parametric study of this problem using a ray tracing technique to predict these changes. The windows studied were a flat plate and a simple cyclinder. For the flat-plate study: (1) surface thickness, (2) beam crossing angle, (3) bisecting line - surface normal angle, and (4) incoming beam plane surface orientation were varied. For the cylindrical window additional parameters were also varied: (1) probe volume immersion, (2) probe volume off-radial position, and (3) probe volume position out of the R-theta plane of the lens. A number of empirical correlations were deduced to aid the interested reader in determining the movement, uncrossing, and change in crossing angle for a particular situation.

A parametric study of the beam refraction problems across laser anemometer windows

NASA Technical Reports Server (NTRS)

Owen, Albert K.

1986-01-01

The experimenter is often required to view flows through a window with a different index of refraction than either the medium being observed or the medium that the laser anemometer is immersed in. The refraction that occurs at the window surfaces may lead to undesirable changes in probe volume position or beam crossing angle and can lead to partial or complete beam uncrossing. This report describes the results of a parametric study of this problem using a ray tracing technique to predict these changes. The windows studied were a flat plate and a simple cylinder. For the flat-plate study: (1) surface thickness, (2) beam crossing angle, (3) bisecting line - surface normal angle, and (4) incoming beam plane surface orientation were varied. For the cylindrical window additional parameters were also varied: (1) probe volume immersion, (2) probe volume off-radial position, and (3) probe volume position out of the r-theta plane of the lens. A number of empirical correlations were deduced to aid the reader in determining the movement, uncrossing, and change in crossing angle for a particular situations.

Ferroelectric tunnel junctions with multi-quantum well structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Zhijun; Zhang, Tianjin, E-mail: zhangtj@hubu.edu.cn; Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Hubei University, Wuhan 430062

Ferroelectric tunnel junctions (FTJs) with multi-quantum well structures are proposed and the tunneling electroresistance (TER) effect is investigated theoretically. Compared with conventional FTJs with monolayer ferroelectric barriers, FTJs with single-well structures provide TER ratio improvements of one order of magnitude, while FTJs with optimized multi-well structures can enhance this improvement by another order of magnitude. It is believed that the increased resonant tunneling strength combined with appropriate asymmetry in these FTJs contributes to the improvement. These studies may help to fabricate FTJs with large TER ratio experimentally and put them into practice.

Echo spectroscopy of TLS of multiwell adiabatic potential for Pr3+ activator centers in Y2SiO5

NASA Astrophysics Data System (ADS)

Malyukin, Yuri V.; Borysov, R. S.; Zhmurin, P. N.; Lebedenko, A. N.; Grinyov, Boris V.; Znamenskii, N. I.; Manykin, Eduard A.; Orlov, Yu. V.; Petrenko, E. A.; Yukina, T. G.

2002-05-01

Based on the investigation of the temperature dependence of the two-pulse phonon echo amplitude on the 3H4 3Po resonant optical transition of the Pr3+ doped ions in the Y2SiO5 crystal, unusual for crystals, low-temperature mechanisms of Pr3+ spectral line broadening caused by the interaction of doped ions with TLS have been found. The constants characterizing the interaction of the doped Pr3+ ions with phonons and TLS have been determined.

Image stacking approach to increase sensitivity of fluorescence detection using a low cost complementary metal-oxide-semiconductor (CMOS) webcam.

PubMed

Balsam, Joshua; Bruck, Hugh Alan; Kostov, Yordan; Rasooly, Avraham

2012-01-01

Optical technologies are important for biological analysis. Current biomedical optical analyses rely on high-cost, high-sensitivity optical detectors such as photomultipliers, avalanched photodiodes or cooled CCD cameras. In contrast, Webcams, mobile phones and other popular consumer electronics use lower-sensitivity, lower-cost optical components such as photodiodes or CMOS sensors. In order for consumer electronics devices, such as webcams, to be useful for biomedical analysis, they must have increased sensitivity. We combined two strategies to increase the sensitivity of CMOS-based fluorescence detector. We captured hundreds of low sensitivity images using a Webcam in video mode, instead of a single image typically used in cooled CCD devices.We then used a computational approach consisting of an image stacking algorithm to remove the noise by combining all of the images into a single image. While video mode is widely used for dynamic scene imaging (e.g. movies or time-lapse photography), it is not used to capture a single static image, which removes noise and increases sensitivity by more than thirty fold. The portable, battery-operated Webcam-based fluorometer system developed here consists of five modules: (1) a low cost CMOS Webcam to monitor light emission, (2) a plate to perform assays, (3) filters and multi-wavelength LED illuminator for fluorophore excitation, (4) a portable computer to acquire and analyze images, and (5) image stacking software for image enhancement. The samples consisted of various concentrations of fluorescein, ranging from 30 μM to 1000 μM, in a 36-well miniature plate. In the single frame mode, the fluorometer's limit-of-detection (LOD) for fluorescein is ∼1000 μM, which is relatively insensitive. However, when used in video mode combined with image stacking enhancement, the LOD is dramatically reduced to 30 μM, sensitivity which is similar to that of state-of-the-art ELISA plate photomultiplier-based readers. Numerous medical diagnostics assays rely on optical and fluorescence readers. Our novel combination of detection technologies, which is new to biodetection may enable the development of new low cost optical detectors based on an inexpensive Webcam (<$10). It has the potential to form the basis for high sensitivity, low cost medical diagnostics in resource-poor settings.

Image stacking approach to increase sensitivity of fluorescence detection using a low cost complementary metal-oxide-semiconductor (CMOS) webcam

PubMed Central

Balsam, Joshua; Bruck, Hugh Alan; Kostov, Yordan; Rasooly, Avraham

2013-01-01

Optical technologies are important for biological analysis. Current biomedical optical analyses rely on high-cost, high-sensitivity optical detectors such as photomultipliers, avalanched photodiodes or cooled CCD cameras. In contrast, Webcams, mobile phones and other popular consumer electronics use lower-sensitivity, lower-cost optical components such as photodiodes or CMOS sensors. In order for consumer electronics devices, such as webcams, to be useful for biomedical analysis, they must have increased sensitivity. We combined two strategies to increase the sensitivity of CMOS-based fluorescence detector. We captured hundreds of low sensitivity images using a Webcam in video mode, instead of a single image typically used in cooled CCD devices.We then used a computational approach consisting of an image stacking algorithm to remove the noise by combining all of the images into a single image. While video mode is widely used for dynamic scene imaging (e.g. movies or time-lapse photography), it is not used to capture a single static image, which removes noise and increases sensitivity by more than thirty fold. The portable, battery-operated Webcam-based fluorometer system developed here consists of five modules: (1) a low cost CMOS Webcam to monitor light emission, (2) a plate to perform assays, (3) filters and multi-wavelength LED illuminator for fluorophore excitation, (4) a portable computer to acquire and analyze images, and (5) image stacking software for image enhancement. The samples consisted of various concentrations of fluorescein, ranging from 30 μM to 1000 μM, in a 36-well miniature plate. In the single frame mode, the fluorometer's limit-of-detection (LOD) for fluorescein is ∼1000 μM, which is relatively insensitive. However, when used in video mode combined with image stacking enhancement, the LOD is dramatically reduced to 30 μM, sensitivity which is similar to that of state-of-the-art ELISA plate photomultiplier-based readers. Numerous medical diagnostics assays rely on optical and fluorescence readers. Our novel combination of detection technologies, which is new to biodetection may enable the development of new low cost optical detectors based on an inexpensive Webcam (<$10). It has the potential to form the basis for high sensitivity, low cost medical diagnostics in resource-poor settings. PMID:23990697

Developing a scalable model of recombinant protein yield from Pichia pastoris: the influence of culture conditions, biomass and induction regime

PubMed Central

Holmes, William J; Darby, Richard AJ; Wilks, Martin DB; Smith, Rodney; Bill, Roslyn M

2009-01-01

Background The optimisation and scale-up of process conditions leading to high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences. Typical experiments rely on varying selected parameters through repeated rounds of trial-and-error optimisation. To rationalise this, several groups have recently adopted the 'design of experiments' (DoE) approach frequently used in industry. Studies have focused on parameters such as medium composition, nutrient feed rates and induction of expression in shake flasks or bioreactors, as well as oxygen transfer rates in micro-well plates. In this study we wanted to generate a predictive model that described small-scale screens and to test its scalability to bioreactors. Results Here we demonstrate how the use of a DoE approach in a multi-well mini-bioreactor permitted the rapid establishment of high yielding production phase conditions that could be transferred to a 7 L bioreactor. Using green fluorescent protein secreted from Pichia pastoris, we derived a predictive model of protein yield as a function of the three most commonly-varied process parameters: temperature, pH and the percentage of dissolved oxygen in the culture medium. Importantly, when yield was normalised to culture volume and density, the model was scalable from mL to L working volumes. By increasing pre-induction biomass accumulation, model-predicted yields were further improved. Yield improvement was most significant, however, on varying the fed-batch induction regime to minimise methanol accumulation so that the productivity of the culture increased throughout the whole induction period. These findings suggest the importance of matching the rate of protein production with the host metabolism. Conclusion We demonstrate how a rational, stepwise approach to recombinant protein production screens can reduce process development time. PMID:19570229

In vitro chondrogenic commitment of human Wharton's jelly stem cells by co-culture with human articular chondrocytes.

PubMed

Pereira, R C; Costa-Pinto, A R; Frias, A M; Neves, N M; Azevedo, H S; Reis, R L

2017-06-01

Wharton's jelly stem cells (WJSCs) are a potential source of transplantable stem cells in cartilage-regenerative strategies, due to their highly proliferative and multilineage differentiation capacity. We hypothesized that a non-direct co-culture system with human articular chondrocytes (hACs) could enhance the potential chondrogenic phenotype of hWJSCs during the expansion phase compared to those expanded in monoculture conditions. Primary hWJSCs were cultured in the bottom of a multiwell plate separated by a porous transwell membrane insert seeded with hACs. No statistically significant differences in hWJSCs duplication number were observed under either of the culture conditions during the expansion phase. hWJSCs under co-culture conditions show upregulations of collagen type I and II, COMP, TGFβ1 and aggrecan, as well as of the main cartilage transcription factor, SOX9, when compared to those cultured in the absence of chondrocytes. Chondrogenic differentiation of hWJSCs, previously expanded in co-culture and monoculture conditions, was evaluated for each cellular passage using the micromass culture model. Cells expanded in co-culture showed higher accumulation of glycosaminoglycans (GAGs) compared to cells in monoculture, and immunohistochemistry for localization of collagen type I revealed a strong detection signal when hWJSCs were expanded under monoculture conditions. In contrast, type II collagen was detected when cells were expanded under co-culture conditions, where numerous round-shaped cell clusters were observed. Using a micromass differentiation model, hWJSCs, previously exposed to soluble factors secreted by hACs, were able to express higher levels of chondrogenic genes with deposition of cartilage extracellular matrix components, suggesting their use as an alternative cell source for treating degenerated cartilage. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Isolation and culture of corneal cells and their interactions with dissociated trigeminal neurons.

PubMed

Chan, K Y; Haschke, R H

1982-08-01

The three cell types of rabbit cornea (epithelium, stromal fibroblasts and endothelium) were isolated by an improved method using both microdissection and selective enzyme treatment. This technique reproducibly resulted in an almost total recovery of each cell type from a given cornea. When maintained in culture, the three cell types showed different morphologic characteristics, each resembling the in vivo counterpart. The epithelial culture consisted of both attached and floating cells. The attached cells located at the marginal area of a colony were irregular in shape and possessed pseudopodia, while those in the confluent area were polygonal. Floating cells were typically vacuolated, curve-shaped and joined in groups of 2-4 cells as a spherical body enclosing a lucent interior. Comparison of mitotic rates, ultrastructure, keratin levels and other cytologic evidence suggested that the attached cells may correspond to the basal cells and less differentiated wing cells, while the floating cells may be analogous to the more differentiated wing cells and superficial cells. Neurons dissociated from neonatal rabbit trigeminal (Gasserian) ganglia were plated into multiwells partially covered with a given corneal cell type. The percentages of viable and neurite-bearing neurons were evaluated on the first three days. When neurons were grown in contact with each of the corneal cell types, neurites were extended in every case. However, when neurons were not in contact with the corneal cells in the coculture, only epithelial cells permitted neurite outgrowth. The data suggested two types of cellular interactions between corneal cells and sensory neurons, one of which may be the specific release of a neuronotrophic factor by epithelial cells. This culture system represents the first step towards developing an in vitro model for studying various cornea-trigeminal interactions.

Increasing the throughput and productivity of Caco-2 cell permeability assays using liquid chromatography-mass spectrometry: application to resveratrol absorption and metabolism.

PubMed

Li, Yongmei; Shin, Young Geun; Yu, Chongwoo; Kosmeder, Jerome W; Hirschelman, Wendy H; Pezzuto, John M; van Breemen, Richard B

2003-12-01

The Caco-2 cell monolayer permeability assay has become a standard model of human intestinal absorption and transport. This paper reviews recent progress in increasing the throughput of Caco-2 cell monolayer assays and in expanding the scope of this assay to include modeling intestinal drug metabolism. The state-of-the-art in Caco-2 cell monolayer permeability assays combines multi-well plates fitted with semi-permeable inserts on which Caco-2 cells have been cultured with liquid chromatography-mass spectrometry (LC-MS) or LC-tandem mass spectrometry (LC-MS-MS) for the quantitative analysis of test compounds and the identification of their intestinal metabolites. After reviewing the progress in increasing the throughput of Caco-2 cell monolayer assays for both modeling human intestinal permeability or transport and the metabolism of xenobiotic compounds, we demonstrate the application of LC-MS and LC-MS-MS to the measurement of resveratrol permeability and metabolism in the Caco-2 model. trans-Resveratrol (trans-3,5,4'-trihydroxystilbene) is a polyphenolic compound occurring in grapes, peanuts and other food sources, that is under investigation as a cancer chemoprevention agent. The apparent permeability coefficient for apical (AP) to basolateral (BL) movement of resveratrol was 2.0 x 10(-5)cm/sec. Resveratrol was not a substrate for P-glycoprotein or the multi-drug resistance associated proteins (MRP). No phase I metabolites were observed, but the phase II conjugates resveratrol-3-glucuronide and resveratrol-3-sulfate was identified based on LC-MS and LC-MS-MS analysis and comparison with synthetic standards. Although these data indicate that resveratrol diffuses rapidly across the intestinal epithelium, extensive phase II metabolism during absorption might reduce resveratrol bioavailability.

ViCAR: An Adaptive and Landmark-Free Registration of Time Lapse Image Data from Microfluidics Experiments

PubMed Central

Hattab, Georges; Schlüter, Jan-Philip; Becker, Anke; Nattkemper, Tim W.

2017-01-01

In order to understand gene function in bacterial life cycles, time lapse bioimaging is applied in combination with different marker protocols in so called microfluidics chambers (i.e., a multi-well plate). In one experiment, a series of T images is recorded for one visual field, with a pixel resolution of 60 nm/px. Any (semi-)automatic analysis of the data is hampered by a strong image noise, low contrast and, last but not least, considerable irregular shifts during the acquisition. Image registration corrects such shifts enabling next steps of the analysis (e.g., feature extraction or tracking). Image alignment faces two obstacles in this microscopic context: (a) highly dynamic structural changes in the sample (i.e., colony growth) and (b) an individual data set-specific sample environment which makes the application of landmarks-based alignments almost impossible. We present a computational image registration solution, we refer to as ViCAR: (Vi)sual (C)ues based (A)daptive (R)egistration, for such microfluidics experiments, consisting of (1) the detection of particular polygons (outlined and segmented ones, referred to as visual cues), (2) the adaptive retrieval of three coordinates throughout different sets of frames, and finally (3) an image registration based on the relation of these points correcting both rotation and translation. We tested ViCAR with different data sets and have found that it provides an effective spatial alignment thereby paving the way to extract temporal features pertinent to each resulting bacterial colony. By using ViCAR, we achieved an image registration with 99.9% of image closeness, based on the average rmsd of 4.10−2 pixels, and superior results compared to a state of the art algorithm. PMID:28620411

Isolation and identification of antimicrobial compound from Mentha longifolia L. leaves grown wild in Iraq.

PubMed

Al-Bayati, Firas A

2009-06-12

Mentha longifolia L. (Lamiaceae) leaves have been traditionally implemented in the treatment of minor sore throat and minor mouth or throat irritation by the indigenous people of Iraq, although the compounds responsible for the medicinal properties have not been identified. In the present study, an antimicrobial compound was isolated and characterized, and its biological activity was assessed. The compound was isolated and characterized from the extracted essential oil using different spectral techniques: TLC, FTIR spectra and HPLC. Antimicrobial activity of the compound was assessed using both disc diffusion and microdilution method in 96 multi-well microtiter plates. A known compound was isolated from the essential oil of the plant and was identified as (-) menthol. The isolated compound was investigated for its antimicrobial activity against seven selected pathogenic and non-pathogenic microorganisms: Staphylococcus aureus, Streptococcus mutans, Streptococcus faecalis, Streptococcus pyogenis, Lactobacillus acidophilus, Pseudomonas aeruginosa and the yeast Candida albicans. Menthol at different concentrations (1:1, 1:5, 1:10, 1:20) was active against all tested bacteria except for P. aeruginosa, and the highest inhibitory effect was observed against S. mutans (zone of inhibition: 25.3 mm) using the disc diffusion method. Minimal inhibitory concentration MIC values ranged from 15.6-125.0 microg/ml, and the most promising results were observed against S. aureus and S. mutans (MIC 15.6 microg/ml) while, S. faecalis, S. pyogenis and L. acidophilus ranked next (MIC 31.2 microg/ml). Furthermore, menthol achieved considerable antifungal activity against the yeast C. albicans (zone of inhibition range: 7.1-18.5 mm; MIC: 125.0). The isolation of an antimicrobial compound from M. longifolia leaves validates the use of this plant in the treatment of minor sore throat and minor mouth or throat irritation.

Real-time Measurements of Amino Acid and Protein Hydroperoxides Using Coumarin Boronic Acid*

PubMed Central

Michalski, Radoslaw; Zielonka, Jacek; Gapys, Ewa; Marcinek, Andrzej; Joseph, Joy; Kalyanaraman, Balaraman

2014-01-01

Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7–23 m−1 s−1) were significantly higher than that measured for H2O2 (1.5 m−1 s−1). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1–1.5 × 103 m−1 s−1. Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems. PMID:24928516

Prediction of metabolism-induced hepatotoxicity on three-dimensional hepatic cell culture and enzyme microarrays.

PubMed

Yu, Kyeong-Nam; Nadanaciva, Sashi; Rana, Payal; Lee, Dong Woo; Ku, Bosung; Roth, Alexander D; Dordick, Jonathan S; Will, Yvonne; Lee, Moo-Yeal

2018-03-01

Human liver contains various oxidative and conjugative enzymes that can convert nontoxic parent compounds to toxic metabolites or, conversely, toxic parent compounds to nontoxic metabolites. Unlike primary hepatocytes, which contain myriad drug-metabolizing enzymes (DMEs), but are difficult to culture and maintain physiological levels of DMEs, immortalized hepatic cell lines used in predictive toxicity assays are easy to culture, but lack the ability to metabolize compounds. To address this limitation and predict metabolism-induced hepatotoxicity in high-throughput, we developed an advanced miniaturized three-dimensional (3D) cell culture array (DataChip 2.0) and an advanced metabolizing enzyme microarray (MetaChip 2.0). The DataChip is a functionalized micropillar chip that supports the Hep3B human hepatoma cell line in a 3D microarray format. The MetaChip is a microwell chip containing immobilized DMEs found in the human liver. As a proof of concept for generating compound metabolites in situ on the chip and rapidly assessing their toxicity, 22 model compounds were dispensed into the MetaChip and sandwiched with the DataChip. The IC 50 values obtained from the chip platform were correlated with rat LD 50 values, human C max values, and drug-induced liver injury categories to predict adverse drug reactions in vivo. As a result, the platform had 100% sensitivity, 86% specificity, and 93% overall predictivity at optimum cutoffs of IC 50 and C max values. Therefore, the DataChip/MetaChip platform could be used as a high-throughput, early stage, microscale alternative to conventional in vitro multi-well plate platforms and provide a rapid and inexpensive assessment of metabolism-induced toxicity at early phases of drug development.

A new simple method with high precision for determining the toxicity of antifouling paints on brine shrimp larvae (Artemia): first results.

PubMed

Castritsi-Catharios, J; Bourdaniotis, N; Persoone, G

2007-04-01

The use of antifouling paints is the only truly effective method for the protection of underwater structures from the development of fouling organisms. In the present study, the surface to volume concept constitutes the basis for the development of a new and improved method for determining the toxicity of antifouling paints on marine organisms. Particular emphasis is placed on the attainment of a standardized uniformity of coated surfaces. Failure to control the thickness of the coat of paint in previous studies of this type, has led to inaccurate evaluation of the relative toxicity of samples. Herein, an attempt is made to solve this problem using a simple technique which gives completely uniform and smooth surfaces. The effectiveness of this technique is assessed through two series of experiments using two different types of test containers: 50 ml modified syringes and 7 ml multiwells. The results of the toxicity experiments follow a normal distribution around the average value which allows to consider these values as reliable for comparison of the level of toxic effect detected with the two types of test containers. The mean lethal concentration L(S/V)(50) in the test series conducted in the multiwells (20.38 mm(2)ml(-1)) does not differ significantly from that obtained in the test series using modified syringes (20.065 mm(2)ml(-1)). It can thus be concluded from this preliminary study that the new method and the two different ways of exposing the test organisms to the antifouling paints and their leachates gave reliable and replicable results.

Microplate Fluorescence Assay for Measurement of the Ability of Strains of Listeria monocytogenes from Meat and Meat-Processing Plants To Adhere to Abiotic Surfaces▿

PubMed Central

Gamble, Rachel; Muriana, Peter M.

2007-01-01

Listeria monocytogenes is a significant food-borne pathogen that is capable of adhering to and producing biofilms on processing equipment, making it difficult to eliminate from meat-processing environments and allowing potential contamination of ready-to-eat (RTE) products. We devised a fluorescence-based microplate method for screening isolates of L. monocytogenes for the ability to adhere to abiotic surfaces. Strains of L. monocytogenes were incubated for 2 days at 30°C in 96-well microplates, and the plates were washed in a plate washer. The retained cells were incubated for 15 min at 25°C with 5,6-carboxyfluorescein diacetate and washed again, and then the fluorescence was read with a plate reader. Several enzymatic treatments (protease, lipase, and cellulase) were effective in releasing adherent cells from the microplates, and this process was used for quantitation on microbiological media. Strongly adherent strains of L. monocytogenes were identified that had 15,000-fold-higher levels of fluorescence and 100,000-fold-higher plate counts in attachment assays than weakly adherent strains. Strongly adherent strains of L. monocytogenes adhered equally well to four different substrates (glass, plastic, rubber, and stainless steel); showed high-level attachment on microplates at 10, 20, 30, and 40°C; and showed significant differences from weakly adherent strains when examined by scanning electron microscopy. A greater incidence of strong adherence was observed for strains isolated from RTE meats than for those isolated from environmental surfaces. Analysis of surface adherence among Listeria isolates from processing environments may provide a better understanding of the molecular mechanisms involved in attachment and suggest solutions to eliminate them from food-processing environments. PMID:17586676

A high throughput screen for biomining cellulase activity from metagenomic libraries.

PubMed

Mewis, Keith; Taupp, Marcus; Hallam, Steven J

2011-02-01

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production (1). Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils (2), buffalo rumen (3) and the termite hind-gut (4) using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood (5)). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio (6). Other methods have been reported (7,8) but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate (9). Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction (10). The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

A high-throughput lab-on-a-chip interface for zebrafish embryo tests in drug discovery and ecotoxicology

NASA Astrophysics Data System (ADS)

Zhu, Feng; Akagi, Jin; Hall, Chris J.; Crosier, Kathryn E.; Crosier, Philip S.; Delaage, Pierre; Wlodkowic, Donald

2013-12-01

Drug discovery screenings performed on zebrafish embryos mirror with a high level of accuracy. The tests usually performed on mammalian animal models, and the fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, conventional methods utilising 96-well microtiter plates and manual dispensing of fish embryos are very time-consuming. They rely on laborious and iterative manual pipetting that is a main source of analytical errors and low throughput. In this work, we present development of a miniaturised and high-throughput Lab-on-a-Chip (LOC) platform for automation of FET assays. The 3D high-density LOC array was fabricated in poly-methyl methacrylate (PMMA) transparent thermoplastic using infrared laser micromachining while the off-chip interfaces were fabricated using additive manufacturing processes (FDM and SLA). The system's design facilitates rapid loading and immobilization of a large number of embryos in predefined clusters of traps during continuous microperfusion of drugs/toxins. It has been conceptually designed to seamlessly interface with both upright and inverted fluorescent imaging systems and also to directly interface with conventional microtiter plate readers that accept 96-well plates. We also present proof-of-concept interfacing with a high-speed imaging cytometer Plate RUNNER HD® capable of multispectral image acquisition with resolution of up to 8192 x 8192 pixels and depth of field of about 40 μm. Furthermore, we developed a miniaturized and self-contained analytical device interfaced with a miniaturized USB microscope. This system modification is capable of performing rapid imaging of multiple embryos at a low resolution for drug toxicity analysis.

Intracellular O2 sensing probe based on cell-penetrating phosphorescent nanoparticles.

PubMed

Fercher, Andreas; Borisov, Sergey M; Zhdanov, Alexander V; Klimant, Ingo; Papkovsky, Dmitri B

2011-07-26

A new intracellular O(2) (icO(2)) sensing probe is presented, which comprises a nanoparticle (NP) formulation of a cationic polymer Eudragit RL-100 and a hydrophobic phosphorescent dye Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP). Using the time-resolved fluorescence (TR-F) plate reader set-up, cell loading was investigated in detail, particularly the effects of probe concentration, loading time, serum content in the medium, cell type, density, etc. The use of a fluorescent analogue of the probe in conjunction with confocal microscopy and flow cytometry analysis, revealed that cellular uptake of the NPs is driven by nonspecific energy-dependent endocytosis and that the probe localizes inside the cell close to the nucleus. Probe calibration in biological environment was performed, which allowed conversion of measured phosphorescence lifetime signals into icO(2) concentration (μM). Its analytical performance in icO(2) sensing experiments was demonstrated by monitoring metabolic responses of mouse embryonic fibroblast cells under ambient and hypoxic macroenvironment. The NP probe was seen to generate stable and reproducible signals in different types of mammalian cells and robust responses to their metabolic stimulation, thus allowing accurate quantitative analysis. High brightness and photostability allow its use in screening experiments with cell populations on a commercial TR-F reader, and for single cell analysis on a fluorescent microscope.

A miniaturized fibrinolytic assay for plasminogen activators

NASA Technical Reports Server (NTRS)

Lewis, M. L.; Nachtwey, D. S.; Damron, K. L.

1991-01-01

This report describes a micro-clot lysis assay (MCLA) for evaluating fibrinolytic activity of plasminogen activators (PA). Fibrin clots were formed in wells of microtiter plates. Lysis of the clots by PA, indicated by change in turbidity (optical density, OD), was monitored with a microplate reader at five minutes intervals. Log-log plots of PA dilution versus endpoint, the time at which the OD value was halfway between the maximum and minimum value for each well, were linear over a broad range of PA concentrations (2-200 International units/ml). The MCLA is a modification and miniaturization of well established fibrinolytic methods. The significant practical advantages of the MCLA are that it is a simple, relatively sensitive, non-radioactive, quantitative, kinetic, fibrinolytic micro-technique which can be automated.

[Book review] Ducks, geese and swans of North America

USGS Publications Warehouse

Krapu, Gary L.

1983-01-01

This is the 3rd edition of the classic work "The Ducks, Geese and Swans of North America," which was first published in December 1942.  The original edition was authored by Francis C. Kortright with color plates by T. M. Shortt. An authoritative reference on North American waterfowl for many years, the book had become outdated as a result of major advances in the field of waterfowl biology. The need to update the 1st edition culminated in the publication in 1976 of a 2nd edition authored by Frank Bellrose. Readers interested in comparing features of the 1976 edition with other major recent works on North American waterfowl by P. A. Johnsgard and R. S. Palmer should read Weller (1977, Auk 94: 173).

Modeling cross-hole slug tests in an unconfined aquifer

DOE PAGES

Malama, Bwalya; Kuhlman, Kristopher L.; Brauchler, Ralf; ...

2016-06-28

Cross-hole slug test date are analyzed with an extended version of a recently published unconfined aquifer model accounting for waterable effects using the linearized kinematic condition. The use of cross-hole slug test data to characterize aquifer heterogeneity and source/observation well oscillation parameters is evaluated. The data were collected in a series of multi-well and multi-level pneumatic slug tests conducted at a site in Widen, Switzerland. Furthermore, the tests involved source and observation well pairs separated by distances of up to 4 m, and instrumented with pressure transducers to monitor aquifer response in discrete intervals.

A high-throughput assay of membrane protein stability.

PubMed

Postis, Vincent L G; Deacon, Sarah E; Roach, Peter C J; Wright, Gareth S A; Xia, Xiaobing; Ingram, Jean C; Hadden, Jonathan M; Henderson, Peter J F; Phillips, Simon E V; McPherson, Michael J; Baldwin, Stephen A

2008-12-01

The preparation of purified, detergent-solubilized membrane proteins in a monodisperse and stable form is usually a prerequisite for investigation not only of their function but also for structural studies by X-ray crystallography and other approaches. Typically, it is necessary to explore a wide range of conditions, including detergent type, buffer pH, and the presence of additives such as glycerol, in order to identify those optimal for stability. Given the difficulty of expressing and purifying membrane proteins in large amounts, such explorations must ideally be performed on as small a scale as practicable. To achieve this objective in the UK Membrane Protein Structure Initiative, we have developed a rapid, economical, light-scattering assay of membrane protein aggregation that allows the testing of 48 buffer conditions in parallel on 6 protein targets, requiring less than 2 mg protein for each target. Testing of the assay on a number of unrelated membrane transporters has shown that it is of generic applicability. Proteins of sufficient purity for this plate-based assay are first rapidly prepared using simple affinity purification procedures performed in batch mode. Samples are then transferred by microdialysis into each of the conditions to be tested. Finally, attenuance at 340 nm is monitored in a 384-well plate using a plate reader. Optimal conditions for protein stability identified in the assay can then be exploited for the tailored purification of individual targets in as stable a form as possible.

Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warner, Marvin G.; Grate, Jay W.; Tyler, Abby J.

2009-09-01

A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment wasmore » demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.« less

Portable Microplate Analyzer with a Thermostatic Chamber Based on a Smartphone for On-site Rapid Detection.

PubMed

Wan, Zijian; Zhong, Longjie; Pan, Yuxiang; Li, Hongbo; Zou, Quchao; Su, Kaiqi; Wang, Ping

2017-01-01

A microplate method provides an efficient way to use modern detection technology. However, there are some difficulties concerning on-site detection, such as being non-portable and time-consuming. In this work, a novel portable microplate analyzer with a thermostatic chamber based on a smartphone was designed for rapid on-site detection. An analyzer with a wide-angle lens and an optical filter provides a proper environment for the microplate. A smartphone app-iPlate Monitor was used for RGB analyze of image. After a consistency experiment with a microtiter plate reader (MTPR), the normalized calibration curves were y = 0.7276x + 0.0243 (R 2 = 0.9906) and y = 0.3207x + 0.0094 (R 2 = 0.9917) with a BCA protein kit as well as y = 0.182x + 0.0134 (R 2 = 0.994) and y = 0.0674x + 0.0003 (R 2 = 0.9988) with a glucose kit. The times for obtaining the detection requirement were 15 and 10 min for the BCA protein kit and the glucose kit at 37°C; in contrast, it required more than 30 and 20 min at ambient temperature. Meanwhile, it also showed good repeatability for detections.

Influence of standard and novel LTB4 analogs on human neutrophil chemotaxis measured by the multiwell cap assay.

PubMed

Psychoyos, S; Uziel-Fusi, S; Bhagwat, S; Morrissey, M M

1989-11-30

Standard and novel LTB4 analogs were tested for neutrophil chemoattractant activity using the multiwell cap assay (Evans et al. (1986) Biosc. Rep. 6, 1041). The assay uses disposable equipment and measures chemotaxis by the number of cells able to migrate across the full thickness of cellulose nitrate filters. Under standard conditions (90 min incubation at 37 degrees C in buffer containing 2% bovine albumin), LTB4 and 6-cis-LTB1 had EC50 values of 3.5 and 15,000 nM, respectively. 20-hydroxy-LTB4 was equipotent with LTB4 and exhibited a similar biphasic chemotactic response, however, only one third of the number of cells migrated through the filter. 20-carboxy-LTB4 was inactive up to 1,000 nM. 5-desoxy-((6,7)-cis-cyclopropyl)-LTB2, (6,7)-benzo-LTB2 and 5-desoxy-(8,10)-LTB2 had EC50 values of 11,300, 50,000 and 84,000 nM, respectively. Checkerboard analysis indicated a chemokinetic component of 42% for LTB4 at a concentration causing peak chemotaxis. Reduction of albumin in the buffer to 0.5% increased the apparent potencies of LTB4 and 6-cis-LTB1 five-fold. Since LTB4 is a mediator of inflammation, various anti-inflammatory agents were tested at peak concentrations observed in vivo for in vitro inhibition of LTB4-stimulated chemotaxis in the presence of 0.5% albumin. Under the conditions of the assay, chloroquine diphosphate, dexamethasone, indomethacin, penicillamine, piroxicam and diclofenac sodium were inactive; gold sodium thiomalate was inhibitory (IC50 = 20 microM).

The in situ aeration in an old landfill in China: Multi-wells optimization method and application.

PubMed

Liu, Lei; Ma, Jun; Xue, Qiang; Shao, Jingbang; Chen, Yijun; Zeng, Gang

2018-06-01

The optimization design of well spacing (WS) and aeration rate (AR) is crucial to the in situ aeration system operation in under long-term and high-efficiency conditions. This optimization design aims to transport additional air into landfills and to develop an improved oxygen environment for enhancing aerobic degradation. Given the specific pore structure distribution within landfills, providing sufficient oxygen in all waste bodies in field sites through gas wells is difficult. The design of well distribution also lacks adequate criteria. In this work, the multi-well optimization aeration method (MWOAM) was proposed to select the WS and AR from prediction results that consider gas transport properties by maximizing oxygen storage ratio (OSR) as the key objective threshold. This method was applied to the aeration restoration engineering in Jinkou landfill, which represents the first full-scale application of an aeration project in China, to optimize the operation scheme of the aeration system. Results of the gas concentration monitoring show that the trend of the OSR with aeration time based on the measurement agrees with the prediction. The oxygen and methane contents remain high and low within the landfill during the aeration process, respectively. Moreover, the temperature in the waste body did not exceed the upper limit value. These results suggested that the MWOAM is an effective means of supplying sufficient oxygen content across the landfill body and extend the aeration system operation for the long term. Therefore, this work provides reliable evidence to support the design and operation management of the aeration systems in full-scale landfills. Copyright © 2018 Elsevier Ltd. All rights reserved.

Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation

PubMed Central

Panpradist, Nuttada; Beck, Ingrid A.; Chung, Michael H.; Kiarie, James N.; Frenkel, Lisa M.; Lutz, Barry R.

2016-01-01

Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories. PMID:26751207

UV-visible spectroscopy as an alternative to liquid chromatography for determination of everolimus in surfactant-containing dissolution media: a useful approach based on solid-phase extraction.

PubMed

Kamberi, Marika; Tran, Thu-Ngoc

2012-11-01

High-throughput 96-well solid phase extraction (SPE) plate with C-18 reversed phase sorbent followed by UV-visible (UV-Vis) microplate reader was applied to the analysis of hydrophobic drugs in surfactant-containing dissolution media, which are often used to evaluate the in-vitro drug release of drug eluting stents (DES). Everolimus and dissolution medium containing Triton X-405 were selected as representatives, and the appropriate SPE conditions (adsorption, washing and elution) were investigated to obtain a practical and reliable sample clean-up. It was shown that the developed SPE procedure was capable of removing interfering components (Triton X-405 and its impurities), allowing for an accurate automated spectrophotometric analysis to be performed. The proposed UV-Vis spectrophotometric method yielded equivalent results compared to a classical LC analysis method. Linear regression analysis indicated that both methods have the ability to obtain test results that are directly proportional to the concentration of analyte in the sample within the selected range of 1.0-10 μg/ml for everolimus, with a coefficient of correlation (r(2)) value of >0.998 and standard deviation of the residuals (Syx) of <2%. The individual recoveries of everolimus ranged from 97 to 104% for the UV-Vis spectrophotometric method and from 98 to 102 for the HPLC method, respectively. The 95% CI of the mean recovery for the UV-Vis spectrophotometric method was 99-102% and for the HPLC method was 99-101%. No statistical difference was found between the mean recoveries of the methods (p=0.42). Hence the methods are free from interference due to Triton and other chemicals present in the dissolution medium. The variation in the amount of everolimus estimated by UV-Vis spectrophotometric and HPLC methods was ≤3.5%, and the drug release profiles obtained by both methods were found to be equivalent by evaluation with two-one-sided t-test (two-tailed, p=0.62; mean of differences, 0.17; 95% CI, 0.62-0.96) and similarity factor f2 (f2 value, 87). The excellent conformity of the results makes UV-Vis spectrophotometer an ideal tool for analyzing the drugs in the media containing surfactants, after SPE. The 96-well SPE plates in combination with UV-Vis microplate reader provide a high throughput method for the determination of in-vitro drug release profile of DES. Switching from HPLC to UV-Vis spectrophotometer microplate reader assay reduces the solvent consumption and labor required for the sample analyses. This directly impacts the profitability of the laboratory. Copyright © 2012 Elsevier B.V. All rights reserved.

High-throughput spectral and lifetime-based FRET screening in living cells to identify small-molecule effectors of SERCA

PubMed Central

Schaaf, Tory M.; Peterson, Kurt C.; Grant, Benjamin D.; Bawaskar, Prachi; Yuen, Samantha; Li, Ji; Muretta, Joseph M.; Gillispie, Gregory D.; Thomas, David D.

2017-01-01

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green (GFP, donor) and red (RFP, acceptor) fluorescent proteins fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA’s ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS. PMID:27899691

Assessing Reservoir Depositional Environments to Develop and Quantify Improvements in CO 2 Storage Efficiency. A Reservoir Simulation Approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okwen, Roland; Frailey, Scott; Leetaru, Hannes

2014-09-30

The storage potential and fluid movement within formations are dependent on the unique hydraulic characteristics of their respective depositional environments. Storage efficiency (E) quantifies the potential for storage in a geologic depositional environment and is used to assess basinal or regional CO 2 storage resources. Current estimates of storage resources are calculated using common E ranges by lithology and not by depositional environment. The objectives of this project are to quantify E ranges and identify E enhancement strategies for different depositional environments via reservoir simulation studies. The depositional environments considered include deltaic, shelf clastic, shelf carbonate, fluvial deltaic, strandplain, reef,more » fluvial and alluvial, and turbidite. Strategies considered for enhancing E include CO 2 injection via vertical, horizontal, and deviated wells, selective completions, water production, and multi-well injection. Conceptual geologic and geocellular models of the depositional environments were developed based on data from Illinois Basin oil fields and gas storage sites. The geologic and geocellular models were generalized for use in other US sedimentary basins. An important aspect of this work is the development of conceptual geologic and geocellular models that reflect the uniqueness of each depositional environment. Different injection well completions methods were simulated to investigate methods of enhancing E in the presence of geologic heterogeneity specific to a depositional environment. Modeling scenarios included horizontal wells (length, orientation, and inclination), selective and dynamic completions, water production, and multiwell injection. A Geologic Storage Efficiency Calculator (GSECalc) was developed to calculate E from reservoir simulation output. Estimated E values were normalized to diminish their dependency on fluid relative permeability. Classifying depositional environments according to normalized baseline E ranges ranks fluvial deltaic and turbidite highest and shelf carbonate lowest. The estimated average normalized baseline E of turbidite, and shelf carbonate depositional environments are 42.5% and 13.1%, with corresponding standard deviations of 11.3%, and 3.10%, respectively. Simulations of different plume management techniques suggest that the horizontal well, multi-well injection with brine production from blanket vertical producers are the most efficient E enhancement strategies in seven of eight depositional environments; for the fluvial deltaic depositional environment, vertical well with blanket completions is the most efficient. This study estimates normalized baseline E ranges for eight depositional environments, which can be used to assess the CO 2 storage resource of candidate formations. This study also improves the general understanding of depositional environment’s influence on E. The lessons learned and results obtained from this study can be extrapolated to formations in other US basins with formations of similar depositional environments, which should be used to further refine regional and national storage resource estimates in future editions of the Carbon Utilization and Storage Atlas of the United States. Further study could consider the economic feasibility of the E enhancement strategies identified here.« less

The tunneling effect for a class of difference operators

NASA Astrophysics Data System (ADS)

Klein, Markus; Rosenberger, Elke

We analyze a general class of self-adjoint difference operators H𝜀 = T𝜀 + V𝜀 on ℓ2((𝜀ℤ)d), where V𝜀 is a multi-well potential and 𝜀 is a small parameter. We give a coherent review of our results on tunneling up to new sharp results on the level of complete asymptotic expansions (see [30-35]).Our emphasis is on general ideas and strategy, possibly of interest for a broader range of readers, and less on detailed mathematical proofs. The wells are decoupled by introducing certain Dirichlet operators on regions containing only one potential well. Then the eigenvalue problem for the Hamiltonian H𝜀 is treated as a small perturbation of these comparison problems. After constructing a Finslerian distance d induced by H𝜀, we show that Dirichlet eigenfunctions decay exponentially with a rate controlled by this distance to the well. It follows with microlocal techniques that the first n eigenvalues of H𝜀 converge to the first n eigenvalues of the direct sum of harmonic oscillators on ℝd located at several wells. In a neighborhood of one well, we construct formal asymptotic expansions of WKB-type for eigenfunctions associated with the low-lying eigenvalues of H𝜀. These are obtained from eigenfunctions or quasimodes for the operator H𝜀, acting on L2(ℝd), via restriction to the lattice (𝜀ℤ)d. Tunneling is then described by a certain interaction matrix, similar to the analysis for the Schrödinger operator (see [22]), the remainder is exponentially small and roughly quadratic compared with the interaction matrix. We give weighted ℓ2-estimates for the difference of eigenfunctions of Dirichlet-operators in neighborhoods of the different wells and the associated WKB-expansions at the wells. In the last step, we derive full asymptotic expansions for interactions between two “wells” (minima) of the potential energy, in particular for the discrete tunneling effect. Here we essentially use analysis on phase space, complexified in the momentum variable. These results are as sharp as the classical results for the Schrödinger operator in [22].

Tissue-equivalent TL sheet dosimetry system for X- and gamma-ray dose mapping.

PubMed

Nariyama, N; Konnai, A; Ohnishi, S; Odano, N; Yamaji, A; Ozasa, N; Ishikawa, Y

2006-01-01

To measure dose distribution for X- and gamma rays simply and accurately, a tissue-equivalent thermoluminescent (TL) sheet-type dosemeter and reader system were developed. The TL sheet is composed of LiF:Mg,Cu,P and ETFE polymer, and the thickness is 0.2 mm. For the TL reading, a square heating plate, 20 cm on each side, was developed, and the temperature distribution was measured with an infrared thermal imaging camera. As a result, linearity within 2% and the homogeneity within 3% were confirmed. The TL signal emitted is detected using a CCD camera and displayed as a spatial dose distribution. Irradiation using synchrotron radiation between 10 and 100 keV and (60)Co gamma rays showed that the TL sheet dosimetry system was promising for radiation dose mapping for various purposes.

Characterization of specific anti-Candida IgM, IgA and IgE: diagnostic value in deep-seated infections.

PubMed

Aubert, D; Puygauthier-Toubas, D; Leon, P; Pignon, B; Foudrinier, F; Marnef, F; Boulant, J; Pinon, J M

1996-01-01

The proposed serological diagnosis of systemic Candida infections is based on a microplate immunocapture technique detecting IgM, IgA and IgE anti-Candida antibodies. Activity is revealed with a suspension of human erythrocytes sensitized with somatic antigen of Candida albicans, and is quantified on an automated plate reader. The sera were obtained from patients with deep-seated (n = 56) and superficial (n = 193) candidosis. We compared this immunological method with a combination of indirect immunofluorescence and co-immunoelectrodiffusion. The immunocapture method was more sensitive (80.4% vs. 48.2% with indirect immunofluorescence and 58.9% with co-immunoelectrodiffusion), and often provided the diagnosis at an earlier stage, with clear therapeutic advantages. The IgA isotype was a particularly valuable marker of deep-seated Candida infections.

Microvolume, kinetic-dependent enzyme-linked immunosorbent assay for amoeba antibodies.

PubMed Central

Mathews, H M; Walls, K W; Huong, A Y

1984-01-01

We describe a microvolume enzyme-linked immunosorbent assay based on enzyme rate kinetics. Antigens from Entamoeba histolytica were adsorbed in wells of disposable polystyrene strips containing 12 flat-bottom wells. After exposure to the serum of a patient and peroxidase-labeled anti-human immunoglobulin G, the rate of color change in specific substrate was determined by eight sequential readings of individual wells over a 2-min period with a microcomputer-controlled model MR-600 automated plate reader. The changes in absorbance readings were converted to slope values for each well by the microcomputer. Thus, 12 samples were read, and results were printed in ca. 3.5 min. Assay conditions are described and data are presented to show that this assay is quantitative for antibody and antigen concentration with a single-tube (well) dilution. PMID:6321547

Alternate reading strategies and variable asymmetry of the planum temporale in adult resilient readers.

PubMed

Welcome, Suzanne E; Leonard, Christiana M; Chiarello, Christine

2010-05-01

Resilient readers are characterized by impaired phonological processing despite skilled text comprehension. We investigated orthographic and semantic processing in resilient readers to examine mechanisms of compensation for poor phonological decoding. Performance on phonological (phoneme deletion, pseudoword reading), orthographic (orthographic choice, orthographic analogy), and semantic (semantic priming, homograph resolution) tasks was compared between resilient, poor and proficient readers. Asymmetry of the planum temporale was investigated in order to determine whether atypical readers showed unusual morphology in this language-relevant region. Resilient readers showed deficits on phonological tasks similar to those shown by poor readers. We obtained no evidence that resilient readers compensate via superior orthographic processing, as they showed neither exceptional orthographic skill nor increased reliance on orthography to guide pronunciation. Resilient readers benefited more than poor or proficient readers from semantic relationships between words and experienced greater difficulty when such relationships were not present. We suggest, therefore, that resilient readers compensate for poor phonological decoding via greater reliance on word meaning relationships. The reading groups did not differ in mean asymmetry of the planum temporale. However, resilient readers showed greater variability in planar asymmetry than proficient readers. Poor readers also showed a trend towards greater variability in planar asymmetry, with more poor readers than proficient readers showing extreme asymmetry. Such increased variability suggests that university students with less reading skill display less well regulated brain anatomy than proficient readers. Copyright 2010 Elsevier Inc. All rights reserved.

Antibacterial properties studies of trunk barks of Terminalia ivorensis, a commercial and medicinal species on some methicillin-resistant Staphylococci species strains.

PubMed

Coulibaly, K; Zirihi, G N; Guessennd-Kouadio, N; Oussou, K R; Dosso, M

2014-09-01

Methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis and coagulase-negative Staphylococcus infections are a worldwide concern. Terminalia ivorensis, of Combretaceae family plant, is widely used traditional medicinal in Côte d'Ivoire to treat dermal diseases (affection in which Staphylococci are implied) including local inflammation and also to treat voice-loss. This study focused to investigate the effect in vitro of the extracts of trunk barks of Terminalia ivorensis on some methicillin/oxacillin-resistant strains of Staphylococcus aureus, S. epidermidis, coagulase-negative S. and reference strain of S. aureus ATCC 25923. Antibacterial activity of aqueous, 70% ethanolic 70% and aqueous residue extracts was assessed using agar disc-diffusion method and liquid medium microdilution method in 96 multi-well micro-titer plates. This method led us to determine minimum inhibition concentration (M.I.C.) and minimum bactericidal concentration (M.B.C.). The presence of chemical groups major was detected qualitatively. Aqueous and 70% ethanolic 70% extracts showed significant activity against all the bacteria except aqueous residue when compared with the standard antibiotic oxacillin (5 µg/ml). M.I.C. for aqueous and 70% ethanolic 70% extracts ranged from 0,83-16,67 mg/ml and 0,156-13,33 mg/ml respectively. Viable cell determination revealed the bactericidal nature of the two barks extracts. The 70% ethanolic 70% extract exhibited the highest activity according to the M.B.C. values. The phytochemical analysis indicates the presence of tannins, saponins, flavonoids, terpen/sterols, coumarins, polyphenols and traces of alkaloid. The in-vitro antibacterial efficacy shown by the barks of this plant and his lushness in chimical compounds, would justify use of this one in the traditional treatment of some diseases of microbial origin. These compounds could be suggested to provide alternative solution to the development of new therapeutic agents.

Isolation and identification of antibacterial compounds from Thymus kotschyanus aerial parts and Dianthus caryophyllus flower buds.

PubMed

Mohammed, Muthanna J; Al-Bayati, Firas A

2009-06-01

The aerial parts of Thymus kotschyanus Boiss. and Hohen. (Lamiaceae) and flower buds of Dianthus caryophyllus L. (Caryophyllaceae) have been traditionally implemented in the treatment of wounds, throat and gum infections and gastro-intestinal disorder by the indigenous people of northern Iraq, although the compounds responsible for the medicinal properties have not been identified. In this study, antibacterial compounds from both plants were isolated and characterized, and the biological activity of each compound was assessed individually and combined. Compounds were isolated and characterized from the extracted essential oils of both plants using different spectral techniques: TLC, FTIR spectra and HPLC. The minimum inhibitory concentrations MIC values for the compounds were assessed individually and combined based on a microdilution and the checkerboard method in 96 multi-well microtiter plates. Two known compounds were isolated from the essential oils of both plants and were identified as thymol and eugenol. The isolated compounds were investigated for their single and combined antibacterial activities against seven selected pathogenic bacteria; Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Thymol MIC values ranged from 15.6 to 250.0 microg/ml and B. cereus was found to be the most sensitive pathogen with a MIC value of 15.6 microg/ml. Eugenol achieved stronger MIC values against most tested pathogens and the best MIC value (15.6 microg/ml) was observed against B. cereus, L. monocytogenes and K. pneumoniae whereas, S. aureus, P. mirabilis and E. coli were inhibited with a MIC value of 31.2 microg/ml. Combination results had antibacterial enhancement against most pathogens and the best synergistic result was seen against P. mirabilis and E. coli. The isolation of two antibacterial compounds from Thymus kotschyanus aerial parts and Dianthus caryophyllus flower buds validates the use of these species in the treatment of throat and gum infections, wound-healing and gastro-intestinal disorder.

Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

PubMed Central

Warren, Sean C.; Margineanu, Anca; Alibhai, Dominic; Kelly, Douglas J.; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Katan, Matilda

2013-01-01

Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment. PMID:23940626

Isolation and identification of antimicrobial compound from Mentha longifolia L. leaves grown wild in Iraq

PubMed Central

Al-Bayati, Firas A

2009-01-01

Background Mentha longifolia L. (Lamiaceae) leaves have been traditionally implemented in the treatment of minor sore throat and minor mouth or throat irritation by the indigenous people of Iraq, although the compounds responsible for the medicinal properties have not been identified. In the present study, an antimicrobial compound was isolated and characterized, and its biological activity was assessed. Methods The compound was isolated and characterized from the extracted essential oil using different spectral techniques: TLC, FTIR spectra and HPLC. Antimicrobial activity of the compound was assessed using both disc diffusion and microdilution method in 96 multi-well microtiter plates. Results A known compound was isolated from the essential oil of the plant and was identified as (-) menthol. The isolated compound was investigated for its antimicrobial activity against seven selected pathogenic and non-pathogenic microorganisms: Staphylococcus aureus, Streptococcus mutans, Streptococcus faecalis, Streptococcus pyogenis, Lactobacillus acidophilus, Pseudomonas aeruginosa and the yeast Candida albicans. Menthol at different concentrations (1:1, 1:5, 1:10, 1:20) was active against all tested bacteria except for P. aeruginosa, and the highest inhibitory effect was observed against S. mutans (zone of inhibition: 25.3 mm) using the disc diffusion method. Minimal inhibitory concentration MIC values ranged from 15.6–125.0 μg/ml, and the most promising results were observed against S. aureus and S. mutans (MIC 15.6 μg/ml) while, S. faecalis, S. pyogenis and L. acidophilus ranked next (MIC 31.2 μg/ml). Furthermore, menthol achieved considerable antifungal activity against the yeast C. albicans (zone of inhibition range: 7.1–18.5 mm; MIC: 125.0). Conclusion The isolation of an antimicrobial compound from M. longifolia leaves validates the use of this plant in the treatment of minor sore throat and minor mouth or throat irritation. PMID:19523224

Humidity assay for studying plant-pathogen interactions in miniature controlled discrete humidity environments with good throughput

PubMed Central

Jiang, Huawei; Sahu, Binod Bihari; Kambakam, Sekhar; Singh, Prashant; Wang, Xinran; Wang, Qiugu; Bhattacharyya, Madan K.; Dong, Liang

2016-01-01

This paper reports a highly economical and accessible approach to generate different discrete relative humidity conditions in spatially separated wells of a modified multi-well plate for humidity assay of plant-pathogen interactions with good throughput. We demonstrated that a discrete humidity gradient could be formed within a few minutes and maintained over a period of a few days inside the device. The device consisted of a freeway channel in the top layer, multiple compartmented wells in the bottom layer, a water source, and a drying agent source. The combinational effects of evaporation, diffusion, and convection were synergized to establish the stable discrete humidity gradient. The device was employed to study visible and molecular disease phenotypes of soybean in responses to infection by Phytophthora sojae, an oomycete pathogen, under a set of humidity conditions, with two near-isogenic soybean lines, Williams and Williams 82, that differ for a Phytophthora resistance gene (Rps1-k). Our result showed that at 63% relative humidity, the transcript level of the defense gene GmPR1 was at minimum in the susceptible soybean line Williams and at maximal level in the resistant line Williams 82 following P. sojae CC5C infection. In addition, we investigated the effects of environmental temperature, dimensional and geometrical parameters, and other configurational factors on the ability of the device to generate miniature humidity environments. This work represents an exploratory effort to economically and efficiently manipulate humidity environments in a space-limited device and shows a great potential to facilitate humidity assay of plant seed germination and development, pathogen growth, and plant-pathogen interactions. Since the proposed device can be easily made, modified, and operated, it is believed that this present humidity manipulation technology will benefit many laboratories in the area of seed science, plant pathology, and plant-microbe biology, where humidity is an important factor that influences plant disease infection, establishment, and development. PMID:27279932

Hybrid magnetite nanoparticles/ Rosmarinus officinalis essential oil nanobiosystem with antibiofilm activity

NASA Astrophysics Data System (ADS)

Chifiriuc, Carmen; Grumezescu, Valentina; Grumezescu, Alexandru Mihai; Saviuc, Crina; Lazăr, Veronica; Andronescu, Ecaterina

2012-04-01

Biofilms formed by fungal organisms are associated with drastically enhanced resistance against most antimicrobial agents, contributing to the persistence of the fungi despite antifungal therapy. The purpose of this study is to combine the unique properties of nanoparticles with the antimicrobial activity of the Rosmarinus officinalis essential oil in order to obtain a nanobiosystem that could be pelliculised on the surface of catheter pieces, in order to obtain an improved resistance to microbial colonization and biofilm development by Candida albicans and C. tropicalis clinical strains. The R. officinalis essential oils were extracted in a Neo-Clevenger type apparatus, and its chemical composition was settled by GC-MS analysis. Functionalized magnetite nanoparticles of up to 20 nm size had been synthesized by precipitation method adapted for microwave conditions, with oleic acid as surfactant. The catheter pieces were coated with suspended core/shell nanoparticles (Fe3O4/oleic acid:CHCl3), by applying a magnetic field on nanofluid, while the CHCl3 diluted essential oil was applied by adsorption in a secondary covering treatment. The fungal adherence ability was investigated in six multiwell plates, in which there have been placed catheters pieces with and without hybrid nanoparticles/essential oil nanobiosystem pellicle, by using culture-based methods and confocal laser scanning microscopy (CLSM). The R. officinalis essential oil coated nanoparticles strongly inhibited the adherence ability and biofilm development of the C. albicans and C. tropicalis tested strains to the catheter surface, as shown by viable cell counts and CLSM examination. Due to the important implications of C andida spp. in human pathogenesis, especially in prosthetic devices related infections and the emergence of antifungal tolerance/resistance, using the new core/shell/coated shell based on essential oil of R. officinalis to inhibit the fungal adherence could be of a great interest for the biomedical field, opening new directions for the design of film-coated surfaces with antibiofilm properties.

A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples

PubMed Central

Kumanan, Vijayarani; Nugen, Sam R.; Baeumner, Antje J.

2009-01-01

A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (101 to 106) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortuitum, M. scrofulaceum, M. intracellulare, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR. PMID:19255522

Spore immobilization and its analytical performance for monitoring of aflatoxin M1 in milk.

PubMed

Singh, V K; Singh, N A; Kumar, N; Raghu, H V; Sharma, Pradeep Kumar; Singh, K P; Yadav, Avinash

2014-12-01

Immobilization of Bacillus megaterium spores on Eppendorf tubes through physical adsorption has been used in the detection of aflatoxin M1 (AFM1) in milk within real time of 45 ± 5 min using visual observation of changes in a chromogenic substrate. The appearance of a sky-blue colour indicates the absence of AFM1 in milk, whereas no colour change indicates the presence of AFM1 in milk at a 0.5 ppb Codex maximum residue limit. The working performance of the immobilized spores was shown to persist for up to 6 months. Further, spores immobilized on 96-well black microtitre plates by physical adsorption and by entrapment on sensor disk showed a reduction in detection sensitivity to 0.25 ppb within a time period of 20 ± 5 min by measuring fluorescence using a microbiological plate reader through the addition of milk and fluorogenic substrate. A high fluorescence ratio indicated more substrate hydrolysis due to spore-germination-mediated release of marker enzymes of spores in the absence of AFM1 in milk; however, low fluorescence ratios indicated the presence of AFM1 at 0.25 ppb. Immobilized spores on 96-well microtitre plates and sensor disks have shown better reproducibility after storage at 4 °C for 6 months. Chromogenic assay showed 1.38% false-negative and 2.77% false-positive results while fluorogenic assay showed 4.16% false-positive and 2.77% false-negative results when analysed for AFM1 using 72 milk samples containing raw, pasteurized, and dried milk. Immobilization of spores makes these chromogenic and fluorogenic assays portable, selective, cost-effective for real-time detection of AFM1 in milk at the dairy farm, reception dock, and manufacturing units of the dairy industry.

Reading strategies of fast and slow readers.

PubMed

Haberlandt, K F; Graesser, A C; Schneider, N J

1989-09-01

In three subject-paced experiments we evaluated reading patterns at the word, line, and sentence level for fast and slow readers. A moving-window method was used to collect word reading times for natural texts. At the word level, reading times of word N were influenced by features of word N-1 for fast readers but not for slow readers. The lag effect exhibited by fast readers indicates that they continue to process a word when it is no longer in view, thus limiting the notion of immediate processing. Contrary to our initial expectation that fast readers would process only a single new argument from a sentence, whereas slow readers would process several new arguments, we found that both reader groups adopted a many-argument strategy. However, fast and slow readers differed in terms of the text units (lines vs. sentences) defining the new-argument effects: Fast readers exhibited greater new-argument effects relative to lines, whereas slow readers exhibited greater new-argument effects relative to sentences. Specifically, slow readers integrated the new arguments primarily at the end of the sentence, whereas fast readers did so at line boundaries. These results are discussed in terms of a buffer-and-integrate model of reading comprehension.

A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

PubMed

Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman

2016-12-15

The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management. Copyright © 2016 Elsevier B.V. All rights reserved.

The Earth story ... a facebook world in the geo blogosphere

NASA Astrophysics Data System (ADS)

Redfern, S. A.

2013-12-01

Facebook has become one of the dominant virtual worlds of our planet, and among the plethora of cute pictures of cats and unintelligible photos of plates of food are a few gems that attract a strong following. I have been contributing as an 'admin' to one facebook community - 'The Earth Story', over the past few months. The initial driver was writing short pieces of geo-news for my first-year undergraduate students, but quickly I discovered that far more people were reading the small newsy items on facebook than would ever hear my lectures or read my academic papers. This is not to negate the latter, but highlights the capacity for short snippets of Earth Science news from the virtual community out there. Each post on 'The Earth Story' (TES) typically gets read by more than 100k people, and the page has more than 0.5 million followers. Such outlets offer great opportunities for conveying the excitement and challenges of our subject, and the responses from readers often take the discussion further. Since contributing to TES I have also had the opportunity to work for 6 weeks at the BBC as a science journalist in BBC world service radio and online news, and again have seen the appetite for readers for good science stories. Here, I reflect on these experiences and consider the challenge of bringing cutting edge discovery to a general audience, and how social media offer routes to discovery that bypass traditional vehicles.

Principles for new optical techniques in medical diagnostics for mHealth applications

NASA Astrophysics Data System (ADS)

Balsam, Joshua Michael

Medical diagnostics is a critical element of effective medical treatment. However, many modern and emerging diagnostic technologies are not affordable or compatible with the needs and conditions found in low-income and middle-income countries and regions. Resource-poor areas require low-cost, robust, easy-to-use, and portable diagnostics devices compatible with telemedicine (i.e. mHealth) that can be adapted to meet diverse medical needs. Many suitable devices will need to be based on optical technologies, which are used for many types of biological analyses. This dissertation describes the fabrication and detection principles for several low-cost optical technologies for mHealth applications including: (1) a webcam based multi-wavelength fluorescence plate reader, (2) a lens-free optical detector used for the detection of Botulinum A neurotoxin activity, (3) a low cost micro-array reader that allows the performance of typical fluorescence based assays demonstrated for the detection of the toxin staphylococcal enterotoxin (SEB), and (4) a wide-field flow cytometer for high throughput detection of fluorescently labeled rare cells. This dissertation discusses how these technologies can be harnessed using readily available consumer electronics components such as webcams, cell phones, CCD cameras, LEDs, and laser diodes. There are challenges in developing devices with sufficient sensitivity and specificity, and approaches are presented to overcoming these challenges to create optical detectors that can serve as low cost medical diagnostics in resource-poor settings for mHealth.

An integrated platform for simultaneous multi-well field potential recording and Fura-2-based calcium transient ratiometry in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes.

PubMed

Rast, Georg; Weber, Jürgen; Disch, Christoph; Schuck, Elmar; Ittrich, Carina; Guth, Brian D

2015-01-01

Human induced pluripotent stem cell-derived cardiomyocytes are available from various sources and they are being evaluated for safety testing. Several platforms are available offering different assay principles and read-out parameters: patch-clamp and field potential recording, imaging or photometry, impedance measurement, and recording of contractile force. Routine use will establish which assay principle and which parameters best serve the intended purpose. We introduce a combination of field potential recording and calcium ratiometry from spontaneously beating cardiomyocytes as a novel assay providing a complementary read-out parameter set. Field potential recording is performed using a commercial multi-well multi-electrode array platform. Calcium ratiometry is performed using a fiber optic illumination and silicon avalanche photodetectors. Data condensation and statistical analysis are designed to enable statistical inference of differences and equivalence with regard to a solvent control. Simultaneous recording of field potentials and calcium transients from spontaneously beating monolayers was done in a nine-well format. Calcium channel blockers (e.g. nifedipine) and a blocker of calcium store release (ryanodine) can be recognized and discriminated based on the calcium transient signal. An agonist of L-type calcium channels, FPL 64176, increased and prolonged the calcium transient, whereas BAY K 8644, another L-type calcium channel agonist, had no effect. Both FPL 64176 and various calcium channel antagonists have chronotropic effects, which can be discriminated from typical "chronotropic" compounds, like (±)isoprenaline (positive) and arecaidine propargyl ester (negative), based on their effects on the calcium transient. Despite technical limitations in temporal resolution and exact matching of composite calcium transient with the field potential of a subset of cells, the combined recording platform enables a refined interpretation of the field potential recording and a more reliable identification of drug effects on calcium handling. Copyright © 2015 Elsevier Inc. All rights reserved.

Design and implementation of relational databases relevant to the diverse needs of a tuberculosis case contact study in the Gambia.

PubMed

Jeffries, D J; Donkor, S; Brookes, R H; Fox, A; Hill, P C

2004-09-01

The data requirements of a large multidisciplinary tuberculosis case contact study are complex. We describe an ACCESS-based relational database system that meets our rigorous requirements for data entry and validation, while being user-friendly, flexible, exportable, and easy to install on a network or stand alone system. This includes the development of a double data entry package for epidemiology and laboratory data, semi-automated entry of ELISPOT data directly from the plate reader, and a suite of new programmes for the manipulation and integration of flow cytometry data. The double entered epidemiology and immunology databases are combined into a separate database, providing a near-real-time analysis of immuno-epidemiological data, allowing important trends to be identified early and major decisions about the study to be made and acted on. This dynamic data management model is portable and can easily be applied to other studies.

Simultaneous Detection of Metalloprotease Activities in Complex Biological Samples Using the PrAMA (Proteolytic Activity Matrix Assay) Method.

PubMed

Conrad, Catharina; Miller, Miles A; Bartsch, Jörg W; Schlomann, Uwe; Lauffenburger, Douglas A

2017-01-01

Proteolytic Activity Matrix Analysis (PrAMA) is a method for simultaneously determining the activities of specific Matrix Metalloproteinases (MMPs) and A Disintegrin and Metalloproteinases (ADAMs) in complex biological samples. In mixtures of unknown proteases, PrAMA infers selective metalloproteinase activities by using a panel of moderately specific FRET-based polypeptide protease substrates in parallel, typically monitored by a plate-reader in a 96-well format. Fluorescence measurements are then quantitatively compared to a standard table of catalytic efficiencies measured from purified mixtures of individual metalloproteinases and FRET substrates. Computational inference of specific activities is performed with an easily used Matlab program, which is provided herein. Thus, we describe PrAMA as a combined experimental and mathematical approach to determine real-time metalloproteinase activities, which has previously been applied to live-cell cultures, cellular lysates, cell culture supernatants, and body fluids from patients.

Detection of Penicillinase in Milk by Sandwich ELISA Based Polyclonal and Monoclonal Antibody.

PubMed

Zhao, Yinli; Li, Guoxi

2016-01-01

A sandwich ELISA has been developed using polyclonal and monoclonal antibody for the determination of penicillinase in milk. For this purpose, specific polyclonal and monoclonal antibodies against penicillinase were generated and characterized. Using penicillinase standards prepared from 1-128 ng/mL, the method indicated that the detection limit of the sandwich ELISA, as measured in an ELISA plate reader, was as low as 0.86 ng/mL of penicillinase. For determine the accuracy, raw milk containing 2, 8, 32, and 64 ng/mL of penicillinase were tested by sandwich ELISA. Recoveries were from 93-97.5%, and the coefficient of variation [CV (%)] were from 5.55-8.38%. For interassay reproducibility, recoveries were from 89.5-95.1%, the coefficient of variation [CV (%)] were from 5.26-9.58%. This sandwich ELISA provides a useful screening method for quantitative detection of penicillinase in milk.

LED instrument approach instruction display

NASA Technical Reports Server (NTRS)

Meredith, B. D.; Kelly, W. L., IV; Crouch, R. K.

1979-01-01

A display employing light emitting diodes (LED's) was developed to demonstrate the feasibility of such displays for presenting landing and navigation information to reduce the workload of general aviation pilots during IFR flight. The display consists of a paper tape reader, digital memory, control electronics, digital latches, and LED alphanumeric displays. A presentable digital countdown clock-timer is included as part of the system to provide a convenient means of monitoring time intervals for precise flight navigation. The system is a limited capability prototype assembled to test pilot reaction to such a device under simulated IFR operation. Pilot opinion indicates that the display is helpful in reducing the IFR pilots workload when used with a runway approach plate. However, the development of a compact, low power second generation display was recommended which could present several instructions simultaneously and provide information update capability. A microprocessor-based display could fulfill these requirements.

High-content screening in microfluidic devices.

PubMed

Cheong, Raymond; Paliwal, Saurabh; Levchenko, Andre

2010-08-01

Miniaturization is the key to advancing the state of the art in high-content screening (HCS) in order to enable dramatic cost savings through reduced usage of expensive biochemical reagents and to enable large-scale screening on primary cells. Microfluidic technology offers the potential to enable HCS to be performed with an unprecedented degree of miniaturization. This perspective highlights a real-world example from the authors’ work of HCS assays implemented in a highly miniaturized microfluidic format. The advantages of this technology are discussed, including cost savings, high-throughput screening on primary cells, improved accuracy, the ability to study complex time-varying stimuli, and ease of automation, integration and scaling. The reader will understand the capabilities of anew microfluidics-based platform for HCS and the advantages it provides over conventional plate-based HCS. Microfluidics technology will drive significant advancements and broader usage and applicability of HCS in drug discovery.

Chasing lava: a geologist's adventures at the Hawaiian Volcano Observatory

USGS Publications Warehouse

Duffield, Wendell A.

2003-01-01

A lively account of the three years (1969-1972) spent by geologist Wendell Duffield working at the Hawaiian Volcano Observatory at Kilauea, one of the world's more active volcanoes. Abundantly illustrated in b&w and color, with line drawings and maps, as well. Volcanologists and general readers alike will enjoy author Wendell Duffield's report from Kilauea--home of Pele, the goddess of fire and volcanoes. Duffield's narrative encompasses everything from the scientific (his discovery that the movements of cooled lava on a lava lake mimic the movements of the earth's crust, providing an accessible model for understanding plate tectonics) to the humorous (his dog's discovery of a snake on the supposedly snake-free island) to the life-threatening (a colleague's plunge into molten lava). This charming account of living and working at Kilauea, one of the world's most active volcanoes, is sure to be a delight.

Measurement of cell respiration and oxygenation in standard multichannel biochips using phosphorescent O2-sensitive probes.

PubMed

Kondrashina, Alina V; Papkovsky, Dmitri B; Dmitriev, Ruslan I

2013-09-07

Measurement of cell oxygenation and oxygen consumption is useful for studies of cell bioenergetics, metabolism, mitochondrial function, drug toxicity and common pathophysiological conditions. Here we present a new platform for such applications which uses commercial multichannel biochips (μ-slides, Ibidi) and phosphorescent O2 sensitive probes. This platform was evaluated with both extracellular and intracellular O2 probes, several different cell types and treatments including mitochondrial uncoupling and inhibition, depletion of extracellular Ca(2+) and inhibition of V-ATPase and histone deacetylases. The results show that compared to the standard microwell plates currently used, the μ-slide platform provides facile O2 measurements with both suspension and adherent cells, higher sensitivity and reproducibility, and faster measurement time. It also allows re-perfusion and multiple treatments of cells and multi-parametric analyses in conjunction with other probes. Optical measurements are conducted on standard fluorescence readers and microscopes.

The Ascent of Science

NASA Astrophysics Data System (ADS)

Silver, Brian L.

2000-04-01

From the revolutionary discoveries of Galileo and Newton to the mind-bending theories of Einstein and Heisenberg, from plate tectonics to particle physics, from the origin of life to universal entropy, and from biology to cosmology, here is a sweeping, readable, and dynamic account of the whole of Western science.In the approachable manner and method of Stephen Jay Gould and Carl Sagan, the late Brian L. Silver translates our most important, and often most obscure, scientific developments into a vernacular that is not only accessible and illuminating but also enjoyable. Silver makes his comprehensive case with much clarity and insight; his book aptly locates science as the apex of human reason, and reason as our best path to the truth. For all readers curious about--or else perhaps intimidated by--what Silver calls "the scientific campaign up to now", The Ascent of Science will be fresh, vivid, and fascinating reading.

Review of Research into the Concept of the Microblowing Technique for Turbulent Skin Friction Reduction

NASA Technical Reports Server (NTRS)

2004-01-01

A new technology for reducing turbulent skin friction, called the Microblowing Technique (MBT), is presented. Results from proof-of-concept experiments show that this technology could potentially reduce turbulent skin friction by more than 50% of the skin friction of a solid flat plate for subsonic and supersonic flow conditions. The primary purpose of this review paper is to provide readers with information on the turbulent skin friction reduction obtained from many experiments using the MBT. Although the MBT has a penalty for obtaining the microblowing air associated with it, some combinations of the MBT with suction boundary layer control methods are an attractive alternative for a real application. Several computational simulations to understand the flow physics of the MBT are also included. More experiments and computational fluid dynamics (CFD) computations are needed for the understanding of the unsteady flow nature of the MBT and the optimization of this new technology.

A Self-Reporting Photocatalyst for Online Fluorescence Monitoring of High Throughput RAFT Polymerization.

PubMed

Yeow, Jonathan; Joshi, Sanket; Chapman, Robert; Boyer, Cyrille Andre Jean Marie

2018-04-25

Translating controlled/living radical polymerization (CLRP) from batch to the high throughput production of polymer libraries presents several challenges in terms of both polymer synthesis and characterization. Although recently there have been significant advances in the field of low volume, high throughput CLRP, techniques able to simultaneously monitor multiple polymerizations in an "online" manner have not yet been developed. Here, we report our discovery that 5,10,15,20-tetraphenyl-21H,23H-porphine zinc (ZnTPP) is a self-reporting photocatalyst that can mediate PET-RAFT polymerization as well as report on monomer conversion via changes in its fluorescence properties. This enables the use of a microplate reader to conduct high throughput "online" monitoring of PET-RAFT polymerizations performed directly in 384-well, low volume microtiter plates. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Correction of microplate location effects improves performance of the thrombin generation test

PubMed Central

2013-01-01

Background Microplate-based thrombin generation test (TGT) is widely used as clinical measure of global hemostatic potential and it becomes a useful tool for control of drug potency and quality by drug manufactures. However, the convenience of the microtiter plate technology can be deceiving: microplate assays are prone to location-based variability in different parts of the microtiter plate. Methods In this report, we evaluated the well-to-well consistency of the TGT variant specifically applied to the quantitative detection of the thrombogenic substances in the immune globulin product. We also studied the utility of previously described microplate layout designs in the TGT experiment. Results Location of the sample on the microplate (location effect) contributes to the variability of TGT measurements. Use of manual pipetting techniques and applications of the TGT to the evaluation of procoagulant enzymatic substances are especially sensitive. The effects were not sensitive to temperature or choice of microplate reader. Smallest location effects were observed with automated dispenser-based calibrated thrombogram instrument. Even for an automated instrument, the use of calibration curve resulted in up to 30% bias in thrombogenic potency assignment. Conclusions Use of symmetrical version of the strip-plot layout was demonstrated to help to minimize location artifacts even under the worst-case conditions. Strip-plot layouts are required for quantitative thrombin-generation based bioassays used in the biotechnological field. PMID:23829491

Correction of microplate location effects improves performance of the thrombin generation test.

PubMed

Liang, Yideng; Woodle, Samuel A; Shibeko, Alexey M; Lee, Timothy K; Ovanesov, Mikhail V

2013-07-05

Microplate-based thrombin generation test (TGT) is widely used as clinical measure of global hemostatic potential and it becomes a useful tool for control of drug potency and quality by drug manufactures. However, the convenience of the microtiter plate technology can be deceiving: microplate assays are prone to location-based variability in different parts of the microtiter plate. In this report, we evaluated the well-to-well consistency of the TGT variant specifically applied to the quantitative detection of the thrombogenic substances in the immune globulin product. We also studied the utility of previously described microplate layout designs in the TGT experiment. Location of the sample on the microplate (location effect) contributes to the variability of TGT measurements. Use of manual pipetting techniques and applications of the TGT to the evaluation of procoagulant enzymatic substances are especially sensitive. The effects were not sensitive to temperature or choice of microplate reader. Smallest location effects were observed with automated dispenser-based calibrated thrombogram instrument. Even for an automated instrument, the use of calibration curve resulted in up to 30% bias in thrombogenic potency assignment. Use of symmetrical version of the strip-plot layout was demonstrated to help to minimize location artifacts even under the worst-case conditions. Strip-plot layouts are required for quantitative thrombin-generation based bioassays used in the biotechnological field.

Interfacing Lab-on-a-Chip Embryo Technology with High-Definition Imaging Cytometry.

PubMed

Zhu, Feng; Hall, Christopher J; Crosier, Philip S; Wlodkowic, Donald

2015-08-01

To spearhead deployment of zebrafish embryo biotests in large-scale drug discovery studies, automated platforms are needed to integrate embryo in-test positioning and immobilization (suitable for high-content imaging) with fluidic modules for continuous drug and medium delivery under microperfusion to developing embryos. In this work, we present an innovative design of a high-throughput three-dimensional (3D) microfluidic chip-based device for automated immobilization and culture and time-lapse imaging of developing zebrafish embryos under continuous microperfusion. The 3D Lab-on-a-Chip array was fabricated in poly(methyl methacrylate) (PMMA) transparent thermoplastic using infrared laser micromachining, while the off-chip interfaces were fabricated using additive manufacturing processes (fused deposition modelling and stereolithography). The system's design facilitated rapid loading and immobilization of a large number of embryos in predefined clusters of traps during continuous microperfusion of drugs/toxins. It was conceptually designed to seamlessly interface with both upright and inverted fluorescent imaging systems and also to directly interface with conventional microtiter plate readers that accept 96-well plates. Compared with the conventional Petri dish assays, the chip-based bioassay was much more convenient and efficient as only small amounts of drug solutions were required for the whole perfusion system running continuously over 72 h. Embryos were spatially separated in the traps that assisted tracing single embryos, preventing interembryo contamination and improving imaging accessibility.

Spectrophotometric determination of various polyanions with polymeric film optodes using microtiter plate reader.

PubMed

Dürüst, Nedime; Meyerhoff, Mark E; Unal, Nazangül; Naç, Sibel

2011-08-05

Polycation-sensitive membrane optodes based on the chromoionophore 2',7'-dichlorofluorescein octadecylester (DCFOE) have previously been developed and used for determination of heparin via a titrimetric method. In this study, it is shown that some other important polyanions such as PPS (pentosan polysulfate), DNA, xanthan, Na-alginate, and carrageenan (food additive) can also be readily determined by using DCFOE-based microtiter plate-format optodes (MPOs) and polycationic titrants that bind these polyanionic species. The optical sensors are prepared with poly(vinyl chloride) (PVC), polyurethane (PU), bis(2-ethylhexyl)sebacate (DOS), and 2',7'-dichlorofluorescein octadecylester (DCFOE) and exhibit reproducible and sensitive absorbance changes in response to the varying polycationic titrant concentrations. Three different polycations; protamine, poly-l-lysine and poly-l-arginine, are employed as titrants. The method has a detection limit of 1 μg mL(-1), and a dynamic range of 1-40 μg mL(-1). After the quantitative determinations are successfully demonstrated in buffered solutions, similar titrations are also performed in real samples. The method is validated by recovery studies in these samples. The average polyanion recoveries were quantitative [99.7(±1.3) % for pastry cream with vanillin (protamine titrant); 100.4 (±3.3) % for pastry gel with strawberry(PLA titrant), and 102.9(±2.0) % for pastry gel with strawberry (PLL titrant)]. Copyright © 2011 Elsevier B.V. All rights reserved.

Can we assess healing of surgically treated long bone fractures on radiograph?

PubMed

Perlepe, V; Omoumi, P; Larbi, A; Putineanu, D; Dubuc, J-E; Schubert, T; Vande Berg, B

2018-06-01

To determine the frequency and causes for limitations in the radiographic evaluation of surgically treated long bone fractures. Six readers separately scored 140 sets of antero-posterior (AP) and lateral radiographs of surgically treated long bone fractures, using a radiographic union score (RUS). We determined the rate of assessability of the fracture edges at each of the four cortical segments (n=560) seen tangentially on the two radiographs and the causes for non-assessability. The rate of feasibility of the RUS (more than two fracture edges assessable per fracture) was determined and compared according to different parameters. Fracture edges were visible in 71% to 81% of the 560 cortical segments. Metal hardware superimposition was the most frequent cause for non-assessability (79-95%). RUS values could be calculated in 58% to 75% of fractures. Scoring was statistically significantly less frequently calculable in plated (31-56%) than in nailed fractures (90-97%), in distal (47-61%) than in proximal (78-89%) bones and in upper (27-49%) than in lower (76-91%) limb bones (P≤0.01). The type of stabilization hardware is the main limiting factor in the radiographic assessment of surgically treated long bone fractures. Scoring was feasible in only 31% to 56% of plated fractures. Copyright © 2018 Société française de radiologie. Published by Elsevier Masson SAS. All rights reserved.

Reader performance in visual assessment of breast density using visual analogue scales: Are some readers more predictive of breast cancer?

NASA Astrophysics Data System (ADS)

Rayner, Millicent; Harkness, Elaine F.; Foden, Philip; Wilson, Mary; Gadde, Soujanya; Beetles, Ursula; Lim, Yit Y.; Jain, Anil; Bundred, Sally; Barr, Nicky; Evans, D. Gareth; Howell, Anthony; Maxwell, Anthony; Astley, Susan M.

2018-03-01

Mammographic breast density is one of the strongest risk factors for breast cancer, and is used in risk prediction and for deciding appropriate imaging strategies. In the Predicting Risk Of Cancer At Screening (PROCAS) study, percent density estimated by two readers on Visual Analogue Scales (VAS) has shown a strong relationship with breast cancer risk when assessed against automated methods. However, this method suffers from reader variability. This study aimed to assess the performance of PROCAS readers using VAS, and to identify those most predictive of breast cancer. We selected the seven readers who had estimated density on over 6,500 women including at least 100 cancer cases, analysing their performance using multivariable logistic regression and Receiver Operator Characteristic (ROC) analysis. All seven readers showed statistically significant odds ratios (OR) for cancer risk according to VAS score after adjusting for classical risk factors. The OR was greatest for reader 18 at 1.026 (95% Cl 1.018-1.034). Adjusted Area Under the ROC Curves (AUCs) were statistically significant for all readers, but greatest for reader 14 at 0.639. Further analysis of the VAS scores for these two readers showed reader 14 had higher sensitivity (78.0% versus 42.2%), whereas reader 18 had higher specificity (78.0% versus 46.0%). Our results demonstrate individual differences when assigning VAS scores; one better identified those with increased risk, whereas another better identified low risk individuals. However, despite their different strengths, both readers showed similar predictive abilities overall. Standardised training for VAS may improve reader variability and consistency of VAS scoring.

On the heteroclinic connection problem for multi-well gradient systems

NASA Astrophysics Data System (ADS)

Zuniga, Andres; Sternberg, Peter

2016-10-01

We revisit the existence problem of heteroclinic connections in RN associated with Hamiltonian systems involving potentials W :RN → R having several global minima. Under very mild assumptions on W we present a simple variational approach to first find geodesics minimizing length of curves joining any two of the potential wells, where length is computed with respect to a degenerate metric having conformal factor √{ W}. Then we show that when such a minimizing geodesic avoids passing through other wells of the potential at intermediate times, it gives rise to a heteroclinic connection between the two wells. This work improves upon the approach of [22] and represents a more geometric alternative to the approaches of e.g. [5,10,14,17] for finding such connections.

On a Minimum Problem in Smectic Elastomers

NASA Astrophysics Data System (ADS)

Buonsanti, Michele; Giovine, Pasquale

2008-07-01

Smectic elastomers are layered materials exhibiting a solid-like elastic response along the layer normal and a rubbery one in the plane. Balance equations for smectic elastomers are derived from the general theory of continua with constrained microstructure. In this work we investigate a very simple minimum problem based on multi-well potentials where the microstructure is taken into account. The set of polymeric strains minimizing the elastic energy contains a one-parameter family of simple strain associated with a micro-variation of the degree of freedom. We develop the energy functional through two terms, the first one nematic and the second one considering the tilting phenomenon; after, by developing in the rubber elasticity framework, we minimize over the tilt rotation angle and extract the engineering stress.

Review-Esso Resources Canada Ltd. , Norman Wells expansion project drilling program

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schaef, D.G.

Esso Resources Canada Limited has embarked on a project to increase production from its Norman Wells Oil field located 145 km south of the Arctic Circle, from 475 m/sup 3//D to 4000 m/sup 3//D of crude oil. This paper provides details on the development drilling portion of the project which is comprised of 150 wells to be drilled in 3 years utilizing 2 drilling rigs from July 1982 through September 1985. The majority of the wells will be directionally drilled from multiwell land pads and artificial islands to shallow reservoir targets underlying the Mackenzie River, a major river intersecting themore » field boundaries. Experience from the initial 27 wells completed is provided.« less

Benefit of computer-aided detection analysis for the detection of subsolid and solid lung nodules on thin- and thick-section CT.

PubMed

Godoy, Myrna C B; Kim, Tae Jung; White, Charles S; Bogoni, Luca; de Groot, Patricia; Florin, Charles; Obuchowski, Nancy; Babb, James S; Salganicoff, Marcos; Naidich, David P; Anand, Vikram; Park, Sangmin; Vlahos, Ioannis; Ko, Jane P

2013-01-01

The objective of our study was to evaluate the impact of computer-aided detection (CAD) on the identification of subsolid and solid lung nodules on thin- and thick-section CT. For 46 chest CT examinations with ground-glass opacity (GGO) nodules, CAD marks computed using thin data were evaluated in two phases. First, four chest radiologists reviewed thin sections (reader(thin)) for nodules and subsequently CAD marks (reader(thin) + CAD(thin)). After 4 months, the same cases were reviewed on thick sections (reader(thick)) and subsequently with CAD marks (reader(thick) + CAD(thick)). Sensitivities were evaluated. Additionally, reader(thick) sensitivity with assessment of CAD marks on thin sections was estimated (reader(thick) + CAD(thin)). For 155 nodules (mean, 5.5 mm; range, 4.0-27.5 mm)-74 solid nodules, 22 part-solid (part-solid nodules), and 59 GGO nodules-CAD stand-alone sensitivity was 80%, 95%, and 71%, respectively, with three false-positives on average (0-12) per CT study. Reader(thin) + CAD(thin) sensitivities were higher than reader(thin) for solid nodules (82% vs 57%, p < 0.001), part-solid nodules (97% vs 81%, p = 0.0027), and GGO nodules (82% vs 69%, p < 0.001) for all readers (p < 0.001). Respective sensitivities for reader(thick), reader(thick) + CAD(thick), reader(thick) + CAD(thin) were 40%, 58% (p < 0.001), and 77% (p < 0.001) for solid nodules; 72%, 73% (p = 0.322), and 94% (p < 0.001) for part-solid nodules; and 53%, 58% (p = 0.008), and 79% (p < 0.001) for GGO nodules. For reader(thin), false-positives increased from 0.64 per case to 0.90 with CAD(thin) (p < 0.001) but not for reader(thick); false-positive rates were 1.17, 1.19, and 1.26 per case for reader(thick), reader(thick) + CAD(thick), and reader(thick) + CAD(thin), respectively. Detection of GGO nodules and solid nodules is significantly improved with CAD. When interpretation is performed on thick sections, the benefit is greater when CAD marks are reviewed on thin rather than thick sections.

Understanding Reading through the Eyes of Third-Grade Struggling Readers

ERIC Educational Resources Information Center

Wiggs, Christine E.

2012-01-01

Within the vast research base on struggling readers, very few studies address the nature of struggling readers from their own perspectives; that is, how struggling readers experience reading instruction. The purpose of this qualitative case study research was to gain a deeper understanding of how three third-grade struggling readers viewed…

Defense Automation Resources Management Manual

DTIC Science & Technology

1988-09-01

Electronic Command Signals Programmer, Plugboard Programmers Punch, Card Punch, Paper Tape Reader, Character Reader-Generator, Time Cards Reader...Multiplexor-Shift Register Group Multiplier Panel Control, Plugboard Panel, Interconnection, Digital Computer Panel, Meter-Attenuator, Tape Recorder PC Cards...Perforator, Tape Plug-In Unit Potentiometer, Coefficient, Analog Computer Programmer, Plugboard Punch, Paper Tape Racks Reader, Time Code Reader

Examining Readers' Evaluations of Objectivity and Bias in News Discourse

ERIC Educational Resources Information Center

Cramer, Peter; Eisenhart, Christopher

2014-01-01

Readers' objectivity and bias evaluations of news texts were investigated in order to better understand the process by which readers make these kinds of judgments and the evidence on which they base them. Readers were primed to evaluate news texts for objectivity and bias, and their selections and metacommentary were analyzed. Readers detected…

Readers' Readings: Applications of Reader-Response Theory.

ERIC Educational Resources Information Center

Steiner, Linda

In the interest of applying reader response theory to journalism this paper posits that readers of newspapers, like readers of literature, take an active role in making meaning from the articles they read, rather than passively accepting news as a finished, static product. Additionally, it proposes that journalism textbooks pay little attention to…

When transparency is opaque: Effects of diacritic marks and vowel letters on dyslexic Hebrew readers.

PubMed

Weiss, Yael; Katzir, Tami; Bitan, Tali

2016-10-01

The current study examined the effects of orthographic transparency and familiarity on brain mechanisms involved in word recognition in adult dyslexic Hebrew readers. We compared functional Magnetic Resonance Imaging (fMRI) brain activation in 21 dyslexic readers and 22 typical readers, and examined the effects of diacritic marks that provide transparent but less familiar information and vowel letters that increase orthographic transparency without compromising familiarity. Dyslexic readers demonstrated reduced activation in left supramarginal gyrus (SMG) as compared to typical readers, as well as different patterns of activation within the left inferior frontal gyrus (IFG). Furthermore, in contrast to typical readers, dyslexic readers did not show increased activation for diacritics in left temporo-parietal junction regions, associated with mapping orthography to phonology. Nevertheless, both groups showed the facilitation effect of vowel letters on regions associated with lexical-semantic access. Altogether the results suggest that while typical readers can compensate for the reduced familiarity of pointed words with increased reliance on decoding of smaller units, dyslexic readers do not, and therefore they show a higher cost. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparison of reading and listening-reading techniques for administration of PIAT Reading Comprehension subtest: justification for the bypass approach.

PubMed

Weinberg, W A; McLean, A; Brumback, R A

1988-04-01

The Peabody Individual Achievement Test (PIAT) Reading Comprehension subtest was administered to a group of 23 learning disabled children in Grades 5 through 7 who had been classified by the clinical Lexical Paradigm as good readers or poor readers. Using standardized test administration, 14 poor readers scored substantially below the 9 good readers; however, when the child was allowed to listen and read silently while the test item was read aloud, poor readers showed marked improvement in performance compared to only moderate improvement shown by the good readers. This difference in improvement between the groups was significant and resulted in poor readers achieving performance similar to that of the good readers administered the test in the standard manner.

Bone metastases in castration-resistant prostate cancer: associations between morphologic CT patterns, glycolytic activity, and androgen receptor expression on PET and overall survival.

PubMed

Vargas, Hebert Alberto; Wassberg, Cecilia; Fox, Josef J; Wibmer, Andreas; Goldman, Debra A; Kuk, Deborah; Gonen, Mithat; Larson, Steven M; Morris, Michael J; Scher, Howard I; Hricak, Hedvig

2014-04-01

To compare the features of bone metastases at computed tomography (CT) to tracer uptake at fluorine 18 fluorodeoxyglucose (FDG) positron emission tomography (PET) and fluorine 18 16β-fluoro-5-dihydrotestosterone (FDHT) PET and to determine associations between these imaging features and overall survival in men with castration-resistant prostate cancer. This is a retrospective study of 38 patients with castration-resistant prostate cancer. Two readers independently evaluated CT, FDG PET, and FDHT PET features of bone metastases. Associations between imaging findings and overall survival were determined by using univariate Cox proportional hazards regression. In 38 patients, reader 1 detected 881 lesions and reader 2 detected 867 lesions. Attenuation coefficients at CT correlated inversely with FDG (reader 1: r = -0.3007; P < .001; reader 2: r = -0.3147; P < .001) and FDHT (reader 1: r = -0.2680; P = .001; reader 2: r = -0.3656; P < .001) uptake. The number of lesions on CT scans was significantly associated with overall survival (reader 1: hazard ratio [HR], 1.025; P = .05; reader 2: HR, 1.021; P = .04). The numbers of lesions on FDG and FDHT PET scans were significantly associated with overall survival for reader 1 (HR, 1.051-1.109; P < .001) and reader 2 (HR, 1.026-1.082; P ≤ .009). Patients with higher FDHT uptake (lesion with the highest maximum standardized uptake value) had significantly shorter overall survival (reader 1: HR, 1.078; P = .02; reader 2: HR, 1.092; P = .02). FDG uptake intensity was not associated with overall survival (reader 1, P = .65; reader 2, P = .38). In patients with castration-resistant prostate cancer, numbers of bone lesions on CT, FDG PET, and FDHT PET scans and the intensity of FDHT uptake are significantly associated with overall survival. RSNA, 2013

Bone Metastases in Castration-Resistant Prostate Cancer: Associations between Morphologic CT Patterns, Glycolytic Activity, and Androgen Receptor Expression on PET and Overall Survival

PubMed Central

Wassberg, Cecilia; Fox, Josef J.; Wibmer, Andreas; Goldman, Debra A.; Kuk, Deborah; Gonen, Mithat; Larson, Steven M.; Morris, Michael J.; Scher, Howard I.; Hricak, Hedvig

2014-01-01

Purpose To compare the features of bone metastases at computed tomography (CT) to tracer uptake at fluorine 18 fluorodeoxyglucose (FDG) positron emission tomography (PET) and fluorine 18 16β-fluoro-5-dihydrotestosterone (FDHT) PET and to determine associations between these imaging features and overall survival in men with castration-resistant prostate cancer. Materials and Methods This is a retrospective study of 38 patients with castration-resistant prostate cancer. Two readers independently evaluated CT, FDG PET, and FDHT PET features of bone metastases. Associations between imaging findings and overall survival were determined by using univariate Cox proportional hazards regression. Results In 38 patients, reader 1 detected 881 lesions and reader 2 detected 867 lesions. Attenuation coefficients at CT correlated inversely with FDG (reader 1: r = −0.3007; P < .001; reader 2: r = −0.3147; P < .001) and FDHT (reader 1: r = −0.2680; P = .001; reader 2: r = −0.3656; P < .001) uptake. The number of lesions on CT scans was significantly associated with overall survival (reader 1: hazard ratio [HR], 1.025; P = .05; reader 2: HR, 1.021; P = .04). The numbers of lesions on FDG and FDHT PET scans were significantly associated with overall survival for reader 1 (HR, 1.051–1.109; P < .001) and reader 2 (HR, 1.026–1.082; P ≤ .009). Patients with higher FDHT uptake (lesion with the highest maximum standardized uptake value) had significantly shorter overall survival (reader 1: HR, 1.078; P = .02; reader 2: HR, 1.092; P = .02). FDG uptake intensity was not associated with overall survival (reader 1, P = .65; reader 2, P = .38). Conclusion In patients with castration-resistant prostate cancer, numbers of bone lesions on CT, FDG PET, and FDHT PET scans and the intensity of FDHT uptake are significantly associated with overall survival. © RSNA, 2013 PMID:24475817

Children and adults both see 'pirates' in 'parties': letter-position effects for developing readers and skilled adult readers.

PubMed

Paterson, Kevin B; Read, Josephine; McGowan, Victoria A; Jordan, Timothy R

2015-03-01

Developing readers often make anagrammatical errors (e.g. misreading pirates as parties), suggesting they use letter position flexibly during word recognition. However, while it is widely assumed that the occurrence of these errors decreases with increases in reading skill, empirical evidence to support this distinction is lacking. Accordingly, we compared the performance of developing child readers (aged 8-10 years) against the end-state performance of skilled adult readers in a timed naming task, employing anagrams used previously in this area of research. Moreover, to explore the use of letter position by developing readers and skilled adult readers more fully, we used anagrams which, to form another word, required letter transpositions over only interior letter positions, or both interior and exterior letter positions. The patterns of effects across these two anagram types for the two groups of readers were very similar. In particular, both groups showed similarly slowed response times (and developing readers increased errors) for anagrams requiring only interior letter transpositions but not for anagrams that required exterior letter transpositions. This similarity in the naming performance of developing readers and skilled adult readers suggests that the end-state skilled use of letter position is established earlier during reading development than is widely assumed. © 2014 John Wiley & Sons Ltd.

Three Readers, Three Languages, Three Texts: The Strategic Reading of Multilingual and Multiliterate Readers

ERIC Educational Resources Information Center

Alsheikh, Negmeldin O.

2011-01-01

This case study investigates the metacognitive reading strategies of three advanced proficient trilingual readers whose native language is Hausa. The study examines the reading strategies employed by the three readers in English, French and Hausa. The aim of the study was to compare the reading strategy profiles of trilingual readers through…

Struggling Readers: Assessment and Instruction in Grades K-6. Solving Problems in the Teaching of Literacy.

ERIC Educational Resources Information Center

Balajthy, Ernest; Lipa-Wade, Sally

This book focuses on three distinct types of struggling readers that teachers will instantly recognize from their own classrooms--the "Catch-On Reader," the "Catch-Up Reader," and the "Stalled Reader." The book provides detailed case studies which bring to life the specific problems these students are likely to face…

"True Wizardry": Readers Theatre in the Classroom. PEN 79.

ERIC Educational Resources Information Center

Robertson, Marion E.

This article describes Readers Theater (a form of group storytelling in which two or more readers present a piece of literature by reading aloud from hand-held scripts) and advocates its use in the classroom. The paper's seven sections are as follows: (1) What is Readers Theater; (2) Readers Theater and Its Conventions (discussing scripts,…

NaturalReader: A New Generation Text Reader

ERIC Educational Resources Information Center

Flood, Jacqueline

2007-01-01

NaturalReader (http://www.naturalreaders.com/) is a new generation text reader, which means that it reads any machine readable text using synthesized speech without having to copy and paste the selected text into the NaturalReader application window. It installs a toolbar directly into all of the Microsoft Office[TM] programs and uses a mini-board…

RFID Reader Antenna with Multi-Linear Polarization Diversity

NASA Technical Reports Server (NTRS)

Fink, Patrick; Lin, Greg; Ngo, Phong; Kennedy, Timothy; Rodriguez, Danny; Chu, Andrew; Broyan, James; Schmalholz, Donald

2018-01-01

This paper describes an RFID reader antenna that offers reduced polarization loss compared to that typically associated with reader-tag communications involving arbitrary relative orientation of the reader antenna and the tag.

Is a "Phoenician" reading style superior to a "Chinese" reading style? Evidence from fourth graders.

PubMed

Bowey, Judith A

2008-07-01

This study compared normally achieving fourth-grade "Phoenician" readers, who identify nonwords significantly more accurately than they do exception words, with "Chinese" readers, who show the reverse pattern. Phoenician readers scored lower than Chinese readers on word identification, exception word reading, orthographic choice, spelling, reading comprehension, and verbal ability. When compared with normally achieving children who read nonwords and exception words equally well, Chinese readers scored as well as these children on word identification, regular word reading, orthographic choice, spelling, reading comprehension, phonological sensitivity, and verbal ability and scored better on exception word reading. Chinese readers also used rhyme-based analogies to read nonwords derived from high-frequency exception words just as often as did these children. As predicted, Phoenician and Chinese readers adopted somewhat different strategies in reading ambiguous nonwords constructed by analogy to high-frequency exception words. Phoenician readers were more likely than Chinese readers to read ambiguous monosyllabic nonwords via context-free grapheme-phoneme correspondences and were less likely to read disyllabic nonwords by analogy to high-frequency analogues. Although the Chinese reading style was more common than the Phoenician style in normally achieving fourth graders, there were similar numbers of poor readers with phonological dyslexia (identifying nonwords significantly more accurately than exception words) and surface dyslexia (showing the reverse pattern), although surface dyslexia was more common in the severely disabled readers. However, few of the poor readers showed pure patterns of phonological or surface dyslexia.

Demonstration of Multi- and Single-Reader Sample Size Program for Diagnostic Studies software.

PubMed

Hillis, Stephen L; Schartz, Kevin M

2015-02-01

The recently released software Multi- and Single-Reader Sample Size Sample Size Program for Diagnostic Studies , written by Kevin Schartz and Stephen Hillis, performs sample size computations for diagnostic reader-performance studies. The program computes the sample size needed to detect a specified difference in a reader performance measure between two modalities, when using the analysis methods initially proposed by Dorfman, Berbaum, and Metz (DBM) and Obuchowski and Rockette (OR), and later unified and improved by Hillis and colleagues. A commonly used reader performance measure is the area under the receiver-operating-characteristic curve. The program can be used with typical common reader-performance measures which can be estimated parametrically or nonparametrically. The program has an easy-to-use step-by-step intuitive interface that walks the user through the entry of the needed information. Features of the software include the following: (1) choice of several study designs; (2) choice of inputs obtained from either OR or DBM analyses; (3) choice of three different inference situations: both readers and cases random, readers fixed and cases random, and readers random and cases fixed; (4) choice of two types of hypotheses: equivalence or noninferiority; (6) choice of two output formats: power for specified case and reader sample sizes, or a listing of case-reader combinations that provide a specified power; (7) choice of single or multi-reader analyses; and (8) functionality in Windows, Mac OS, and Linux.

Engaging Literacy: A Case Study in the Use of e-Reader Devices

ERIC Educational Resources Information Center

Lettenmaier, Kris

2013-01-01

The purpose of this case study was to determine to what extent e-readers affected struggling readers as a part of reading instruction at the middle school level to improve students' literacy skills of comprehension, fluency, and vocabulary, and to show how e-readers affected engagement and self-efficacy of struggling readers to read and use…

Cognitive Processes in Discourse Comprehension: Passive Processes, Reader-Initiated Processes, and Evolving Mental Representations

ERIC Educational Resources Information Center

van den Broek, Paul; Helder, Anne

2017-01-01

As readers move through a text, they engage in various types of processes that, if all goes well, result in a mental representation that captures their interpretation of the text. With each new text segment the reader engages in passive and, at times, reader-initiated processes. These processes are strongly influenced by the readers'…

How Do Fluent and Poor Readers' Endurance Differ in Reading?

ERIC Educational Resources Information Center

Bastug, Muhammet; Keskin, Hasan Kagan; Akyol, Mustafa

2017-01-01

It was observed in this research how endurance status of fluent readers and poor readers changed as the text became longer. 40 students of the primary school 4th-grade, 20 were fluent readers and other 20 were poor readers, participated in the research. A narrative text was utilised in the data collection process. Students' oral readings were…

The Impact of Morphological Awareness on Word Reading and Dictation in Chinese Early Adolescent Readers With and Without Dyslexia.

PubMed

Kalindi, Sylvia Chanda; Chung, Kevin Kien Hoa

2018-01-01

This study investigated the role of morphological awareness in understanding Chinese word reading and dictation among Chinese-speaking adolescent readers in Hong Kong as well as the cognitive-linguistic profile of early adolescent readers with dyslexia. Fifty-four readers with dyslexia in Grades 5 and 6 were compared with 54 chronological age-matched (CA) typical readers on the following measures of cognitive-linguistic and literacy skills: morphological awareness, phonological awareness, visual-orthographic knowledge, rapid naming, vocabulary knowledge, verbal short-term memory (STM), Chinese word reading, and dictation (or spelling). The results indicated that early adolescent readers with dyslexia performed less well than the typical readers on all cognitive-linguistic and literacy measures except the phonological measures. Both groups' scores showed substantial correlations between morphological awareness and Chinese word reading and dictation. Visual-orthographic knowledge and rapid naming were also associated with dictation in early adolescent readers with and without dyslexia, respectively. Moderated multiple regression analyses further revealed that morphological awareness and rapid naming explained unique variance in word reading and dictation for the readers with dyslexia and typical readers separately after controlling readers' age and group effect. These results highlight the potential importance of morphological awareness and rapid naming in Chinese word reading and writing in Chinese early adolescents' literacy development and impairment.

An Analysis of the Reading Strategies Used by Deaf and Hearing Adults: Similarities and Differences in Phonological Processing and Metacognition

ERIC Educational Resources Information Center

Silvestri, Julia A.

2016-01-01

This study is a mixed methods analysis of reading processes and language experiences of deaf and hearing readers. The sample includes four groups each with fifteen adults--identified as: deaf/high-achieving readers, deaf/struggling/non-academic readers, hearing/high-achieving readers, and hearing/non-academic readers. The purpose of this study is…

Using Twitter to Teach Reader-Oriented Biblical Interpretation: "Tweading" the Gospel of Mark

ERIC Educational Resources Information Center

Williamson, Robert, Jr.

2013-01-01

Twitter offers an engaging way to introduce students to reader-oriented interpretation of the Bible. The exercise described here introduces students to the idea that the reader has a role in the production of a text's meaning, which thus varies from reader to reader. Twitter enables us to capture the real-time thoughts of a variety of…

Visual assessment of breast density using Visual Analogue Scales: observer variability, reader attributes and reading time

NASA Astrophysics Data System (ADS)

Ang, Teri; Harkness, Elaine F.; Maxwell, Anthony J.; Lim, Yit Y.; Emsley, Richard; Howell, Anthony; Evans, D. Gareth; Astley, Susan; Gadde, Soujanya

2017-03-01

Breast density is a strong risk factor for breast cancer and has potential use in breast cancer risk prediction, with subjective methods of density assessment providing a strong relationship with the development of breast cancer. This study aims to assess intra- and inter-observer variability in visual density assessment recorded on Visual Analogue Scales (VAS) among trained readers, and examine whether reader age, gender and experience are associated with assessed density. Eleven readers estimated the breast density of 120 mammograms on two occasions 3 years apart using VAS. Intra- and inter-observer agreement was assessed with Intraclass Correlation Coefficient (ICC) and variation between readers visualised on Bland-Altman plots. The mean scores of all mammograms per reader were used to analyse the effect of reader attributes on assessed density. Excellent intra-observer agreement (ICC>0.80) was found in the majority of the readers. All but one reader had a mean difference of <10 percentage points from the first to the second reading. Inter-observer agreement was excellent for consistency (ICC 0.82) and substantial for absolute agreement (ICC 0.69). However, the 95% limits of agreement for pairwise differences were -6.8 to 15.7 at the narrowest and 0.8 to 62.3 at the widest. No significant association was found between assessed density and reader age, experience or gender, or with reading time. Overall, the readers were consistent in their scores, although some large variations were observed. Reader evaluation and targeted training may alleviate this problem.

The N170 ERP component differs in laterality, distribution, and association with continuous reading measures for deaf and hearing readers.

PubMed

Emmorey, Karen; Midgley, Katherine J; Kohen, Casey B; Sehyr, Zed Sevcikova; Holcomb, Phillip J

2017-11-01

The temporo-occipitally distributed N170 ERP component is hypothesized to reflect print-tuning in skilled readers. This study investigated whether skilled deaf and hearing readers (matched on reading ability, but not phonological awareness) exhibit similar N170 patterns, given their distinct experiences learning to read. Thirty-two deaf and 32 hearing adults viewed words and symbol strings in a familiarity judgment task. In the N170 epoch (120-240ms) hearing readers produced greater negativity for words than symbols at left hemisphere (LH) temporo-parietal and occipital sites, while deaf readers only showed this asymmetry at occipital sites. Linear mixed effects regression was used to examine the influence of continuous measures of reading, spelling, and phonological skills on the N170 (120-240ms). For deaf readers, better reading ability was associated with a larger N170 over the right hemisphere (RH), but for hearing readers better reading ability was associated with a smaller RH N170. Better spelling ability was related to larger occipital N170s in deaf readers, but this relationship was weak in hearing readers. Better phonological awareness was associated with smaller N170s in the LH for hearing readers, but this association was weaker and in the RH for deaf readers. The results support the phonological mapping hypothesis for a left-lateralized temporo-parietal N170 in hearing readers and indicate that skilled reading is characterized by distinct patterns of neural tuning to print in deaf and hearing adults. Copyright © 2017 Elsevier Ltd. All rights reserved.

Deaf Readers’ Response to Syntactic Complexity: Evidence from Self-Paced Reading

PubMed Central

Traxler, Matthew J.; Corina, David P.; Morford, Jill P.; Hafer, Sarah; Hoversten, Liv J.

2013-01-01

This study was designed to determine the feasibility of using self-paced reading methods to study deaf readers and to assess how deaf readers respond to two syntactic manipulations. Three groups of participants read the test sentences: deaf readers, hearing monolingual English readers, and hearing bilingual readers whose second language was English. In Experiment 1, participants read sentences containing subject relative or object relative clauses. The test sentences contained semantic information that influences on-line processing outcomes (Traxler et al., 2002; 2005). All of the participant groups had greater difficulty processing sentences containing object relative clauses. This difficulty was reduced when helpful semantic cues were present. In Experiment 2, participants read active voice and passive voice sentences. The sentences were processed similarly by all three groups. Comprehension accuracy was higher in hearing readers than in deaf readers. Within deaf readers, native signers read the sentences faster and comprehended them to a higher degree than did non-native signers. These results indicate that self-paced reading is a useful method for studying sentence interpretation among deaf readers. PMID:23868696

An analysis of the reading strategies used by adult and student deaf readers.

PubMed

Banner, Alyssa; Wang, Ye

2011-01-01

The purpose of this study was to identify and examine effective reading strategies used by adult deaf readers compared with student deaf readers. There were a total of 11 participants: 5 deaf adults ranging from 27 to 36 years and 6 deaf students ranging from 16 to 20 years. Assessment methods included interview and think-aloud procedures in which individuals were interrupted 3 times during the reading of a text to answer questions about their internal cognitive processes. It was found that both student and adult groups had highly skilled readers who demonstrated higher level reading strategies and less skilled readers who demonstrated lower level strategies, and only the highest skilled reader demonstrated both breadth and depth of strategies in all three categories: "constructing meaning," "monitoring and improving comprehension," and "evaluating comprehension." The study contributes evidence toward two identified gaps in the existing body of research: (a) the lack of investigation into the reading strategies utilized by deaf readers in text comprehension and (b) the overemphasis of most research on studying less skilled deaf readers while overlooking highly proficient deaf readers.

Using Randomized Clinical Trials to Determine the Impact of Reading Intervention on Struggling Adolescent Readers: Reports of Research from Five Nationally Funded Striving Readers Grants

ERIC Educational Resources Information Center

Loadman, William; Sprague, Kim; Hamilton, Jennifer; Coffey, Deb; Faddis, Bonnie

2010-01-01

In 2005, the U.S. Department of Education awarded eight Striving Readers grants to projects around the country. The goal of Striving Readers is to improve literacy skills and achievement for struggling readers in middle and high school and to increase the research base using randomized clinical research to address improvement in adolescent…

Utility of CT Findings in the Diagnosis of Cecal Volvulus.

PubMed

Dane, Bari; Hindman, Nicole; Johnson, Evan; Rosenkrantz, Andrew B

2017-10-01

The objective of our study was to assess the utility of CT features in the diagnosis of cecal volvulus. Forty-three patients undergoing CT for cecal volvulus and with surgical or clinical follow-up were included. Two radiologists (11 years and 1 year of experience) evaluated CT examinations for the following: whirl sign, abnormal cecal position, "bird beak" sign, severe cecal distention, mesenteric engorgement, a newly described "central appendix" sign (defined as abnormal appendix position near midline), and overall impression for cecal volvulus. Univariable and multivariable assessments were performed. Patients with CT examinations in which the appendix was not visible were excluded from calculations involving the central appendix sign. Fifty-one percent (n = 22) of patients had cecal volvulus. All CT findings were significantly more common in patients with cecal volvulus (p < 0.01) other than mesenteric engorgement for reader 1 (p = 0.332). Readers 1 and 2 identified the central appendix sign in 92.9% and 92.3% of patients with volvulus versus in 37.5 and 31.1% of patients without volvulus. The whirl sign exhibited a sensitivity for cecal volvulus of 90.9% for reader 1 and 95.5% for reader 2, and a specificity of 61.9% for both readers. Abnormal cecal position exhibited a sensitivity of 90.0% for reader 1 and 100.0% for reader 2 and a specificity of 66.7% and 38.1%. The bird beak sign exhibited a sensitivity of 86.4% for reader 1 and 100.0% for reader 2 and a specificity of 85.7% and 71.4%. Severe cecal distention exhibited a sensitivity of 100.0% for both readers and a specificity of 81.0% and 61.9%. Mesenteric engorgement exhibited a sensitivity of 40.9% for reader 1 and 100.0% for reader 2 and a specificity of 76.2% and 71.4%. The central appendix sign exhibited a sensitivity of 92.9% for reader 1 and 92.3% for reader 2 and a specificity of 62.5% and 68.8%. Overall impression exhibited a sensitivity of 100.0% for both readers and a specificity of 76.2% and 57.1%. At multivariable analysis, the AUC for cecal volvulus ranged from 0.787 to 0.931, and the whirl sign was an independent predictor of volvulus for both readers (p ≤ 0.014); the central appendix sign was also an independent predictor in patients with a visualized appendix for reader 2 (p ≤ 0.001). CT exhibited high diagnostic performance and very high sensitivity for cecal volvulus. The whirl sign was a significant independent predictor of volvulus for both readers.

Reading Proficiency and Adaptability in Orthographic Processing: An Examination of the Effect of Type of Orthography Read on Brain Activity in Regular and Dyslexic Readers

PubMed Central

Bar-Kochva, Irit; Breznitz, Zvia

2014-01-01

Regular readers were found to adjust the routine of reading to the demands of processing imposed by different orthographies. Dyslexic readers may lack such adaptability in reading. This hypothesis was tested among readers of Hebrew, as Hebrew has two forms of script differing in phonological transparency. Event-related potentials were recorded from 24 regular and 24 dyslexic readers while they carried out a lexical decision task in these two forms of script. The two forms of script elicited distinct amplitudes and latencies at ∼165 ms after target onset, and these effects were larger in regular than in dyslexic readers. These early effects appeared not to be merely a result of the visual difference between the two forms of script (the presence of diacritics). The next effect of form of script was obtained on amplitudes elicited at latencies associated with orthographic-lexical processing and the categorization of stimuli, and these appeared earlier in regular readers (∼340 ms) than in dyslexic readers (∼400 ms). The behavioral measures showed inferior reading skills of dyslexic readers compared to regular readers in reading of both forms of script. Taken together, the results suggest that although dyslexic readers are not indifferent to the type of orthography read, they fail to adjust the routine of reading to the demands of processing imposed by both a transparent and an opaque orthography. PMID:24465844

[Survey among readers of the Tijdschrift voor Psychiatrie].

PubMed

Brandt-Dominicus, J C; van Harten, P N

2006-01-01

The editorial board has defined the objectives of this journal as follows: to provide its readers with refreshing information, to report the results of scientific research and to build bridges between research and everyday practice. To find out what readers want and whether they support the board's objectives, and to use this information in planning the contents of future issues of the journal. All readers were invited to complete a questionnaire either on paper or via the website. Questionnaires were completed by 255 readers (response rate 5.7%). The average rating on a 5-point Likert scale was 3.8. Thematic issues and issues devoted to book reviews were given a high rating. There seems to be a demandfor evidence-based medicine, the inclusion of scientific results reported in other journals and practice-based articles. No significant differences were found between the views expressed by readers who had previously submitted an article for publication and those who had not, nor between Flemish readers and Dutch readers. The website was not visited very often. The Tijdschrift voor Psychiatrie is greatly appreciated by its readers. They are largely in agreement with the objectives formulated by the editorial board. Thematic issues are highly rated and many subjects are mentioned by the readers for future issues. The low response to the survey can affect its representativity. The editorial board will take readers' wishes into consideration when selecting material for future issues of the journal.

Evidence for Deficits in the Temporal Attention Span of Poor Readers

PubMed Central

Visser, Troy A. W.

2014-01-01

Background While poor reading is often associated with phonological deficits, many studies suggest that visual processing might also be impaired. In particular, recent research has indicated that poor readers show impaired spatial visual attention spans in partial and whole report tasks. Given the similarities between competition-based accounts for reduced visual attention span and similar explanations for impairments in sequential object processing, the present work examined whether poor readers show deficits in their “temporal attention span” – that is, their ability to rapidly and accurately process sequences of consecutive target items. Methodology/Principal Findings Poor and normal readers monitored a sequential stream of visual items for two (TT condition) or three (TTT condition) consecutive target digits. Target identification was examined using both unconditional and conditional measures of accuracy in order to gauge the overall likelihood of identifying a target and the likelihood of identifying a target given successful identification of previous items. Compared to normal readers, poor readers showed small but consistent deficits in identification across targets whether unconditional or conditional accuracy was used. Additionally, in the TTT condition, final-target conditional accuracy was poorer than unconditional accuracy, particularly for poor readers, suggesting a substantial cost arising from processing the previous two targets that was not present in normal readers. Conclusions/Significance Mirroring the differences found between poor and normal readers in spatial visual attention span, the present findings suggest two principal differences between the temporal attention spans of poor and normal readers. First, the consistent pattern of reduced performance across targets suggests increased competition amongst items within the same span for poor readers. Second, the steeper decline in final target performance amongst poor readers in the TTT condition suggests a reduction in the extent of their temporal attention span. PMID:24651313

Evidence for deficits in the temporal attention span of poor readers.

PubMed

Visser, Troy A W

2014-01-01

While poor reading is often associated with phonological deficits, many studies suggest that visual processing might also be impaired. In particular, recent research has indicated that poor readers show impaired spatial visual attention spans in partial and whole report tasks. Given the similarities between competition-based accounts for reduced visual attention span and similar explanations for impairments in sequential object processing, the present work examined whether poor readers show deficits in their "temporal attention span"--that is, their ability to rapidly and accurately process sequences of consecutive target items. Poor and normal readers monitored a sequential stream of visual items for two (TT condition) or three (TTT condition) consecutive target digits. Target identification was examined using both unconditional and conditional measures of accuracy in order to gauge the overall likelihood of identifying a target and the likelihood of identifying a target given successful identification of previous items. Compared to normal readers, poor readers showed small but consistent deficits in identification across targets whether unconditional or conditional accuracy was used. Additionally, in the TTT condition, final-target conditional accuracy was poorer than unconditional accuracy, particularly for poor readers, suggesting a substantial cost arising from processing the previous two targets that was not present in normal readers. Mirroring the differences found between poor and normal readers in spatial visual attention span, the present findings suggest two principal differences between the temporal attention spans of poor and normal readers. First, the consistent pattern of reduced performance across targets suggests increased competition amongst items within the same span for poor readers. Second, the steeper decline in final target performance amongst poor readers in the TTT condition suggests a reduction in the extent of their temporal attention span.

Reliability of semiquantitative assessment of osteophytes and subchondral cysts on tomosynthesis images by radiologists with different levels of expertise.

PubMed

Hayashi, Daichi; Xu, Li; Gusenburg, Jeffrey; Roemer, Frank W; Hunter, David J; Li, Ling; Guermazi, Ali

2014-01-01

We aimed to assess reliability of the evaluation of osteophytes and subchondral cysts on tomosynthesis images when read by radiologists with different levels of expertise. Forty subjects aged >40 years had both knees evaluated using tomosynthesis. Images were read by an "experienced" reader (musculoskeletal radiologist with prior experience) and an "inexperienced" reader (radiology resident with no prior experience). Readers graded osteophytes from 0 to 3 and noted the presence/absence of subchondral cysts in four locations of the tibiofemoral joint. Twenty knees were randomly selected and re-read. Inter- and intrareader reliabilities were calculated using overall exact percent agreement and weighted κ statistics. Diagnostic performance of the two readers was compared against magnetic resonance imaging readings by an expert reader (professor of musculoskeletal radiology). The experienced reader showed substantial intrareader reliability for graded reading of osteophytes (90%, κ=0.93), osteophyte detection (95%, κ=0.86) and cyst detection (95%, κ=0.83). The inexperienced reader showed perfect intrareader reliability for cyst detection (100%, κ=1.00) but intrareader reliability for graded reading (75%, κ=0.79) and detection (80%, κ=0.61) of osteophytes was lower than the experienced reader. Inter-reader reliability was 61% (κ=0.72) for graded osteophyte reading, 91% (κ=0.82) for osteophyte detection, and 88% (κ=0.66) for cyst detection. Diagnostic performance of the experienced reader was higher than the inexperienced reader regarding osteophyte detection (sensitivity range 0.74-0.95 vs. 0.54-0.75 for all locations) but diagnostic performance was similar for subchondral cysts. Tomosynthesis offers excellent intrareader reliability regardless of the reader experience, but experience is important for detection of osteophytes.

Reliability of semiquantitative assessment of osteophytes and subchondral cysts on tomosynthesis images by radiologists with different levels of expertise

PubMed Central

Hayashi, Daichi; Xu, Li; Gusenburg, Jeffrey; Roemer, Frank W.; Hunter, David J.; Li, Ling; Guermazi, Ali

2014-01-01

PURPOSE We aimed to assess reliability of the evaluation of osteophytes and subchondral cysts on tomosynthesis images when read by radiologists with different levels of expertise. MATERIALS AND METHODS Forty subjects aged >40 years had both knees evaluated using tomosynthesis. Images were read by an “experienced” reader (musculoskeletal radiologist with prior experience) and an “inexperienced” reader (radiology resident with no prior experience). Readers graded osteophytes from 0 to 3 and noted the presence/absence of subchondral cysts in four locations of the tibiofemoral joint. Twenty knees were randomly selected and re-read. Inter- and intrareader reliabilities were calculated using overall exact percent agreement and weighted κ statistics. Diagnostic performance of the two readers was compared against magnetic resonance imaging readings by an expert reader (professor of musculoskeletal radiology). RESULTS The experienced reader showed substantial intrareader reliability for graded reading of osteophytes (90%, κ=0.93), osteophyte detection (95%, κ=0.86) and cyst detection (95%, κ=0.83). The inexperienced reader showed perfect intrareader reliability for cyst detection (100%, κ=1.00) but intrareader reliability for graded reading (75%, κ=0.79) and detection (80%, κ=0.61) of osteophytes was lower than the experienced reader. Inter-reader reliability was 61% (κ=0.72) for graded osteophyte reading, 91% (κ=0.82) for osteophyte detection, and 88% (κ=0.66) for cyst detection. Diagnostic performance of the experienced reader was higher than the inexperienced reader regarding osteophyte detection (sensitivity range 0.74–0.95 vs. 0.54–0.75 for all locations) but diagnostic performance was similar for subchondral cysts. CONCLUSION Tomosynthesis offers excellent intrareader reliability regardless of the reader experience, but experience is important for detection of osteophytes. PMID:24834489

DOE Office of Scientific and Technical Information (OSTI.GOV)

St James, S; Argento, D; DeWitt, D

Purpose: Fast neutron therapy is offered at the University of Washington Medical Center for treatment of selected cancers. The hardware and control systems of the UW Clinical Neutron Therapy System are undergoing upgrades to enable delivery of IMNT. To clinically implement IMNT, dose verification tools need to be developed. We propose a portal imaging system that relies on the creation of positron emitting isotopes ({sup 11}C and {sup 15}O) through (n, 2n) reactions with a PMMA plate placed below the patient. After field delivery, the plate is retrieved from the vault and imaged using a reader that detects the annihilationmore » photons. The pattern of activity produced in the plate provides information to reconstruct the neutron fluence map that can be compared to fluence maps from Monte Carlo (MCNP) simulations to verify treatment delivery. We have previously performed Monte Carlo simulations of the portal imaging system (GATE simulations) and the beam line (MCNP simulations). In this work, initial measurements using a prototype system are presented. Methods: Custom electronics were developed for BGO detectors read out with photomultiplier tubes (previous generation PET detectors from a CTI ECAT 953 scanner). Two detectors were placed in coincidence, with a detector separation of 2 cm. Custom software was developed to create the crystal look up tables and perform a limited angle planar reconstruction with a stochastic normalization. To test the initial capabilities of the system, PMMA squares were irradiated with neutrons at a depth of 1.5 cm and read out using the prototype system. Doses ranging from 10–200 cGy were delivered. Results: Using the prototype system, dose differences in the therapeutic range could be determined. Conclusion: The prototype portal imaging system is capable of detecting neutron doses as low as 10–50 cGy and shows great promise as a patient QA tool for IMNT.« less

Comparative Evaluation of Antibacterial Effect of Dental Luting Cements on Streptococcus mutans and Lactobacillus acidophilus: An In vitro Study.

PubMed

Feroz, Sma; Bhoyar, A; Khan, S

2016-12-01

This study aimed at evaluating and comparing the antibacterial activity of six types of dental luting cements on Streptococcus mutans and Lactobacillus acidophilus using the agar diffusion test (ADT) and the direct-contact test (DCT). The antibacterial activity in ADT was measured based on the diameter of the zone of inhibition formed, whereas in DCT the density of the bacterial suspension was measured. The lower the density of the suspension, the more antibacterial activity the cement possesses. Agar diffusion test was carried out on the bacteria. After an incubation period of 24 hours, the plates were checked for the presence of zone of inhibition. In DCT the cement was mixed and applied. Once the cement was set, bacterial suspension and brain-heart infusion medium was poured and incubated for 24 hours. After 24 hours, the plate was placed in the enzyme-linked immunosorbent assay plate reader, which measured the optical density of the fluid. The first set of data was recorded approximately 1 hour after incubation. Overall, three sets of data were recorded. Additional experiments were performed on set test materials that were allowed to age for 24 hours, 1 week, 1, 3, and 6 months. When using ADT only two cements zinc oxide eugenol (ZOE) and zinc polycarboxylate (ZPC) cement showed antibacterial activity against the test organisms. When using DCT, all cements showed some amount of antibacterial activity. Zinc oxide eugenol and ZPC cement showed highest amount of antibacterial activity against S. mutans and L. acidophilus respectively. Within the limitations of study, ZOE cement and ZPC cement were most effective against the tested microorganisms followed by the newer resin cement. The glass ionomer cement was the weakest of all. Patients with high caries index can be treated more effectively using the abovementioned cements.

EST Readers: Some Principles for Their Design and Use.

ERIC Educational Resources Information Center

Moore, Christopher J.

1983-01-01

The editor of "Heinemann's Science and Technology Readers" responds to questions concerning the objectives and design of the series. The market for technical readers; reader format and difficulty level; illustrations, technical exercises, and the language teacher; and the intended classroom use are discussed. (MSE)

Mapping the reading circuitry for skilled deaf readers: an fMRI study of semantic and phonological processing.

PubMed

Emmorey, Karen; Weisberg, Jill; McCullough, Stephen; Petrich, Jennifer A F

2013-08-01

We examined word-level reading circuits in skilled deaf readers whose primary language is American Sign Language, and hearing readers matched for reading ability (college level). During fMRI scanning, participants performed a semantic decision (concrete concept?), a phonological decision (two syllables?), and a false-font control task (string underlined?). The groups performed equally well on the semantic task, but hearing readers performed better on the phonological task. Semantic processing engaged similar left frontotemporal language circuits in deaf and hearing readers. However, phonological processing elicited increased neural activity in deaf, relative to hearing readers, in the left precentral gyrus, suggesting greater reliance on articulatory phonological codes, and in bilateral parietal cortex, suggesting increased phonological processing effort. Deaf readers also showed stronger anterior-posterior functional segregation between semantic and phonological processes in left inferior prefrontal cortex. Finally, weaker phonological decoding ability did not alter activation in the visual word form area for deaf readers. Copyright © 2013 Elsevier Inc. All rights reserved.

Promoting an Addiction to Reading

ERIC Educational Resources Information Center

Preddy, Leslie B.

2007-01-01

The author has identified three kinds of readers: (1) resistive and struggling; (2) apathetic and reluctant; and (3) effective and motivated. The resistive and struggling reader is often the reader who needs help building reading competency and confidence while creating ample opportunity for practice. These readers often respond to relationships…

Diagnostic accuracy of mammography readers and their memory performance have no correlation with each other.

PubMed

Kok, P; Pitman, A G; Cawson, J N; Gledhill, S; Kremer, S; Lawson, J; Mehta, K; Mercuri, V; Shnier, D; Taft, R; Zentner, L

2010-08-01

The study aims to determine if any association exists between visual memory performance and diagnostic accuracy performance in a group of radiologist mammogram readers. One hundred proven mammograms (23 with cancers) were grouped into 5 sets of 20 cases, with sets being of equal difficulty. Pairs of sets were presented in 5 reads (40 cases per read, order random) to a panel of 8 radiologist readers (either present or past screening readers, with experience range from <1 year to >20 years). The readers were asked to either 'clear' or 'call back' cases depending on need for further workup, and at post-baseline reads to indicate whether each case was 'new' or 'old' (i.e. remembered from prior read). Two sets were presented only at baseline (40 cases per reader), and were used to calculate the reader's false recollection rate. Three sets were repeated post-baseline once or twice (100 cases per reader). Reading conditions were standardised. Memory performance differed markedly between readers. The number of correctly remembered cases (of 100 'old' cases) had a median of 10.5 and range of 0-58. The observed number of false recollections (of 40 'totally new' cases) had a median of 2 and range of 0-17. Diagnostic performance measures were mean (range): sensitivity 0.68 (0.54-0.81); specificity 0.82 (0.74-0.91); positive predictive value (PPV) 0.55 (0.50-0.65); negative predictive value (NPV) 0.89 (0.86-0.93) and accuracy 0.78 (0.76-0.83). Confidence intervals (CIs; 95%) for each reader overlapped for all the diagnostic parameters, indicating a lack of statistically significant difference between the readers at the 5% level. The most sensitive and the most specific reader showed a trend away from each other on sensitivity, specificity, NPV and PPV; their accuracies were 0.76 and 0.82, respectively, and their accuracy 95% CIs overlapped considerably. Correlation analysis by reader showed no association between observed memory performance and any of the diagnostic accuracy measures in our group of readers. In particular, there was no correlation between diagnostic accuracy and memory performance. There was no association between visual memory performance and diagnostic accuracy as a screening mammographer in our group of eight representative readers. Whether a radiologist has a good or a bad visual memory for cases, and in particular mammograms, should not impact on his or her performance as a radiologist and mammogram reader.

Long-Term Performance of Readers Trained in Grading Crohn Disease Activity Using MRI.

PubMed

Puylaert, Carl A J; Tielbeek, Jeroen A W; Bipat, Shandra; Boellaard, Thierry N; Nio, C Yung; Stoker, Jaap

2016-12-01

We aim to evaluate the long-term performance of readers who had participated in previous magnetic resonance imaging (MRI) reader training in grading Crohn disease activity. Fourteen readers (8 women; 12 radiologists, 2 residents; mean age 40; range 31-59), who had participated in a previous MRI reader training, participated in a follow-up evaluation after a mean interval of 29 months (range 25-34 months). Follow-up evaluation comprised 25 MRI cases of suspected or known Crohn disease patients with direct feedback; cases were identical to the evaluation set used in the initial reader training (of which readers were unaware). Grading accuracy, overstaging, and understaging were compared between training and follow-up using a consensus score by two experienced abdominal radiologists as the reference standard. In the follow-up evaluation, overall grading accuracy was 73% (95% confidence interval [CI]: 62%-81%), which was comparable to reader training grading accuracy (72%, 95% CI: 61%-80%) (P = .66). Overstaging decreased significantly from 19% (95% CI: 12%-27%) to 13% (95% CI: 8%-21%) between training and follow-up (P = .03), whereas understaging increased significantly from 9% (95% CI: 4%-21%) to 14% (95% CI: 7%-26%) (P < .01). Readers have consistent long-term accuracy for grading Crohn disease activity after case-based reader training with direct feedback. Copyright © 2016 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.

Low self-concept in poor readers: prevalence, heterogeneity, and risk.

PubMed

McArthur, Genevieve; Castles, Anne; Kohnen, Saskia; Banales, Erin

2016-01-01

There is evidence that poor readers are at increased risk for various types of low self-concept-particularly academic self-concept. However, this evidence ignores the heterogeneous nature of poor readers, and hence the likelihood that not all poor readers have low self-concept. The aim of this study was to better understand which types of poor readers have low self-concept. We tested 77 children with poor reading for their age for four types of self-concept, four types of reading, three types of spoken language, and two types of attention. We found that poor readers with poor attention had low academic self-concept, while poor readers with poor spoken language had low general self-concept in addition to low academic self-concept. In contrast, poor readers with typical spoken language and attention did not have low self-concept of any type. We also discovered that academic self-concept was reliably associated with reading and receptive spoken vocabulary, and that general self-concept was reliably associated with spoken vocabulary. These outcomes suggest that poor readers with multiple impairments in reading, language, and attention are at higher risk for low academic and general self-concept, and hence need to be assessed for self-concept in clinical practice. Our results also highlight the need for further investigation into the heterogeneous nature of self-concept in poor readers.

Low self-concept in poor readers: prevalence, heterogeneity, and risk

PubMed Central

Castles, Anne; Kohnen, Saskia; Banales, Erin

2016-01-01

There is evidence that poor readers are at increased risk for various types of low self-concept—particularly academic self-concept. However, this evidence ignores the heterogeneous nature of poor readers, and hence the likelihood that not all poor readers have low self-concept. The aim of this study was to better understand which types of poor readers have low self-concept. We tested 77 children with poor reading for their age for four types of self-concept, four types of reading, three types of spoken language, and two types of attention. We found that poor readers with poor attention had low academic self-concept, while poor readers with poor spoken language had low general self-concept in addition to low academic self-concept. In contrast, poor readers with typical spoken language and attention did not have low self-concept of any type. We also discovered that academic self-concept was reliably associated with reading and receptive spoken vocabulary, and that general self-concept was reliably associated with spoken vocabulary. These outcomes suggest that poor readers with multiple impairments in reading, language, and attention are at higher risk for low academic and general self-concept, and hence need to be assessed for self-concept in clinical practice. Our results also highlight the need for further investigation into the heterogeneous nature of self-concept in poor readers. PMID:27867764

Skilled deaf readers have an enhanced perceptual span in reading.

PubMed

Bélanger, Nathalie N; Slattery, Timothy J; Mayberry, Rachel I; Rayner, Keith

2012-07-01

Recent evidence suggests that, compared with hearing people, deaf people have enhanced visual attention to simple stimuli viewed in the parafovea and periphery. Although a large part of reading involves processing the fixated words in foveal vision, readers also utilize information in parafoveal vision to preprocess upcoming words and decide where to look next. In the study reported here, we investigated whether auditory deprivation affects low-level visual processing during reading by comparing the perceptual span of deaf signers who were skilled and less-skilled readers with the perceptual span of skilled hearing readers. Compared with hearing readers, the two groups of deaf readers had a larger perceptual span than would be expected given their reading ability. These results provide the first evidence that deaf readers' enhanced attentional allocation to the parafovea is used during complex cognitive tasks, such as reading.

Parallel versus sequential processing in print and braille reading.

PubMed

Veispak, Anneli; Boets, Bart; Ghesquière, Pol

2012-01-01

In the current study we investigated word, pseudoword and story reading in Dutch speaking braille and print readers. To examine developmental patterns, these reading skills were assessed in both children and adults. The results reveal that braille readers read less accurately and fast than print readers. While item length has no impact on word reading accuracy and speed in the group of print readers, it has a significant impact on reading accuracy and speed in the group of braille readers, particularly in the younger sample. This suggests that braille readers rely more strongly on an enduring sequential reading strategy. Comparison of the different reading tasks suggests that the advantage in accuracy and speed of reading in adult as compared to young braille readers is achieved through semantic top-down processing. Copyright © 2012 Elsevier Ltd. All rights reserved.

21 CFR 886.5800 - Ophthalmic bar reader.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Ophthalmic bar reader. 886.5800 Section 886.5800...) MEDICAL DEVICES OPHTHALMIC DEVICES Therapeutic Devices § 886.5800 Ophthalmic bar reader. (a) Identification. An ophthalmic bar reader is a device that consists of a magnifying lens intended for use by a...

What Can Readers Read after Graded Readers?

ERIC Educational Resources Information Center

McQuillan, Jeff

2016-01-01

Nation (2014) concluded that most of the vocabulary one needs to read challenging texts in English can be acquired incidentally through voluminous reading. This study examines possible texts that second language (L2) readers can use to move from controlled-vocabulary materials such as graded readers, which go up through approximately the…

Benefit of the Doubt. Reader Reflections

ERIC Educational Resources Information Center

Mathematics Teacher, 2016

2016-01-01

The editors of Mathematics Teacher appreciate the interest of readers and value the views of those who write in with comments. The editors ask that name and affiliation including email address be provided at the end of their letters. This September 2016 Reader Reflections, provides reader comments on the following articles: (1) "Innocent…

Moving Readers from Struggling to Proficient

ERIC Educational Resources Information Center

Wolter, Deborah

2017-01-01

If we want to move children from struggling to read to being proficient readers, we must address the disparate ways that teachers respond to readers with varying abilities. Restorative practices, akin to restorative justice, build relationships, make connections, and foster a reader's sense of ownership and empowerment. What would happen if…

Semantic and Phonological Coding in Poor and Normal Readers.

ERIC Educational Resources Information Center

Vellutino, Frank R.; And Others

1995-01-01

Using poor and normal readers, three studies evaluated semantic coding and phonological coding deficits as explanations for reading disability. It was concluded that semantic coding deficits are unlikely causes of difficulties in poor readers in early stages but accrue with prolonged reading difficulties in older readers. Phonological coding…

Actual Readers versus Implied Readers: Role Conflicts in Office 97.

ERIC Educational Resources Information Center

Shroyer, Roberta

2000-01-01

Explains the controversy surrounding the Office Assistant ("Paper-Clip") in Microsoft's Office 97. Discusses why actual readers rejected the default Office Assistant's role as implied writer and rebelled against the reader role implied for them. Notes users resented its intrusive behavior, rejected its implied writer role, and refused to…

Using Readers' Theatre in the Classroom.

ERIC Educational Resources Information Center

Annarella, Lorie A.

Reader's Theatre can be used to combine basic literature and writing instruction with creative arts. Improvisational playmaking by students, using literature in the form of plays, prose, and poetry, forms the basis of Reader's Theatre. Use of Reader's Theatre in the classroom can: (1) foster deeper understanding of character, setting, and plot…

Evolution of multi-well pad development and influence of well pads on environmental violations and wastewater volumes in the Marcellus shale (USA).

PubMed

Manda, Alex K; Heath, Jamie L; Klein, Wendy A; Griffin, Michael T; Montz, Burrell E

2014-09-01

A majority of well pads for unconventional gas wells that are drilled into the Marcellus shale (northeastern USA) consist of multiple wells (in some cases as many as 12 wells per pad), yet the influence of the evolution of well pad development on the extent of environmental violations and wastewater production is unknown. Although the development of multi-well pads (MWP) at the expense of single well pads (SWP) has been mostly driven by economic factors, the concentrated nature of drilling activities from hydraulic fracturing and horizontal drilling operations on MWP suggests that MWP may create less surface disturbance, produce more volumes of wastewater, and generate more environmental violations than SWP. To explore these hypotheses, we use geospatial techniques and statistical analyses (i.e., regression and Mann-Whitney tests) to assess development of unconventional shale gas wells, and quantify environmental violations and wastewater volumes on SWP and MWP in Pennsylvania. The analyses include assessments of the influence of different types of well pads on potential, minor and major environmental events. Results reveal that (a) in recent years, a majority of pads on which new wells for unconventional gas were drilled are MWP, (b) on average, MWP have about five wells located on each pad and thus, had the transition to MWP not occurred, between two and four times as much land surface disturbance would have occurred per year if drilling was relegated to SWP, (c) there were more environmental violations on MWP than SWP, but when the number of wells were taken into account, fewer environmental violations per well were observed on MWP than on SWP, (d) there were more wastewater and recycled wastewater volumes per pad and per well produced on MWP than on SWP, and (e) the proportion of wastewater that was recycled was higher on MWP than SWP. This study sheds light on how the evolution from SWP to MWP has influenced environmental violations and wastewater production in a field that has undergone rapid development in recent years. Copyright © 2014 Elsevier Ltd. All rights reserved.

Livermore time-sharing system. Part I. Octopus. Chapter 5. Card reader/card punch. [Description of card reader and formats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lawrence, J.D.

1970-03-12

The Control Data 405 card reader, modified by the Control Data 3649 card read controller, is the primary mechanism for transferring information from a deck of punched cards into the CDC 6600 and CDC 7600 computers of the LLL Octopus system. The card reader operates at a maximum rate of 1200 cards per minute. A description of the card reader and its operation is given. A discussion of formates is included. (RWR)

A Sign Language Screen Reader for Deaf

NASA Astrophysics Data System (ADS)

El Ghoul, Oussama; Jemni, Mohamed

Screen reader technology has appeared first to allow blind and people with reading difficulties to use computer and to access to the digital information. Until now, this technology is exploited mainly to help blind community. During our work with deaf people, we noticed that a screen reader can facilitate the manipulation of computers and the reading of textual information. In this paper, we propose a novel screen reader dedicated to deaf. The output of the reader is a visual translation of the text to sign language. The screen reader is composed by two essential modules: the first one is designed to capture the activities of users (mouse and keyboard events). For this purpose, we adopted Microsoft MSAA application programming interfaces. The second module, which is in classical screen readers a text to speech engine (TTS), is replaced by a novel text to sign (TTSign) engine. This module converts text into sign language animation based on avatar technology.

What are the early indicators of persistent word reading difficulties among Chinese readers in elementary grades?

PubMed

Yeung, Pui-Sze; Ho, Connie Suk-Han; Chan, David Wai-Ock; Chung, Kevin Kien-Hoa

2014-05-01

To identify the indicators of persistent reading difficulties among Chinese readers in early elementary grades, the performance of three groups of Chinese children with different reading trajectories ('persistent poor word readers', 'improved poor word readers' and 'skilled word readers') in reading-related measures was analysed in a 3-year longitudinal study. The three groups were classified according to their performance in a standardized Chinese word reading test in Grade 1 and Grade 4. Results of analysis of variance and logistic regression on the reading-related measures revealed that rapid naming and syntactic skills were important indicators of early word reading difficulty. Syntactic skills and morphological awareness were possible markers of persistent reading problems. Chinese persistent poor readers did not differ significantly from skilled readers on the measures of phonological skills. Copyright © 2014 John Wiley & Sons, Ltd.

Type Testing of Model 7200 Automatic TLD Reader.

PubMed

Malek Mohammadi, M; Hosseini Pooya, S M

2017-04-20

The type testing of measuring devices is one of the most important parts of a quality management system in a personal dosimetry services program. In this study, based upon the International Electrotechnical Commission (IEC) 62387 criteria, a reader-testing program was performed for a home-made personal thermoluminescent dosimetry (TLD) reader. The stability of the reader, the effects of light exposure, temperature and fluctuations of primary power supply on TLD read-outs as the main parameters were investigated in this program. Moreover, this study assesses some important criteria of dosimetry system including the non-linearity of response, reusability, after effect and overload that may include significant contribution in the performance of a reader. The results showed that the TLD reader met all requirements of the IEC for the reader tests by a large margin. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

One-week 96-well soft agar growth assay for cancer target validation.

PubMed

Ke, Ning; Albers, Aaron; Claassen, Gisela; Yu, De-hua; Chatterton, Jon E; Hu, Xiuyuan; Meyhack, Bernd; Wong-Staal, Flossie; Li, Qi-Xiang

2004-05-01

Soft agar growth, used to measure cell anchorage-independent proliferation potential, is one of the most important and most commonly used assays to detect cell transformation. However, the traditional soft agar assay is time-consuming, labor-intensive, and plagued with inconsistencies due to individual subjectivity. It does not, therefore, meet the increasing demands of today's oncology drug target screening or validation processes. This report describes an alternative 96-well soft agar growth assay that can function as a replacement for the traditional method and overcomes the aforementioned limitations. It offers the following advantages: a shortened assay duration (1 week instead of 4 weeks) that makes transient transfection or treatment possible; plate reader quantification of soft agar growth (measuring cloning efficiency and colony size); and a significant reduction in required labor. Higher throughput also makes it possible to process large numbers of samples and treatments simultaneously and in a much more efficient manner, while saving precious workspace and overall cost.

Correlation of cholinergic drug induced quenching of acetylcholinesterase bound thioflavin-T fluorescence with their inhibition activity

NASA Astrophysics Data System (ADS)

Islam, Mullah Muhaiminul; Rohman, Mostofa Ataur; Gurung, Arun Bahadur; Bhattacharjee, Atanu; Aguan, Kripamoy; Mitra, Sivaprasad

2018-01-01

The development of new acetylcholinesterase inhibitors (AChEIs) and subsequent assay of their inhibition efficiency is considered to be a key step for AD treatment. The fluorescence intensity of thioflavin-T (ThT) bound in the active site of acetylcholinesterase (AChE) quenches substantially in presence of standard AChEI drugs due to the dynamic replacement of the fluorophore from the AChE active site as confirmed from steady state emission as well as time-resolved fluorescence anisotropy measurement and molecular dynamics simulation in conjunction with docking calculation. The parametrized % quenching data for individual system shows excellent correlation with enzyme inhibition activity measured independently by standard Ellman AChE assay method in a high throughput plate reader system. The results are encouraging towards design of a fluorescence intensity based AChE inhibition assay method and may provide a better toolset to rapidly evaluate as well as develop newer AChE-inhibitors for AD treatment.

Peptide-based, two-fluorophore, ratiometric probe for quantifying mobile zinc in biological solutions.

PubMed

Zhang, Daniel Y; Azrad, Maria; Demark-Wahnefried, Wendy; Frederickson, Christopher J; Lippard, Stephen J; Radford, Robert J

2015-02-20

Small-molecule fluorescent sensors are versatile agents for detecting mobile zinc in biology. Capitalizing on the abundance of validated mobile zinc probes, we devised a strategy for repurposing existing intensity-based sensors for quantitative applications. Using solid-phase peptide synthesis, we conjugated a zinc-sensitive Zinpyr-1 derivative and a zinc-insensitive 7-hydroxycoumarin derivative onto opposite ends of a rigid P9K peptide scaffold to create HcZ9, a ratiometric fluorescent probe for mobile zinc. A plate reader-based assay using HcZ9 was developed, the accuracy of which is comparable to that of atomic absorption spectroscopy. We investigated zinc accumulation in prostatic cells and zinc levels in human seminal fluid. When normal and tumorigenic cells are bathed in zinc-enriched media, cellular mobile zinc is buffered and changes slightly, but total zinc levels increase significantly. Quantification of mobile and total zinc levels in human seminal plasma revealed that the two are positively correlated with a Pearson's coefficient of 0.73.

Effect of microbubbled water on the removal of a biofilm attached to orthodontic appliances--an in vitro study.

PubMed

Mukumoto, Mio; Ohshima, Tomoko; Ozaki, Miwa; Konishi, Hirokazu; Maeda, Nobuko; Nakamura, Yoshiki

2012-01-01

Orthodontic appliances often cause oral diseases such as dental caries and gingivitis due to the attachment of an oral biofilm. However, there are few reliable methods to remove the biofilm from the orthodontic appliances. The aim of this study was to investigate the effects of microbubbled water on the removal of biofilms made with Streptococcus mutans or Candida albicans on orthodontic appliances. The orthodontic appliances with biofilm were immersed with microbubbled water and the remaining biofilm on the appliances was detected and measured using a micro-plate reader and an absorbance meter. The microbubbled water had a sufficient effect on the removal of biofilm from orthodontic appliances. The effects of microbubbled water were significantly higher than those of tap water (S. mutans: p<0.05, C. albicans: p<0.01). The results of this study suggest that microbubbled water is effective in the removal of biofilm from the mouth of orthodontic patients.

Computer Games versus Maps before Reading Stories: Priming Readers' Spatial Situation Models

ERIC Educational Resources Information Center

Smith, Glenn Gordon; Majchrzak, Dan; Hayes, Shelley; Drobisz, Jack

2011-01-01

The current study investigated how computer games and maps compare as preparation for readers to comprehend and retain spatial relations in text narratives. Readers create situation models of five dimensions: spatial, temporal, causal, goal, and protagonist (Zwaan, Langston, & Graesser 1995). Of these five, readers mentally model the spatial…

Cognitive Development in Early Readers.

ERIC Educational Resources Information Center

Briggs, Chari; Elkind, David

Some studies of early readers are discussed. It is pointed out that study of early readers has relevance for practical and theoretical issues in psychology and education. Of interest in this document are the following questions: (1) Are there any special talents or traits distinguishing early from non-early readers? (2) Do children who read early…

French Immersion Experience and Reading Skill Development in At-Risk Readers

ERIC Educational Resources Information Center

Kruk, Richard S.; Reynolds, Kristin A. A.

2012-01-01

We tracked the developmental influences of exposure to French on developing English phonological awareness, decoding and reading comprehension of English-speaking at-risk readers from Grade 1 to Grade 3. Teacher-nominated at-risk readers were matched with not-at-risk readers in French immersion and English language programs. Exposure to spoken…

Reader Theories and Educational Media Analysis.

ERIC Educational Resources Information Center

Becker, Ann DeVaney

Post structural reader theories--i.e., theories that focus on the reader/viewer rather than the text as the creator of meaning--are considered in this paper in terms of their application to educational media research. Some key concepts of reader theories are defined as follows: (1) reading is the process of creating meaning while viewing an…

21 CFR 886.5810 - Ophthalmic prism reader.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Ophthalmic prism reader. 886.5810 Section 886.5810...) MEDICAL DEVICES OPHTHALMIC DEVICES Therapeutic Devices § 886.5810 Ophthalmic prism reader. (a) Identification. An ophthalmic prism reader is a device intended for use by a patient who is in a supine position...

21 CFR 886.5810 - Ophthalmic prism reader.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Ophthalmic prism reader. 886.5810 Section 886.5810...) MEDICAL DEVICES OPHTHALMIC DEVICES Therapeutic Devices § 886.5810 Ophthalmic prism reader. (a) Identification. An ophthalmic prism reader is a device intended for use by a patient who is in a supine position...

21 CFR 886.5810 - Ophthalmic prism reader.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Ophthalmic prism reader. 886.5810 Section 886.5810...) MEDICAL DEVICES OPHTHALMIC DEVICES Therapeutic Devices § 886.5810 Ophthalmic prism reader. (a) Identification. An ophthalmic prism reader is a device intended for use by a patient who is in a supine position...

21 CFR 886.5810 - Ophthalmic prism reader.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Ophthalmic prism reader. 886.5810 Section 886.5810...) MEDICAL DEVICES OPHTHALMIC DEVICES Therapeutic Devices § 886.5810 Ophthalmic prism reader. (a) Identification. An ophthalmic prism reader is a device intended for use by a patient who is in a supine position...

E-Readers: Powering up for Engagement

ERIC Educational Resources Information Center

Miranda, Twyla; Johnson, Kary A.; Rossi-Williams, Dara

2012-01-01

E-readers like the Amazon Kindle and the Barnes and Noble Nook are beginning to make their way into school libraries and classrooms. It's about time. E-readers have tremendous potential to entice reluctant readers to read more. A study that the authors recently conducted among low-reading-ability middle school students demonstrated that potential.…

"Science in Society, Omnibus Pack, Readers M-P."

ERIC Educational Resources Information Center

Association for Science Education, Cambridge (England).

Four additional readers have been written for use in the Science in Society general studies project. Three of the readers discuss the applications and importance of engineering in the world. They include: Engineering 1 (Reader M), which discusses such topics as the role of engineering in society, structural design and engineering, the engineering…

Reader Engagement in English and Persian Applied Linguistics Articles

ERIC Educational Resources Information Center

Ansarin, Ali Akbar; Tarlani-Aliabdi, Hassan

2011-01-01

There is an increasing interest in the way academic writers establish the presence of their readers over the past few years. Establishing the presence of readers or what Kroll (1984, p.181) calls imagining "a second voice" is accomplished when a writer refers "explicitly" to their readers using explicit linguistic resources…

Abdullah's Genuine Indonesian Curry Powder--about Early Readers and Differentiated Teaching.

ERIC Educational Resources Information Center

Hansen, Vagn Rabol; Robenhagen, Ole

A study established some characteristics of early readers and their homes and families, how they learned to read, and how their surroundings, and particularly the school, reacted. Approximately 300 Danish early readers and their families were interviewed and completed questionnaires. Results indicated that: (1) half the early readers had either a…

The Troubled Reader: Access to Intervention for the School Psychologist.

ERIC Educational Resources Information Center

Meredith, Kurt; Steele, Jeannie

Intended for school psychologists, the guidelines suggest ways of intervening with troubled readers. An initial chapter notes the size of the problem and asserts that school psychologists have some training to work with troubled readers. Chapter 2 reviews the reading process, addressing such aspects as the interaction of text, reader, and content…

Comparisons of Learning Ease and Transfer Propensity in Poor and Average Readers.

ERIC Educational Resources Information Center

Day, Jeanne D.; Zajakowski, Amy

1991-01-01

Assisted and unassisted performance of 14 average readers and 14 readers with learning disabilities (ages 10-13) were compared on a reading comprehension task. The groups differed in how easily they learned to find the main idea under different topic sentence placement conditions, with the learning-disabled readers requiring significantly more…

Bimodal Reading: Benefits of a Talking Computer for Average and Less Skilled Readers.

ERIC Educational Resources Information Center

Montali, Julie; Lewandowski, Lawrence

1996-01-01

Eighteen average readers and 18 less-skilled readers (grades 8 and 9) were presented with social studies and science passages via a computer either visually (on screen), auditorily (read by digitized voice), or bimodally (on screen, highlighted while being voiced). Less-skilled readers demonstrated comprehension in the bimodal condition equivalent…

Becoming a Reader: Significant Social Influences on Avid Book Readers

ERIC Educational Resources Information Center

Merga, Margaret K.

2017-01-01

Understanding how social influences can foster avid book reader identification is a key research goal that warrants further investigation beyond a limited early-years lens. The author's 2015 International Study of Avid Book Readers (ISABR) explored, as one of its key research questions, the influence positive social agents can have on avid book…

RESOLV: Readers' Representation of Reading Contexts and Tasks

ERIC Educational Resources Information Center

Rouet, Jean-François; Britt, M. Anne; Durik, Amanda M.

2017-01-01

We introduce RESOLV, a theoretical model to account for readers' construction and management of goals during text comprehension and use. RESOLV focuses on readers' experience of their physical, social, and communicative context prior to actually engaging with texts. RESOLV assumes that readers construct two types of mental models prior to reading:…

The Effects of Surface Structure Variables on Performance in Reading Comprehension Tests.

ERIC Educational Resources Information Center

Drum, Priscilla; And Others

1981-01-01

Concludes that reading comprehension tests that are valid for beginning readers should incorporate different factors than tests appropriate for upper elementary readers, since word recognition and word meaning are prime sources of difficulty for younger readers while content density depresses the performance of readers in upper elementary grades.…

Reader Use and Understanding of Newspaper Informational Graphics.

ERIC Educational Resources Information Center

Pasternack, Steve; Utt, Sandra H.

While the use of informational graphics in newspapers is increasing, little is known regarding how well readers can understand them and how readers use them. A study investigated whether readers of newspapers read graphics before or after they read the headline/text, and whether people decide to read a graphic device for appearance-related or…

What Predicts Adult Readers' Understanding of STEM Texts?

ERIC Educational Resources Information Center

Follmer, D. Jake; Fang, Shin-Yi; Clariana, Roy B.; Meyer, Bonnie J. F.; Li, Ping

2018-01-01

The current study examined the relations among key variables that underlie reading comprehension of expository science texts in a diverse population of adult native English readers. Using Mechanical Turk to sample a range of adult readers, the study also examined the effect of text presentation on readers' comprehension and knowledge structure…

Reading and Spelling Error Analysis of Native Arabic Dyslexic Readers

ERIC Educational Resources Information Center

Abu-rabia, Salim; Taha, Haitham

2004-01-01

This study was an investigation of reading and spelling errors of dyslexic Arabic readers ("n"=20) compared with two groups of normal readers: a young readers group, matched with the dyslexics by reading level ("n"=20) and an age-matched group ("n"=20). They were tested on reading and spelling of texts, isolated…

Reading anxiety, classroom anxiety, language motivation, reader self-perception, and arabic achievement of Arab-American students learning arabic as a second language.

PubMed

Alkhateeb, Haitham M

2014-12-01

The present study assessed the relations between reading anxiety, classroom anxiety, language motivation, and readers' self-perception for a sample of Arab-American students in Arabic classes. The effects of sex, grade, and years studying Arabic on academic achievement were examined as well. Measures were administered to 118 middle school students (56 boys, 62 girls; M age = 13.0 yr., SD = 0.8), and teachers reported academic grades in Arabic. Reading anxiety was significantly correlated with classroom anxiety and reader self-perception. Classroom anxiety scores were significantly correlated with motivation and reader self-perception. Significant positive correlations were found between language motivation and reader self-perception scores, and between years studying Arabic and reader self-perception scores. Boys in the second year of Arabic had significantly lower classroom anxiety than girls, and students in Grade 7 had higher reader self-perception than those in Grade 8. Classroom anxiety, language motivation, and reader self-perception significantly predicted Arabic achievement. Pedagogical implications are discussed.

RAC-multi: reader anti-collision algorithm for multichannel mobile RFID networks.

PubMed

Shin, Kwangcheol; Song, Wonil

2010-01-01

At present, RFID is installed on mobile devices such as mobile phones or PDAs and provides a means to obtain information about objects equipped with an RFID tag over a multi-channeled telecommunication networks. To use mobile RFIDs, reader collision problems should be addressed given that readers are continuously moving. Moreover, in a multichannel environment for mobile RFIDs, interference between adjacent channels should be considered. This work first defines a new concept of a reader collision problem between adjacent channels and then suggests a novel reader anti-collision algorithm for RFID readers that use multiple channels. To avoid interference with adjacent channels, the suggested algorithm separates data channels into odd and even numbered channels and allocates odd-numbered channels first to readers. It also sets an unused channel between the control channel and data channels to ensure that control messages and the signal of the adjacent channel experience no interference. Experimental results show that suggested algorithm shows throughput improvements ranging from 29% to 46% for tag identifications compared to the GENTLE reader anti-collision algorithm for multichannel RFID networks.

Geometric Distribution-Based Readers Scheduling Optimization Algorithm Using Artificial Immune System.

PubMed

Duan, Litian; Wang, Zizhong John; Duan, Fu

2016-11-16

In the multiple-reader environment (MRE) of radio frequency identification (RFID) system, multiple readers are often scheduled to interrogate the randomized tags via operating at different time slots or frequency channels to decrease the signal interferences. Based on this, a Geometric Distribution-based Multiple-reader Scheduling Optimization Algorithm using Artificial Immune System (GD-MRSOA-AIS) is proposed to fairly and optimally schedule the readers operating from the viewpoint of resource allocations. GD-MRSOA-AIS is composed of two parts, where a geometric distribution function combined with the fairness consideration is first introduced to generate the feasible scheduling schemes for reader operation. After that, artificial immune system (including immune clone, immune mutation and immune suppression) quickly optimize these feasible ones as the optimal scheduling scheme to ensure that readers are fairly operating with larger effective interrogation range and lower interferences. Compared with the state-of-the-art algorithm, the simulation results indicate that GD-MRSOA-AIS could efficiently schedules the multiple readers operating with a fairer resource allocation scheme, performing in larger effective interrogation range.

Interference with the operation of medical devices resulting from the use of radio frequency identification technology.

PubMed

Houliston, Bryan; Parry, David; Webster, Craig S; Merry, Alan F

2009-06-19

To replicate electromagnetic interference (EMI) with a common drug infusion device resulting from the use of radio frequency identification (RFID) technology in a simulated operating theatre environment. An infusion pump, of a type previously reported as having failed due to RFID EMI, was placed in radio frequency (RF) fields of various strengths, and its operation observed. Different strength RF fields were created by varying the number of RFID readers, the use of a high-gain RFID antenna, the distance between the reader(s) and the infusion pump, and the presence of an RFID tag on the infusion pump. The infusion pump was not affected by low-power RFID readers, even when in direct contact. The pump was disrupted by a high-power reader at 10 cm distance when an RFID tag was attached, and by a combination of high-power and low-power readers at 10 cm distance. Electronic medical devices may fail in the presence of high-power RFID readers, especially if the device is tagged. However, low-power RFID readers appear to be safer.

Geometric Distribution-Based Readers Scheduling Optimization Algorithm Using Artificial Immune System

PubMed Central

Duan, Litian; Wang, Zizhong John; Duan, Fu

2016-01-01

In the multiple-reader environment (MRE) of radio frequency identification (RFID) system, multiple readers are often scheduled to interrogate the randomized tags via operating at different time slots or frequency channels to decrease the signal interferences. Based on this, a Geometric Distribution-based Multiple-reader Scheduling Optimization Algorithm using Artificial Immune System (GD-MRSOA-AIS) is proposed to fairly and optimally schedule the readers operating from the viewpoint of resource allocations. GD-MRSOA-AIS is composed of two parts, where a geometric distribution function combined with the fairness consideration is first introduced to generate the feasible scheduling schemes for reader operation. After that, artificial immune system (including immune clone, immune mutation and immune suppression) quickly optimize these feasible ones as the optimal scheduling scheme to ensure that readers are fairly operating with larger effective interrogation range and lower interferences. Compared with the state-of-the-art algorithm, the simulation results indicate that GD-MRSOA-AIS could efficiently schedules the multiple readers operating with a fairer resource allocation scheme, performing in larger effective interrogation range. PMID:27854342

RAC-Multi: Reader Anti-Collision Algorithm for Multichannel Mobile RFID Networks

PubMed Central

Shin, Kwangcheol; Song, Wonil

2010-01-01

At present, RFID is installed on mobile devices such as mobile phones or PDAs and provides a means to obtain information about objects equipped with an RFID tag over a multi-channeled telecommunication networks. To use mobile RFIDs, reader collision problems should be addressed given that readers are continuously moving. Moreover, in a multichannel environment for mobile RFIDs, interference between adjacent channels should be considered. This work first defines a new concept of a reader collision problem between adjacent channels and then suggests a novel reader anti-collision algorithm for RFID readers that use multiple channels. To avoid interference with adjacent channels, the suggested algorithm separates data channels into odd and even numbered channels and allocates odd-numbered channels first to readers. It also sets an unused channel between the control channel and data channels to ensure that control messages and the signal of the adjacent channel experience no interference. Experimental results show that suggested algorithm shows throughput improvements ranging from 29% to 46% for tag identifications compared to the GENTLE reader anti-collision algorithm for multichannel RFID networks. PMID:22315528

Vessel suppressed chest Computed Tomography for semi-automated volumetric measurements of solid pulmonary nodules.

PubMed

Milanese, Gianluca; Eberhard, Matthias; Martini, Katharina; Vittoria De Martini, Ilaria; Frauenfelder, Thomas

2018-04-01

To evaluate whether vessel-suppressed computed tomography (VSCT) can be reliably used for semi-automated volumetric measurements of solid pulmonary nodules, as compared to standard CT (SCT) MATERIAL AND METHODS: Ninety-three SCT were elaborated by dedicated software (ClearRead CT, Riverain Technologies, Miamisburg, OH, USA), that allows subtracting vessels from lung parenchyma. Semi-automated volumetric measurements of 65 solid nodules were compared between SCT and VSCT. The measurements were repeated by two readers. For each solid nodule, volume measured on SCT by Reader 1 and Reader 2 was averaged and the average volume between readers acted as standard of reference value. Concordance between measurements was assessed using Lin's Concordance Correlation Coefficient (CCC). Limits of agreement (LoA) between readers and CT datasets were evaluated. Standard of reference nodule volume ranged from 13 to 366 mm 3 . The mean overestimation between readers was 3 mm 3 and 2.9 mm 3 on SCT and VSCT, respectively. Semi-automated volumetric measurements on VSCT showed substantial agreement with the standard of reference (Lin's CCC = 0.990 for Reader 1; 0.985 for Reader 2). The upper and lower LoA between readers' measurements were (16.3, -22.4 mm 3 ) and (15.5, -21.4 mm 3 ) for SCT and VSCT, respectively. VSCT datasets are feasible for the measurements of solid nodules, showing an almost perfect concordance between readers and with measurements on SCT. Copyright © 2018 Elsevier B.V. All rights reserved.

Comprehensive automatic assessment of retinal vascular abnormalities for computer-assisted retinopathy grading.

PubMed

Joshi, Vinayak; Agurto, Carla; VanNess, Richard; Nemeth, Sheila; Soliz, Peter; Barriga, Simon

2014-01-01

One of the most important signs of systemic disease that presents on the retina is vascular abnormalities such as in hypertensive retinopathy. Manual analysis of fundus images by human readers is qualitative and lacks in accuracy, consistency and repeatability. Present semi-automatic methods for vascular evaluation are reported to increase accuracy and reduce reader variability, but require extensive reader interaction; thus limiting the software-aided efficiency. Automation thus holds a twofold promise. First, decrease variability while increasing accuracy, and second, increasing the efficiency. In this paper we propose fully automated software as a second reader system for comprehensive assessment of retinal vasculature; which aids the readers in the quantitative characterization of vessel abnormalities in fundus images. This system provides the reader with objective measures of vascular morphology such as tortuosity, branching angles, as well as highlights of areas with abnormalities such as artery-venous nicking, copper and silver wiring, and retinal emboli; in order for the reader to make a final screening decision. To test the efficacy of our system, we evaluated the change in performance of a newly certified retinal reader when grading a set of 40 color fundus images with and without the assistance of the software. The results demonstrated an improvement in reader's performance with the software assistance, in terms of accuracy of detection of vessel abnormalities, determination of retinopathy, and reading time. This system enables the reader in making computer-assisted vasculature assessment with high accuracy and consistency, at a reduced reading time.

The role of feedback and differences between good and poor decoders in a repeated word reading paradigm in first grade.

PubMed

van Gorp, Karly; Segers, Eliane; Verhoeven, Ludo

2017-04-01

The direct, retention, and transfer effects of repeated word and pseudoword reading were studied in a pretest, training, posttest, retention design. First graders (48 good readers, 47 poor readers) read 25 CVC words and 25 CVC pseudowords in ten repeated word reading sessions, preceded and followed by a transfer task with a different set of items. Two weeks after training, trained items were assessed again in a retention test. Participants either received phonics feedback, in which each word was spelled out and repeated; word feedback, in which each word was repeated; or no feedback. During the training, both good and poor readers improved in accuracy and speed. The increase in speed was stronger for poor readers than for good readers. The good readers demonstrated a stronger increase for pseudowords than for words. This increase in speed was most prominent in the first four sessions. Two weeks after training, the levels of accuracy and speed were retained. Furthermore, transfer effects on speed were found for pseudowords in both groups of readers. Good readers performed most accurately during the training when they received no feedback while poor readers performed most accurately during the training with the help of phonics feedback. However, feedback did not differentiate for reading speed or for effects after the training. The effects of repeated word reading were found to be stronger for poor readers than for good readers. Moreover, these effects were found to be stronger for pseudowords than for words. This indicates that repeated word reading can be seen as an important trigger for the improvement of decoding skills.

Assessing operating characteristics of CAD algorithms in the absence of a gold standard

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roy Choudhury, Kingshuk; Paik, David S.; Yi, Chin A.

2010-04-15

Purpose: The authors examine potential bias when using a reference reader panel as ''gold standard'' for estimating operating characteristics of CAD algorithms for detecting lesions. As an alternative, the authors propose latent class analysis (LCA), which does not require an external gold standard to evaluate diagnostic accuracy. Methods: A binomial model for multiple reader detections using different diagnostic protocols was constructed, assuming conditional independence of readings given true lesion status. Operating characteristics of all protocols were estimated by maximum likelihood LCA. Reader panel and LCA based estimates were compared using data simulated from the binomial model for a range ofmore » operating characteristics. LCA was applied to 36 thin section thoracic computed tomography data sets from the Lung Image Database Consortium (LIDC): Free search markings of four radiologists were compared to markings from four different CAD assisted radiologists. For real data, bootstrap-based resampling methods, which accommodate dependence in reader detections, are proposed to test of hypotheses of differences between detection protocols. Results: In simulation studies, reader panel based sensitivity estimates had an average relative bias (ARB) of -23% to -27%, significantly higher (p-value <0.0001) than LCA (ARB -2% to -6%). Specificity was well estimated by both reader panel (ARB -0.6% to -0.5%) and LCA (ARB 1.4%-0.5%). Among 1145 lesion candidates LIDC considered, LCA estimated sensitivity of reference readers (55%) was significantly lower (p-value 0.006) than CAD assisted readers' (68%). Average false positives per patient for reference readers (0.95) was not significantly lower (p-value 0.28) than CAD assisted readers' (1.27). Conclusions: Whereas a gold standard based on a consensus of readers may substantially bias sensitivity estimates, LCA may be a significantly more accurate and consistent means for evaluating diagnostic accuracy.« less

A Combinatorial Platform for the Optimization of Peptidomimetic Methyl-Lysine Reader Antagonists

NASA Astrophysics Data System (ADS)

Barnash, Kimberly D.

Post-translational modification of histone N-terminal tails mediates chromatin compaction and, consequently, DNA replication, transcription, and repair. While numerous post-translational modifications decorate histone tails, lysine methylation is an abundant mark important for both gene activation and repression. Methyl-lysine (Kme) readers function through binding mono-, di-, or trimethyl-lysine. Chemical intervention of Kme readers faces numerous challenges due to the broad surface-groove interactions between readers and their cognate histone peptides; yet, the increasing interest in understanding chromatin-modifying complexes suggests tractable lead compounds for Kme readers are critical for elucidating the mechanisms of chromatin dysregulation in disease states and validating the druggability of these domains and complexes. The successful discovery of a peptide-derived chemical probe, UNC3866, for the Polycomb repressive complex 1 (PRC1) chromodomain Kme readers has proven the potential for selective peptidomimetic inhibition of reader function. Unfortunately, the systematic modification of peptides-to-peptidomimetics is a costly and inefficient strategy for target-class hit discovery against Kme readers. Through the exploration of biased chemical space via combinatorial on-bead libraries, we have developed two concurrent methodologies for Kme reader chemical probe discovery. We employ biased peptide combinatorial libraries as a hit discovery strategy with subsequent optimization via iterative targeted libraries. Peptide-to-peptidomimetic optimization through targeted library design was applied based on structure-guided library design around the interaction of the endogenous peptide ligand with three target Kme readers. Efforts targeting the WD40 reader EED led to the discovery of the 3-mer peptidomimetic ligand UNC5115 while combinatorial repurposing of UNC3866 for off-target chromodomains resulted in the discovery of UNC4991, a CDYL/2-selective ligand, and UNC4848, a MPP8 and CDYL/2 ligand. Ultimately, our efforts demonstrate the generalizability of a peptidomimetic combinatorial platform for the optimization of Kme reader ligands in a target class manner.

Linking embryo toxicity with genotoxic responses in the freshwater snail Physa acuta: single exposure to benzo(a)pyrene, fluoxetine, bisphenol A, vinclozolin and exposure to binary mixtures with benzo(a)pyrene.

PubMed

Sánchez-Argüello, Paloma; Aparicio, Natalia; Fernández, Carlos

2012-06-01

Genotoxic effects on fauna after waterborne pollutant exposure have been demonstrated by numerous research programmes. Less effort has been focused on establishing relationship between genotoxicity and long-term responses at higher levels of biological organization. Taking into account that embryos may be more sensitive indicators of reproductive impairment than alterations in fertility, we have developed two assays in multiwell plates to address correlations between embryo toxicity and genotoxicity. The potential teratogenicity was assessed by analyzing abnormal development and mortality of Physa acuta at embryonic stage. Genotoxicity was measured by the micronucleus (MN) test using embryonic cells. Our results showed that linkage between genotoxicity and embryo toxicity depends on mechanisms of action of compounds under study. Embryo toxic responses showed a clear dose-related tendency whereas no clear dose-dependent effect was observed in micronucleus induction. The higher embryo toxicity was produced by benzo(a)pyrene exposure followed by fluoxetine and bisphenol A. Vinclozolin was the lower embryo toxic compound. Binary mixtures with BaP always resulted in higher embryo toxicity than single exposures but antagonistic effects were observed for MN induction. Benzo(a)pyrene produced the higher MN induction at 0.04 mg/L, which also produced clear embryo toxic effects. Fluoxetine did not induce cytogenetic effects but 0.25mg/L altered embryonic development. Bisphenol A significantly reduced hatchability at 0.5mg/L while MN induction appeared with higher treatments than those that start causing teratogenicity. Much higher concentration of vinclozolin (5mg/L) reduced hatchability and induced maximum MN formation. In conclusion, while validating one biomarker of genotoxicity and employing one ecologically relevant effect, we have evaluated the relative sensitivity of a freshwater mollusc for a range of chemicals. The embryo toxicity test is a starting point for the development of a life cycle test with freshwater snails even for undertaking multigeneration studies focused on transgenerational effects. Copyright © 2012 Elsevier Inc. All rights reserved.

Assessment of biochar safety via its leachate characterization using physicochemical and biological assays

NASA Astrophysics Data System (ADS)

Dailianis, Stefanos; Tsouloufa, Argyro; Antonopoulou, Maria; Konstantinou, Ioannis; Karapanagioti, Hrissi K.; Manariotis, Ioannis D.

2016-04-01

The present study investigates the physicochemical composition of water aliquots derived from biochars produced from the pyrolysis of malt spent rootlets, in combination with the concomitant toxicological profile in each case. Specifically, physicochemical parameters and heavy metal ions were determined in aliquots of six (6) serial washes of biochar (1.5 g of solid was added in column and washed 6 times with 40 mL of distilled water per wash). The chemical analysis of each aliquot showed increased levels of PO4-3, Cl-, NO3-, SO4-2, F- and Br- in the first wash aliquot, followed by a significant decrease over washes. Non-detectable concentrations were observed after 3 washes in almost all cases. Similarly, the increased levels of Zn, Be, Cs, Mn, V and Se determined in the first wash aliquot were eliminated followed successive washes. In parallel, the toxic potency of each wash aliquot was recorded by (a) a multi-well test plate bioassay, using instars II-III larvae of the fairy shrimp Thamnocephalus platyurus, hatched from cysts derived from Screening Toxicity test supplied by MicroBio Tests Inc. (Thamnotoxkit FTM) and (b) the Microtox bioassay, using bioluminescent bacteria Vibrio fischeri. According to the results, first and second wash aliquots were toxic for T. platyurus (LC50 values of 22.12 and 68.28% v/v, respectively), followed by a significant elimination of toxicity after further washes in all cases. Similarly, the Microtox bioassay showed a significant inhibition of Vibrio luminescence after treatment for a period of 5-90 min (98-100% inhibition of luminescence) with the first wash aliquot (EC50 ≤ 0.01 % v/v), with no toxicity to be observed after successive washes. According to the results, at least one wash of biochar is prerequisite for improving its safety for further use. Moreover, the removal of both inorganic and organic, such as metal ions, substances commonly washed by the biochar, could be a crucial step for its sustainable use and final application, thus avoiding the induction of adverse effects on biota.

Patients with inflammatory bowel disease (IBD) reveal increased induction capacity of intracellular interferon-gamma (IFN-γ) in peripheral CD8+ lymphocytes co-cultured with intestinal epithelial cells

PubMed Central

Bisping, G; Lügering, N; Lütke-Brintrup, S; Pauels, H -G; Schürmann, G; Domschke, W; Kucharzik, T

2001-01-01

Intestinal epithelial cells seem to play a key role during IBD. The network of cellular interactions between epithelial cells and lamina propria mononuclear cells is still incompletely understood. In the following co-culture model we investigated the influence of intestinal epithelial cells on cytokine expression of T cytotoxic and T helper cells from patients with IBD and healthy controls. Peripheral blood mononuclear cells (PBMC) were purified by a Ficoll–Hypaque gradient followed by co-incubation with epithelial cells in multiwell cell culture insert plates in direct contact as well as separated by transwell filters. We used Caco-2 cells as well as freshly isolated colonic epithelia obtained from surgical specimens. Three-colour immunofluorescence flow cytometry was performed after collection, stimulation and staining of PBMC with anti-CD4, anti-CD8, anti-IFN-γ and anti-IL-4. Patients with IBD (Crohn's disease (CD), n = 12; ulcerative colitis (UC), n = 16) and healthy controls (n = 10) were included in the study. After 24 h of co-incubation with Caco-2 cells we found a significant increase of IFN-γ-producing CD8+ lymphocytes in patients with IBD. In contrast, healthy controls did not respond to the epithelial stimulus. No significant differences could be found between CD and UC or active and inactive disease. A significant increase of IFN-γ+/CD8+ lymphocytes in patients with UC was also seen after direct co-incubation with primary cultures of colonic crypt cells. The observed epithelial–lymphocyte interaction seems to be MHC I-restricted. No significant epithelial cell-mediated effects on cytokine expression were detected in the PBMC CD4+ subsets. Patients with IBD—even in an inactive state of disease—exert an increased capacity for IFN-γ induction in CD8+ lymphocytes mediated by intestinal epithelial cells. This mechanism may be important during chronic intestinal inflammation, as in the case of altered mucosal barrier function epithelial cells may become targets for IFN-γ-producing CD8+ lymphocytes. PMID:11167992

My Journey as a Reader

ERIC Educational Resources Information Center

Cevellos, Tatiana

2008-01-01

In this case history, the author describes how her journey as a reader evolved from a poor reader who did not like to read in elementary school into an avid trilingual reader in graduate school. Once she discovered the joy of reading, each language in which she read had its own purpose and emotional connection. She credits self-selected reading…

Recall of English Function Words and Inflections by Skilled and Average Deaf Readers.

ERIC Educational Resources Information Center

Kelly, Leonard P.

1993-01-01

The performance of 17 youth on a verbatim recall task indicated that skilled deaf readers are more able than average deaf readers to sustain a record of English function words and inflections. The relative speed of skilled readers when making lexical decisions about phonologically similar word pairs indicated greater access to phonological…

Evoking/Interpreting Literature through Visualizing Roles.

ERIC Educational Resources Information Center

Karolides, Nicholas J.

Differences in readers' interpretations of a given text illustrate premises of the transactional or reader response theory of literature. The theory holds that: (1) meaning resides in the coming together of reader and text; (2) the reader affects the reading of the text and is affected by the text; and (3) there are potentially as many meanings to…

Rereading Multicultural Readers: What Definition of Multicultural Are We Buying?

ERIC Educational Resources Information Center

Shapiro, Nancy

A flood of new multicultural readers and textbooks are hitting the market for writing and literature courses at the college level. Yet there has been no systematic examination of how these readers are being used, the purposes and audiences for which they are written, or the critical reception they have received. Multicultural readers distinguish…

The Role of the Teacher in Reader's Theater Instruction

ERIC Educational Resources Information Center

Rasisnski, Timothy; Stokes, Faida; Young, Chase

2017-01-01

Reader's Theater is a transformative and influential instructional tool for reading with far-reaching benefits for all students. Teachers are critical players in the use of Readers Theater in classroom; the effects of Reader's Theater are a direct result of a teacher's involvement. These effects include an increase in word recognition, fluency,…

Choosing the Adequate Level of Graded Readers--Preliminary Study

ERIC Educational Resources Information Center

Prtljaga, Jelena; Palinkaševic, Radmila; Brkic, Jovana

2015-01-01

Graded readers have been used as second language teaching material since the end of the Second World War. They are an important source of simplified material which provides comprehensible input on all levels. It is of crucial importance for a successful usage of graded readers in the classroom and in studies which focus on graded readers, that an…

Predicting Text Comprehension, Processing, and Familiarity in Adult Readers: New Approaches to Readability Formulas

ERIC Educational Resources Information Center

Crossley, Scott A.; Skalicky, Stephen; Dascalu, Mihai; McNamara, Danielle S.; Kyle, Kristopher

2017-01-01

Research has identified a number of linguistic features that influence the reading comprehension of young readers; yet, less is known about whether and how these findings extend to adult readers. This study examines text comprehension, processing, and familiarity judgment provided by adult readers using a number of different approaches (i.e.,…

Contributions of Syntactic Awareness to Reading in Chinese-Speaking Adolescent Readers with and without Dyslexia

ERIC Educational Resources Information Center

Chung, Kevin K. H.; Ho, Connie S.-H.; Chan, David W.; Tsang, Suk-Man; Lee, Suk-Han

2013-01-01

This study investigated the relative contribution of syntactic awareness to Chinese reading among Chinese-speaking adolescent readers with and without dyslexia. A total of 78 junior high school students in Hong Kong, 26 dyslexic adolescent readers, 26 average adolescent readers of the same age (chronological age control group) and 26 younger…

Where to Put the Manicules: A Theory of Expert Reading

ERIC Educational Resources Information Center

Horning, Alice S.

2011-01-01

Manicules are hand-drawn symbols used by medieval readers to mark important parts of a text. Knowing where to place manicules is one characteristic of an expert reader. A meta-cognitive theory of expert reading helps to account for what readers know that allows them to place manicules appropriately. This theory proposes that expert readers are…

Reading Comprehension and Working Memory in Learning-Disabled Readers: Is the Phonological Loop More Important Than the Executive System?

ERIC Educational Resources Information Center

Swanson, H. Lee

1999-01-01

Investigated the contribution of two working-memory systems (the articulatory loop and the central executive) to the performance differences between learning-disabled and skilled readers. Found that, compared to skilled readers, learning-disabled readers experienced constraints in the articulatory and long-term memory system, and suffered…

The Effect of Script on Poor Readers' Sensitivity to Dynamic Visual Stimuli

ERIC Educational Resources Information Center

Kim, Jeesun; Davis, Chris; Burnham, Denis; Luksaneeyanawin, Sudaporn

2004-01-01

The current research examined performance of good and poor readers of Thai on two tasks that assess sensitivity to dynamic visual displays. Readers of Thai, a complex alphabetic script that nonetheless has a regular orthography, were chosen in order to contrast patterns of performance with readers of Korean Hangul (a similarly regular language but…

Gifted Male Readers: Current Understandings and Suggestions for Future Research

ERIC Educational Resources Information Center

Pagnani, Alexander R.

2013-01-01

Research literature concerning gifted male readers relies primarily on more extensive bodies of work regarding gifted males and male readers. Studied as a whole, the two halves portray a worrisome state of affairs for gifted male readers, who lag behind their female counterparts in the same patterns found across the ability spectrum. This literacy…

A General Audiovisual Temporal Processing Deficit in Adult Readers with Dyslexia

ERIC Educational Resources Information Center

Francisco, Ana A.; Jesse, Alexandra; Groen, Margriet A.; McQueen, James M.

2017-01-01

Purpose: Because reading is an audiovisual process, reading impairment may reflect an audiovisual processing deficit. The aim of the present study was to test the existence and scope of such a deficit in adult readers with dyslexia. Method: We tested 39 typical readers and 51 adult readers with dyslexia on their sensitivity to the simultaneity of…

Differences in the Use of Selective Attention by More Successful and Less Successful Tenth-Grade Readers.

ERIC Educational Resources Information Center

Reynolds, Ralph E.; And Others

1990-01-01

Differences in use of selective attention by 64 more successful and 48 less successful readers in the tenth grade were studied. More successful readers are more aware of how and when to use the selective attention strategy and use more conceptual attention while reading than do less successful readers. (SLD)

The Reader, the Text, the Poem: The Transactional Theory of the Literary Work.

ERIC Educational Resources Information Center

Rosenblatt, Louise M.

Transactional literary theory centers on the reader's contribution in the two-way relationship with the literary text, lifting the reader to a prominent, essential position along with the author and the text. It develops the premise that the reader evokes a literary work through selective attention to the details of the author's "paper and…

Does Feeling Come First? How Poetry Can Help Readers Broaden Their Understanding of Metacognition

ERIC Educational Resources Information Center

Eva-Wood, Amy L.

2008-01-01

Assuming that readers' emotional responses can enhance readers' metacognitive experiences and inform literary analysis, this study of 11th-grade poetry readers features instruction that models both cognitive and affective reading processes. The author: (1) Presents a case for more explicit attention to emotion in language arts classrooms; (2)…

Different Orthographies Different Context Effects: The Effects of Arabic Sentence Context in Skilled and Poor Readers.

ERIC Educational Resources Information Center

Rabia, Salim Abu; Siegel, Linda S.

1995-01-01

Investigates whether Arabic orthography differs from an alphabetic orthography regarding context effects among poor and skilled readers. Finds that skilled as well as poor readers significantly improved their reading accuracy when they read voweled and unvoweled words in context and that skilled readers significantly improved their reading voweled…

A demonstration of lack of variability among six tuberculin skin test readers.

PubMed Central

Perez-Stable, E J; Slutkin, G

1985-01-01

The variability of tuberculin skin test readings among six trained and experienced readers was evaluated using a modified sliding caliper method. Each of 537 tests were read independently by two readers. There were 23 disagreements between paired readers resulting in an overall interobserver reliability of 95.7 per cent. In 82 per cent of the paired readings the results were different by 2 mm or less. The observer lack of variability was likely due to the training and experience of the readers. PMID:4051078

Pedagog II Realization.

DTIC Science & Technology

1979-12-01

BE2 CD66 ALl SGG/110(14) 5/AS2 SGG/79(14) AMI 110(13) 5/AU2 78(16) NI KRIRF/lll(2) ANl PRIRF/lll(l) ARI IPC/lll(ll) 7/BPl IPC/144(4) ASI KRF/106(9...KRS 6034 Read a character from the keyboard/reader buffer. The keyboard/reader flag is set when the operation is completed. KIE 6035 Enable the...keyboard/ reader flag is a 1. KCC 6032 Clear the AC and the keyboard/reader flag. KRS 6034 Read a character from the keyboard/reader buffer. The keyboard

Synthesis and dual PPARalpha/delta agonist effects of 1,4-disubstituted 1,2,3-triazole analogues of GW 501516.

PubMed

Ciocoiu, Calin C; Nikolić, Natasa; Nguyen, Huyen Hoa; Thoresen, G Hege; Aasen, Arne J; Hansen, Trond Vidar

2010-07-01

Ten 1,4-disubstituted 1,2,3-triazoles 2a-2j were prepared and tested for their ability to increase oleic acid oxidation in human myotubes using a high-throughput multiwell assay. Compounds 2e (2-{4-[(1-(3-fluoro-4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methylthio]-2-methylphenoxy}acetic acid) and 2i (2-{4-[(1-(3-chloro-4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-4-yl)methylthio]-2-methylphenoxy}acetic acid) exhibited potent agonist activities. Compounds 2e and 2i also exhibited powerful agonist effects for both PPARalpha and PPARdelta in a luciferase-based assay. Consequently, these triazoles can be categorized as dual PPAR agonists. Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.

An MILP-based cross-layer optimization for a multi-reader arbitration in the UHF RFID system.

PubMed

Choi, Jinchul; Lee, Chaewoo

2011-01-01

In RFID systems, the performance of each reader such as interrogation range and tag recognition rate may suffer from interferences from other readers. Since the reader interference can be mitigated by output signal power control, spectral and/or temporal separation among readers, the system performance depends on how to adapt the various reader arbitration metrics such as time, frequency, and output power to the system environment. However, complexity and difficulty of the optimization problem increase with respect to the variety of the arbitration metrics. Thus, most proposals in previous study have been suggested to primarily prevent the reader collision with consideration of one or two arbitration metrics. In this paper, we propose a novel cross-layer optimization design based on the concept of combining time division, frequency division, and power control not only to solve the reader interference problem, but also to achieve the multiple objectives such as minimum interrogation delay, maximum reader utilization, and energy efficiency. Based on the priority of the multiple objectives, our cross-layer design optimizes the system sequentially by means of the mixed-integer linear programming. In spite of the multi-stage optimization, the optimization design is formulated as a concise single mathematical form by properly assigning a weight to each objective. Numerical results demonstrate the effectiveness of the proposed optimization design.

Exploring e-readers to support clinical medical education: two case studies*†

PubMed Central

von Isenburg, Megan

2011-01-01

Question: Can e-readers loaded with medical textbooks and other relevant material benefit medical students, residents, and preceptors in clinical settings? Settings: The settings are North Carolina community clinics served by Duke University Medical Center and St. Joseph's Hospital in Bryan, Texas, and Scott and White Memorial Hospital in Temple, Texas. Methods: Duke University: Twenty second-year medical students and fourteen family medicine clerkship preceptors used Kindle e-readers in clinics during eight months of rotations. Students and preceptors provided feedback through an anonymous online survey. Texas A&M University: Nine fourth-year medical students in an elective compared medical textbooks in print, online, and on a Kindle. Six residents at a local hospital completed an anonymous online survey after a three-week loan of a Kindle loaded with medical textbooks. Results: The e-reader's major advantages in clinical settings are portability and searchability. The selected e-reader's limitations include connection speed, navigation, and display. User preferences varied, but online resources were preferred. Participants suggested additional uses for Kindles in medical education. Conclusions: The selected e-reader's limitations may be resolved with further development of the device. Investigation of other e-readers is needed. Criteria for evaluating e-readers in clinical settings should include portability, searchability, speed, navigation, and display. Research comparing e-readers and mobile devices in clinical education is also warranted. PMID:21464848

An MILP-Based Cross-Layer Optimization for a Multi-Reader Arbitration in the UHF RFID System

PubMed Central

Choi, Jinchul; Lee, Chaewoo

2011-01-01

In RFID systems, the performance of each reader such as interrogation range and tag recognition rate may suffer from interferences from other readers. Since the reader interference can be mitigated by output signal power control, spectral and/or temporal separation among readers, the system performance depends on how to adapt the various reader arbitration metrics such as time, frequency, and output power to the system environment. However, complexity and difficulty of the optimization problem increase with respect to the variety of the arbitration metrics. Thus, most proposals in previous study have been suggested to primarily prevent the reader collision with consideration of one or two arbitration metrics. In this paper, we propose a novel cross-layer optimization design based on the concept of combining time division, frequency division, and power control not only to solve the reader interference problem, but also to achieve the multiple objectives such as minimum interrogation delay, maximum reader utilization, and energy efficiency. Based on the priority of the multiple objectives, our cross-layer design optimizes the system sequentially by means of the mixed-integer linear programming. In spite of the multi-stage optimization, the optimization design is formulated as a concise single mathematical form by properly assigning a weight to each objective. Numerical results demonstrate the effectiveness of the proposed optimization design. PMID:22163743

Neuroimaging reveals dual routes to reading in simultaneous proficient readers of two orthographies

PubMed Central

Das, T.; Padakannaya, P.; Pugh, K. R.; Singh, N. C.

2012-01-01

Orthographic differences across languages impose differential weighting on distinct component processes, and consequently on different pathways during word-reading tasks. Readers of transparent orthographies such as Italian and Hindi are thought to rely on spelling-to-sound assembly and show increased activation in phonologically-tuned areas along the dorsal pathway, whereas reading an opaque orthography such as English is thought to rely more on lexically-mediated processing associated with increased activation of semantically-tuned regions along the ventral pathway. To test if biliterate Hindi/English readers exhibit orthography-specific reading pathways, we used behavioural measures and functional neuroimaging. Reaction times and activation patterns of monolingual English and Hindi readers were compared to two groups of adult biliterates; 14 simultaneous readers who learnt to read both languages at age 5 and 10 sequential readers who learnt Hindi at 5 and English at 10. Simultaneous, but not sequential readers demonstrated relative activation differences of dorsal and ventral areas in the two languages. Similar to native counterparts, simultaneous readers preferentially activated the left inferior temporal gyrus for English and left inferior parietal lobule (L-IPL) for Hindi, whereas, sequential readers showed higher activation along the L-IPL for reading both languages. We suggest that early simultaneous exposure to reading distinct orthographies results in orthography-specific plasticity that persists through adulthood. PMID:20854914

Could We Make Diverse Learning Materials Compatible with E-Readers Used in Classroom Learning Settings?

ERIC Educational Resources Information Center

Young, Shelley Shwu-Ching; Lin, Wei-Lin

2012-01-01

This study explores how to make diverse learning/instructional materials compatible with e-readers when the instructor pioneered to adopt e-readers into a course of the graduate level. What problems did the instructor encounter when she used the e-readers as a major tool to deliver learning contents, such as the process of converting the…

Early Cognitive and Linguistic Profiles of Different Types of 7- to 8-Year-Old Readers

ERIC Educational Resources Information Center

Potocki, Anna; Ecalle, Jean; Magnan, Annie

2017-01-01

The aim of this study was to investigate the early characteristics of four profiles of readers established in second grade (7-8 years of age): good readers, specific poor decoders, specific poor comprehenders and general poor readers. These profiles were compared retrospectively on a range of measures administered 2 years earlier, in kindergarten.…

Follow the Reader: An Effective Strategy to Support Students Reading More Complex Text

ERIC Educational Resources Information Center

Klvacek, Michelle L.; Monroe, Eula Ewing; Wilcox, Brad; Hall-Kenyon, Kendra M.; Morrison, Timothy G.

2017-01-01

This article describes how one second-grade teacher implemented Follow the Reader, her term for dyad reading. Common Core expects students to read increasingly complex texts. Teachers can implement dyad reading with this end in mind. It is a modified version of the neurological impress method in which a lead reader and an assisted reader sit side…

Are You a Reader? 5th Graders Respond

ERIC Educational Resources Information Center

Barone, Diane; Barone, Rebecca

2016-01-01

The author tells the story of a 5th-grade teacher who challenges her class to take on self-identities as readers. Students defined seven characteristics of what it means to be a good reader and considered whether those characteristics applied to them: Good readers read for fun, talk about books, usually finish the book they're reading, can relate…

What Strategies Do Resistive Readers Use to Fake Their Way through School Assigned Texts?

ERIC Educational Resources Information Center

Krieger, Courtney M.

2012-01-01

A trend among adolescent readers is the practice of fake-reading. Fake-reading occurs when students who can read choose not to, and develop strategies to fake their way through school-assigned texts to earn good grades. This study examines four resistive readers, or fake-readers, assigned to read Neal Shusterman's "Unwind" (2007)…

Readers and Texts in the Primary Years. Rethinking Reading.

ERIC Educational Resources Information Center

Martin, Tony; Leather, Bob

Exploring the responses of a variety of readers from three-year-old Dominic sharing a picture book with his father to adults reading a poem by Ted Hughes, this book examines the ways in which various readers respond to different texts. The aim of the book is to develop an awareness of the issues involved in readers' responses for primary teachers.…

A Study of How the Family Is Portrayed in Basal Readers.

ERIC Educational Resources Information Center

Evans, Nancy J.

A study examined the ways that basal readers portrayed the family. It was expected that the readers would give only token representation of both parents working or of one-parent or mixed marriage families, and that very few portrayals of reading or of reading materials would be found. Eight basal readers from 6 publishing companies for grades 1…

eBooks--Ready for School Libraries?

ERIC Educational Resources Information Center

Pappas, Marjorie

2009-01-01

For those who tend to purchase many books for personal or professional use, the eBook reader would easily pay for itself within a year. The two leading brands of eBook readers today are the Amazon Kindle and the Sony Book Reader. Both are similar in size, weight, and purchase price. The Kindle includes a keyboard while the Reader provides access…

The Phenomenology of Composition: The Application of Certain Principles of Reader-Response Criticism to the Teaching of Composition.

ERIC Educational Resources Information Center

Meyers, G. Douglas

An application of reader response criticism, with its abundance of ways of construing readers, permits writing teachers to identify sets of readers for students more effectively than simply exhorting them to remember their audience while writing. Composition teachers can employ the concept of "narratee" (the author's alter ego) as a…

The Negative Consequences of becoming a Good Reader: Identity Theory as a Lens for Understanding Struggling Readers, Teachers, and Reading Instruction

ERIC Educational Resources Information Center

Hall, Leigh A.

2010-01-01

Background/Context: The majority of middle school students in U.S. schools are struggling readers and lack the reading abilities needed to successfully comprehend texts, complete reading-related assignments, and learn subject matter content. Researchers have suggested that struggling readers' comprehension abilities can be improved if their…

The Development and Use of Cheremic, Graphemic, and Phonological Coding in Deaf High School-Aged Readers.

ERIC Educational Resources Information Center

O'Connor, Katherine; Taylor, Linda S.

This report outlines the a study that investigated the reading skills of high school students (ages 14-19) with severe or profound prelingual deafness, including 6 skilled readers with parents with deafness, 6 skilled readers with hearing parents, and 6 average readers with hearing parents. The study determined short-term memory strategies used by…

Finding a (W)hole in the Text: A Case Study of Four Readers Reading.

ERIC Educational Resources Information Center

Nance, Mary Moore

The purpose of the study was to examine: (1) which perspectives of Reader Response Theory were most applicable in this study; (2) which factors influenced reader responses; and (3) how readers' responses changed over time. The four participants for this case study were chosen from a subject pool of 10 initiate adult full-time divinity students in…

A General Audiovisual Temporal Processing Deficit in Adult Readers With Dyslexia.

PubMed

Francisco, Ana A; Jesse, Alexandra; Groen, Margriet A; McQueen, James M

2017-01-01

Because reading is an audiovisual process, reading impairment may reflect an audiovisual processing deficit. The aim of the present study was to test the existence and scope of such a deficit in adult readers with dyslexia. We tested 39 typical readers and 51 adult readers with dyslexia on their sensitivity to the simultaneity of audiovisual speech and nonspeech stimuli, their time window of audiovisual integration for speech (using incongruent /aCa/ syllables), and their audiovisual perception of phonetic categories. Adult readers with dyslexia showed less sensitivity to audiovisual simultaneity than typical readers for both speech and nonspeech events. We found no differences between readers with dyslexia and typical readers in the temporal window of integration for audiovisual speech or in the audiovisual perception of phonetic categories. The results suggest an audiovisual temporal deficit in dyslexia that is not specific to speech-related events. But the differences found for audiovisual temporal sensitivity did not translate into a deficit in audiovisual speech perception. Hence, there seems to be a hiatus between simultaneity judgment and perception, suggesting a multisensory system that uses different mechanisms across tasks. Alternatively, it is possible that the audiovisual deficit in dyslexia is only observable when explicit judgments about audiovisual simultaneity are required.

Modifying the brain activation of poor readers during sentence comprehension with extended remedial instruction: a longitudinal study of neuroplasticity.

PubMed

Meyler, Ann; Keller, Timothy A; Cherkassky, Vladimir L; Gabrieli, John D E; Just, Marcel Adam

2008-08-01

This study used fMRI to longitudinally assess the impact of intensive remedial instruction on cortical activation among 5th grade poor readers during a sentence comprehension task. The children were tested at three time points: prior to remediation, after 100 h of intensive instruction, and 1 year after the instruction had ended. Changes in brain activation were also measured among 5th grade good readers at the same time points for comparison. The central finding was that prior to instruction, the poor readers had significantly less activation than good readers bilaterally in the parietal cortex. Immediately after instruction, poor readers made substantial gains in reading ability, and demonstrated significantly increased activation in the left angular gyrus and the left superior parietal lobule. Activation in these regions continued to increase among poor readers 1 year post-remediation, resulting in a normalization of the activation. These results are interpreted as reflecting changes in the processes involved in word-level and sentence-level assembly. Areas of overactivation were also found among poor readers in the medial frontal cortex, possibly indicating a more effortful and attentive guided reading strategy.

Modifying the Brain Activation of Poor Readers during Sentence Comprehension with Extended Remedial Instruction: A Longitudinal Study of Neuroplasticity

PubMed Central

Meyler, Ann; Keller, Timothy A.; Cherkassky, Vladimir L.; Gabrieli, John D. E.; Just, Marcel Adam

2008-01-01

This study used fMRI to longitudinally assess the impact of intensive remedial instruction on cortical activation among 5th grade poor readers during a sentence comprehension task. The children were tested at 3 time points: prior to remediation, after 100 hours of intensive instruction, and 1 year after the instruction had ended. Changes in brain activation were also measured among 5th grade good readers at the same time points for comparison. The central finding was that prior to instruction, the poor readers had significantly less activation than good readers bilaterally in the parietal cortex. Immediately after instruction, poor readers made substantial gains in reading ability, and demonstrated significantly increased activation in the left angular gyrus and the left superior parietal lobule. Activation in these regions continued to increase among poor readers 1 year post-remediation, resulting in a normalization of the activation. These results are interpreted as reflecting changes in the processes involved in word-level and sentence-level assembly. Areas of overactivation were also found among poor readers in the medial frontal cortex, possibly indicating a more effortful and attentionally-guided reading strategy. PMID:18495180

Differential cognitive and perceptual correlates of print reading versus braille reading.

PubMed

Veispak, Anneli; Boets, Bart; Ghesquière, Pol

2013-01-01

The relations between reading, auditory, speech, phonological and tactile spatial processing are investigated in a Dutch speaking sample of blind braille readers as compared to sighted print readers. Performance is assessed in blind and sighted children and adults. Regarding phonological ability, braille readers perform equally well compared to print readers on phonological awareness, better on verbal short-term memory and significantly worse on lexical retrieval. The groups do not differ on speech perception or auditory processing. Braille readers, however, have more sensitive fingers than print readers. Investigation of the relations between these cognitive and perceptual skills and reading performance indicates that in the group of braille readers auditory temporal processing has a longer lasting and stronger impact not only on phonological abilities, which have to satisfy the high processing demands of the strictly serial language input, but also directly on the reading ability itself. Print readers switch between grapho-phonological and lexical reading modes depending on the familiarity of the items. Furthermore, the auditory temporal processing and speech perception, which were substantially interrelated with phonological processing, had no direct associations with print reading measures. Copyright © 2012 Elsevier Ltd. All rights reserved.

Semantic and visual memory codes in learning disabled readers.

PubMed

Swanson, H L

1984-02-01

Two experiments investigated whether learning disabled readers' impaired recall is due to multiple coding deficiencies. In Experiment 1, learning disabled and skilled readers viewed nonsense pictures without names or with either relevant or irrelevant names with respect to the distinctive characteristics of the picture. Both types of names improved recall of nondisabled readers, while learning disabled readers exhibited better recall for unnamed pictures. No significant difference in recall was found between name training (relevant, irrelevant) conditions within reading groups. In Experiment 2, both reading groups participated in recall training for complex visual forms labeled with unrelated words, hierarchically related words, or without labels. A subsequent reproduction transfer task showed a facilitation in performance in skilled readers due to labeling, with learning disabled readers exhibiting better reproduction for unnamed pictures. Measures of output organization (clustering) indicated that recall is related to the development of superordinate categories. The results suggest that learning disabled children's reading difficulties are due to an inability to activate a semantic representation that interconnects visual and verbal codes.

Can We 'Read' the Eye-Movement Patterns of Readers? Unraveling the Relationship Between Reading Profiles and Processing Strategies.

PubMed

Koornneef, Arnout; Mulders, Iris

2017-02-01

In an eye-tracking experiment we examined the risky reading hypothesis, in which long saccades and many regressions are considered to be indicative of a proactive reading style (Rayner et al. in Psychol Aging 21(3):448, 2006; Psychol Aging 24(3):755, 2009). We did so by presenting short texts-that confirmed or disconfirmed verb-based implicit causality expectations-to two types of readers: proactive readers (long saccades, many regressions) and conservative readers (short saccades, few regressions). Whereas proactive readers used implicit causality information to predict upcoming referents, and slowed down immediately when they encountered a pronoun that was inconsistent with these verb-based expectations, the conservative readers slowed down much later in the sentence. These findings were consistent with the predictions of the risky reading hypothesis and as such presented novel evidence for the general idea that the eye-movement profile of readers reveals valuable information about their processing strategy.

Skilled Deaf Readers have an Enhanced Perceptual Span in Reading

PubMed Central

Bélanger, Nathalie N.; Slattery, Timothy J.; Mayberry, Rachel I.; Rayner, Keith

2013-01-01

Recent evidence suggests that deaf people have enhanced visual attention to simple stimuli in the parafovea in comparison to hearing people. Although a large part of reading involves processing the fixated words in foveal vision, readers also utilize information in parafoveal vision to pre-process upcoming words and decide where to look next. We investigated whether auditory deprivation affects low-level visual processing during reading, and compared the perceptual span of deaf signers who were skilled and less skilled readers to that of skilled hearing readers. Compared to hearing readers, deaf readers had a larger perceptual span than would be expected by their reading ability. These results provide the first evidence that deaf readers’ enhanced attentional allocation to the parafovea is used during a complex cognitive task such as reading. PMID:22683830

Compact mobile-reader system for two-way wireless communication, tracking and status monitoring for transport safety and security

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsai, Han-Chung; Liu, Yung Y.; Lee, Hok L.

A system for monitoring a plurality radio frequency identification tags is described. The system uses at least one set of radio frequency identification tags. Each tag is attached to a container and includes several sensors for detecting physical conditions of said container. The system includes at least one autonomous intermediate reader in wireless communication with the frequency identification tags. The intermediate reader includes external wireless communication system, intermediate reader logic controller, and a self-contained rechargeable power supply. The system uses a central status reporting system in communication the intermediate reader.

Reader Training for the Restaging of Biochemically Recurrent Prostate Cancer Using 18F-Fluciclovine PET/CT.

PubMed

Miller, Matthew P; Kostakoglu, Lale; Pryma, Daniel; Yu, Jian Qin; Chau, Albert; Perlman, Eric; Clarke, Bonnie; Rosen, Donald; Ward, Penelope

2017-10-01

18 F-Fluciclovine is a novel PET/CT tracer. This blinded image evaluation (BIE) sought to demonstrate that, after limited training, readers naïve to 18 F-fluciclovine could interpret 18 F-fluciclovine images from subjects with biochemically recurrent prostate cancer with acceptable diagnostic performance and reproducibility. The primary objectives were to establish individual readers' diagnostic performance and the overall interpretation (2/3 reader concordance) compared with standard-of-truth data (histopathology or clinical follow-up) and to evaluate interreader reproducibility. Secondary objectives included comparison to the expert reader and assessment of intrareader reproducibility. Methods: 18 F-Fluciclovine PET/CT images ( n = 121) and corresponding standard-of-truth data were collected from 110 subjects at Emory University using a single-time-point static acquisition starting 5 min after injection of approximately 370 MBq of 18 F-fluciclovine. Three readers were trained using standardized interpretation methodology and subsequently evaluated the images in a blinded manner. Analyses were conducted at the lesion, region (prostate, including bed and seminal vesicle, or extraprostatic, including all lymph nodes, bone, or soft-tissue metastasis), and subject level. Results: Lesion-level overall positive predictive value was 70.5%. The readers' positive predictive value and negative predictive value were broadly consistent with each other and with the onsite read. Sensitivity was highest for readers 1 and 2 (68.5% and 63.9%, respectively) whereas specificity was highest for reader 3 (83.6%). Overall, prostate-level sensitivity was high (91.4%), but specificity was moderate (48.7%). Interreader agreement was 94.7%, 74.4%, and 70.3% for the lesion, prostate, and extraprostatic levels, respectively, with associated Fleiss' κ-values of 0.54, 0.50, and 0.57. Intrareader agreement was 97.8%, 96.9%, and 99.1% at the lesion level; 100%, 100%, and 91.7% in the prostate region; and 83.3%, 75.0%, and 83.3% in the extraprostatic region for readers 1, 2, and 3, respectively. Concordance between the BIE and the onsite reader exceeded 75% for each reader at the lesion, region, and subject levels. Conclusion: Specific training in the use of standardized interpretation methodology for assessment of 18 F-fluciclovine PET/CT images enables naïve readers to achieve acceptable diagnostic performance and reproducibility when staging recurrent prostate cancer. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Future of the Book? Challenge of the Digital World

ERIC Educational Resources Information Center

Pledger, Pat

2010-01-01

In the last ten years there has been much speculation about the role of e-books and e-book readers. This paper will look at the impact of e-book readers on publishing and reading, the types of e-book readers, their advantages and disadvantages. Some ideas for future e-books and e-book readers and their use in the library and classroom will be…

Breaking off Engagement: Readers' Disengagement as a Function of Reader and Text Characteristics

ERIC Educational Resources Information Center

Goedecke, Patricia J.; Dong, Daqi; Shi, Genghu; Feng, Shi; Risko, Evan; Olney, Andrew M.; D'Mello, Sidney K.; Graesser, Arthur C.

2015-01-01

Engagement during reading can be measured by the amount of time readers invest in the reading process. It is hypothesized that disengagement is marked by a decrease in time investment as compared with the demands made on the reader by the text. In this study, self-paced reading times for screens of text were predicted by a text complexity score…

Have You Heard the Word? For a Low-Budget Way to Get Kids Wild about Reading, Try Readers Theater

ERIC Educational Resources Information Center

Avi

2009-01-01

In its simplest sense, readers theater is the presentation of written work in a dramatic format. The fundamental difference between readers theater and traditional theater is that performers read their lines. With readers theater there's no memorization, and usually there are no sets or costumes. There's also little or no stage movement. It's…

Handheld E-Book Readers and Scholarship Report and Reader Survey: ACLS Humanities E-Book. White Paper No. 3

ERIC Educational Resources Information Center

Gielen, Nina

2010-01-01

This report describes a conversion experiment and subsequent reader survey conducted by the American Council of Learned Societies (ACLS) Humanities E-Book (HEB) in late 2009 and early 2010 to assess the viability of using scholarly monographs with handheld e-readers. As sample content, HEB selected six titles from its own online collection, three…

Eight Simple Secrets to More Persuasive Writing.

ERIC Educational Resources Information Center

Caswell, Donald

The goal of persuasive writing is to move the reader to action or to get the reader to refrain from action, and most of the secrets of persuasion can work either way. To get readers to take action, a writer has to appeal to the emotions as much as possible. To get readers to refrain from action, a writer must appeal to the intellect. The secrets…

An Investigation of Two Instructional Settings in the Use of Semantic Mapping with Poor Readers. Program Report 85-4.

ERIC Educational Resources Information Center

Pittelman, Susan D.; And Others

A study investigated whether semantic mapping is more effective for poor readers instructed in a small group of poor readers or in a class of students with mixed reading abilities. Students in five fourth-grade classes served as the control, receiving no semantic mapping instruction. Subjects, 39 fourth-grade poor readers, were presented semantic…

"It's All Coming Together": An Encounter between Implied Reader and Actual Reader in the Australian Rainforest

ERIC Educational Resources Information Center

Williams, Sandra J.

2008-01-01

In this paper I discuss how taking a particular literary theory--the implied reader--serves to offer a focus for the teacher's initial reading of a text and provides a formative assessment tool. Iser's Implied Reader theory is discussed, after which a picture book, "Where the Forest Meets the Sea" by Jeannie Baker, is analysed from this…

Prostate cancer localization with endorectal MR imaging and MR spectroscopic imaging: effect of clinical data on reader accuracy.

PubMed

Dhingsa, Rajpal; Qayyum, Aliya; Coakley, Fergus V; Lu, Ying; Jones, Kirk D; Swanson, Mark G; Carroll, Peter R; Hricak, Hedvig; Kurhanewicz, John

2004-01-01

To determine the effect of digital rectal examination findings, sextant biopsy results, and prostate-specific antigen (PSA) levels on reader accuracy in the localization of prostate cancer with endorectal magnetic resonance (MR) imaging and MR spectroscopic imaging. This was a retrospective study of 37 patients (mean age, 57 years) with biopsy-proved prostate cancer. Transverse T1-weighted, transverse high-spatial-resolution, and coronal T2-weighted MR images and MR spectroscopic images were obtained. Two independent readers, unaware of clinical data, recorded the size and location of suspicious peripheral zone tumor nodules on a standardized diagram of the prostate. Readers also recorded their degree of diagnostic confidence for each nodule on a five-point scale. Both readers repeated this interpretation with knowledge of rectal examination findings, sextant biopsy results, and PSA level. Step-section histopathologic findings were the reference standard. Logistic regression analysis with generalized estimating equations was used to correlate tumor detection with clinical data, and alternative free-response receiver operating characteristic (AFROC) curve analysis was used to examine the overall effect of clinical data on all positive results. Fifty-one peripheral zone tumor nodules were identified at histopathologic evaluation. Logistic regression analysis showed awareness of clinical data significantly improved tumor detection rate (P <.02) from 15 to 19 nodules for reader 1 and from 13 to 19 nodules for reader 2 (27%-37% overall) by using both size and location criteria. AFROC analysis showed no significant change in overall reader performance because there was an associated increase in the number of false-positive findings with awareness of clinical data, from 11 to 21 for reader 1 and from 16 to 25 for reader 2. Awareness of clinical data significantly improves reader detection of prostate cancer nodules with endorectal MR imaging and MR spectroscopic imaging, but there is no overall change in reader accuracy, because of an associated increase in false-positive findings. A stricter definition of a true-positive result is associated with reduced sensitivity for prostate cancer nodule detection. Copyright RSNA, 2004

Estimating age of sea otters with cementum layers in the first premolar

USGS Publications Warehouse

Bodkin, James L.; Ames, J.A.; Jameson, R.J.; Johnson, A.M.; Matson, G.M.

1997-01-01

We assessed sources of variation in the use of tooth cementum layers to determine age by comparing counts in premolar tooth sections to known ages of 20 sea otters (Enhydra lutris). Three readers examined each sample 3 times, and the 3 readings of each sample were averaged by reader to provide the mean estimated age. The mean (SE) of known age sample was 5.2 years (1.0) and the 3 mean estimated ages were 7.0 (1.0), 5.9 (1.1) and, 4.4 (0.8). The proportion of estimates accurate to within +/- 1 year were 0.25, 0.55, and 0.65 and to within +/- 2 years 0.65, 0.80, and 0.70, by reader. The proportions of samples estimated with >3 years error were 0.20, 0.10, and 0.05. Errors as large as 7, 6, and 5 years were made among readers. In few instances did all readers uniformly provide either accurate (error 1 yr) counts. In most cases (0.85), 1 or 2 of the readers provided accurate counts. Coefficients of determination (R2) between known ages and mean estimated ages were 0.81, 0.87, and 0.87, by reader. The results of this study suggest that cementum layers within sea otter premolar teeth likely are deposited annually and can be used for age estimation. However, criteria used in interpreting layers apparently varied by reader, occasionally resulting in large errors, which were not consistent among readers. While large errors were evident for some individual otters, there were no differences between the known and estimated age-class distribution generated by each reader. Until accuracy can be improved, application of this ageing technique should be limited to sample sizes of at least 6-7 individuals within age classes of >/=1 year.

Protocols development for security and privacy of radio frequency identification systems

NASA Astrophysics Data System (ADS)

Sabbagha, Fatin

There are benefits to adopting radio frequency identification (RFID) technology, although there are methods of attack that can compromise the system. This research determined how that may happen and what possible solutions can keep that from happening. Protocols were developed to implement better security. In addition, new topologies were developed to handle the problems of the key management. Previously proposed protocols focused on providing mutual authentication and privacy between readers and tags. However, those protocols are still vulnerable to be attacked. These protocols were analyzed and the disadvantages shown for each one. Previous works assumed that the channels between readers and the servers were secure. In the proposed protocols, a compromised reader is considered along with how to prevent tags from being read by that reader. The new protocols provide mutual authentication between readers and tags and, at the same time, remove the compromised reader from the system. Three protocols are proposed. In the first protocol, a mutual authentication is achieved and a compromised reader is not allowed in the network. In the second protocol, the number of times a reader contacts the server is reduced. The third protocol provides authentication and privacy between tags and readers using a trusted third party. The developed topology is implemented using python language and simulates work to check the efficiency regarding the processing time. The three protocols are implemented by writing codes in C language and then compiling them in MSP430. IAR Embedded workbench is used, which is an integrated development environment with the C/C++ compiler to generate a faster code and to debug the microcontroller. In summary, the goal of this research is to find solutions for the problems on previously proposed protocols, handle a compromised reader, and solve key management problems.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alvarez, P; Gonzalez, S; McDonald, L

Purpose: Analysis of the performance of the microSTARii reader for optically stimulated luminescence dosimeters (OSLD) used by the IROC Houston Quality Assurance Center (IROC HQAC) for external audits compare to the characteristics of the Microstar reader. Methods: The IROC HQAC uses the Microstar reader for the OSLD program for verification of output of photon, electron and proton beams. The calculation of dose from the OSLD system is based on a group of factors defined at time of the commissioning of a batch of detectors. Factors like system sensitivity (SS), depletion (KD), element correction factor (ECF), linearity (KL), energy correction (KE).more » The new microSTARii unit presents many hardware and software upgrades that were considered useful for this program. Based on these changes many factors, that were considered reader dependent, were revised in order to analyze the effect of the new reading process. The SS, KD, ECF and KL were evaluated and compared with data defined based on reading done on Microstar reader. Results: The SS is reader specific and specified at 100 cGy dose level. This value is define per reading session and monitored over time. The KD factor was found to be different because of reading procedure are different. The ECF values changed for a group of nano dots compare to values defined based on reading done on the Microstar reader. The KL was defined for the reader. Conclusion: The new microSTARii reader presents new features that improve the efficiency of the OSLD program at the IROC HQAC. New characterization is needed before final implementation is done to match the requirements of the existing OSLD system defined for the Microstar reader. Changes in uncertainty of the results has not been analized.« less

Semantic and phonological coding in poor and normal readers.

PubMed

Vellutino, F R; Scanlon, D M; Spearing, D

1995-02-01

Three studies were conducted evaluating semantic and phonological coding deficits as alternative explanations of reading disability. In the first study, poor and normal readers in second and sixth grade were compared on various tests evaluating semantic development as well as on tests evaluating rapid naming and pseudoword decoding as independent measures of phonological coding ability. In a second study, the same subjects were given verbal memory and visual-verbal learning tasks using high and low meaning words as verbal stimuli and Chinese ideographs as visual stimuli. On the semantic tasks, poor readers performed below the level of the normal readers only at the sixth grade level, but, on the rapid naming and pseudoword learning tasks, they performed below the normal readers at the second as well as at the sixth grade level. On both the verbal memory and visual-verbal learning tasks, performance in poor readers approximated that of normal readers when the word stimuli were high in meaning but not when they were low in meaning. These patterns were essentially replicated in a third study that used some of the same semantic and phonological measures used in the first experiment, and verbal memory and visual-verbal learning tasks that employed word lists and visual stimuli (novel alphabetic characters) that more closely approximated those used in learning to read. It was concluded that semantic coding deficits are an unlikely cause of reading difficulties in most poor readers at the beginning stages of reading skills acquisition, but accrue as a consequence of prolonged reading difficulties in older readers. It was also concluded that phonological coding deficits are a probable cause of reading difficulties in most poor readers.